IMPACT DES INHIBITEURS DE LA VOIE mTOR SUR LA

UNIVERSITE DE FRANCHE-COMTE
UFR SCIENCES MEDICALES ET PHARMACEUTIQUES DE BESANCON
ECOLE DOCTORALE "ENVIRONNEMENT, SANTE"
Thèse en vue de l’obtention du titre de docteur en
SCIENCES DE LA VIE
Discipline: Immunologie
IMPACT DES INHIBITEURS DE LA VOIE mTOR SUR
LA REPONSE IMMUNITAIRE T ANTI-TUMORALE
Présentée et soutenue publiquement le 30 Octobre 2015 par
Laurent BEZIAUD
Sous la direction de M. le Professeur Olivier ADOTEVI
JURY
Docteur Nathalie BONNEFOY, Université de Montpellier
Docteur Christophe CAUX, Université de Lyon 1
Rapporteur
Rapporteur
Professeur Philippe SAAS, Université de Franche-Comté, Besançon
Docteur Corinne TANCHOT, Université Paris Descartes
Professeur Pierre TIBERGHIEN, Université de Franche-Comté, Besançon
Examinateur
Examinateur
Examinateur
2
REMERCIEMENTS
Ce travail de thèse a été effectué au sein du laboratoire "Interaction Hôte-GreffonTumeur et Ingénierie Cellulaire et Génique" de l'unité UMR1098. Je tiens ainsi à remercier le
Professeur Philippe SAAS et le Professeur Christophe BORG qui m'ont accueilli au sein de
leur équipe.
Je souhaite également témoigner ma reconnaissance aux membres du jury qui m'ont
fait l'honneur d'accepter de juger de ce travail. Merci en particulier au Docteur Nathalie
BONNEFOY et au Docteur Christophe CAUX qui ont consacré de leur temps à la lecture de
ce manuscrit et à l'évaluation de la qualité du travail effectué durant cette thèse. Merci
également au Professeur Pierre TIBERGHIEN d'avoir présidé ce jury de thèse.
Je souhaite remercier la Ligue Nationale Contre le Cancer pour leur soutien financier
en dernière année qui m'a permis de finaliser cette thèse.
Je remercie tout particulièrement mon directeur de thèse, le Professeur Olivier
ADOTEVI, qui m'a guidé pendant ces quatre années de thèse. Je le remercie pour son soutien,
la confiance qu'il m'a accordée en me faisant venir de Paris, puis tout au long de la thèse, son
enthousiasme et sa bonne humeur. Tu as toujours été présent durant ces quatre années, et m'as
toujours poussé vers le haut. J'ai beaucoup appris à tes côtés, et je sors grandis de cette thèse
grâce toi.
Je tiens également à remercier le Docteur Corinne TANCHOT qui a accepté de faire
partie de mon jury de thèse, mais plus particulièrement pour m'avoir fait découvrir le monde
de la recherche. Tu m'as fait aimer la recherche et l'Immuno, et surtout tu m'as toujours
soutenu et as tout fait pour m'aider à entrer en thèse. C'était important pour moi que tu sois
présente le jour de ma thèse.
Je tiens à adresser toute ma gratitude aux personnes qui m'ont permis de mener à bien
ces travaux de recherche. Je souhaite remercier en particulier Caroline, la grande chef de la
Plateforme. En plus de gérer les prélèvements, tu as toujours été là pour me donner des
conseils en cytométrie, et surtout tu as toujours été une épaule pour m'écouter et discuter
quand j'en avais besoin. Et en prime, toujours un petit chocolat pour moi. Je remercie
également Yann, le grand chef conseiller des thésards. Tu es toujours de bons conseils, et as
toujours une réponse à nos questions. J'ai énormément apprécié ta bonne humeur et ton
humour, même si on ne comprend pas toujours tes blagues! Je souhaite également remercier
Lise, la grande chef du L0. Merci de ton aide précieuse pour les innombrables Elisa
simultanés, et surtout c'était toujours agréable de te voir pour discuter au bureau.
Une petite pensée pour la relève, Elodie LMJ, qui entre en thèse et donne un petit coup
de main pour finaliser ce projet mTOR après mon départ. Merci de ta gentillesse, je ne suis
plus ton maître maintenant, c'est à toi de devenir la boss. Bon courage pour ta thèse, tout ira
bien, tu as le potentiel mais arrête d'être trop gentille!
3
Je souhaite également remercier tous les membres du labo que j'ai pu côtoyer dans le
laboratoire et qui m'ont toujours encouragé et permis de travailler dans une super ambiance:
Jeremy le footballeur, tu as toujours le mot pour rire et m'as permis de toucher encore le
ballon à Besançon. Jeanne ma voisine, ma fournisseuse officielle de chocobons. Sindy, qui
aime se déguiser sans lunettes pour ne pas que je la retrouve au labo. Fanny, sans qui je ne
pourrais rentrer le weekend à Créteil. Sans oublier Carron, Geoffrey, Adam, Clémentine,
Kiki, Claire, Chantal de l'anim, Idir, JMC, Elodie BR, Marie, Laurie S, Afag, JR, Kamal,
Marine, Tristan, Sabéha, Eléonore, Gaëlle, Adeline R, Anne Dup', Patricia, Sarah, Laurie R,
Francis, Adeline B, Charline T…
C'est également important pour moi de remercier les anciens thésards que j'ai côtoyé,
qui sont mes amis et qui ont été mes mentors. Romain "Jobs", la star de l'UMR, qui m'a
énormément aidé et conseillé dans cette dernière ligne droite. Magalie, à qui j'ai succédé et
qui a été mon modèle. J'espère avoir fait aussi bien que toi. Charline, qui m'a appris beaucoup
de manips in vitro, et que j'ai toujours appelé à l'aide en cas d'oubli. Merci de ta sympathie.
Enfin, les thésards de mes anciens labo, Sara qui a toujours été de bons conseils, même de
loin, et mon grand ami Fédérico, que j'admire et qui a toujours été là quand il le fallait, et sans
qui je n'aurai jamais pu venir à Besançon. C'est grâce à toi que j'ai pu réaliser cette thèse.
Je souhaite également remercier tous mes proches hors du labo, qui m'ont également
aidé, au moins par leur présence, et par les bons moments passés ensembles, agréables durant
les difficiles années de thèse. Une petite pensée pour mes amis de Créteil, les Gibbons. La
Brèche m'a vraiment manqué. Je pense également à mes amis de Fac, les B3 et leur entourage,
ainsi que mes amis du master ITB, et mon amie Nathalie, qui m'a touché en réussissant à
venir de loin pour assister à la thèse. Enfin, merci à ma mère qui a toujours été là quand il le
fallait, et une pensée pour Bà, qui restera toujours dans un coin de ma tête.
A Emilie, mon bras droit au labo et amie. Je n'oublierai pas les bons moments de
rigolade au labo qui m'ont permis de tenir bon durant les moments difficiles. Quelques
missions resteront mythiques. Merci de ton soutien constant. Notre amitié sincère a été une
belle découverte durant cette thèse. J'espère que tu garderas un bon souvenir de mon passage
au labo et ne me remplaceras pas trop vite…!
Enfin, je remercie du fond du cœur Laura, ma deuxième moitié, avec qui j'ai partagé
ce projet de bout en bout, et bien plus. Merci de ton aide, de ta présence et de ton soutien, que
ce soit durant les bons moments ou les périodes difficiles. Tu m'as beaucoup apporté, tant
professionnellement que personnellement, et m'as permis d'arriver au bout de cette thèse.
Mais ce n'est que le début du chemin. TVB. A toi de jouer maintenant, la suite est entre de
bonnes mains!
Merci.
4
RESUME
La voie de signalisation mTOR (mammalian Target Of Rapamycin) joue un rôle central
dans la croissance cellulaire, le métabolisme, et l’homéostasie des lymphocytes T (LT). Lors de la
transplantation d'organes, l'administration de rapamycine, un inhibiteur de mTOR (mTORi),
bloque l'activation des LT et promeut la polarisation des lymphocytes T CD4 régulateur (Treg). En
cancérologie, des mTORi sont utilisés pour leur action inhibitrice sur la prolifération et
l'angiogenèse tumorales. Cependant l'immunosuppression via l’induction de T reg nécessaire à la
prévention du rejet de greffe pourrait être délétère pour la réponse anti-tumorale. Notre hypothèse
est que l’efficacité clinique des mTORi serait également dépendante de la modulation de
l’immunité adaptative T induite par ces traitements chez les patients atteints de cancer.
Au cours de cette thèse, nous avons abordé cette question immunologique dans une
cohorte prospective de patients atteints de cancer rénal métastatique (mRCC) traités par
évérolimus. L'analyse du taux de Treg et de la réponse spontanée T CD4 Th1 anti-tumorale (antitélomérase TERT) par Elispot-IFN-g a été effectuée au moment de l'inclusion des patients et tous
les deux mois après le début du traitement. Nous avons observé chez la majorité des patients une
augmentation du taux de Treg après traitement par évérolimus. Ces Treg expriment Hélios,
suggérant un phénotype Treg naturel. La fréquence et la qualité de la réponse Th1 anti-TERT sont
également augmentées suite au traitement. Nous avons montré que conjointement ces deux
paramètres immunologiques corrèlent avec l’efficacité clinique du traitement. Les patients
présentant précocement une diminution des Treg associée à une augmentation des Th1 anti-TERT
ont une meilleure survie par rapport aux patients dont les paramètres immunitaires ne variaient
pas, ou variaient dans une même direction (13,2 mois vs 8 et 4 mois). De plus, au moment de la
progression la plupart des patients perdaient leur réponse Th1 anti-TERT, et cet effet était associé
à une augmentation des Treg. Les Treg traités par mTORi in vitro inhibent plus fortement la
prolifération de LT allogéniques, par un mécanisme contact dépendant. Par l'utilisation d'anticorps
monoclonaux déplétant les LT chez la souris et par l'utilisation de souris DEREG, nous avons
montré que la présence de Treg in vivo altère l’efficacité anti-tumorale des mTORi, par un
mécanisme impliquant l’inhibition des réponses T CD8 anti-tumorales. En conséquence,
l’efficacité des mTORi a pu être augmentée par sa combinaison avec des agents bloquant les T reg.
En addition, l’administration de temsirolimus améliore l’efficacité anti-tumorale d’un vaccin
thérapeutique, en favorisant la différenciation des LT CD8 anti-tumoraux centraux mémoires
(CD62L+CD127+) et précurseurs mémoires (CD127+KLRG1lo) induits par la vaccination.
En conclusion, ces études ont montré pour la première fois le rôle de l’immunité T antitumorale sur l’efficacité clinique des mTORi et soulignent ainsi l’intérêt potentiel de combiner les
mTORi avec des immunothérapies anti-tumorales.
5
6
ABSTRACT
The mammalian Target Of Rapamycin (mTOR) pathway plays a central role in cell
growth, metabolism and T lymphocytes homeostasis. In organ transplant patients, the mTOR
inhibitor (mTORi) rapamycin is prescribed to inhibit T cell activation and promote FoxP3+
regulatory CD4 T cells (Tregs). In cancer patients, mTORi are administrated for their antiproliferative and anti-angiogenic properties on tumor cells. However, the immunosuppressive
profile required to prevent graft rejection could be detrimental for anti-tumor responses. We
hypothesized that clinical efficacy of mTORi could be dependant of an adaptive T immunity
modulation in cancer patients.
Here we performed a dynamic immunomonitoring study in metastatic renal cell
carcinoma (mRCC) patients treated with everolimus. The monitoring of Tregs and the
spontaneous tumor-specific Th1 (anti-telomerase TERT) response evaluated by using IFN-gELISPOT were performed within blood at baseline and every two months after the beginning
of everolimus treatment.
Concomitant increase of Tregs and anti-TERT Th1 response occurred during
everolimus treatment in most patients. We showed that patients' Tregs express Helios,
suggesting a natural Tregs phenotype. The frequency and quality of anti-TERT Th1 responses
are increased following treatment. We identified three immune groups based on the early
modulation of both Treg and anti-tumor Th1 cells and found that patients with {low Tregs plus
high anti-tumor Th1 cells} show the best survival. At disease progression, most patients loose
the anti-tumor Th1 response and this is associated with an increase of Tregs. In vitro mTORiexposed Tregs show higher immunosuppressive properties compared to untreated Tregs by
inhibiting T cells proliferation, in a contact-dependant manner. By using monoclonal
depleting antibodies in mice and by using the DEREG transgenic mouse model, we show that
the presence of Tregs in vivo alters the responses to mTORi via a mechanism involving the
inhibition of antitumor CD8 T cell responses. Furthermore the efficacy of mTORi is improved
by combination with Tregs depleting agents such as sunitinib and a CCR4 antagonist. In
addition, temsirolimus treatment increases the antitumor efficacy of therapeutic vaccine by
promoting tumor-specific central memory (CD62L+CD127+) and precursor memory
(CD127+KLRG1lo) CD8 T cell differentiation.
Altogether, our results describe for the first time a dual impact of host adaptive
antitumor T cell immunity on the clinical effectiveness of mTORi and prompt their
association with immunotherapies.
7
8
TABLE DES MATIERES
LISTE DES ABREVIATIONS .................................................................................... 11
LISTE DES FIGURES ET TABLEAUX..................................................................... 13
CONTEXTE SCIENTIFIQUE ..................................................................................... 15
Chapitre I: LA VOIE mTOR .................................................................................... 17
I. LA VOIE DE SIGNALISATION PI3K/AKT/mTOR ....................................... 19
1. Découverte ..................................................................................................... 19
2. Les composants de la voie mTOR ................................................................. 21
3. Régulation de la voie mTOR ......................................................................... 21
4. Fonctions régulées par la voie mTOR ........................................................... 24
4.1. Synthèse protéique et lipidique ............................................................... 24
4.2. Métabolisme et survie cellulaire ............................................................. 25
4.3. Autophagie .............................................................................................. 26
II. mTOR ET REPONSES IMMUNITAIRES ...................................................... 27
1. Rôle de la voie mTOR dans la régulation lymphocytaire T .......................... 27
1.1. Homéostasie lymphocytaire T ................................................................ 27
1.2. Activation des lymphocytes T ................................................................ 28
1.3. Migration des lymphocytes T ................................................................. 29
2. Importance de mTOR dans la différenciation lymphocytaire T CD4 ........... 30
2.1. mTOR et différenciation des lymphocytes T CD4 Th1, Th2 et Th17 .... 32
2.2. mTOR et différentiation des LT CD4 régulateurs .................................. 34
2.2.1. mTOR et promotion des Treg .......................................................... 34
2.2.2. Implication de mTOR dans la régulation fonctionnelle des Treg .... 36
3. Rôle de mTOR dans la différenciation des LT CD8 ..................................... 37
3.1. Les LT CD8 mémoires ........................................................................... 37
3.2. L’inhibition de mTOR favorise la génération des LT CD8 mémoires ... 39
4. Rôle de mTOR sur les autres cellules du système immunitaire .................... 41
4.1. mTOR et lymphocytes B ........................................................................ 41
4.2. mTOR et cellules dendritiques ............................................................... 42
4.3. mTOR et cellules myéloïdes suppressives .............................................. 43
4.4. mTOR et cellules natural killer (NK) ..................................................... 44
9
III. CIBLAGE THERAPEUTIQUE DE LA VOIE mTOR .................................. 45
1. Ciblage de mTOR en transplantation d’organes ........................................... 45
1.1. La rapamycine......................................................................................... 45
1.2. Immunosuppression induite par la rapamycine ...................................... 46
1.3. Rapamycine et induction de Treg ............................................................. 47
2. Ciblage de mTOR dans les cancers ............................................................... 48
2.1. Dérégulations de la voie mTOR dans le cancer ...................................... 49
2.1.1. Mutations activatrices ..................................................................... 49
2.1.2. Conséquences de la sur-activation de mTOR ................................. 50
2.2. Blocage de la voie mTOR en cancérologie ............................................ 51
2.2.1. La rapamycine et ses analogues ..................................................... 51
2.2.2. Efficacité anti-tumorale des rapalogues ......................................... 52
Chapitre II : IMMUNITE ET CANCER .................................................................. 53
I. IMMUNITE T ANTI-TUMORALE ................................................................. 55
1. Immunosurveillance des cancers et Immunoedition ..................................... 55
2. Rôles des LT CD4 et cancer ......................................................................... 56
2.1. Rôle des LT CD4 Th1 dans la réponse anti-tumorale ............................ 56
2.2. Rôle des Treg dans la réponse pro-tumorale ............................................ 57
2.3. Rôles des autres LT CD4 helper dans la réponse anti-tumorale ............. 60
II. CANCER DU REIN ET IMMUNITE ............................................................. 63
1. Généralités ..................................................................................................... 63
2. Le cancer du rein, une tumeur immunogène ................................................. 64
3. Les traitements anti-mTOR dans le RCC ...................................................... 65
4. Immunothérapies dans le RCC ...................................................................... 66
RATIONNEL ET OBJECTIFS .................................................................................... 69
RESULTATS ............................................................................................................... 73
ARTICLE 1: The efficacy of rapalogs everolimus and temsirolimus relies on a
drastic modulation of adaptive antitumor T cell immunity .................................................. 75
ARTICLE 2: A CCR4 antagonist combined with mTOR inhibitors improves
vaccination-induced antitumor memory CD8 T cell responses .......................................... 113
DISCUSSION............................................................................................................. 135
CONCLUSION ET PERSPECTIVES ....................................................................... 153
BIBLIOGRAPHIE ..................................................................................................... 157
ANNEXES ................................................................................................................. 183
10
LISTE DES ABREVIATIONS
A
M
AMPK: AMP-activated protein kinase
ARN: Acide Ribonucléique
ATP: Adénosine Triphosphate
Bcl2: B-cell leukemia/lymphoma-2
MAPK: Mitogen-activated protein kinase
MLST8: Mammalian lethal with sec-13 protein 8
mRCC:
Carcinome
des
cellules
rénales
métastatique
mTOR: mammalian Target Of Rapamycin
mTORi: inhibiteur de mTOR
C
P
CD: Cluster Differentiation
CMH: Complexe Majeur Histocompatibilité
CPA: Cellules présentatrices d’Antigènes
CXCR: Chemokine Receptor
PD-1: Programmed Death 1
PDCD4: Programmed cell death 4
PDGF1: Platelet-derived growth factor
PDL1: Programmes Death Ligand 1
PI3K: Phosphoinositide 3 kinase
PIP2: Phosphatidylinositol 3,4-bisphosphate
PP2A: Phospho-protein phosphatase 2A
Pras40: prolin-rich Akt substrate 40kDa
PTEN: phosphatase and tensin homolog deleted on
chromosome 10
Protor 1/2: protein observed with rictor 1 and 2
B
D
DC: Cellule Dendritique
Deptor: DEP domain containing mTOR-interacting
protein
E
4EBP: eIF4E binding protein
eEF2K: Eukaryotic Elongation Factor-2 Kinase
EGF: Epidermal Growth Factor
eIF4: eukaryotic initiation factor 4
ERK: Extracellular signal-regulated kinase
F
FAT: Focal adhesion targeting
FIP200: Focal adhesion kinase family–interacting
protein of 200 kDa
FOXO: Forkhead family of transcription factors
FoxP3: Forkhead box P3
FRB: FKBP12-rapamycin binding domain
G
GAP: GTPase-activating protein
H
HIF1a: Hypoxia-Inducible Factor-1
I
IFN-g: Interféron gamma
IGF1: Insulin Growth Factor 1
IKKb: IkappaB kinase
IL: Interleukine
IRF1: Interferon regulatory factor 1
IRS: Insulin Substrat Receptor
K
R
RAG: Recombinaison activating gene
Raptor:
regulatory-associated
protein
of
mammalian target of rapamycin
Rictor: rapamycin-insensitive companion of mTOR
Rheb: Ras homolog enriched in brain
RORgT: Retinoic acid receptor-related orphan
receptor gamma
RSK1: Ribosomal protein S6 kinase
S
S6K: Small subunit ribosomal protein S6 Kinase
SOCS: Suppressor of cytokine signalling
STAT3: Signal transducer and activator of
transcription
T
TCM: Lymphocyte T central mémoire
TCR: T cell receptor
TEM: Lymphocyte T effecteur mémoire
TERT: Telomérase reverse transcriptase
Tfh: follicular helper T cell
Th: T helper
TNF-a: Tumor Necrosis Factor
TSC: Tuberous sclerosis complex
TSCM: Lymphocyte T souche mémoire
V
VEGF:
Vascular
Endothelial
Growth
Factor
KLF2: Kruppel-like factor 2
11
12
LISTE DES FIGURES ET TABLEAUX
Figure 1: Structure de TOR et de la rapamycine ...................................................................... 20
Figure 2: Complexes mTOR et rôle central dans l’homéostasie cellulaire .............................. 20
Figure 3: Mécanismes de régulation de la voie de signalisation mTOR .................................. 22
Figure 4: Fonctions régulées par la voie mTOR. ..................................................................... 26
Figure 5 Les 3 signaux impliqués dans l’activation lymphocytaire T (Halloran, 2004) .......... 28
Figure 6: Impact de mTOR dans la migration lymphocytaire T .............................................. 30
Figure 7: Polarisation des différentes sous-populations de LT CD4 ....................................... 31
Figure 8: Implication des molécules en aval de la voie mTOR dans la différenciation LT CD4
helper Th1, Th2 et Th17 ........................................................................................................... 33
Figure 9: Mécanismes de la différenciation Treg mTOR-dépendante ....................................... 35
Figure 10: Cinétique de la réponse T CD8 anti-virale ............................................................. 37
Figure 11: Les différentes sous-populations LT mémoires ...................................................... 38
Figure 12: Impact de la rapamycine dans la génération de LT CD8 mémoires (Araki et al.,
2010)......................................................................................................................................... 40
Figure 13: mTOR dans la différenciation LT CD8 mémoire ................................................... 41
Figure 14: Mode d'action des immunosuppresseurs utilisés en transplantation sur les 3 signaux
d’activation lymphocytaire T ................................................................................................... 46
Figure 15: Impact de la rapamycine sur la différentiation des LT CD4 helper........................ 47
Figure 16: Dérégulation de la voie mTOR et oncogenèse ....................................................... 51
Figure 17: Treg et microenvironnement tumoral. ...................................................................... 58
Figure 18: Mécanismes de suppression utilisés par les Treg. .................................................... 60
Figure 19: Rôle des différentes sous-populations lymphocytaires T CD4 dans le contrôle de
l’immunité anti-tumorale.......................................................................................................... 62
Figure 20: Modèle suggéré de la modulation des réponses immunitaire anti-tumorale médiée
par les mTORi ........................................................................................................................ 155
Tableau 1: Comparaison des nTreg et iTreg (Bilate and Lafaille, 2012) .................................... 34
13
14
CONTEXTE SCIENTIFIQUE
15
16
Chapitre I:
LA VOIE mTOR
17
18
I. LA VOIE DE SIGNALISATION PI3K/AKT/mTOR
La mammalian (ou "mechanistic") Target Of Rapamycin (mTOR) est une protéine
impliquée dans une voie de signalisation cruciale pour un transit efficace entre métabolisme
anabolique (synthèse des protéines, de lipides, stockage de nutriments) et catabolique
(dégradation des protéines, autophagie). En réponse à des signaux environnementaux, mTOR
contrôle ainsi divers processus permettant de générer ou d'utiliser de grandes quantités
d'énergie et de nutriments, et impacte sur la plupart des fonctions cellulaires majeures, telles
que la croissance ou la prolifération cellulaire.
1. Découverte
La mammalian Target Of Rapamycin est une sérine-thréonine kinase de 289 kDa
appartenant à la famille des phosphatidylinositol 3-kinase-related kinases (PIKK), située en
aval de la signalisation PI3K-Akt. Tel que son nom l'indique, la découverte de mTOR est liée
à celle de la rapamycine, molécule dont elle est la cible. La rapamycine est un macrolide
antifongique produit par la bactérie Streptomyces hygroscopicus isolée en 1965 sur l'île Rapa
Nui (île de Pâques) (Vézina et al., 1975). Au début des années 1990, des criblages génétiques
chez la levure Saccharomyces cerevisiae ont permis d’identifier les gènes DRR1 (ou TOR1)
et DRR2 (ou TOR2) comme médiateurs des effets toxiques de la rapamycine sur la levure
(Heitman et al., 1991; Kunz et al., 1993). TOR et les mécanismes d'action de la rapamycine
ont ensuite été découverts comme conservés de la levure à l'Homme et des approches
biochimiques ont permis d’isoler pour la première fois mTOR, cible physique de la
rapamycine chez les mammifères (Brown et al., 1994; Sabatini et al., 1994; Sabers et al.,
1995). Bien que sa découverte initiale chez la levure ait permis d'identifier deux gènes, dans
la plupart des organismes, TOR est encodé par un seul gène contenant de multiples domaines.
L'extrémité N-terminale contient jusqu'à 20 répétitions en tandem HEAT (un domaine
d'hélices-a antiparallèles, le facteur d'élongation 3, PP2A et TOR) importantes pour les
interactions protéine-protéine, suivie par un domaine FAT (FRAP, ATM, TRRAP). La partie
C-terminale de TOR contient le domaine kinase et le domaine de liaison (FRB) de la
rapamycine. L'extrémité C-terminale contient un domaine FATC, qui est associé au domaine
FAT dans toutes les protéines de la famille PIKK pour moduler leur activité kinase (Figure 1).
19
Figure 1: Structure de TOR et de la
rapamycine
(Benjamin et al., 2011)
De par ses larges propriétés anti-prolifératives, la découverte de la rapamycine a attiré
l'attention sur le rôle et l'importance de mTOR. En plus d'être hautement conservée et
constitutivement exprimée par toutes les cellules eucaryotes, il est apparu que la protéine
mTOR possède un rôle central dans l'homéostasie cellulaire: en réponse à des signaux
environnementaux indiquant la disponibilité en nutriments, en facteurs de croissance ou en
énergie, ou le stress subit par la cellule, mTOR contrôle la croissance, la prolifération et le
métabolisme cellulaire, l'autophagie ou l'angiogenèse (Figure 2).
Dommages
à l’ADN
Facteurs de
croissance
Hypoxie
Nutriments
(acides aminés)
Energie
(ATP)
Stress
mTOR
Raptor
Pras40
mLST8
mTOR Tti1
Deptor
Tel2
Rictor mLST8
mSin1 mTOR Tti1
Tel2
Protor1/2 Deptor
mTORC1
mTORC2
Autophagie
Prolifération
Organisation du
cytosquelette
Angiogénèse
Survie
Métabolisme
Croissance
Figure 2: Complexes mTOR et rôle central dans l’homéostasie cellulaire
20
2. Les composants de la voie mTOR
La sérine thréonine kinase mTOR exerce ses fonctions à travers deux principaux
complexes, mTOR complexe 1 (mTORC1) et 2 (mTORC2) (Figure 2).
mTORC1. C'est le complexe le mieux caractérisé parmi les deux. Il est formé de cinq
protéines: Raptor, Pras40, mLST8, Deptor et le complexe Tti1/Tel2 (Hara et al., 2002;
Kaizuka et al., 2010; Kim et al., 2003; Peterson et al., 2009; Vander Haar et al., 2007). Raptor
est une protéine de 150 kDa qui se fixe au complexe et permet la fixation et la
phosphorylation de protéines en aval, telles que S6K, 4EBP ou STAT3. Raptor est également
important dans la régulation de la localisation intracellulaire de mTORC1 (Kim et al., 2002;
Sancak et al., 2008). mLST8 est un régulateur positif de mTORC1 (Kim et al., 2003), tandis
que Pras40 et Deptor sont des régulateurs négatifs de mTORC1. Deptor régule négativement
mTORC1 à travers le domaine FAT (FRAP-ATM-TTRAP) (Peterson et al., 2009). Lorsque
mTORC1 est activé, il peut phosphoryler ces deux composants, et réduire leur interaction
avec mTORC1(Peterson et al., 2009). Tti1/Tel2 a principalement pour rôle d'assurer
l'assemblage et la stabilité du complexe mTORC1 (Kaizuka et al., 2010).
mTORC2. En plus de posséder en commun avec mTORC1 les molécules mLST8,
Deptor et Tti1/Tel2, le complexe mTORC2 est également composé de Rictor, mSin1 et
protor1/2 (Jacinto et al., 2006; Pearce et al., 2007; Sarbassov et al., 2005). mLST8, Deptor et
Tti1/Tel2 ont un rôle similaire à celui qu'ils tiennent dans le complexe mTORC1. Rictor est
essentiel à l'activité catalytique de mTORC2, et pourrait agir dans le recrutement des autres
substrats composant le complexe (Sarbassov et al., 2004). Il se lie à mSin1 et ces protéines
s'aident à se stabiliser (Jacinto et al., 2006).
3. Régulation de la voie mTOR
Un environnement cellulaire suffisamment riche en facteurs de croissance, oxygène,
acides aminés ou énergie, active la voie mTOR qui permet en conséquence à la cellule de
synthétiser les protéines nécessaires pour croître et survivre. Au contraire, un appauvrissement
de l'environnement ou un état de stress cellulaire inhibe mTOR qui fait transiter la cellule vers
un métabolisme catabolique et un arrêt de sa croissance. Les mécanismes de régulation de la
voie mTOR et les fonctions en résultant sont présentés dans les figures 3 et 4 respectivement.
21
INHIBITION mTOR
Hypoxie
ACTIVATION mTOR
Energie
Diminution ATP
Insuline, IGF1
PDGF1, EGF
AMPK
TNF-a
Acides
aminés
IKKb
Rag-GDP
IRS
Dommages
À l’ADN
REDD1
Facteurs de
croissance
PI2P
p53
Ras
PI3K
PTEN
PI3P
Raf
mTORC2
PDK1
MEK 1/2
Akt
ERK 1/2
+
RSK
-
-
TSC1/2
-
+
Rheb-GTP
Rheb-GDP
-
+
Rag-GTP
mTORC1
Figure 3: Mécanismes de régulation de la voie de signalisation mTOR
22
Le complexe TSC1/TSC2. Les travaux explorant la régulation de mTOR ont porté plus
particulièrement sur l'étude de la signalisation impliquant le complexe mTORC1. L'activation
de mTORC1 est principalement dépendante de l'hétérodimère composé de TSC1 (tuberous
sclerosis, ou hamartin) et TSC2 (tuberin), situé juste en amont de mTORC1 et qui joue un rôle
clé dans la régulation du complexe. TSC1/TSC2 inhibe mTORC1 en fonctionnant comme une
GTPase-activating protein (GAP) pour convertir Rheb-GTP en Rheb-GDP. Rheb-GTP
interagit directement et active fortement mTORC1. Lorsque le complexe TSC1/TSC2 est
phosphorylé, sa fonction répressive est absente et permet à Rheb-GDP de réguler
négativement mTORC1 (Inoki et al., 2003; Tee et al., 2002).
Insuline et facteurs de croissance "insulin-like". L'activité mTOR est fortement liée à
la signalisation PI3K. L’insuline, ou des membres de la famille " insulin-like growth factors "
tels qu'IGF1 (insulin-like growth factor 1), PDGF1 (platelet-derived growth factor) ou EGF
(epidermal growth factor) vont se fixer sur des récepteurs ayant une activité tyrosine kinase et
induire une signalisation via PI3K. Une phosphorylation des résidus tyrosine du récepteur
activé va permettre l'activation d'effecteurs en aval, dont IRS (insulin substrate receptor) qui
va se fixer à PI3K et l'activer. PI3K induit la phosphorylation de PIP2 en PIP3, tandis que
PTEN (phosphatase and tensin homolog deleted on chromosome 10) s'oppose à l'activité
PI3K en déphosphorylant PIP3 en PIP2. PIP3 peut directement activer mTORC2 (Zinzalla et
al., 2011) ou recruter la sérine thréonine kinase Akt à proximité de la membrane plasmique,
où elle est à son tour phosphorylée et activée par la kinase PDK1. Akt induit alors l'activation
de mTORC1 en phosphorylant et provoquant la dissociation de raptor et PRAS40, un
inhibiteur de mTORC1 (Vander Haar et al., 2007; Wang et al., 2007).
Facteurs de croissance. Les facteurs de croissance peuvent également activer
mTORC1 via l'accumulation d'acides phosphatidiques, qui vont se fixer sur mTORC1 et
activer la signalisation en aval (Fang et al., 2003). Akt/PKB, ERK1/2 et RSK1 activés par la
cascade de signalisation induite par les facteurs de croissance vont directement phosphoryler
et inhiber le complexe TSC1/TSC2, et induire l'activation de mTORC1 (Inoki et al., 2002;
Potter et al., 2002).
Cytokines pro-inflammatoires et acides aminés. Des cytokines pro-inflammatoires
telles que le TNF-a peuvent activer mTORC1 en bloquant TSC1/TSC2 via IKKb (Lee et al.,
2007). Certains acides aminés, en particulier la leucine et l'arginine, peuvent également
23
activer mTOR. La signalisation via les Rag GTPase est alors impliquée, favorisant la liaison
avec Raptor, qui va activer mTORC1 via Rheb (Hara et al., 1998).
Stress cellulaire. Au contraire, un stress cellulaire dû à un faible taux énergétique ou
d'oxygène, ou dû à des dommages à l'ADN va agir sur la voie mTOR et cette fois-ci l'inhiber
et ainsi diminuer la consommation énergétique et nutritionnelle. Lorsque le niveau d'ATP est
bas ou en cas d'hypoxie, l'AMPK va phosphoryler TSC2 et augmenter l'activité GAP sur Rheb
et ainsi inhiber mTORC1 (Inoki et al., 2006). L'AMPK peut également agir directement sur
mTORC1 en phosphorylant Raptor et inhiber allostériquement le complexe (Gwinn et al.,
2008). L'hypoxie peut également induire l'expression de REDD1 (transcriptional regulation of
DNA damage response I), qui va activer le complexe TSC1/TSC2 (Brugarolas et al., 2004).
Des dommages à l'ADN peuvent aussi inhiber mTORC1 via des mécanismes impliquant une
transcription p53-dépendante. p53 va activer l'AMPK via un mécanisme dépendant de Sestrin
1/2, conduisant à l'activation de TSC2 et l'inhibition de l'activité mTORC1 (Budanov and
Karin, 2008). p53 peut également activer directement TSC2, ainsi que PTEN, et inhiber
l'activation de mTOR (Feng et al., 2005).
4. Fonctions régulées par la voie mTOR
4.1. Synthèse protéique et lipidique
La synthèse protéique est le processus contrôlé par mTORC1 le mieux caractérisé.
mTORC1 va moduler la traduction d'ARNm spécifique en régulant la phosphorylation de
différentes protéines impliquées dans la traduction, notamment S6K1, 4E-BP1 et eEF2K (Ma
and Blenis, 2009). S6K1 régule la taille cellulaire, la traduction des protéines et la
prolifération cellulaire (Holz, 2012). L'activation de S6K1 par mTORC1 va induire
l'augmentation de la synthèse protéique via la régulation de différents substrats: S6K1 peut
induire la phase d'élongation de la traduction en phosphorylant et inhibant eEF2K, un
répresseur d'eEF2. S6K1 permet également l'activation d'eIF4A, une hélicase ARN qui
favorise le mouvement du ribosome et permet une traduction efficace. S6K1 phosphoryle et
permet la dégradation de PDCD4 (programmed cell death 4), un inhibiteur d'eIF4G (Dorrello
et al., 2006) et recrute eIF4B (eukaryotic initiation factor 4B) qui active eIF4A (Raught et al.,
2004). Enfin, S6K1 va phosphoryler S6, un composant de la sous unité ribosomale 40S et va
induire une augmentation de la synthèse protéique. L'activation de mTORC1 et la
phosphorylation de 4E-BP1 tiennent un rôle important dans la synthèse de protéines
impliquées dans la croissance cellulaire et dans la progression du cycle cellulaire. Ainsi,
24
l'inhibition par phosphorylation de 4E-BP1 permet l'activation d'eIF4E qui se fixe sur eIF4G.
Ce complexe dirige l'appareil traductionnel, et initie la traduction en particulier de c-myc,
cycline D1 et ornithine decarboxylase. D'autres mécanismes sont également impliqués, avec
l'activation de TIF-1A qui va promouvoir l'interaction avec ARN Pol I et l'expression d'ARN
ribosomique (Mayer et al., 2004) ou la phosphorylation et l'inhibition de Maf1 un répresseur
de l'ARN Pol III, qui induit l'ARN ribosomique 5S et la transcription d'ARN de transfert
(Kantidakis et al., 2010). La voie mTOR régule également l'expression d'HIF1a (hypoxia
inducible factor 1a), qui joue un rôle clé dans l'angiogenèse. HIF1a augmente l'expression du
VEGF, qui active la prolifération et la migration de cellules endothéliales (Carmeliet and Jain,
2000).
Une synthèse lipidique est également un mécanisme important pour la génération des
membranes des cellules en prolifération (Laplante and Sabatini, 2009). mTORC1 active le
facteur de transcription SREBP1/2 qui contrôle l'expression de gènes impliqués dans la
synthèse d'acides gras et de cholestérol. En réponse à une diminution en stérol ou insuline,
SREBP va libérer une forme active qui va entrer dans le noyau pour activer la transcription
(Wang et al., 2011a). De plus, mTORC1 peut phosphoryler Lipin-1 et empêcher son entrée
dans le noyau pour supprimer l'activité de SREBP1/2 (Peterson et al., 2011).
4.2. Métabolisme et survie cellulaire
L'activation de mTORC1 régule également positivement le métabolisme cellulaire et
la production d'ATP. mTORC1 augmente le flux glycolytique en activant la transcription et la
traduction d'HIF1a, un régulateur de plusieurs gènes glycolytiques (Düvel et al., 2010). Une
autre étude montre également que mTORC1 peut augmenter l'expression de PGC-1a, un coactivateur de transcription jouant un rôle central dans la régulation du métabolisme
énergétique, en stimulant la biogénèse mitochondriale (Cunningham et al., 2007).
L'activation de mTOR permet également d'accroitre la survie cellulaire. L'activation de
S6K1, qui peut se lier aux membranes mitochondriales, permet de phosphoryler les molécules
pro-apoptotiques Bad, de dissocier le complexe Bad/Bcl2 et Bad/Bcl-xl, et permettre la
libération de protéines anti-apoptotiques (Laplante and Sabatini, 2012). De plus, le complexe
mTORC2 joue un rôle dans la survie cellulaire. Lorsqu'il est activé, il est phosphorylé sur
Ser2481 (Copp et al., 2009), et régule la survie et la prolifération cellulaire, le métabolisme et
l'organisation du cytosquelette. L'activation de mTORC2 régule des kinases de la sous famille
AGC, incluant Akt, SGK1 et PKC-a. L'activation de PKC-a, paxilin et Rho GTPase module
25
l'actine du cytosquelette (Jacinto et al., 2004). La sérine thréonine kinase Akt est activée en
aval de la signalisation PI3K, après phosphorylation sur ser473 par mTORC2, et est important
pour la survie, le métabolisme et la prolifération cellulaire et permet d'activer mTORC1.
4.3. Autophagie
L'activation de mTORC1 inhibe l'autophagie, le processus central de dégradation de la
cellule. Ainsi, une diminution de l'activité mTOR due à un appauvrissement en nutriments ou
en facteurs de croissance, ou lors de stress cellulaire, induit l'autophagie. Cela permet de créer
et utiliser l'énergie intracellulaire par le recyclage des protéines cytoplasmiques et organites
dans le lysosome, et adapter l'organisme et la cellule à l'environnement (Codogno and Meijer,
2005). Ainsi, ULK1/Atg13/FIP200, non phosphorylé lorsque la voie mTOR est inhibée, initie
l'autophagie (Ganley et al., 2009).
mTORC1
Akt
mTORC2
SURVIE
Paxilin
Rho
GTPase
PKC-a
CYTOSQUELETTE
SREBP1/2
inactif
S6K1
eEF2K
SREBP1/2
actif
eEF2
4EBP1
eIF4B
Maf1
ULK1/Atg13/FIP200
PDCD4
TIF-1A
eIF4G
eIF4A
Elongation
traduction
SYNTHESE
LIPIDIQUE
ARN Pol I
rRNA
Initiation
traduction
ARN Pol III
rRNA + tRNA
SYNTHESE PROTEIQUE
HIF1a
PGC-1a
Angiogenèse
AUTOPHAGIE
Cycline D1, c-myc,
ornithine carboxilase
Progression
Cycle cellulaire
VEGF
Acides gras
Cholestérol
eIF4E
Métabolisme
mitochondrial
et glycolytique
Croissance
cellulaire
Figure 4: Fonctions régulées par la voie mTOR.
26
II. mTOR ET REPONSES IMMUNITAIRES
En plus de tenir un rôle crucial dans le contrôle de la prolifération et de la croissance
cellulaire, la voie de signalisation mTOR est également un régulateur clé des réponses
immunitaires, en particulier de l'homéostasie lymphocytaire T. mTOR est ainsi un nœud
central dans la cascade de signalisation qui découle de l'intégration des signaux du
microenvironnement immunitaire. De nombreuses études ont mis en évidence l'importance de
mTOR comme déterminant fondamental de la différenciation des lymphocytes T CD4 et T
CD8.
1. Rôle de la voie mTOR dans la régulation lymphocytaire T
Les lymphocytes T (LT) sont des acteurs clés de l'immunité adaptative. Le maintien de
ces cellules dans un état quiescent ou leur engagement dans une réponse immunitaire va
dépendre de la détection de signaux antigéniques et inflammatoires. Ainsi la protéine mTOR
dans les LT, en plus d'intégrer les signaux environnementaux ou métaboliques communs à
toutes les cellules eucaryotes, va également contrôler l'homéostasie, intégrer des stimuli
immunitaires conduisant à l'activation des LT et réguler la migration cellulaire vers les sites
inflammatoires.
1.1. Homéostasie lymphocytaire T
A l'état de repos, les LT matures circulent dans l'organisme vers les zones T des
organes lymphoïdes secondaires (ganglions, rate, tissus lymphoïdes des muqueuses) dans un
stade quiescent (phase G0) et sont caractérisés par une petite taille et une faible activité
métabolique. Ces lymphocytes sont cataboliques et utilisent principalement l'autophagie pour
produire les molécules nécessaires à la synthèse protéique et énergétique. Leur survie repose
sur l'engagement du TCR à des peptides du soi présentés sur des complexes majeurs
d'histocompatibilités (CMH) et sur l'intégration de l'IL-7 (Surh and Sprent, 2008). Ces
signaux pouvant potentiellement résulter en une activation inappropriée, cet état quiescent
doit être activement maintenu. Ainsi, les facteurs de transcription KLF2 et FOXO induisent
l'expression d'inhibiteurs de l'activation cellulaire (Modiano et al., 2008). En absence
d'activation, TSC1 maintient la quiescence des LT en inhibant mTORC1 et en contrôlant ainsi
la taille de la cellule, l'entrée en cycle cellulaire et la réponse à une stimulation antigénique.
L'absence de TSC1 conduit à l'apoptose des LT et à la perte des LT conventionnels circulants,
27
montrant ainsi l'importance de la voie mTOR dans la régulation de l'état de quiescence des LT
(Wu et al., 2011; Yang et al., 2011).
Lorsqu'un LT naïf s'active, il entre en expansion clonale et passe d'un programme
métabolique catabolique à anabolique. Même en présence d'un taux adéquat d'oxygène dans
l'environnement, les LT activés utilisent la glycolyse pour générer de l'énergie, par un
phénomène appelé l'effet Warburg (Pearce, 2010). mTOR est un régulateur central de la
glycolyse et du métabolisme cellulaire. L'arrivée de signaux d'activation immunologiques
conduisent à l’activation de PI3K, puis d'Akt qui en retour va promouvoir l'expression de
transporteurs de glucose (Frauwirth et al., 2002). mTORC1, via HIF1a, promeut l'expression
de protéines impliquées dans la glycolyse et la consommation de glucose, tandis que
l'activation de SREBP1 conduit à l'augmentation de protéines critiques pour la voie pentose
phosphate et des acides gras (Düvel et al., 2010). Ainsi, en cohérence avec le fait que la
génération des LT effecteurs nécessite une augmentation de la synthèse protéique, il a été
observé que la reconnaissance d’un antigène par des LT CD8 conduit à la phosphorylation de
la protéine S6 par mTORC1 (Salmond et al., 2009). Une inhibition de ces voies métaboliques,
conduisant à l’inhibition de mTOR, bloque l'activation et la fonction lymphocytaire T et
induit une anergie (Jhun et al., 2005; Zheng et al., 2009).
La cytokine homéostatique IL-7 induit un signal PI3K-Akt retardé mais maintenu, et
une activation de mTOR. Cette activation dépend de l'activité transcriptionnelle de STAT5, et
contribue à l'assimilation du glucose et au maintien de l’homéostasie des cellules T (Wofford
et al., 2008).
1.2. Activation des lymphocytes T
mTOR
Figure 5 Les 3 signaux impliqués dans l’activation lymphocytaire T (Halloran, 2004)
28
Un LT naïf s’active suite à sa stimulation par un peptide antigénique présenté par une
cellule présentatrice d’antigène (CPA), et à la réception de signaux immunologiques. Ces
signaux conduisent à l’activation de la voie mTOR, qui en réponse induit la prolifération et la
différenciation du LT (Figure 5).
La présentation d’un peptide antigénique sur le CMH d'une CPA au TCR d'une cellule
T constitue le « signal 1 », transduit par le complexe CD3. mTORC1 et mTORC2 sont activés
dans les minutes suivant la stimulation du TCR, et la magnitude de cette activation est
directement corrélée à la quantité d’antigène et à la durée de l’interaction LT-CPA (Katzman
et al., 2010; Turner et al., 2009). Des signaux de co-stimulation ou « signal 2 » sont délivrés
par la suite, lorsque les molécules CD80, CD86 ou CD40 exprimés par les CPA se fixent sur
le CD28 ou le CD40L du LT. Ces signaux sont nécessaires pour que l’activation se poursuive.
CD28 est un signal classique d’activation de la voie PI3K-Akt, qui en retour va augmenter
l’activation de mTOR déjà induite par la présentation antigénique et faciliter l’activation du
LT (Colombetti et al., 2006). OX40, une autre molécule co-stimulatrice de la famille des
TNFR, permet également l’augmentation de la signalisation dépendante d’Akt (So et al.,
2011), tandis que l’axe PD1-PDL1, régulateur négatif des réponses lymphocytaires T diminue
l’activité mTOR (Francisco et al., 2009). Les signaux 1 et 2 activent les voies
calcium/calcineurine, MAP kinase, et NF-kB. Ces voies de signalisation activent des facteurs
de transcription et déclenchent l’expression d’interleukines, ou de récepteurs tels que CD25
ou CD40L. Les cytokines, en particulier l’IL-2, activent la voie de signalisation mTOR et
fournissent le « signal 3 » d’activation lymphocytaire T, déclenchant la prolifération cellulaire
(Halloran, 2004).
1.3. Migration des lymphocytes T
La signalisation mTOR joue un rôle important dans la régulation de la migration des
LT. Les LT naïfs circulent vers les organes lymphoïdes secondaires grâce aux récepteurs de
homing CD62L, CD127 ou CCR7 (Finlay and Cantrell, 2010). De même, S1PR1 est un
récepteur couplé à une protéine G spécifique du lipide S1P (shingosine-1-phosphate) qui est
un régulateur crucial de la recirculation des LT. L’activation de S1PR1 par un agoniste induit
une séquestration des LT dans le thymus ou les organes lymphoïdes secondaires (Mandala et
al., 2002). Après l’activation lymphocytaire T, et l’activation de mTOR, les LT diminuent
l’expression de ces récepteurs, et ainsi facilitent leur sortie de l’organe lymphoïde secondaire
pour migrer en périphérie vers les sites de l'infection (Sinclair et al., 2008). Ainsi, les LT
quiescents et activés sont caractérisés par des fonctions migratoires différentes, et une bonne
29
régulation de cette migration est essentielle pour une réponse immunitaire efficace.
L’expression des récepteurs de homing est liée aux facteurs de transcription FOXO et KLF2,
responsables de l'état quiescent des LT et inhibés suite à l'activation de mTORC2 via Akt
(Kerdiles et al., 2009). De même, la molécule S1P1 qui joue un rôle critique dans la sortie des
LT des ganglions, est également régulée par KLF2 (Carlson et al., 2006). mTOR est
également requis pour l’expression de T-bet (Rao et al., 2010), dont l’une des fonctions est
d’induire l’expression de récepteurs de chimiokines pro-inflammatoires, tel que CXCR3, qui
coordonne la migration des LT effecteurs (Taqueti et al., 2006) (Figure 6).
mTORC1
mTORC2
AKT
T-bet
Migration vers les sites
inflammatoires
(CXCR3)
KLF2
Figure 6: Impact de mTOR dans
la migration lymphocytaire T
FOXO
Migration homéostatique vers
les organes lymphoïdes
(CD62L, CCR7 and S1PR1)
2. Importance de mTOR dans la différenciation lymphocytaire T CD4
Les LT CD4 représentent un bras important du système immunitaire adaptatif car ils
orchestrent à la fois la réponse immunitaire cellulaire et humorale face à des pathogènes.
Parmi les sous-populations lymphocytaires T CD4, les LT CD4 régulateurs (Treg) sont requis
pour le maintien de la tolérance au soi et pour inhiber les réponses immunitaires pouvant
altérer l’hôte. La rencontre d’un LT CD4 naïf avec son antigène combinée aux signaux
d'activation 2 et 3 lui permet alors d'entamer un programme de différenciation aboutissant à la
formation de LT effecteurs ou mémoires. Initialement, deux sous-populations de LT CD4
helper ont été identifiées, les LT CD4 Th1 (T helper-1) sécrétant l’IFN-g contre les
pathogènes intracellulaires et les LT CD4 Th2 sécrétant l’IL-4 ciblant les parasites
extracellulaires. Aujourd'hui, au moins 7 sous-populations distinctes de LT CD4, déterminées
par les cytokines qu’elles sécrètent et les facteurs de transcription spécifiques des lignées
qu’elles expriment après activation, ont été identifiées: les Th1, Th2, Th9, Th17, Th22, TFH
(follicular helper T cell) et les iTreg (induced-regulatory T cell) (O’Shea and Paul, 2010)
(Figure 7).
30
IL-12
Th1
T-bet
Th2
GATA3
Th9
PU.1
IRF4
Th17
RORgT
Treg
FoxP3
TFH
Bcl6
Th22
?
STAT4
IL-4
STAT6
IL-4, TGF-b
STAT6
TCR-Ag
LT CD4
LT CD4
naïf Co-stimulations activé
IL-6, TGF-b
STAT3
TGF-b
STAT5
IL-6, IL-21
STAT3
?
IFN-g
TNF-a
IL-2
IL-4
IL-5
IL-13
IL-9
IL-10
IL-17
IL-21
IL-22
TGF-b
IL-10
IL-35
IL-21
IL-4
IL-22
TNF-a
IL-13
Figure 7: Polarisation des différentes sous-populations de LT CD4
Il existe une grande plasticité entre les LT CD4 à l'égard de leur capacité à se
différencier en sous-ensembles effecteurs uniques. En dépit de sa spécificité, la
reconnaissance de l'antigène par le TCR fournit peu d'indications en termes d'engagement
dans une lignée effectrice. Au contraire, le microenvironnement immunitaire joue un rôle
critique dans cette différenciation suite à la reconnaissance de l'antigène. In vitro, des LT CD4
naïfs sont facilement poussés vers une population effectrice spécifique grâce à l'utilisation de
concentrations élevées de cytokines associées à des anticorps anti-cytokines antagonistes.
Cependant in vivo, les LT CD4 sont confrontés à des concentrations plus subtiles de
cytokines, souvent avec des effets opposés. De même, l'activation initiale des LT CD4 naïfs
peut donner lieu à l'expression simultanée des facteurs de transcription spécifiques de chaque
sous-population. Ainsi, les LT CD4 doivent intégrer des signaux divers et parfois opposés
pour fournir l’instruction de leur engagement vers une lignée effectrice. mTOR, de par sa
capacité à détecter et intégrer divers signaux environnementaux et sa capacité à réguler le
métabolisme et l'activation des LT, tient un rôle central dans l'intégration des signaux du
microenvironnement immunitaire des LT CD4 pour instruire leur différenciation en souspopulation de LT helper ou Treg.
31
2.1. mTOR et différenciation des lymphocytes T CD4 Th1, Th2 et Th17
L’implication de la voie mTOR a été particulièrement étudiée dans la différenciation
des LT CD4 helper Th1, Th2 et Th17, les trois sous-populations Th les plus anciennement
décrites.
Initialement décrits par les travaux de Mossmann et al., les Th1 et Th2 se différencient
par leurs fonctions effectrices et leur profil de production de cytokines (Mosmann et al.,
1986). Des LT CD4 naïfs non différenciés activés par l’IL-12 sécrétés par des cellules
dendritiques (DC) spécialisées vont acquérir la capacité de produire de l’INF-g, de l’IL-2 et
du TNF-a. Au contraire, les LT CD4 naïfs activés en présence d’IL-4 vont produire de l’IL-4,
de l’IL-5 et de l’IL-13, mais pas d’IFN-g. La génération des Th1 en réponse à l’IL-12 va
conduire à l’expression de STAT4 indispensable à la médiation des signaux vers Th1 et du
facteur de transcription T-bet, tandis que la génération des Th2 en réponse à l’IL-4 va
conduire à l’expression de STAT6 et du facteur de transcription GATA-3 (Zhu et al., 2010).
En 2003, une troisième sous-population lymphocytaire T CD4 effectrice appelée LT
CD4 Th17 a été caractérisée (Aggarwal et al., 2003). Ces Th17 produisent de l’IL-17A, de
l’IL-17F, de l’IL-21 et de l’IL-22 (Harrington et al., 2005). La signalisation induite par le
TGF-b et l’IL-6 passent par la protéine STAT3 pour induire la génération des Th17 et induire
l’expression du facteur de transcription RORgT (Zhu et al., 2010).
Le rôle de mTOR dans l’intégration des signaux induits par les récepteurs
cytokiniques et dans la régulation de la différenciation des LT CD4 helper a été largement
exploré à l’aide de souris déficientes en composants essentiels de la voie mTOR. Grâce à
l’utilisation de LT invalidés pour Frap1, le gène codant pour la protéine mTOR, l'équipe de
G. Delgoffe et J. Powell a démontré en 2009 que l’activation de mTOR est cruciale pour
l’intégration des signaux conduisant à la différenciation des LT CD4 naïfs en Th1, Th2 ou
Th17 (Delgoffe et al., 2009). Malgré un développement et une capacité d’activation non
altérés, les LT CD4 déficients en mTOR perdent leur habilité à répondre à une stimulation du
TCR et à des molécules de co-stimulation en présence d’IFN-g (Th1), d’IL-4 (Th2) ou d’IL-6
(Th17). Ces LT présentent en outre une diminution de l'activation de STAT4, STAT6 et
STAT3 et de l'expression des facteurs de transcription T-bet, GATA-3 et RORgT dans des
conditions de stimulation Th1, Th2 et Th17 respectivement.
L’utilisation de souris déficientes en mTORC1 (Rheb-/-) ou mTORC2 (Rictor-/-)
spécifiquement sur les LT CD4 a permis à cette même équipe en 2011 d’étudier plus
32
précisément le rôle des composants de mTOR dans la régulation de la différenciation des LT
CD4 helper. En absence de mTORC1, les LT CD4 ne parviennent pas à sécréter d’IFN-g ou
d’IL-17 dans des conditions de différenciation Th1 ou Th17 respectivement, mais conservent
leur capacité à se différencier en Th2. Par contre la délétion en mTORC2 altère la capacité des
LT CD4 à sécréter de l’IL-4 en réponse à une stimulation spécifique Th2, mais n’empêche pas
une différenciation vers un profil Th1 ou Th17. La signalisation cytokinique passe par
l’activation des facteurs de transcription STAT, dont l’expression est régulée par les protéines
inhibitrices SOCS (Yu et al., 2003). Les LT CD4 déficients en mTORC1 activent plus
fortement SOCS3, conduisant à une inhibition des STAT4 et STAT3, tandis que l’absence en
mTORC2 augmente l’activation de SOCS5, et permet l’inhibition de STAT6 (Delgoffe et al.,
2011). L’impact de l’absence de mTORC2 sur la différenciation Th2 peut être annulé par
l’activation de la protéine kinase C, une molécule impliquée dans la différenciation
lymphocytaire T CD4, via NFkB (Lee et al., 2010b). Les travaux pionniers de G. Delgoffe et
J. Powell ont ainsi montré le lien entre activation de mTOR et la différenciation des LT CD4
helper. L’activation de mTORC1 est requise pour la différenciation des LT CD4 naïfs en Th1
et Th17, via l’inhibition de SOCS5 et l’activation de STAT4 et STAT3, par contre l’activation
de mTORC2 est requise pour la différenciation des LT CD4 naïfs en Th2, via l’inhibition de
SOCS3 et l’activation de STAT6 (Figure 8).
TCR et co-stimulation
Rictor mLST8
mLST8
Raptor
mTOR Tti1
Pras40
Tel2
Deptor
mSin1 mTOR Tti1
Tel2
Protor1/2 Deptor
MTORC1
MTORC2
IL-12
IL-4
IL-6, TGF-b
SOCS5
SOCS3
STAT4
STAT3
STAT6
T-bet
RORgT
GATA-3
Différenciation
Th1
Différenciation
Th17
Différenciation
Th2
Figure 8: Implication des molécules en
aval de la voie mTOR dans la
différenciation LT CD4 helper Th1,
Th2 et Th17
33
2.2. mTOR et différentiation des LT CD4 régulateurs
Les Treg CD4+CD25+ découverts en 1995 par l'équipe de S. Sakaguchi sont des LT
suppresseurs importants pour la régulation de l'homéostasie des réponses immunitaires et le
maintien de la tolérance immunitaire centrale et périphérique (Sakaguchi et al., 1995).
L'identification du marqueur FoxP3 en 2003 a permis une meilleure caractérisation
phénotypique et fonctionnelle de cette sous-population LT CD4 (Hori et al., 2003). Ce
marqueur est spécifique des Treg chez la souris mais chez l’homme, l’activation de LT peut
induire l’expression transitoire de ce marqueur (Walker et al., 2003). La discrimination entre
Treg et LT activés se fait par le marqueur CD127, exprimé faiblement sur les Treg et fortement
sur les LT activés. De plus, au niveau génique, les Treg présentent une déméthylation des
motifs CpG en amont de l’exon 1 du locus FoxP3 qui n’est pas retrouvé chez les LT activés
(O’Shea and Paul, 2010). Les Treg représentent environ 1 à 5% de la population lymphocytaire
T CD4 chez l'homme (Sakaguchi, 2004) et parmi eux, il faut distinguer les Treg dit naturels
(nTreg), issus de la différenciation thymique, des Treg induits (iTreg), issus de la différenciation
de LT CD4 conventionnels en réponse à une stimulation antigénique dans un environnement
suppresseur. Le TGF-b est la cytokine critique de la différenciation Treg, tandis que la
présence d’IL-2 est également nécessaire afin d’induire STAT5 et d’augmenter l’expression
de FoxP3 (Bilate and Lafaille, 2012).
Caractéristiques
Treg naturels
Treg induits
Génération
Lieu d’induction
Thymus
Signal de co-stimulation associé
Environnement cytokinique
CD28
TGF-b, IL-2 ou IL-15
Fonctions et phénotypes
Contact-dépendant
CD45RA+/- CD25hi FoxP3hi CD39+
Constitutive
Forte
Complète
Mécanisme suppression
Marqueurs
Expression de FoxP3, CTLA-4, GITR
Niveau d’expression de CD25
Déméthylation de la région TSDR du
locus FoxP3
Autres marqueurs
Spécificité
Helios, Nrp1,PD-1, Swap70
Antigènes du soi
Organes lymphoïdes secondaires/
Tissus enflammés
CTLA-4
TGF-b, IL-2
Cytokine-dépendant?
CD45RA- CD25low FoxP3low CD39+
Induite
Variable
Partielle
Dapl1, Igfbp4
Antigènes du soi et exogènes
Tableau 1: Comparaison des nTreg et iTreg (Bilate and Lafaille, 2012)
2.2.1. mTOR et promotion des Treg
Les précédents travaux de G. Delgoffe et J. Powell utilisant des souris déficientes en
mTOR (Frap-/-), ont montré que l'activation de la voie mTOR est un déterminent négatif des
Treg. En absence de mTOR, les LT CD4 naïfs se différencient en Treg, même dans un
microenvironnement destiné à une polarisation Th1, Th2 ou Th17. Les iTreg induits en
34
absence de mTOR expriment un taux de FoxP3 équivalent à celui des nTreg et conservent leur
pouvoir suppressif in vitro. Les LT CD4 naïfs stimulés en absence de mTOR sont capables de
répondre à des taux très faibles de TGF-b pour se différencier en iTreg, via un mécanisme
impliquant Smad3. Cette molécule joue un rôle critique dans la promotion des Treg en réponse
au TGF-b pour induire NF-AT qui contribue à l’expression de FoxP3 (Tone et al., 2008).
L’activation de mTOR est antagoniste à Smad3, et les LT CD4 déficients en mTOR
présentent une forte phosphorylation de Smad3 à l’état basal, les rendant ainsi plus sensibles à
l’activation par le TGF-b et donc plus disposés à une différentiation en Treg, comparé à des LT
non déficients en mTOR (Delgoffe et al., 2009). En complément des mécanismes impliquant
Smad3, l’effet inhibiteur de la voie mTOR sur la différenciation des Treg implique aussi les
protéines FOXO1 et FOXO3, qui permettent l’induction de FoxP3. Ces molécules sont
inactivées par la voie Akt dépendante de mTORC2 (Merkenschlager and von Boehmer, 2010;
Ouyang et al., 2010). L’utilisation de souris déficientes en mTORC1 ou mTORC2 confirme
que la différenciation des LT CD4 naïfs en iTreg nécessite l’inhibition simultanée des deux
complexes (Delgoffe et al., 2011) (Figure 9).
Récepteur TGF-b
Treg
TCR et
co-stimulation
Récepteur IL-2
PTEN
SMAD3
PI2P
SMAD4
PI3K
mTORC1
AKT
P
P
SMAD3
FOXO1
SMAD4
FOXO3
PI3P
PDK1
Figure 9: Mécanismes de la
différenciation Treg mTORdépendante
mTORC2
FoxP3
Inversement, une augmentation de l’activité de la signalisation mTOR inhibe
l’induction des iTreg. Une activité du signal TCR et de l’axe PI3K/Akt/mTOR rendue
constitutive par la délétion de PTEN inhibe l’induction de FoxP3. Cette inhibition
s’effectuerait par un mécanisme au niveau chromatique, avec des méthylations de la lysine 4
de l’histone H3 (H3K4me2 and -3), proche du site de départ (TSS) de la transcription de
FoxP3 (Sauer et al., 2008). De même, l’activité Akt rendue constitutive par transduction
35
rétrovirale d’un de ses allèles confirme Akt et mTOR comme fort répresseur de la génération
des iTreg in vitro ou in vivo. L'activation d'Akt sur les précurseurs thymiques avant
l’expression de FoxP3 est délétère pour la génération thymique des nTreg. Par contre,
l’activation constitutive d’Akt et de mTOR sur des Treg n'altère pas cette population
(Haxhinasto et al., 2008).
2.2.2. Implication de mTOR dans la régulation fonctionnelle des Treg
En plus du rôle crucial de la voie de signalisation mTOR dans la génération de Treg, il
a été récemment montré qu’une modulation de l’activité de mTOR peut également réguler les
fonctions des Treg.
L’axe PD1/PD-L1, important dans la régulation négative de la réponse immunitaire,
est impliqué dans l’inhibition de la voie mTOR. De plus, cet axe régule la génération et la
fonction Treg. En ce sens, Francisco et al. ont montré qu’une activation de la voie mTOR suite
à une déficience en PD-L1 sur des CPA prévient de la différenciation des LT CD4 naïfs en
Treg. Une stimulation par des billes recouvertes de PD-L1 peut induire directement des Treg in
vitro, tandis qu’in vivo des souris déficientes en PD-L1 et PD-L2 présentent de fortes
altérations dans le pourcentage et la fonction des iTreg (Francisco et al., 2009).
A l’aide de LT CD4 transgéniques (par approches de gain et de perte de fonction),
l’équipe d’H. Chi a décrit l’importance de l’axe S1PR1-mTOR dans la génération thymique,
la maintenance périphérique et l’activité suppressive des Treg. S1PR1 est un régulateur crucial
de la recirculation des LT. Par la fixation de son ligand S1P, S1PR1 induit la signalisation
Akt/mTOR et diminue ainsi l’activation de Smad3. L’activation de S1PR1-mTOR empêche
alors la stimulation du LT CD4 par le TGF-b, et bloque la différentiation et l’activité
suppressive des Treg (Liu et al., 2009, 2010).
Cette même équipe a par la suite exploré plus précisément l'impact de la voie mTOR
sur l'homéostasie et la fonction des Treg. Malgré qu'il ait été largement démontré que l'axe
PI3K-Akt-mTOR est un régulateur négatif de la différenciation des Treg, mTORC1 est un
déterminent positif de la fonction suppressive des Treg en couplant signaux métaboliques et
immunologiques. Des signaux antigéniques (TCR) et cytokiniques (IL-2) activent mTORC1
qui en retour programme les fonctions suppressives des Treg. L’activation de l’axe mTORC1,
en augmentant le métabolisme lipidique et du cholestérol, coordonne la prolifération des Treg
et augmente l’expression des molécules suppressives CTLA-4 et ICOS. En ce sens, une
déplétion en mTORC1 sur les Treg (Raptor-/- sur les FoxP3+) conduit à une profonde perte de
l'activité suppressive des Treg et au développement de syndromes inflammatoire chez ces
36
souris transgéniques (Zeng et al., 2013). Néanmoins, une activation constitutive de mTORC1,
via la délétion spécifique de son régulateur négatif TSC1, résulte en la perte des fonctions in
vivo des Treg dans des conditions inflammatoires (Park et al., 2013).
La voie mTOR est ainsi cruciale dans les mécanismes de régulation fonctionnelle des
Treg. mTORC1 est nécessaire à leur activité suppressive, néanmoins, une activation trop forte
de mTOR, via S1PR1 ou l’absence de TSC1, altère les fonctions des Treg, suggérant que la
force du signal mTOR régule l’activé des Treg. Un faible signal mTOR serait ainsi bénéfique
pour la fonction des Treg. En ce sens, Procaccini et al. ont montré qu’une inhibition transitoire
de mTOR par la rapamycine induit une prolifération des Treg, tandis qu’un traitement
chronique bloque cette prolifération. Ces travaux suggèrent ainsi l’existence d’une boucle
oscillatoire de l’activité de mTOR impactant sur la réponse lymphocytaire T régulatrice
(Procaccini et al., 2010).
3. Rôle de mTOR dans la différenciation des LT CD8
3.1. Les LT CD8 mémoires
Les LT CD8 sont des composants essentiels de la réponse immunitaire adaptative,
générant à la fois les cellules effectrices nécessaires à la clairance d’une infection virale ou
parasitaire, des bactéries intracellulaires, et à la mise en place de lymphocytes mémoires à
longue durée de vie permettant une réponse secondaire rapide (Kaech and Cui, 2012). Suite à
leur rencontre initiale avec un antigène, les LT CD8 naïfs prolifèrent rapidement, avec une
phase d’expansion clonale, et génèrent des lymphocytes effecteurs. Durant la phase de
contraction qui suit, la plupart des LT CD8 meurent par apoptose, tandis qu’une petite souspopulation de LT CD8 spécifiques d’antigènes se développe en une population de
lymphocytes mémoires à longue durée de vie (Williams and Bevan, 2007) (Figure 10).
Figure 10: Cinétique de la réponse T
CD8 anti-virale
(Williams and Bevan, 2007)
37
Les LT mémoires (CD4 ou CD8) expriment le récepteur à l’IL-7 (CD127) et la chaîne
b du récepteur à l’IL-2 (CD122), reflétant le rôle de l’IL-7 et de l’IL-15 dans la génération de
cette sous-population. Au niveau transcriptionnel, cette différenciation d’un lymphocyte
effecteur vers un lymphocyte mémoire est facilitée par la transition de l’expression du facteur
de transcription T-bet vers le facteur de transcription Eomes (Intlekofer et al., 2005). Les LT
mémoires qui expriment des molécules telles que CD62L et CCR7 permettant une migration
efficace vers les ganglions lymphatiques sont appelés les LT centraux mémoires (TCM). Ceux
qui n’expriment pas ces molécules sont situés dans les tissus non lymphoïdes et sont appelés
les LT effecteurs mémoires (TEM). Ces deux sous-populations sont néanmoins présentes dans
le sang et la rate. Les TEM présentent des fonctions effectrices plus rapides que les TCM, tandis
que les TCM présentent une capacité de survie et de prolifération plus élevée, en addition
d’une capactié d’auto-renouvellement. Il semblerait que les TCM soient les précurseurs des
TEM (Sallusto et al., 1999; Wherry et al., 2003). Récemment, l'équipe de N. Restifo a
caractérisé une sous-population de LT mémoires ayant des propriétés de renouvellement
importantes, qualifiée de cellules souche mémoires (TSCM). Cette population présente le même
phénotype que des LT naïfs (CD45RA+ CD45RO- CCR7+ CD62L+ CD27+ CD28+) mais
exprime également CD95, particulièrement présent sur les LT mémoires. Ces TSCM sont
multipotents, capables de générer l’ensemble des groupes de LT mémoires, dont les TCM
(Gattinoni et al., 2011).
Figure 11: Les différentes sous-populations LT mémoires
(Gattinoni and Restifo, 2013)
38
Contrairement aux LT CD8 effecteurs qui ont une demande métabolique énorme et
reposent sur un métabolisme anabolique, les LT CD8 mémoires ont des besoins métaboliques
plus faibles et basculent vers un métabolisme catabolique (Gerriets and Rathmell, 2012). De
par son importance dans la régulation des signaux d’activation lymphocytaire et du
métabolisme cellulaire, mTOR impacte sur la différenciation des LT CD8 mémoires.
3.2. L’inhibition de mTOR favorise la génération des LT CD8 mémoires
Araki et al. ont été les premiers à avoir démontré le rôle de mTOR dans la génération
des LT CD8 mémoires. L'inhibition de la voie mTOR par la rapamycine chez des souris
infectées par le virus de la chorioméningite lymphocytaire (LCMV) augmente les réponses T
CD8 mémoires spécifiques de LCMV (Araki et al., 2009). L’implication de la voie mTOR
dans la différenciation des LT CD8 mémoires semble corrélée aux différentes phases
(expansion et contraction) de la réponse immunitaire T CD8. Une administration de
rapamycine durant la phase d'expansion de la réponse immunitaire anti-virale augmente le
nombre de LT CD8 précurseurs mémoires par une diminution de l'apoptose durant la phase de
contraction immunitaire. Au contraire, un traitement durant la phase de contraction améliore
la qualité de la réponse T CD8 mémoire, en accélérant la transition vers une différenciation
lymphocytaire T CD8 mémoire à longue durée de vie (CD127+KLRG1lo et CD127+CD62L+).
Un traitement continu par la rapamycine pendant la phase d'expansion et de contraction de la
réponse immunitaire anti-virale permet d'augmenter à la fois la magnitude et la qualité des LT
CD8 mémoires. Ce phénomène est intrinsèque au LT CD8 spécifiques de l'antigène, et est
dépendant de mTORC1, des résultats similaires étant retrouvés avec des souris déficientes en
Raptor. Néanmoins, une inhibition trop forte de mTOR avec une dose élevée de rapamycine
altère les réponses T CD8 (Araki et al., 2009) (Figure 12). La promotion d’une réponse T
CD8 antivirale mémoire par inhibition de mTOR a également été décrite chez des primates
non humains (Turner et al., 2011).
39
Figure 12: Impact de la rapamycine dans la génération de LT CD8 mémoires (Araki et al., 2010)
Suites aux travaux pionniers de K. Araki, de nombreuses études ont été poursuivies
dans le but de décrire les mécanismes par lesquels l'inhibition de mTOR induit la génération
de LT CD8 mémoires. Pearce et al. suggèrent un mécanisme lié à la modulation du
métabolisme induite par mTOR. Une incapacité à oxyder les acides gras et à basculer vers un
métabolisme catabolique chez des LT CD8 déficients en TRAF6, un régulateur en aval de
mTOR, empêche la génération de LT CD8 mémoires contre une infection à Listeria
monocytogenes. La réponse T CD8 effectrice n'est quant à elle pas altérée, et l'inhibition de
mTOR par la rapamycine restaure la génération de LT CD8 mémoires (Pearce et al., 2009).
L'équipe de P. Shrikant a démontré que mTOR régule la détermination des phénotypes LT
CD8 effecteurs et mémoires par la modulation de l'expression des facteurs de transcription Tbet et Eomes. L'activation de la voie mTOR dans les LT CD8 par la stimulation du TCR et les
signaux de co-stimulations aboutit à l'expression de T-bet et d'un phénotype effecteur,
maintenu par une stimulation avec de l'IL-12. FOXO1, réprimé par mTOR, est un régulateur
clé de l'expression de T-bet et Eomes dans les LT. L'inhibition de mTOR par la rapamycine
va activer FOXO1 et promouvoir l'expression d'Eomes, dont l'expression est associée à un
phénotype T CD8 mémoire (Li et al., 2011b; Rao et al., 2010, 2012) (Figure 13).
40
Activation mTOR
Inhibition mTOR
T-bet
LT CD8 naïf
LT CD8 activé
FOXO1
LT CD8 effecteur
Eomes
LT CD8 mémoire
Figure 13: mTOR dans la différenciation LT CD8 mémoire
Comme pour l’étude de son implication dans la différenciation LT CD4, la création de
souris génétiquement invalidées a permis une meilleure compréhension de l’implication de la
voie mTOR sur la régulation des réponses mémoires T CD8. L'équipe de G. Delgoffe et J.
Powell a montré que mTORC1 et mTORC2 régulent différemment la génération des LT CD8
effecteurs et mémoires. Une activation constitutive de mTORC1 par la délétion de TSC2 sur
les LT résulte en la génération de LT CD8 effecteurs efficaces. Comparé à des LT CD8 non
mutés, les LT CD8 déficients en TSC2 présentent après stimulation une augmentation de la
prolifération et de la sécrétion cytokinique, et ont un métabolisme glycolytique plus élevé.
Néanmoins, ces LT CD8 sont incapables de se différencier en LT CD8 mémoires. Au
contraire, des LT CD8 déficients en mTORC1 (Rheb-/-) présentent les capacités de longue
durée de vie et de faible métabolisme habituelles des LT mémoires, mais échouent à répondre
à une restimulation. Des LT CD8 déficients en mTORC2 (Rictor-/-) conservent leur capacité
effectrice, mais peuvent également se différencier en LT CD8 mémoires efficaces, capables
de répondre à une restimulation. Ces travaux montrent que mTORC1 est nécessaire à la
réponse T CD8 effectrice, tandis que mTORC2 régule la génération des LT CD8 mémoires,
par une reprogrammation métabolique (Pollizzi et al., 2015). Ces très récentes observations
confirment les précédents travaux qui montraient également l'impact direct de mTOR sur la
génération de LT CD8 mémoires par l'utilisation de modèles déficients en TSC1 (Krishna et
al., 2014; Shrestha et al., 2014).
4. Rôle de mTOR sur les autres cellules du système immunitaire
4.1. mTOR et lymphocytes B
Au contraire des nombreux travaux portant sur les LT, le rôle de mTOR dans les
lymphocytes B (LB) a été peu étudié. L'inhibition de mTOR par la rapamycine altère la
prolifération des LB et leur différenciation en plasmocytes (Aagaard-Tillery and Jelinek,
1994; Donahue and Fruman, 2007; Sakata et al., 1999). Des études plus récentes utilisant des
modèles de souris génétiquement déficientes en mTOR ont montré qu'une diminution de
l'activité mTOR empêche le développement et la maturation des LB, ainsi que leur
41
différenciation en plasmocytes (Zhang et al., 2011). De manière contradictoire, une seconde
équipe travaillant avec un modèle de souris déficient en TSC1, a montré qu'une activation
constitutive de mTOR altère également la différenciation en plasmocytes après immunisation
(Benhamron and Tirosh, 2011). Ces différents travaux suggèrent qu'une réponse anticorps
optimale requiert un taux d'activation mTOR spécifique, c’est-à-dire qu'une activité mTOR
trop élevée ou trop faible altère la différenciation en plasmocytes. L'impact de mTOR
spécifiquement aux différents stades de la différenciation des LB (activation du LB, formation
du centre germinal, différenciation en plasmocytes) reste à étudier.
4.2. mTOR et cellules dendritiques
Le rôle de la voie mTOR dans le développement et la fonction des DC a été
majoritairement étudié chez la souris. Trois populations de DC sont décrites chez la souris :
les cellules dendritiques conventionnelles (cDC) CD8- ou CD8+, et les cellules dendritiques
plasmacytoïdes (pDC). Le facteur de croissance Flt3l contrôle le développement des DC à
partir d’un précurseur commun dérivé de la moelle osseuse, et est particulièrement important
pour la différenciation des pDC et cDC CD8+ (Sathaliyawala et al., 2010). En addition, les
cellules de Langerhans sont une sous-population spécialisée de DC présentes dans l’épiderme.
L’injection de Flt3l recombinant active mTORC1 dans les DC, et l’inhibition in vitro de
mTOR par la rapamycine inhibe le développement Flt3l-dépendant des pDC et cDC CD8+.
Au contraire, la sur-activation de l'axe PI3K-Akt-mTOR par la délétion de PTEN facilite le
développement des DC in vitro et in vivo (Sathaliyawala et al., 2010). L’inhibition de
mTORC1 mais pas de mTORC2 conduit à une diminution des cellules de Langerhans dans la
peau (Kellersch and Brocker, 2013). La prolifération des précurseurs et le développement de
l’ensemble des populations de DC humaines requièrent également une signalisation
PI3K/Akt/mTOR intacte. Les cellules progénitrices hématopoïétiques CD34+ se développent
en DC myéloïdes CD11c+ (homologues des DC CD8- murins), DC myéloïdes CD141+
(homologues des DC CD8+ murins), pDC et cellules de Langherans (Laar et al., 2010, 2012).
Les DC dérivés des monocytes (moDC) par le GM-CSF et l’IL-4 dépendent également de
mTORC1 (Haidinger et al., 2010). La sécrétion coordonnée de cytokines pro- et antiinflammatoires par les DC est indispensable à l’efficacité de la réponse immunitaire. La voie
de signalisation mTOR est impliquée dans la production de cytokines par les DC. La
stimulation des DC par du LPS active mTORC1, et une inhibition de mTORC1 par la
rapamycine pendant une stimulation des TLR augmente la sécrétion de la cytokine proinflammatoire IL-12, et inhibe la sécrétion d’IL-10. Les DC traitées par la rapamycine
42
induisent ainsi une différenciation vers des LT CD4 Th1 (Ohtani et al., 2008; Turnquist et al.,
2010; Weichhart et al., 2008). Les pDC traitées avec la rapamycine perdent leur capacité à
sécréter de l'interféron de type I, via la suppression de l'activité IRF7 (Cao et al., 2008).
Un autre effet important de mTOR sur les DC est la modulation de leur capacité à
présenter des peptides antigéniques aux LT. Amiel et al. ont montré que des DC exposées à la
rapamycine durant leur stimulation par des agonistes TLR ont une durée de vie prolongée et
expriment des molécules de co-stimulations, favorisant ainsi fortement l'activation de LT CD8
(Amiel et al., 2012). L'activité autophagique, une voie de dégradation lysosomale inhibée par
mTOR, a été démontrée comme importante dans les DC pour une présentation optimale de
peptides antigéniques sur les CMH de classe II pour stimuler les LT CD4 (Lee et al., 2010a).
Une augmentation de la présentation antigénique par des DC via l'induction d'autophagie
après traitement par la rapamycine a été décrite dans des expériences in vitro. De plus, une
vaccination in vivo à base de DC pré-incubées avec la rapamycine induit une meilleure
présentation antigénique résultant en une réponse Th1 plus forte, protégeant contre
Mycobacterium tuberculosis (Jagannath et al., 2009). De manière intéressante, il a été montré
qu'au contraire, lors de l'activation de la voie mTOR dans le contexte de l'infection au VIH,
les DC des sites muqueux présentent des défauts de présentations antigéniques médiées par
l'autophagie et une diminution de la réponse T CD4 spécifique au VIH (Blanchet et al., 2010).
4.3. mTOR et cellules myéloïdes suppressives
Les cellules myéloïdes suppressives (MDSC) sont une population hétérogène de
cellules d'origine myéloïdes, comprenant des cellules myéloïdes progénitrices, ainsi que des
macrophages immatures, granulocytes immatures et DC immatures. Chez la souris, ces
cellules sont caractérisées par l'expression des molécules CD11b et GR1, tandis que chez
l'homme, le phénotype de ces cellules est: LIN-HLA-DR-CD33+ ou CD11b+CD14-CD33+. A
l'état quiescent, les MDSC sont dépourvues d'activité suppressive et sont présentes dans la
moelle osseuse. Les MDSC activées s'accumulent dans les organes lymphoïdes secondaires
ou les sites inflammatoires, et sont de puissants suppresseurs de la fonction des LT, se
caractérisant par une production accrue d'espèces réactives d'oxygène ou d'azote, et d'arginase
(Gabrilovich and Nagaraj, 2009).
L’impact de la voie mTOR sur l’homéostasie des MDSC n’est pas bien défini,
néanmoins des études récentes ont décrit un rôle de mTOR dans la fonction des MDSC. Une
inhibition de la voie mTOR induit une accumulation de MDSC très fonctionnelles dans les
sites inflammatoires (Makki et al., 2014; Nakamura et al., 2015). De manière contradictoire,
43
dans des modèles de souris LAL-/- (souris déficientes pour une enzyme permettant la synthèse
d’acides gras et de cholestérol), dans lesquels les MDSC sont très fonctionnelles et
contribuent à la génération de syndromes métaboliques ou à une prolifération tumorale, une
inhibition de mTOR bloque la fonction suppressive des MDSC (Ding et al., 2014, 2015; Zhao
et al., 2015).
4.4. mTOR et cellules natural killer (NK)
Les cellules natural killer (NK) sont des cellules lymphoïdes du système immunitaire
inné (ILC) impliquées dans l'immunosurveillance des cancers et dans le contrôle précoce des
infections par des pathogènes intracellulaires. Elles ont des propriétés cytotoxiques et
produisent un taux élevé d'IFN-g après activation. Chez la souris, les cellules NK matures sont
CD11bhiCD27lo. Chez l'homme, elles sont CD16+ et CD56bright ou CD56dim. L’activation des
récepteurs cytokiniques à la surface des cellules NK module considérablement leur
développement et activation. L'IL-2, -12, -15, -18, et -21 sont considérés comme étant des
stimulateurs de la fonction des cellules NK alors que le TGF-" et l'IL-10 sont connus comme
étant des régulateurs négatifs (Ali et al., 2015; Vivier et al., 2008). Parmi les cytokines
stimulatrices, l'IL-15 est la plus importante et contrôle à la fois l'homéostasie et l'activation en
périphérie des cellules NK. L'engagement de l'IL-15 sur son récepteur exprimé par les cellules
NK active en parallèle la voie de signalisation des MAP kinase, de PI3K-Akt-mTOR et une
transduction de signaux par STAT5 (Ma et al., 2006). Par l'utilisation de souris déficientes en
mTOR sur les cellules NK, Marçais et al. ont montré un rôle crucial de mTOR dans le
contrôle de la prolifération et de la fonction des cellules NK lors d'une inflammation ou d'une
infection virale. Tandis qu'une faible concentration d'IL-15 active seulement la
phosphorylation de STAT5, une forte concentration active la voie de signalisation mTOR, qui
stimule la croissance des cellules NK et leur assimilation de nutriments, et induit un
rétrocontrôle positif sur le récepteur à l'IL-15 (Marçais et al., 2014). Une inhibition de la voie
mTOR altère la cytotoxicité des cellules NK à la fois chez l'homme et chez la souris
(Donnelly et al., 2014; Marçais et al., 2014).
44
III. CIBLAGE THERAPEUTIQUE DE LA VOIE mTOR
La voie de signalisation mTOR est ainsi impliquée dans de nombreux processus, allant
du métabolisme et de la prolifération cellulaire, à la régulation de la fonction des cellules
immunitaires. En conséquence, mTOR est apparue comme une cible thérapeutique
importante, et sa régulation par des agents inhibiteurs est aujourd'hui couramment recherchée
dans divers domaines cliniques, tels que la prévention du rejet de greffe ou le traitement de
certains cancers.
1. Ciblage de mTOR en transplantation d’organes
L’une des premières propriétés de l'inhibiteur de mTOR (mTORi) rapamycine mise à
profit pour un développement clinique a été son puissant potentiel d'inhibition de la
prolifération des LT activés, lui conférant une activité immunosuppressive. Cette propriété est
recherchée dans le cadre de la prévention du rejet aigu d'allogreffe, le greffon étant reconnu et
considéré comme un corps étranger par le système immunitaire. En pratique clinique, les
molécules de la famille des inhibiteurs de la calcineurine (cyclosporine et tacrolimus)
demeurent la base de la grande majorité des protocoles d’immunosuppression en
transplantation d’organes solides, mais sont caractérisées par une néphrotoxicité. La
rapamycine est administrée principalement en association avec ces inhibiteurs pour en
diminuer leur dose, ou bien en substitution pour limiter leurs effets indésirables à long terme.
1.1. La rapamycine
La rapamycine (ou sirolimus) est le premier mTORi décrit. Il s’agit d’un macrolide
découvert au début des années 1970, à partir de prélèvements de sol de l'île Rapa-Nui (île de
Pâques) dans le cadre de recherche de molécules antifongique. Il est utilisé comme agent
antifongique contre Candida albicans, Cryptococcus neoformans et Aspergillus fumigatus
(Vézina et al., 1975). En se fixant à FKBP12, la rapamycine forme un complexe qui va se lier
à FRB (FKBP12-rapamycin binding domain) sur mTORC1 et l'inhiber (Brown et al., 1994;
Sabatini et al., 1994). La façon dont la fixation de FKBP12-rapamycine puisse inactiver
l'activité de mTORC1 reste néanmoins inconnue. L'altération de l'intégrité structurale de
mTORC1 ou la modification de l'allostérie pourrait être la cause de la réduction d'activité de
la kinase. Il en résulte une diminution de la phosphorylation des cibles en aval: 4EBP1 et
S6K1. Ces molécules sont métabolisées par le cytochrome CYP3A, puis sont éliminées
45
essentiellement par le foie. mTORC2 est insensible à la rapamycine mais certaines études
montrent qu'une longue exposition à la rapamycine peut également réduire le signal de
mTORC2 dans certains types cellulaires (Sarbassov et al., 2006). L'inhibition de mTORC1
peut conduire à la stabilisation de p27Kip1, une kinase inhibitrice de la cycline-CDK2, ainsi
qu’à la suppression de la cycline D1, et en conséquence arrêter le cycle cellulaire en phase G1
et stopper la prolifération cellulaire (Nourse et al., 1994).
1.2. Immunosuppression induite par la rapamycine
Les patients transplantés sont traités avec des immunosuppresseurs nécessaires à une
prise de greffe de longue durée et fonctionnelle. Les effets immunosuppressifs de la
rapamycine ont été reconnus dès les années 1970, bien avant l'identification de sa cible
mTOR et de l'étude de l'implication de cette voie de signalisation sur l'homéostasie et la
fonction des LT CD4. Depuis son autorisation en 1999 par la FDA (Groth et al., 1999), la
rapamycine est utilisée pour prévenir du rejet de greffe de rein grâce à ses effets suppressifs
sur l'activation et la prolifération des LT.
Le mécanisme d’action de la rapamycine est distinct des autres immunosuppresseurs
utilisés en transplantation, tels que la cyclosporine A, le tacrolimus, l’azathioprine (AZA) ou
le mycophenolate mofetil (MMF). La cyclosporine A et le tacrolimus, qui se fixent
respectivement à CsA et FKBP12 (FKBP12 est également le site de liaison de la rapamycine)
agissent après le signal 1 et inhibent l’axe TCR-NFAT. Ces deux médicaments bloquent la
signalisation calcium dépendante et l’activation de la calcineurine en aval de la stimulation du
TCR et inhibe l’expression de gènes codant pour l'IL-2, l'IFN-g ou le TNF-a, ou les gènes liés
à NFAT (tels que Cbl-b, GRAIL, DGK). AZA et MMF sont des anti-métabolites qui inhibent
directement la synthèse des acides nucléiques, étape nécessaire à toute multiplication
cellulaire (Jung et al., 2015).
mTOR
Rapamycine
Figure 14: Mode d'action
des immunosuppresseurs
utilisés en transplantation
sur les 3 signaux
d’activation lymphocytaire
T
(Halloran, 2004)
Cyclosporine
Tacrolimus
Azathioprine
MMF
46
1.3. Rapamycine et induction de Treg
La cyclosporine et le tacrolimus bloquent plus précocément l’activation lymphocytaire
T (après le signal 1) et inhibent toutes les populations de LT. La rapamycine, par l’inhibition
de la voie mTOR, induit l'expansion des nTreg et la génération des iTreg au détriment des LT
CD4 Th1, Th2 ou Th17, ce qui a donc suscité un grand intérêt dans son utilisation pour
promouvoir une tolérance après la transplantation (Figure 15).
Effector and regulatory CD4 T cell differentiation
Th1, Th2, Th17 versus Treg
+ Rapamycine
Figure 15: Impact de la rapamycine sur la différentiation des LT CD4 helper
(Araki et al., 2012)
Ainsi, les patients traités par la rapamycine pour prévenir du rejet de greffe de rein
présentent une fréquence de Treg plus élevée que les patients traités par les inhibiteurs de la
calcineurine (Hendrikx et al., 2009; Noris et al., 2007; Sabbatini et al., 2015; Vallotton et al.,
2011). Contrairement à la rapamycine, les traitements par inhibiteurs de calcineurine bloquent
la transcription de l’IL-2, une cytokine nécessaire au développement et à l’expression de
FoxP3 (Thornton et al., 2004). Cette contribution à un maintien de la greffe suggère que la
rapamycine induit une augmentation de Treg fonctionnels chez les patients (Noris et al., 2007).
En 2005, Battaglia et al. ont été les premiers à avoir rapporté que l’inhibition de
mTOR par la rapamycine en présence d'IL-2 supprime la prolifération des LT effecteurs mais
promeut celle de nTreg fonctionnels, en plus d'induire la génération de iTreg (Battaglia et al.,
2005). Depuis, de nombreuses études ont confirmé que la rapamycine expand les Treg (Kim et
al., 2010; Qian et al., 2011; Zhang et al., 2010a), au détriment des autres populations LT
47
helper (Kopf et al., 2007). Dans plusieurs études, une co-culture de courte durée (moins de 2
semaines) avec la rapamycine n’induit pas l’expansion des Treg (Battaglia et al., 2006; Coenen
et al., 2006; Zeiser et al., 2008). Néanmoins, lorsque les cultures sont supérieures à 3
semaines, la rapamycine induit une forte augmentation de la prolifération Treg (Strauss et al.,
2007, 2009), suggérant que les Treg activés en présence de rapamycine pourrait présenter une
cinétique de prolifération retardée. La fonction des Treg est quant à elle augmentée après
traitement avec la rapamycine, avec une plus forte expression de FoxP3 et une plus grande
activité suppressive, par rapport à des Treg non traités (Basu et al., 2008; Bocian et al., 2010;
Haxhinasto et al., 2008; Strauss et al., 2007). Par la suite, des études ont porté sur l’impact de
différentes doses et cinétiques d’administration de la rapamycine sur la prolifération et la
fonction des Treg en complément d'une stimulation du TCR. Un traitement par la rapamycine
sur des LT CD4 préalablement stimulés induit une expression maximale de FoxP3 (Sauer et
al., 2008). Procaccini et al. ont montré qu’au contraire une réduction de l’activité mTOR
avant l’engagement du TCR est nécessaire à l’entrée des Treg dans le cycle cellulaire. En effet,
un traitement transitoire (1h) par la rapamycine en absence d’IL-2 suivi d’une stimulation des
LT CD4 résulte en une robuste prolifération des Treg. Par contre, un traitement chronique
durant la stimulation du TCR inhibe la prolifération des Treg (Procaccini et al., 2010).
Chez la souris, le taux de Treg mesuré après traitement par la rapamycine varie en
fonction du moment de leur mesure, et en fonction du dosage. L’impact de la rapamycine sur
l'induction et le maintien des Treg est temps-dépendant. Une administration à des jours alternés
ou une administration ponctuelle de la rapamycine est plus efficace pour l'induction et la
prolifération des Treg par rapport à une administration continue (Lu et al., 2010; Shin et al.,
2011; Wang et al., 2012a, 2011b). L’évérolimus, un analogue de la rapamycine également
prescrit chez les patients transplantés, induit dans des modèles murins la conversion de LT
CD4 en Treg et est efficace pour induire une tolérance immunologique après combinaison avec
une vaccination Treg (Daniel et al., 2010). De manière surprenante, certaines études montrent
qu’un traitement par la rapamycine induit des réponses Th1 et Th2 spécifiques, capables de
prévenir du rejet de greffe (Foley et al., 2005; Jung et al., 2006; Mariotti et al., 2008).
2. Ciblage de mTOR dans les cancers
La transformation d'une cellule normale vers une cellule cancéreuse est un processus
accompagné d'altérations génétiques impliquant l'activation d'oncogènes et la perte de gènes
suppresseurs de tumeurs, permettant à la cellule cancéreuse de proliférer, survivre ou former
de nouveaux vaisseaux sanguins. En 2000, Hanahan et Weinberg ont proposé six
48
caractéristiques fondamentales (Hallmarks) du Cancer, qui sont des capacités acquises par
une cellule normale la conduisant à évoluer progressivement vers une cellule tumorale
capable de croître et d'avoir un pouvoir invasif (Hanahan and Weinberg, 2000).
La voie de signalisation mTOR joue un rôle central de régulateur de l’oncogénèse. Les
tumeurs sont intrinsèquement présentes dans un environnement stressant (caractérisés par une
limite en nutriments ou en oxygène, et un faible taux de pH), qui conduirait normalement à
diminuer l'activité de la voie de signalisation mTOR. Ainsi, les différentes étapes de la
cascade de transduction du signal en amont de mTOR peuvent être le siège d'anomalies dans
les cancers humain, la conséquence étant l'activation constitutive de mTOR (Shaw and
Cantley, 2006). mTOR est ainsi apparue comme une cible thérapeutique importante pour le
traitement anti-cancer.
2.1. Dérégulations de la voie mTOR dans le cancer
2.1.1. Mutations activatrices
La modulation des récepteurs tyrosine kinase, due à une activation indépendante de la
fixation de ligands ou la surexpression de ligands permet d'induire l'activation de Ras et de
PI3K. De plus, une perte des mécanismes de rétrocontrôle négatif de ces voies peut conduire à
des signaux de prolifération. Une activation prolongée de Ras, via la perte de NF1 et une
accumulation de Ras-GTP est observée dans des cancers (Cichowski and Jacks, 2001). De
même, la perte du régulateur PTEN va amplifier la signalisation PI3K, et promouvoir la
tumorigénèse, et sa mutation est retrouvée dans plusieurs type de cancers (Cantley and Neel,
1999; Yuan and Cantley, 2008). La perte de p53, très commune dans le cancer, promeut
l'activation de mTORC1 (Feng et al., 2005). Ainsi, l'activation de la voie de signalisation
mTOR par ces mutations est observée dans de nombreux cancers et favorise leur prolifération,
mais elle n'est pas suffisante à elle seule pour induire un cancer.
TSC1/TSC2, dont le rôle principal est d'inhiber mTORC1, est un complexe
suppresseur de tumeur, dont les mutations conduisent à la sclérose tubéreuse, un syndrome
caractérisé par le développement de tumeurs bénignes dans de nombreux organes (van
Slegtenhorst et al., 1997). Des modèles murins déficients en TSC1 présentent le
développement de carcinomes hépatocellulaires caractérisés par une activation chronique de
mTORC1 (Menon et al., 2012).
49
2.1.2. Conséquences de la sur-activation de mTOR
L'activation oncogénique de la signalisation mTOR va être à l'origine de divers
processus. Il devient de plus en plus démontré que la dérégulation de la synthèse protéique en
aval de mTORC1 au niveau de 4E-BP1/eIF4E joue un rôle central dans la formation des
tumeurs. La perte de 4E-BP1 et l'activation en conséquence de la traduction de protéines
promeut la progression du cycle cellulaire et la prolifération des cellules in vitro (Dowling et
al., 2010). En réponse à l'activation oncogénique d'Akt, 4E-BP1/eIF4E va aussi contrôler la
traduction d'ARNm codant pour des protéines pro-oncogéniques, la croissance cellulaire et la
progression tumorale (Hsieh et al., 2010). De plus l'augmentation de la biogénèse
ribosomique liée à l'activation de mTOR promeut la prolifération cellulaire en fournissant la
machinerie nécessaire pour soutenir de haut niveau de croissance cellulaire. Une
augmentation de novo de la synthèse lipidique est également importante pour la prolifération
des cellules cancéreuses afin de produire les acides gras nécessaires à la synthèse de leur
membranes (Menendez and Lupu, 2007). Ainsi, une activation oncogénique de la voie PI3K
permet l'activation de SREBP1 via mTORC1, et la synthèse lipidique (Düvel et al., 2010),
ainsi que la synthèse de la cycline D1 qui a pour conséquence la dérégulation du cycle
cellulaire et l’augmentation de la prolifération des tumeurs (Nourse et al., 1994).
L'augmentation de la synthèse d'HIF1a augmente l'expression du VEGF, qui tient un rôle clé
dans la néo-angiogénèse, en activant la prolifération et la migration de cellules endothéliales
(Carmeliet and Jain, 2000).
L'activation constitutive de la signalisation PI3K-Akt-mTORC1 dans les cellules
cancéreuses va fortement inhiber l'autophagie. Les conséquences sont mal définies dans le
cadre du cancer, avec un rôle dichotomique sur la tumorigénèse, l’autophagie agissant à la
fois comme répresseur de tumeur et comme protecteur de la survie des cellules cancéreuses.
Ainsi, il a pu être observé que des souris dépourvues en éléments essentiels pour l'autophagie
présentent un taux élevé de développement de tumeurs (Yang and Klionsky, 2010). De
manière contradictoire, l'absence en autophagie peut également réduire les capacités des
cellules cancéreuses à survivre dans des conditions pauvres en énergie et en nutriments.
L’inhibition de l’autophagie par la chloroquine dans des modèles pré-cliniques favorise la
réponse anti-tumorale induite par des agents alkylants (Rebecca et al., 2014).
50
Prolifération
Tumorigénèse
Biogénèse ribosomale
Traduction
S6K
Activation oncogènes
Raf
PTEN
p53
mTORC1
HIF1a
Angiogenèse
SREBP1
Lipides/ Nucléotides
Ras
NF1
Métastases
Akt
PI3K
RTK
Survie cellulaire
4EBP1
Signal Oncogenèse
Prolifération
Autophagie
LKB1
TSC1/2
Inhibition
Gènes suppresseurs
de tumeurs
mTORC2
Akt
Survie
FOXO1
SGK1
Prolifération
Figure 16: Dérégulation de la voie mTOR et oncogenèse
2.2. Blocage de la voie mTOR en cancérologie
Le lien évident entre l'activation de mTOR et le cancer a généré un intérêt significatif
pour le ciblage de cette voie de signalisation pour la thérapie anti-cancer. De plus, parmi la
signalisation PI3K/Akt/mTOR, aucune mutation n'a été observée sur les complexes mTORC1
et mTORC2, faisant de la protéine mTOR une cible intéressante. Plusieurs classes
d'inhibiteurs de la voie mTOR ont été testées dans des modèles précliniques ou dans des
essais cliniques: la rapamycine et ses analogues, qui sont des inhibiteurs allostériques de
mTORC1, les doubles inhibiteurs ciblant simultanément les sites catalytiques de PI3K et des
complexes mTORC1-2, et les inhibiteurs compétiteurs d'ATP, ciblant le site catalytique de
mTORC1 et mTORC2. Seuls les analogues de la rapamycine, le temsirolimus et l'évérolimus,
ont aujourd'hui obtenus leur autorisation de mise sur le marché pour le traitement de patients
atteints de cancer.
2.2.1. La rapamycine et ses analogues
La rapamycine a été utilisée dès les années 1980 comme agent anti-cancer dans des
modèles in vitro (Douros and Suffness, 1981). Son développement en oncologie s'effectua
dans les années 1990. La rapamycine a été associée à une induction d'apoptose dans différents
systèmes tumoraux (Hosoi et al., 1999). De plus, l'inhibition de mTOR sensibilise les cellules
tumorales à l'apoptose conférée par des chimiothérapies conventionnelles altérant l'ADN in
vivo (Bruns et al., 2004). D'autres études ont également montré le pouvoir anti-tumoral de la
rapamycine par l'inhibition de l'angiogenèse, l’inhibition de la prolifération des cellules
51
tumorales ou l'induction de l'apoptose (Guba et al., 2002; Namba et al., 2006; Wu et al.,
2007). Malgré ses propriétés anti-tumorales, la rapamycine présente une faible
biodisponibilité et est insoluble dans l'eau, et les résultats des essais cliniques chez les patients
atteints de cancer se sont avérés décevants (Benjamin et al., 2011; Cloughesy et al., 2008).
Ainsi plusieurs analogues de la rapamycine (ou rapalogues) ont été développés, de structure
moléculaires et de mode d'action similaires, mais avec quelques modifications chimiques
rendant ces formulations solubles dans l'eau et facilitant leur administration pour un usage en
cancérologie (Faivre et al., 2006). Ainsi, trois analogues de la rapamycine ont été évalués
dans plusieurs essais de phase III, le temsirolimus, l'évérolimus et le ridaforolimus.
Temsirolimus. Le temsirolimus (ou CCI-779, torisel®) est une pro-drogue dérivée de
la rapamycine. Contrairement aux autres rapalogues, le temsirolimus est hydrolisé en
quelques minutes et transformé en sirolimus. Il est administré en intra-veineux une fois par
semaine chez des patients atteints de cancer du rein avancé de mauvais pronostic, ainsi que
chez les patients de lymphome du manteau (mantle cell lymphoma MLC).
Evérolimus. L'évérolimus (ou RAD001, affinitor®) est un dérivé de la rapamycine,
administré oralement et approuvé pour le traitement du cancer du rein avancé, des tumeurs
neuroendocrines, et du cancer du sein. Il est également administré chez des patients atteints de
lymphangioleiomyomatose et d'astrocytome, deux pathologies caractérisées par l'activation
constitutive de la voie mTOR après dérégulation du complexe TSC1/TSC2.
2.2.2. Efficacité anti-tumorale des rapalogues
Le temsirolimus et l’évérolimus ont montré un réel impact sur l’amélioration de la
survie chez les patients atteints de cancer. Néanmoins, ces traitements induisent des taux de
réponses variables, et favorisent une stabilisation de la maladie plutôt qu’une régression
tumorale (Coppin et al., 2008; Hudes et al., 2007; Motzer et al., 2008). La boucle de
rétrocontrôle négatif de la voie mTOR qui est supprimée après inhibition de mTORC1, va
stimuler la signalisation PI3K-Akt. Des prélèvements de tissus chez des patients atteints de
cancer du sein prélevés après un mois de traitement par évérolimus ont montré un niveau
élevé de protéine Akt activé comparé à des prélèvements avant le début du traitement
(O’Reilly et al., 2006). Une persistance de la phosphorylation de 4EBP1 peut être observée
durant un traitement de longue durée par la rapamycine et peut conduire à une persistance de
la synthèse protéique et de la croissance cellulaire (Choo et al., 2008; Dowling et al., 2010).
Malgré leur potentiel comme stratégie anti-cancer, les mécanismes expliquant l’absence de
réponse complète après traitement par temsirolimus ou évérolimus restent inconnus.
52
Chapitre II :
IMMUNITE ET CANCER
53
54
I. IMMUNITE T ANTI-TUMORALE
L’immunologie des tumeurs tient aujourd’hui une place cruciale dans la recherche
anti-tumorale, avec la démonstration du concept d'immunosurveillance des cancers (Dunn et
al., 2004) et du rôle majeur de l'infiltration des tumeurs par des LT sur les pronostiques
cliniques des cancers (Fridman et al., 2012).
1. Immunosurveillance des cancers et Immunoedition
En 1957, L. Thomas et F. Burnet ont introduit l’hypothèse de l’immunosurveillance
des cancers, stipulant que tout au long de la vie, des cellules effectrices du système
immunitaire patrouillent continuellement dans les tissus et détruisent des cellules
génétiquement altérées et malignes (Burnet, 1957). Bien que cette hypothèse ait longtemps été
contestée, une meilleure connaissance du système immunitaire et particulièrement l’apport de
modèles de souris immunodéficentes dans les années 1990 ont permis de réévaluer le rôle de
l’immunité, en particulier des LT CD4 et T CD8 dans la reconnaissance et l’élimination des
cancers (Dighe et al., 1994; Hung et al., 1998; Shankaran et al., 2001). La notion que le
système immunitaire ne protège pas seulement de la formation des tumeurs mais qu’il puisse
aussi modeler l’immunogénicité tumorale (Shankaran et al., 2001) est une des base de
l’hypothèse de l’Immunoédition des cancers, qui prolonge le concept d’immunosurveillance.
Cette hypothèse démontre le double impact de l’immunité sur le développement des cancers,
à la fois en protégeant l’hôte et en favorisant le développement des cancers et son
échappement aux mécanismes d’élimination (Dunn et al., 2004). Elle se déroule en trois
phases: L’Elimination, au cours de laquelle les cellules tumorales émergeantes sont reconnues
et éliminées par les différents acteurs du système immunitaire ; l’Equilibre, phase
correspondant à un état d’équilibre dynamique entre l’expansion des cellules tumorales ayant
survécu à l’élimination, et le système immunitaire qui la maintient en échec ; l’Echappement,
durant laquelle les cellules cancéreuses échappent à l’élimination en limitant l’expression
d’antigènes et en induisant divers mécanismes de tolérance immunitaire. Cette phase
d’échappement à la destruction immunitaire est aujourd’hui inclue dans les nouvelles
caractéristiques fondamentales (hallmarks) du cancer par Hanahan et Weinberg (Hanahan and
Weinberg, 2011).
55
2. Rôles des LT CD4 et cancer
Une forte infiltration lymphocytaire a été rapportée comme associée à une bonne
efficacité clinique. Pendant longtemps, le rôle des LT CD8 cytotoxiques dans les cancers a été
le plus étudié de par leurs propriétés cytotoxiques sur les cellules tumorales (Vesely et al.,
2011). Ainsi, une forte densité de LT CD8 mémoires est clairement associée à une meilleure
survie dans la majorité des cancers (Fridman et al., 2012). Néanmoins, il est maintenant
clairement établi que les LT CD4 jouent un rôle crucial dans le contrôle de l’immunité antitumorale.
2.1. Rôle des LT CD4 Th1 dans la réponse anti-tumorale
Le rôle anti-tumoral majeur joué par les Th1 a été clairement établi depuis plusieurs
années (Kennedy and Celis, 2008; Ostrand-Rosenberg, 2005). En effet, les réponses Th1 sont
fortement associées à une bonne efficacité clinique et une survie prolongée (Fridman et al.,
2012). Leur action s'effectue principalement via l'aide qu'elles apportent aux autres effecteurs
de l'immunité anti-tumorale: les LT CD8, DC, cellules NK et macrophages.
Les mécanismes de l'aide fournie par les Th1 pour la génération et l'augmentation des
réponses LT CD8 anti-tumorales sont les plus connus et constituent un paramètre important
pour prévenir l'induction d'une tolérance prématurée des LT CD8 (Bourgeois et al., 2002a;
Shafer-Weaver et al., 2009). Un modèle communément admis repose sur la capacité des LT
CD4 Th1 via l'expression transitoire de CD40L à favoriser la maturation des DC qui par la
suite activent les LT CD8 (Ridge et al., 1998). Un autre modèle suggère que les LT CD8,
exprimant transitoirement CD40, puissent recevoir directement l'aide des LT CD4 pour leur
différenciation en LT CD8 mémoires (Bourgeois et al., 2002b). Un autre type d'aide repose
sur les différentes cytokines produites par les Th1, en particulier l'IL-2 et l'IFN-g, qui
facilitent l'expansion et la différenciation des LT CD8 anti-tumoraux. L'IL-2 fonctionne
comme un facteur de prolifération et d'activation qui favorise une réponse LT CD8
cytotoxique efficace (Bos and Sherman, 2010). L'IFN-g favorise quant à lui l'expression des
molécules du CMH par les cellules tumorales conduisant à une reconnaissance accrue par les
Th1 et LT CD8. De plus il induit la sécrétion de chimioattractants (CXCL9, CXCL10…) par
les cellules tumorales pour attirer les LT CD8 spécifiques de la tumeur (Bos and Sherman,
2010; Marzo et al., 2000).
56
En plus de leur action sur les LT CD8, les Th1 sont capables directement ou
indirectement de lyser les cellules tumorales. Les Th1 jouent un rôle dans la stimulation des
cellules de l'immunité innée capables d'exercer une activité anti-tumorale contre la tumeur,
via l'activation des macrophages par l'IFN-g ou des cellules NK par l'IL-2 et l'IFN-g (van den
Broeke et al., 2003; Hung et al., 1998). Par ailleurs les Th1 spécifiques de la tumeur peuvent
eux-mêmes exercer un effet lytique sur les cellules tumorales (Schultz et al., 2000). La
production d'IFN-g ou de TNF-a agit comme un facteur anti-tumoral. De même les Th1
provoquent l'apoptose des cellules tumorales via les voies Fas/FasL ou TRAIL (Hahn et al.,
1995; Qin and Blankenstein, 2000).
2.2. Rôle des Treg dans la réponse pro-tumorale
Le développement de la tumeur est accompagné par l'accumulation de cellules
suppressives dans le microenvironnement tumoral, telles que les Treg (Schreiber et al., 2012).
Un taux élevé de Treg est ainsi retrouvé dans les tissus tumoraux de nombreux types de
cancers, tels que les cancer du sein, des poumons, du foie ou des mélanomes (Nishikawa and
Sakaguchi, 2010). La plupart des études ont montré une corrélation entre l'infiltration des
tumeurs par des Treg avec une faible survie dans les cancers ovariens, du sein ou du rein
(Curiel et al., 2004; Liotta et al., 2011; Merlo et al., 2009). De plus, une diminution du ratio
de LT CD8 par rapport aux Treg parmi les lymphocytes infiltrant les tumeurs (TIL) est associé
à un mauvais pronostic dans plusieurs types de cancer (Nishikawa and Sakaguchi, 2010).
Le recrutement des Treg au niveau des tumeurs se fait principalement par la production
de la chimiokine CCL22 par les cellules tumorales ou les macrophages infiltrant les tumeurs,
qui attire les Treg activés et fonctionnels exprimant CCR4 (Faget et al., 2011; Gobert et al.,
2009). D'autres combinaisons de chimiokines et de récepteurs de chimiokines, tels que
CCL28/CCR10 induits par hypoxie, et CXCL9-10-11/CXCR3, contribuent également à
l'infiltration des Treg dans les tumeurs (Facciabene et al., 2011; Redjimi et al., 2012).
Une conversion de LT CD4 en iTreg en réponse à une stimulation antigénique faible en
présence de TGF-b ou une prolifération des nTreg se produit au niveau de la tumeur, par
l'intermédiaire de DC immatures, rendues tolérogènes par l'IL-10, le TGF-b ou VEGF
sécrétés dans le microenvironnement tumoral (Ghiringhelli et al., 2005).
57
Tumeur
1.Migration Treg
Th effecteur
CCL22
Treg
3. Fonction suppressive médiée par Treg
IL-10
CCR4
TGF-b
VEGF
+
Sang périphérique
Ganglions
2.Prolifération et différenciation Treg
Figure 17: Treg et microenvironnement tumoral.
1) Migration des Treg : Le recrutement des T reg activés exprimant CCR4 est médié par la production de CCL22
par les cellules tumorales. 2) Prolifération et différentiation des T reg. La sécrétion cytokinique de VEGF, IL-10 et
TGF-b par les cellules tumorales entraîne un blocage de la maturation des DC responsables de l’induction des
Treg et de leur prolifération. 3) Fonctions suppressives des T reg sur les lymphocytes T effecteurs infiltrant la
tumeur.
Les différents mécanismes de suppression utilisés par les Treg peuvent être groupés en
quatre « modes d’actions » : une suppression par des cytokines inhibitrices, par une lyse
cellulaire, par une perturbation métabolique et par une modulation de la maturation ou de la
fonction des DC (Vignali et al., 2008).
Suppression par des cytokines inhibitrices. Les Treg ont la capacité de sécréter les
cytokines immunosuppressives TGF-b, IL-10 et IL-35. Un défaut en sécrétion de TGF-b par
les LT induit des syndromes lympho-prolifératifs similaires à ceux observés chez les souris
déficientes en FoxP3 (Li et al., 2006). Le TGF-b présent à une forte concentration à la surface
des Treg et non sécrété contribue également à l’activité suppressive (Nakamura et al., 2001).
Cette cytokine est également capable d’induire l’expression du dérivé oxygéné IDO par les
DC, capable de moduler les réponses immunitaires (Pallotta et al., 2011). Le TGF-b induit
également un rétrocontrôle positif sur le développement des Treg et leur pouvoir suppressif, en
permettant l’induction et la maintenance de l’expression du FoxP3 (Li et al., 2006). L’IL-10
est une autre cytokine immunosuppressive pouvant être produite par les Treg. Des souris
transgéniques dont les Treg sont déficient en IL-10 sont sujets à un développement de maladies
autoimmunes (Rubtsov et al., 2008). Les Treg secrétant l’IL-10 peuvent également rendre les
DC tolérogènes avec des fonctions régulatrices (Steinbrink et al., 1997). L’IL-35 est une
58
cytokine immunosuppressive sécrétée par les Treg plus récemment décrite, et montrant des
propriétés inhibitrices directes sur la prolifération de LT (Collison et al., 2007).
Suppression par lyse cellulaire. La lyse cellulaire est médiée par la sécrétion de
granzymes et de perforines. Les Treg activés induisent la mort cellulaire par le granzyme A et
la perforine, via l'adhésion par CD18 (Grossman et al., 2004). Par l'utilisation d'un modèle de
transplantation de lymphocytes, Gondek et al. ont montré que la tolérance induite par les Treg
dépend également du granzyme B (Gondek et al., 2005). D’autres molécules comme
galectine-1 ou galectine-10 ont démontré un rôle dans les mécanismes de suppression utilisés
par les Treg, capables d’induire l’apoptose de LT (Garín et al., 2007; Kubach et al., 2007).
Suppression par perturbation métabolique. FoxP3 réprime l'expression autocrine d'IL2 par les Treg, et le CD25 (récepteur à l'IL-2) exprimé par les Treg entre en compétition avec les
molécules CD25 exprimées par les LT conventionnels pour la capture de l'IL-2 exogène. Le
récepteur CD25 étant exprimé plus fortement par les Treg par rapport aux LT conventionnels,
une privation en IL-2 pour les LT conventionnels se produit et aboutit à l'apoptose de ces
cellules (Pandiyan et al., 2007). Deaglio et al. ont montré le rôle clé de la signalisation CD39CD73-adénosine dans les fonctions suppressives des Treg. L'ATP est disséminée par des
cellules endommagées ou activées et induit des réactions immunes. L'ectonucleosidase CD39
exprimée par les Treg hydrolyse l'ATP et l'ADP extracellulaires en AMP. Ensuite, l'AMP est
transformée par CD73 en adénosine, qui est immunosuppressive et régule les réponses
immunitaires innées et adaptatives (Deaglio et al., 2007). En se fixant sur les récepteurs A2A,
l'adénosine induit une accumulation intracellulaire d'AMPc, empêchant l'expression de CD25
par l'activation des LT et la sécrétion de cytokines inflammatoires (Huang et al., 1997).
Suppression par ciblage des DC. Les Treg peuvent également moduler la maturation ou
la fonction des DC. Le récepteur inhibiteur CTLA-4, exprimé par les Treg, se fixe au CD80 et
CD86 sur les DC, et provoque l'expression d'IDO, qui induit la suppression des LT (Mellor
and Munn, 2004). Les Treg peuvent également diminuer la capacité des DC à activer les LT,
en diminuant l'expression des molécules de co-stimulation CD80 et CD86 (Cederbom et al.,
2000). LAG3, un homologue de CD4, peut se fixer au CMH II et bloquer la maturation des
DC (Liang et al., 2008). De plus la neuropilline-1 prolonge les interactions entre les Treg et les
DC immatures, et empêche l'activation de LT naïfs (Sarris et al., 2008).
Les Treg présentent également des propriétés immunosuppressives contact-dépendant
avec les LT au niveau de la synapse immunologique. CTLA-4 se fixe sur CD28, et empêche
59
la co-stimulation requise pour l'activation et la prolifération de LT naïfs, avec une meilleure
affinité par rapport à CD80 et CD86 (Yokosuka et al., 2010).
Figure 18: Mécanismes de suppression utilisés par les Treg.
Les différents mécanismes de suppression utilisés par les T reg peuvent être groupés en quatre « modes
d’actions » : une suppression par des cytokines inhibitrices, par une lyse cellulaire, par une perturbation
métabolique et par une modulation de la maturation ou de la fonction des DC (Vignali et al., 2008).
De manière contradictoire, il a été démontré dans certains cas un rôle bénéfique des
Treg. Des recherches sur les transformations malignes induites par un microenvironnement
inflammatoire ont montré que les Treg peuvent protéger l'organisme contre ces tumeurs en
diminuant cette inflammation (Gounaris et al., 2009). Ainsi, des rapports ont démontré une
corrélation clinique entre le taux de Treg infiltrant les tumeurs et le contrôle immunitaire des
tumeurs de patients atteints de cancers de la tête et du cou (Zhang et al., 2010b), et entre le
taux de Treg infiltrant les tumeurs et la survie globale dans le lymphome ou le cancer
colorectal (Salama et al., 2009; Tzankov et al., 2008).
2.3. Rôles des autres LT CD4 helper dans la réponse anti-tumorale
LT CD4 Th2. Contrairement aux Th1, l'immunité médiée par les Th2 est
traditionnellement considérée comme un facteur aggravant la croissance tumorale, favorisant
l'angiogenèse, et inhibant l'immunité cellulaire (De Monte et al., 2011). L'induction de Th2
60
spécifiques d'antigènes pancréatiques a montré une promotion de la transformation
néoplasique et de la croissance tumorale (Ochi et al., 2012). La fréquence de Th2 sécrétant de
l'IL-5 a été corrélée à la progression du cancer du rein et du mélanome (Tatsumi et al., 2002).
Néanmoins, la contribution des Th2 à l'immunité anti-tumorale est contradictoire. La cytokine
IL-4 sécrétée par cette population présente des effets anti-tumoraux avec une augmentation de
l'infiltration des éosinophiles et macrophages dans la tumeur (Tepper et al., 1989, 1992).
LT CD4 Th17. Le rôle des Th17 dans l'immunité des cancers reste encore mal compris
et controversé (Muranski and Restifo, 2009; Wilke et al., 2011). Chez l'homme et la souris,
les Th17 sont négativement corrélés à la présence de Treg et positivement corrélés à la
présence de cellules immunitaires effectrices (LT CD8 cytotoxiques, cellules NK…) (Curiel
et al., 2004; Kryczek et al., 2009). Chez la souris déficiente en IL-17, la croissance tumorale
et le développement de métastases pulmonaires sont accélérés. Inversement, l'expression
ectopique d'IL-17 dans les tumeurs inhibe la progression tumorale (Martin-Orozco et al.,
2009). Plusieurs études ont également mis en évidence une activité pro-tumorale des Th17.
Les premières études ont montré que l'IL-17 favorise la croissance tumorale et les
mécanismes d'angiogenèse, en particulier chez les souris immunodéficientes (Tartour et al.,
1999). L'IL-17 induit la production d'IL-6 par les cellules tumorales, conduisant à
l'augmentation
de
l'expression
de
gènes
pro-angiogéniques
et
à
l'expression
d'ectonucléosidase (Chalmin et al., 2012; Wang et al., 2009).
LT CD4 Th9. Le rôle des Th9 et de l'IL-9 dans l'immunité anti-tumorale a été
récemment mis en évidence (Schmitt and Bopp, 2012). Le transfert adoptif de Th9 chez des
souris sauvages ou immunodéficientes est capable d'inhiber la croissance d'un mélanome
(Purwar et al., 2012). Une seconde étude a confirmé l'activité anti-tumorale des Th9, qui s'est
même avérée supérieure à celle des Th1, Th2 et Th17 (Lu et al., 2012). Des travaux très
récents ont montré que les propriétés anti-tumorales des Th9 sont dictées par le facteur de
transcription IRF1. L'IL-1b induit la phosphorylation de STAT1 et l'expression consécutive
d'IRF1, qui se fixe sur le promoteur Il9 et Il21 et induit la production d'IL-9 et d'IL-21
(Végran et al., 2014).
LT CD4 TFH. Certaines études ont associé la réponse humorale avec la promotion de
la croissance tumorale (Qin et al., 1998; de Visser et al., 2005). Néanmoins de récentes études
ont montré un rôle clé des LT CD4 folliculaires helper dans le recrutement des cellules
immunitaires dans la tumeur et dans la formation de structures folliculaires intra-tumorales,
qui corrèlent avec un bon pronostic (Coppola et al., 2011; Gu-Trantien et al., 2013). Il a
61
également été récemment suggéré qu'une augmentation de l'activité des TFH et de la
production d'anticorps thérapeutiques altèrent les cellules immunosuppressives CD8
régulatrices (Alvarez Arias et al., 2014).
IL-17A, IL-17F
IL-21
IFN-g
Th17
Th1
Recrutement DC,
CD8, NK
Inhibition
Angiogenèse
Angiogenèse
IL-17
Suppression
TIL
IL-9
Th9
Treg
TGF-b
Recrutement
Eosinophiles, macrophages
IL-4
IL-13
Th2
IL-5
Tumeur
Prolifération
tumorale
Lymphocytes B
Anticorps
Anti-antigènes
de tumeur
IL-21
TFH
Figure 19: Rôle des différentes sous-populations lymphocytaires T CD4 dans le contrôle de l’immunité
anti-tumorale.
.
62
II. CANCER DU REIN ET IMMUNITE
Au cours des dernières années, une meilleure compréhension de la biologie du cancer
du rein a entrainé des progrès majeurs dans le traitement des patients atteints de carcinome
des cellules rénales métastatiques (mRCC). Cette tumeur immunogène était traitée par
immunothérapie avec l'IL-2 et l'IFN-a, avant l'introduction de nouveaux agents ciblant
l'angiogenèse et la voie de signalisation mTOR. Cependant, ces agents ciblés induisent
rarement des réponses complètes, et des nouvelles approches d'immunothérapies sont mises
en place pour contrer l'échappement immunitaire de la tumeur.
1. Généralités
Le cancer du rein représente environ 3% des tumeurs malignes chez l’adulte (Gupta et
al., 2008). Parmi les cancers du rein, le carcinome des cellules rénales (RCC) à cellules claires
est le sous-type le plus commun (70-80% des cas), devant les papillaires (10-15%) et les
chromophobes (3-5%) (Ljungberg et al., 2010). Le RCC à cellules claires se développe à
partir de cellules du néphron du tubule proximal par l'activation des voies de l'hypoxie. Il
apparait majoritairement après des mutations somatiques inactivant le gène suppresseur de
tumeur VHL (Von Hippel-Lindau), résultant en une surexpression du facteur de transcription
HIF1a, qui joue un rôle central dans la réponse cellulaire à l'hypoxie et l'induction de
l'expression de VEGF pour une néo-angiogenèse (Nickerson et al., 2008). Les cellules
stromales infiltrantes ont été identifiées comme la source principale du VEGF, et un taux
élevé de l’expression du récepteur au VEGF est associé à une diminution du taux de survie
après résection du RCC (Li et al., 2011a; Rivet et al., 2008). Bien que les patients avec un
RCC localisé soient considérés comme curables, les patients atteints de RCC métastatiques
(mRCC) sont de très mauvais pronostics. Approximativement 25% des patients avec un
cancer du rein sont métastatiques d'emblée au diagnostic (Janzen et al., 2003). Il existe
diverses classification pronostiques dans ces situations métastatiques: le mRCC est classifié
en risque pronostic favorable, intermédiaire ou mauvais, selon un modèle validé par Heng et
al. basé sur des critères cliniques tels que l’état général du patient, le délai entre diagnostic et
début du traitement ou des critères biologiques (Heng et al., 2009).
Les chimiothérapies cytotoxiques sont peu efficaces dans le traitement des RCC,
probablement car ils dérivent des cellules luminales du tubule proximal exprimant un taux
élevé de protéines de résistance aux drogues (MDR-1) (Dutcher and Nanus, 2011; Nanus et
63
al., 2004; Oudard et al., 2007). L'exérèse chirurgicale de la tumeur rénale primitive est le
traitement standard des maladies localisées ou localement avancées (Lam et al., 2008).
Toutefois, malgré la néphrectomie, le risque de rechute selon un mode métastatique survient
chez environ 20-40% des patients (Janzen et al., 2003). En termes de recherche contre le
cancer, une avancée prometteuse a été la validation clinique de molécules capables de cibler
et d'inhiber des voies métaboliques pro-oncogéniques, entrainant moins d'effets secondaires.
La meilleure compréhension des voies oncogéniques, notamment de la voie VHL-HIF1aangiogénèse, a conduit à l'utilisation de thérapies ciblées dans la prise en charge du cancer du
rein. Les inhibiteurs des tyrosines kinases des récepteurs au VEGF (TKI : sorafenib, sunitinib,
pazopanib et axitinib) et les mTORi (évérolimus et temsirolimus) ont été développés et ont
remplacé l’immunothérapie par IL-2 et IFN-a comme standards de traitement pour les RCC.
Le bévacizumab, un anticorps monoclonal anti-VEGF est également validé en combinaison
avec l’IFN-a chez les patients atteints de RCC (Inman et al., 2013).
2. Le cancer du rein, une tumeur immunogène
Le RCC est considéré comme une tumeur immunogène et présente un fort infiltrat de
cellules immunitaires, composé de LT, cellules NK, DC et macrophages. En dépit de ce fort
infiltrat, la réponse immunitaire dirigée contre les RCC n'est pas efficace et tend vers un profil
immunosuppressif et un échappement à la réponse immunitaire.
Malgré que la présence de cellules NK et de LT CD4 Th1 dans la tumeur soit associée
à un bon pronostic (Eckl et al., 2012; Kondo et al., 2006), un taux élevé de TIL est de manière
surprenante corrélé à un mauvais pronostic et à une plus faible survie chez les patients atteints
de RCC (Fridman et al., 2012). Le fort infiltrat de LT CD8 dans les RCC est ainsi associé à un
mauvais pronostic (Nakano et al., 2001). L'analyse de la clonalité des LT CD8 révèle un
faible taux d'expansion dans les RCC par rapport aux autres tumeurs solides (Sittig et al.,
2013). Seuls 20% des LT CD8 parmi les TIL reconnaissent des cellules tumorales autologues
(Markel et al., 2009), suggérant un défaut de la reconnaissance de la tumeur. Récemment, il a
été montré une forte expression de la molécule inhibitrice PD-1 ou des récepteurs inhibiteurs
KIR sur les LT CD8 infiltrant les RCC (Gati et al., 2001; Giraldo et al., 2015). La tumeur
exprime également un taux élevé de molécules inhibitrices telles que PD-L1 ou HLA-G
pouvant affecter ces LT CD8 (Dunker et al., 2008; Wang et al., 2012b). Un autre mécanisme
impliqué dans l'échappement de la tumeur à la réponse immunitaire est la modulation de la
64
maturation des DC induite par la tumeur, provoquant une anergie des LT (Noessner et al.,
2012).
Des altérations de l'immunité dans les RCC modulent également la sécrétion de
cytokines par la tumeur, et induisent un développement des Treg (Finke et al., 2008) qui sont
comme dans la plupart des cancers, associés à un mauvais pronostic (Griffiths et al., 2007;
Liotta et al., 2011). En addition, les Treg isolés des TIL démontrent une plus forte activité
immunosuppressive par rapport aux Treg circulants (Asma et al., 2015). La présence de MDSC
est également décrite chez les patients atteints de RCC, et participe à l'altération des réponses
immunitaires anti-tumorales (Finke et al., 2011). Le microenvironnement des RCC induit
l'activation et la différenciation de macrophages associées aux tumeurs (TAM), dérivés de
monocytes migrant dans la tumeur, impliqués dans la progression tumorale (Daurkin et al.,
2011). Ces TAM sont significativement corrélés à la densité des vaisseaux tumoraux et au
niveau de VEGF (Toge et al., 2009).
3. Les traitements anti-mTOR dans le RCC
Temsirolimus. Le temsirolimus a été approuvé pour le traitement du cancer du rein
avancé en 2007 sur la base des résultats positifs obtenus dans un essai de phase III randomisé,
contrôlé, du temsirolimus seul ou en combinaison avec l'IFN-a (Hudes et al., 2007). Dans
cette étude, 626 patients avec un cancer du rein métastatique non préalablement testé ont été
randomisés et traités avec 25mg de temsirolimus en intra-veineux chaque semaine, ou 3MU
d'IFN-a en sous-cutané 3 fois par semaine, ou une combinaison de 15mg de temsirolimus par
semaine et 6MU d'IFN-a 3 fois par semaine. Les patients ayant reçu temsirolimus ont
présenté une meilleure survie globale et une meilleure survie sans progression par rapport aux
patients traités avec l'IFN-a seul, tandis qu'il n'y avait pas de différence sur la survie globale
entre la combinaison des deux thérapies et le groupe IFN-a (Figure).
Everolimus. L'évérolimus a été approuvé pour le traitement du cancer du rein de stade
avancé en seconde ligne après échecs des traitements par les inhibiteurs de récepteurs tyrosine
kinase sunitinib et/ou sorafenib, suite à l'essai clinique appelé RECORD-1 (Motzer et al.,
2008). Dans cet essai de phase III double aveugle, randomisé avec contrôle placébo, 416
patients ont reçu 10mg d'évérolimus par jour ou un placébo. L'évérolimus montra une
amélioration de la survie sans progression comparé au groupe placébo, sans rapport avec leur
traitement précédent.
65
Chez les patients atteints de mRCC avec un risque pronostique favorable ou
intermédiaire (selon Heng et al.), la thérapie de première ligne couramment utilisée est la
monothérapie avec sunitinib ou pazopanib, ou une combinaison d'IFN-a et de bevacizumab.
Le mTORi évérolimus est recommandé en seconde ligne de traitement pour les patients
progressant après une première ligne avec un inhibiteur de TKI ou du VEGF. La prise en
charge de première intention des patients avec un risque pronostique défavorable réside sur le
sunitinib ou le mTORi temsirolimus (Ljungberg et al., 2010).
4. Immunothérapies dans le RCC
Avant le développement des thérapies ciblées, les cytokines IFN-a et IL-2 étaient les
principaux traitements standars pour le RCC avancé. Ells ont été utilisées pour stimuler à la
fois le système immunitaire inné et adaptatif. Elles induisent la prolifération et la
différenciation des cellules NK, et promeuvent la survie, la prolifération et la différenciation
des LT CD4 helper (Vacchelli et al., 2014). Une forte dose d’IL-2 a démontré des activités
anti-tumorales chez certains patients, avec une réponse durable chez un faible pourcentage
(Fyfe et al., 1995; Klapper et al., 2008; McDermott et al., 2005). Néanmoins, ces doses
présentent de fortes toxicités, et ces traitements sont réservés aux sous-groupes de patients
capables de supporter cette thérapie (Ljungberg et al., 2010).
Il y a un fort rationnel à l'utilisation d'immunothérapies pour le traitement de patients
atteints de RCC. L'observation anecdotique de patients atteints de mRCC présentant une
rémission spontanée dans les bras placébo des essais cliniques (Elhilali et al., 2000; Gleave et
al., 1998), et une forte infiltration des cancers par des cellules immunitaires suggèrent un rôle
important des mécanismes immunitaires dans l'évolution naturelle de la maladie. De plus, le
microenvironnement tumoral de ces cas de régression spontanée était composé d'antigènes
associés aux tumeurs, et les tumeurs étaient infiltrées par des LT CD4 et CD8 effecteurs
mémoires spécifiques de ces antigènes (Gleave et al., 1998). Actuellement, les cytokines
recombinantes sont les seules immunothérapies approuvées pour les RCC. Des observations
émergeants d'essais cliniques suggèrent que de nouvelles immunothérapies ont le potentiel
d'améliorer la survie des patients atteint de RCC. Différentes approches d'immunothérapies
sont actuellement investiguées, comprenant le ciblage des mécanismes immunosuppressifs et
les vaccins:
Ciblage des mécanismes immunosuppressifs. La progression tumorale est en partie due
à l'échappement à l'immunosurveillance, et le ciblage des mécanismes immunosuppressifs est
66
une nouvelle dynamique dans la thérapie anti-cancer. Les inhibiteurs de checkpoint
immunitaires, en particulier les anticorps anti-PD-1/PD-L1 ont montré des réponses
impressionnantes dans le mélanome métastatique (Hodi et al., 2010). PD-1 est un récepteur
inhibiteur membre de la famille B7-CD28 jouant un rôle crucial dans la régulation négative de
l'activation lymphocytaire T (Keir et al., 2008). Les patients dont les tumeurs contiennent des
TIL PD-1+ sont plus susceptibles de présenter des grosses tumeurs, un stade avancé de RCC
ou une différenciation sarcomateuse élevée par rapport aux patients sans TIL PD-1+,
suggérant que l'utilisation d'anticorps bloquant PD-1 puisse contrôler la croissance tumorale et
présenter un potentiel thérapeutique intéressant (Thompson et al., 2007). En addition, 1/3 des
patients atteints de RCC présentent une forte expression de PD-L1, qui se caractérise par une
tumeur plus agressive et un pronostic défavorable (Thompson et al., 2006). Récemment, les
résultats d'un essai de phase II ont montré une réponse objective chez des patients atteints de
mRCC traités avec Nivolumab (Motzer et al., 2015). L'induction d'une régression tumorale
suite à l'administration d'un anticorps anti-PD-L1 a été observé dans un essai chez des patients
atteints de RCC avancés (Brahmer et al., 2012).
Vaccins. Malgré le renouveau de l’immunothérapie anti-cancer, aucun vaccin n’est
parvenu à fournir des résultats cliniques assez satisfaisants pour être approuvé dans le
traitement de RCC. Les nouvelles approches en cours d’investigations exploitent les effets
immunomodulateurs des agents anti-angiogéniques pour combiner avec les vaccins
thérapeutiques. Dans l’essai de phase III IMPRINT, 340 patients HLA-A*02 ont été vaccinés
avec le vaccin IMA-901, développé sur la base d’une sélection de peptides associés à des
antigènes de tumeurs (comprenant MUC1, MMP7, MET, APOL1…) en combinaison avec du
sunitinib (NCT01265901). La combinaison du vaccin AGS-003, à base de DC transfectées
avec CD40L et de l’ARNm tumoral, avec du sunitinib est actuellement évaluée chez des
patients atteints de mRCC avec un risque pronostic défavorable dans l’essai de phase III
ADAPT (NCT01582672).
67
68
RATIONNEL ET OBJECTIFS
69
70
HYPOTHESE DE RECHERCHE
La rapamycine et ses analogues l'évérolimus et le temsirolimus sont des inhibiteurs de
la voie de signalisation mTOR. Ils sont couramment prescrits dans plusieurs applications
cliniques, incluant la prévention du rejet de greffe et le traitement de cancers. Dans le cadre de
la transplantation d'organes, l'administration de la rapamycine génère un environnement
suppressif par l'inhibition de l'activation lymphocytaire T et la promotion des Treg. Chez les
patients atteints de cancer, l'évérolimus et le temsirolimus sont administrés pour leur pouvoir
anti-prolifératif et leur capacité à inhiber l'angiogenèse. Or ces deux indications de
d'administration
des
mTORi
apparaissent
en
contradiction
l'une
avec
l'autre,
l'immunosuppression et la tolérance immunitaire nécessaires à prévenir du rejet de greffe
étant délétères pour la réponse anti-tumorale (Gaumann et al., 2008).
De manière surprenante, en comparaison aux autres immunosuppresseurs (tacrolimus,
cyclosporine…) l’administration de la rapamycine a montré une diminution de l'incidence des
cancers parmi les patients transplantés (Alberú et al., 2011; Campistol et al., 2006; Euvrard et
al., 2012; Vajdic CM et al., 2006). Par ailleurs, l’impact de la voie mTOR dans la génération
des LT CD8 mémoires a été clairement établi. Ainsi, la rapamycine est apparue comme un
outil très intéressant en association avec des vaccins pour générer des LT CD8 mémoires antitumoraux plus efficaces (Amiel et al., 2012; Diken et al., 2013; Li et al., 2012; Wang et al.,
2011c, 2014). Cela suggère que les mTORi, en plus de leur activité immunosuppressive,
présenteraient des propriétés stimulatrices de la réponse immunitaire anti-tumorale (Law,
2005).
Cependant, l’impact de l’inhibition de la voie mTOR sur l’immunité T anti-tumorale
chez les patients atteints de cancer est peu connu. Notre hypothèse est que, en plus de leur
effet anti-prolifératif sur les tumeurs, une modulation de l’immunité T anti-tumorale induite
par les mTORi pourrait influencer leur efficacité thérapeutique chez les patients atteints de
cancer. Récemment, notre équipe a rapporté le cas d'une patiente atteinte d'un cancer rénal
métastatique ayant une réponse durable sous évérolimus. Nous avons observé une corrélation
entre l'efficacité clinique, le taux de Treg et la réponse Th1 spécifique de tumeur. Ainsi une
forte réponse Th1 anti-tumorale associée à un faible taux de Treg était observée pendant la
phase de contrôle de la maladie. En revanche, ces deux paramètres ont inversement évolué
lors de la progression tumorale (Thiery-Vuillemin et al., 2014a). Cette observation renforce
notre hypothèse et a suscité l’intérêt d’étudier les modifications des réponses T anti-tumorales
chez les patients atteints de cancer et traités par mTORi.
71
OBJECTIFS:
Au cours de ces travaux de thèse, nous avons mis en place une analyse des réponses
immunitaires T anti-tumorales chez des patients atteints de cancer rénal métastatique traités
par évérolimus. De plus, des modèles in vivo ont été utilisés pour disséquer l'implication des
différentes populations de LT anti-tumoraux dans l’efficacité des mTORi et pour évaluer la
combinaison de ces derniers avec des immunothérapies.
Les objectifs de l’étude des réponses immunitaires chez les patients traités par évérolimus
sont:
i) Analyser phénotypiquement et fonctionnellement les Treg.
ii) Evaluer les réponses Th1 spécifiques de tumeur.
iii) Déterminer l’implication de la réponse immunitaire anti-tumorale induite par les mTORi
sur l’évolution de la maladie.
Les objectifs dans les modèles in vivo sont :
i) Etudier le rôle de l’immunité adaptative T CD4 et CD8 sur l’efficacité des mTORi dans
différents modèles de tumeurs transplantables chez la souris.
ii) Analyser le rôle immunosuppresseur des Treg chez des souris traitées par les mTORi.
iii) Evaluer l’efficacité de la combinaison des mTORi avec un vaccin thérapeutique antitumoral.
72
RESULTATS
73
74
ARTICLE 1: The efficacy of rapalogs everolimus and temsirolimus relies on a drastic
modulation of adaptive antitumor T cell immunity
Laurent Beziaud, Laura Mansi, Patrice Ravel, Caroline Laheurte, Lise Queiroz, Sindy
Vrecko, Francis Bonnefoy, Clémentine Gamonet, Jean-René Pallandre, Tristan Maurina,
Guillaume Mouillet, Thierry Nguyen Tan Hon, Elsa Curtit, Béatrice Gaugler, Jagadeesh
Bayry, Eric Tartour, Bernard Royer, Antoine Thiery-Vuillemin, Xavier Pivot, Yann Godet,
Christophe Borg and Olivier Adotévi.
Article soumis à Cancer Research
L’objectif principal de cet article a été d’étudier la modulation des réponses T CD4
anti-tumorales chez des patients atteints de cancer rénal métastatique (mRCC) traités par
évérolimus.
Au cours de cette étude nous avons ainsi analysé les Treg et les réponses Th1 antitumorales (anti-télomérase TERT) chez 23 patients atteints de mRCC au moment de leur
inclusion et tous les deux mois jusqu’à la fin de leur traitement par évérolimus. Nous avons
observé une augmentation concomitante des Treg et de la réponse Th1 anti-TERT chez la
plupart des patients suivant le traitement. Un modèle mathématique basé sur le taux de
variation des réponses Th1 anti-TERT et Treg durant les deux premiers mois de traitement par
évérolimus a été utilisé pour évaluer l’implication de l’immunité T anti-tumorale sur
l’efficacité clinique du traitement. Les patients présentant une diminution de la variation des
Treg et simultanément une augmentation de la variation des Th1 anti-TERT présentaient une
meilleure survie par rapport aux patients dont les paramètres immunitaires ne variaient pas, ou
variaient dans une même direction. L’analyse des réponses immunitaires au moment de la
progression tumorale a montré que la plupart des patients perdaient leurs réponses Th1 antiTERT, et que cet effet était associé à une augmentation des Treg.
Les Treg issus des patients expriment Hélios, suggérant un phénotype Treg naturel. Des
expériences
in
vitro
ont
mis
en
évidence
une
augmentation
des
propriétés
immunosuppressives des Treg exposés à temsirolimus ou évérolimus, comparé aux Treg non
traités. Ainsi, les Treg exposés in vitro à l'évérolimus ou au temsirolimus inhibaient plus
fortement la prolifération et la production d’IL-2 et d’IFN-g de LT allogéniques. Nous avons
montré par des expériences de transwell que la propriété immunosuppressive des Treg exposés
aux mTORi était contact-dépendante.
75
Enfin, l'étude plus poussée des relations entre la réponse T anti-tumorale et l’efficacité
des mTORi anti-cancer a été réalisée à l'aide de différents modèles de cancer transplatables
chez la souris par des expériences de déplétion de LT. L'utilisation en particulier du modèle
de souris transgénique DEREG permettant la déplétion spécifique des Treg après injection de
la toxine diphtérique a démontré que la présence de Treg in vivo altérait l’efficacité antitumorale des mTORi, par un mécanisme impliquant l’inhibition des réponses T CD8 antitumorales. De plus, l’efficacité des mTORi était augmentée par la combinaison avec des
agents bloquant les Treg, tels que le sunitib ou un antagoniste du CCR4.
En conclusion, cette étude a montré pour la première fois le rôle de l’immunité T antitumorale sur l’efficacité clinique des mTORi, qui induisent une réponse T anti-tumorale
efficace mais inhibée par une expansion de Treg plus immunosuppressifs. Ces résultats
soulignent l’intérêt potentiel de combiner les mTORi avec des immunothérapies antitumorales.
76
The efficacy of rapalogs everolimus and temsirolimus relies on a drastic
modulation of adaptive antitumor T cell immunity
Laurent Beziaud1,2, Laura Mansi1,2,3, Patrice Ravel4, Caroline Laheurte1,5, Lise Queiroz1,
Sindy Vrecko1,2, Francis Bonnefoy1, Clémentine Gamonet1,2, Jean-René Pallandre1, Tristan
Maurina3, Guillaume Mouillet3, Thierry Nguyen Tan Hon3, Elsa Curtit1,2,3, Béatrice Gaugler1,
Jagadeesh Bayry6, Eric Tartour7, Bernard Royer1,8, Antoine Thiery-Vuillemin1,2,3, Xavier
Pivot1,2,3, Yann Godet1,2, Christophe Borg1,2,3 and Olivier Adotévi*,1,2,3.
1
INSERM UMR1098, LabEx LipSTIC, Besançon, France.
University of Bourgogne Franche-Comté, UMR1098, Besançon, France.
3
Department of Medical Oncology, University Hospital of Besançon, Besançon, France.
4
IRCM - INSERM U1194, Institut de Recherche en Cancérologie de Montpellier, Equipe
Bioinformatique et biologie des systèmes du cancer, Montpellier, France.
5
EFS Bourgogne Franche-Comté, Plateforme de Biomonitoring, INSERM CIC1431,
Besançon, France.
6
INSERM U1138, Centre de Recherche des Cordeliers; Université Pierre et Marie Curie;
Université Paris Descartes; Paris, France
7
INSERM UMR970, Hôpital Européen Georges Pompidou. Department of Biological
Immunology, Assistance Publique-Hôpitaux de Paris. University Paris Descartes, Sorbonne
Paris Cité, Paris, France
8
Department of Pharmacology, University Hospital of Besançon, Besançon, France.
2
Running title: Immune-mediated antitumor efficacy of mTOR inhibitors
Key words: mTOR inhibitors, renal cell carcinoma, regulatory T cells, Th1, CD8 T cells
*Corresponding author:
Pr Olivier Adotévi
INSERM UMR1098 EFS Bourgogne Franche-Comté, 8, rue du Docteur Jean-FrançoisXavier Girod BP 1937 25020 Besançon Cedex France. Phone: +33 3 81 66 93 51 Fax: +33 3
81 66 87 08
E-mail: [email protected]
Conflict of interest: The authors have declared that no conflict of interest exists.
Abstract: 196 words
Word count: 5,053 (introduction, methods, results, discussion)
Number of figures: 7 figures
Number of references: 50
77
ABSTRACT
Although the mTOR inhibitors (mTORi) everolimus and temsirolimus are used as anticancer
drugs, the contribution of an immune modulation mediated by these drugs to their clinical
efficacy has not been investigated. Here we performed a dynamic immunomonitoring study in
metastatic renal cell carcinoma (mRCC) patients treated with everolimus. Concomitant
modulation of FoxP3+ regulatory CD4 T cells (Tregs) and tumor-specific CD4 Th1 response
occurred during everolimus treatment in most patients. We identified three immune groups
based on the early modulation of both Treg and anti-tumor Th1 cells and found that patients
with {low Tregs plus high anti-tumor Th1 cells} showed the best survival. At the disease
progression, most patients lost the anti-tumor Th1 response and this was associated with an
increase of suppressive Tregs. The studies conducted in mice demonstrated that the presence of
Tregs in vivo altered the responses to mTORi via a mechanism involving the inhibition of
antitumor CD8 T cell responses. Furthermore the efficacy of mTORi was improved by
combination with Tregs depleting agents. Altogether, our results describe for the first time a
dual impact of host adaptive antitumor T cell immunity on the clinical effectiveness of
mTORi and prompt their association with immunotherapies.
78
INTRODUCTION
The mTOR protein is a conserved serine/threonine kinase involved in the regulation of cell
growth, metabolism and apoptosis (1). It exerts its physiological functions through two
distinct complexes named mTOR complex 1 (mTORC1) and 2 (mTORC2) downstream of the
PI3K/AKT pathway (1). Oncogenic activation of mTOR signaling induces several processes
required for cancer cells growth, survival, and proliferation (2). Thus mTOR inhibition has
gained great interest in cancer therapy and many rapamycin analogs (rapalogs) are now being
used in clinical settings (3). Everolimus and temsirolimus are two mTOR inhibitors (mTORi)
approved for breast cancer, neuroendocrine carcinoma treatments and relapsing metastatic
renal cell carcinoma (mRCC) patients after anti-angiogenic therapy (4–7).
The mTOR complexes also represent a key regulator of immune responses. Notably, these
pathways are determinant for the differentiation, homeostasis, and functional regulation of
both CD4 and CD8 T cell subsets (8). The lack of mTOR in naïve CD4 T cells has been
shown to promote preferentially forkhead box transcription factor (FoxP3+) regulatory T cells
(Tregs) to the detriment of Th1, Th2 or Th17 differentiation (9,10). In solid organ
transplantation, rapalogs promote Tregs induction and create an immunosuppressive
environment required to prevent graft rejection (11,12). Interestingly, it has been recently
reported that organ transplant recipients treated with mTORi have lower risk of developing
cancer suggesting an impact of mTORi on antitumor immune responses (13). Indeed, recent
immunological studies showed that blocking mTOR signaling can also promote memory T
cell functions and tumor immunity in animal models (14–16). However the mTORi-mediated
modulation of antitumor T immunity and its interaction with treatment efficacy have not been
investigated in cancer patients.
Based on the critical role played by adaptive T cell immunity in cancer (17,18), we
hypothesized that anticancer rapalogs could promote suppressive Tregs which in turn could be
detrimental for host antitumor T cell immunity. In this regard, we recently described a striking
modulation of T cell responses in a mRCC patient treated with everolimus (19). This patient
presented at the time of disease control a strong antitumor Th1 response, which was
completely lost at the progression when high Tregs expansion occurred.
Here, we performed a dynamic monitoring of both Tregs and tumor-specific Th1 responses in a
cohort of mRCC patients treated with everolimus. The clinical impact of the everolimus79
mediated immune modulation was investigated by using a model based on both Tregs and
antitumor Th1 responses evolution. Furthermore, we used various mouse tumor models to
dissect more precisely the role of adaptive antitumor T cells on the efficacy of everolimus and
temsirolimus.
MATERIALS AND METHODS
Patients and sample collections
Metastatic renal cell carcinoma patients treated with everolimus after failure of antiangiogenic
treatment had been enrolled after the signature of informed consent at the University Hospital
Jean Minjoz (Besançon, France) between November 2011 and January 2015. Everolimus was
administrated 10 mg daily, and decreased to 5 mg daily when occurrence of adverse events.
Blood samples were collected at baseline and every 2 months. Peripheral blood mononuclear
cells (PBMC) were isolated by density centrifugation on Ficoll-Hyperpaque gradient (SigmaAldrich, Saint Louis, USA) and frozen until use. Diseases were classified as defined by Heng
et al. (20) and evaluation of response was performed according to RECIST (Response
Evaluation Criteria in Solid Tumors).
Monitoring of Tregs and telomerase-specific Th1 responses in mRCC patients
Tregs staining protocol is detailed in supplementary materials section. Samples were acquired
on a Facs Canto II (BD Biosciences, Le Pont de Claix, France) and analyzed with the Diva or
FlowJo softwares. The spontaneous antitumor Th1 responses were assessed after in vitro
stimulation of PBMC with a mixture of HLA-DR restricted peptides derived from telomerase
(5 µg/mL) during 7 days as previously described (21). The presence of specific T cells was
measured by IFN-#-ELISPOT assay (Diaclone, Besançon, France) as previously reported
(21). Spot-forming cells were counted using the C.T.L. Immunospot system (Cellular
Technology Ltd, Bonn, Germany). The number of specific T cells expressed as spot-forming
cells per 105 cells was calculated after subtracting negative control values (background).
Responses were positive when IFN-# spots number was higher than 10 and more than twice
the background.
80
In vitro Treg culture with mTORi and isolation
Temsirolimus was provided by the Pharmacy unit of the University Hospital of Besançon and
everolimus was kindly obtained from Novartis (Basel, Switzerland). PBMCs were collected
from anonymous healthy donors at the Etablissement Français du Sang (EFS, Besançon,
France) as apheresis kit preparations after the signature of informed consent and following
EFS guidelines. PBMC were cultured with IL-2 (250 UI/mL), in presence or not of either
everolimus or temsirolimus used at 100 ng/mL per day and 500 ng/mL every three days,
respectively. These drugs were used at concentrations based on their pharmacokinetics data of
in patients (22). After 10 days of culture, Tregs from PBMC cultures were sorted using the
EasySep human CD4+CD25high T cell isolation kit (StemCell technologies, Vancouver,
Canada) according to the manufacturer’s protocol. Purity of Tregs was greater than 90%.
Mice
Female C57BL/6NCrl and BALB/cAnCrl mice, 6-8 weeks old, were purchased from Charles
River laboratories (L’Arbresle, France) and housed under pathogen-free conditions. FoxP3eGFP and DEREG transgenic mice (23) were kindly provided by Dr. Perruche (INSERM
UMR1098, Besançon, France). All experiments were carried out according to the good
laboratory practices defined by the animal experimentation rules in France.
Tumor cell lines
The murine renal cell carcinoma RENCA and the melanoma-B16F10 cells transfected with
ovalbumin (B16-OVA) were kindly provided by Pr. Tartour (INSERM U970, Paris, France).
The murine mammary carcinoma cell line 4T1 was kindly provided by Dr. Apetoh (INSERM
U866, Dijon, France).
Tumor challenge and treatment
BALB/cAnCrl mice were subcutaneously (s.c.) injected with 5.105 RENCA or 105 4T1 cells
in 100µL of saline buffer in the abdominal flank or in the mammary zone, respectively.
C57BL/6NCrl, FoxP3-eGFP or DEREG mice were subcutaneously injected with 2.105 B1681
OVA cells in 100µL of saline buffer in the abdominal flank. Tumor growth was monitored
every 2 or 3 days using a caliper and mice were euthanized when tumor mass reached an area
bigger than 300 mm2. Tumor-bearing mice were treated either with 2 mg/kg of temsirolimus
intra-peritoneally (i.p.) every three days or with everolimus administrated orally everyday by
gavage at 0.65 mg/kg. The mTORi were used at concentrations based on the study of their
pharmacokinetic in patients (22). Mice from control groups were injected with the solvent
used to dissolve drugs. Rapamycin (Sigma-Aldrich) was administrated i.p. at 75 µg/kg/day.
The sunitinib provided by Pr. Tartour (INSERM U970, Paris) was administered by gavage at
40 mg/kg/day. The CCR4 antagonist (AF399/420/18 025) provided by Dr. Bayry (INSERM
U872, Paris) was injected i.p. at 1.5µg/3 days per mice.
T cell depletion experiments
To study the implication of immune cells on the antitumor effect of mTORi, mice were
injected intra-peritoneally before tumor graft then every 2 weeks with 200 µg of monoclonal
depleting antibodies (mAb). Anti-CD4 (clone GK1.5), CD8 (2.43) and CD25 (PC61.5)
antibodies or isotype controls were purchased from BioXcell (West Lebanon, NH). In order to
deplete Tregs, mice were injected i.p. twice (Day -4 and Day 0) before tumor graft with 250 µg
of PC61.5 mAb. DEREG mice were i.p. injected with 80 µg/kg of diphtheria toxin (Sigma
Aldrich) in order to specifically deplete Tregs. Depletion efficiency was regularly checked in
the blood.
Assessment of OVA-specific CD8 T cell responses
The ovalbumin-specific CD8 T cells were analyzed ex vivo in the spleen or within the tumor.
The tumor infiltrating lymphocytes (TIL) were recovered after tumor treatment with DNAse,
hyaluronidase and collagenase (Sigma-Aldrich). The OVA257–264 (SIINFEKL, SL8) KbDextramer (Immudex, Denmark) staining was used to quantify OVA-specific CD8 T cells.
Functionality of OVA257–264-specific CD8 T cells was analyzed by IFN-g-ELISPOT on
spleen-isolated CD8+ T cells (Miltenyi Biotec, Paris, France) (24).
82
Statistics
Data are presented as means +/- SEM Statistical comparison between groups was based on
Student t test using Prism 6 GraphPad Software (San Diego, CA). P values lower than 0.05
(*) were considered significant.
Patients: We assessed progression-free survival (PFS) and overall survival (OS) for each
patient. PFS was calculated from the start of everolimus treatment until the date of
progression or death. OS was calculated from the start of everolimus treatment until the date
of last follow-up or death. Data cutoff for survival analysis was January 7th 2015. Patients’
survival was estimated using the Kaplan-Meier method. To determine the impact of the
everolimus-mediated immune modulation on patient survival, we used a mathematical model
based on the normalized variation after two months of both immune variables Treg ($Treg) and
anti-TERT Th1 ($anti-TERT Th1). The normalized variation rate is calculated as follows:
(value at 2 months – value at baseline)/maximum of values for each variable. Patients were
classified into three groups according to the product of $Treg x $anti-TERT Th1. An absolute
value of $Treg x $anti-TERT Th1 lower than 5% was considered as insignificant. The group
$pos represents the patients whose $Treg x $anti-TERT Th1 is positive meaning that Tregs and
anti-TERT Th1 evolve towards the same direction (growth or decline). The group $null
represents the patients whose $Treg x $anti-TERT Th1 tends towards 0. It may mean that the
Tregs and anti-TERT Th1 are rather stable through time or that the $Treg or $anti-TERT Th1 is
insignificant. The group $neg represents the patients whose $Treg x $anti-TERT Th1 is
negative shows patients for whom Treg greatly decrease and anti-TERT Th1 response
significantly increase. Patients' survival probabilities were estimated using the Kaplan-Meier
method. The log rank tests were used to compare survival distribution in the groups. The
significant level of tests used was 0.05. Stagraphics Centurion software was used for the
statistical analysis.
Mice: The exponential regression model (Y = K Exp (a x (T-T0)) was used to fit the
experimental data of the tumor growth. "T0" is a constant time corresponding to the first
apparition of measurable tumor. "T" is the time of the tumor growth during the experiment so
T>= T0. "K" is the size of tumor at T0. The slope "a" is associated to the growth rate of the
tumor size, which is supposed to be constant. The steeper was the slope, the fastest was the
growth. The exponential model used was good (r2>0.8). A statistical test of comparison of
two slopes was computed to make pairwise comparisons among the different sets. This test
83
was used to take into account the repeated measures that were realized from the different
groups of mice. Mice survival was estimated using the Kaplan-Meier method and the log-rank
test.
RESULTS
Concurrent expansion of FoxP3+ Tregs and spontaneous TERT-specific Th1 responses in
mRCC patients treated with everolimus
A prospective immunomonitoring study was conducted in 23 mRCC patients treated with
everolimus. The patients’ main disease characteristics are depicted in Supplementary Table
S1. The monitoring of FoxP3+ Tregs was performed within blood at baseline and every two
months (Supplementary Fig. S1 for Tregs gating strategy). We observed that both percentage
and absolute number of FoxP3+ Tregs gradually increased (at least > 20%) after treatment in
21/23 patients (91.3%) compared to baseline (Fig. 1A and B) (3.5 vs 6.5% P = 0.0002 and 46
vs 75.106 Tregs per liter P =0.0006 respectively, between baseline and 6 months). In seven
patients, a first drop of Tregs was observed before their increase. The total lymphocytes counts
and the circulating CD3+ T subsets were not modified by everolimus treatment
(Supplementary Fig. S2). Tregs presented the phenotype of natural Tregs (nTreg):
CD25hiCD127loFoxP3+Helios+ and expressed CTLA-4 and ICOS (25). Furthermore, higher
expression of Ki67 in Tregs was detected following everolimus treatment suggesting a
proliferation of this population in vivo (Fig. 1C and D).
The spontaneous tumor-specific Th1 response was also evaluated by using IFN-g-ELISPOT.
To this end, we assessed the reactivity of patients’ T lymphocytes against a mixture of pan
HLA-DR-restricted peptides derived from telomerase reverse transcriptase (TERT), a shared
tumor antigen overexpressed in RCC (26). At baseline, 11/23 patients (47.8%) had
spontaneous anti-TERT Th1 response and this frequency reached 17/23 patients (73.9%) two
months after the beginning of treatment (Fig. 1E). Furthermore, we showed that the
magnitude of the IFN-g-producing anti-TERT Th1 cells increased in patients after treatment
(42 vs 105 anti-TERT IFN-g spots/105 cells, P = 0.01) (Fig. 1F). The impact of everolimus
blood concentration (EBC) on immune responses was also evaluated. The EBC weighted to
posologie in the cohort was fairly similar except in three patients and the median EBC was
10.3 µg/L (range 3.90-53.70 µg/L) (Fig. 1G). As shown in Figure 1H, Treg and anti-TERT
84
Th1 rates were quite similar in patients with EBC < 10.3 µg/L or EBC > 10.3 µg/L. Thus a
significant expansion of FoxP3+ Tregs and tumor-specific Th1 responses concomitantly occur
in mRCC patients treated with everolimus.
Impact of everolimus-mediated immune modulation on clinical outcome
At the time of this analysis, the median PFS in this cohort was 10.97 months [IC 95% (4.8712.83)] and median OS was 26.60 months [IC 95% (12.03 – not reached)] (Supplementary
Fig. S3). Treatment was ongoing for 3 patients, stopped for toxicity for 1 patient and 19
patients had disease progression.
To investigate the impact of everolimus-mediated immune modulation on patients' survival,
we used a model based on the early variation (between baseline and two months) of both Tregs
and anti-TERT Th1 cells to classify patients into three immune groups (Fig. 2A). Two
patients, #1 (no data available at M2) and #10 (no anti-TERT response analysis available at
baseline) were excluded of the analysis. In patients belonging to the group 1 ($pos), Tregs and
anti-TERT Th1 cells evolved towards the same direction (growth or decline) (n=6). Group 2
($null) represents patients for whom the two immune parameters are rather stable through time
or that the Tregs or anti-TERT Th1 variation was insignificant (n=11). In the third group of
patients ($neg), the Tregs values of all patients (n=4) decreased whereas the anti-TERT Th1
values greatly increased. The patients belonging to the immune group 3 showed a longer PFS
(median PFS = 13.2 months) than in the others two groups (4.1 and 8 months for group 1 and
group 2 respectively), log rank test P = 0.02 (Fig. 2B). However, this early immune
modulation had no significant impact on OS (not shown). At the time of disease progression,
the majority of patients treated with everolimus (17/19) had a marked increase of circulating
Tregs (Fig. 2C). This was associated with a loss of the anti-TERT Th1 response in most
patients (10/13 responding patients) (Fig. 2D and Supplementary Fig. S4). The antiviral T cell
responses measured at the same time were slightly reduced but still conserved in most patients
(Supplementary Fig. S4). Thus during everolimus treatment, patients for whom {low Treg
values and strong anti-TERT Th1 response} early occurred showed a better survival.
85
Everolimus or temsirolimus-exposed Tregs demonstrate higher inhibitory capacity
Results in mRCC patients suggested that inhibitory Tregs could compete with host antitumor
Th1 immunity after treatment with everolimus. To test this hypothesis, we performed in vitro
lymphocyte cultures in the presence of mTORi. Both everolimus and temsirolimus inhibited
the phosphorylation of S6 ribosomal protein (ser235) in T cells but not Akt (ser473) the
downstream targets of mTORC1 and mTORC2 respectively (Supplementary Fig. S5).
Although the Tregs rate was significantly increased after culture, neither quantitative nor
phenotypic differences were shown between these two drugs (Fig. 3A and B). The rapalogexposed Tregs expressed CTLA-4, ICOS, GITR, CD39 and CCR4 but not PD-1, LAG-3 or
GP-96 (Fig. 3B). Tregs were then assayed for their capacity to suppress allogenic T cell
proliferation and cytokine production. As compared to non-treated Tregs, everolimus or
temsirolimus-exposed Tregs strongly inhibited allogenic T cell proliferation and decreased the
production of IL-2 and IFN-g (Fig. 3C to E). In addition, we showed that the inhibitory
capacity of these Tregs required cell contact. Indeed, the inhibition of both T cells proliferation
and Th1 cytokines production was impaired when Tregs were separated from T cells in
transwell assay (Fig. 3F to H).
Adaptive T cells contribute to mTORi efficacy in an immunogenic murine tumor model
To analyze more extensively the role of antitumor adaptive T cells during mTORi treatment,
we performed in vivo T cell subsets depletion experiments in three murine tumor models:
renal adenocarcinoma RENCA, mammary carcinoma 4T1 and melanoma B16-F10 expressing
chicken ovalbumin (B16-OVA). We showed that RENCA and 4T1 tumors grew
independently of the presence of T cells in vivo. Accordingly, T cells depletion did not impact
the ability of mTORi treatment to inhibit the growth of these tumors (Supplementary Fig. S6).
Inversely, in B16-OVA-bearing mice, T cells depletion reduced the temsirolimus antitumor
efficacy (Fig. 4A). Similar T cell dependency was observed in B16-OVA-bearing mice
treated with everolimus (data not shown) or rapamycin which is not used as anticancer drug
(Fig. 4B). Next, we performed similar experiments by depleting only CD8 T cells. As
expected, both RENCA and 4T1 tumors growth were CD8 T cell independent and
consequently depletion of these cells had no impact on temsirolimus antitumor efficacy
(Supplementary Fig. S6). In contrast, the effect of temsirolimus or everolimus on B16-OVA
growth was significantly impaired in absence of CD8 T cells (P < 0.05) (Fig. 4C to E). Thus,
86
in an immunogenic tumor model such as B16-OVA, the absence of T cells in vivo strongly
decreased everolimus or temsirolimus antitumor efficacy.
CD4 T cells prevented mTORi efficacy in vivo
We further addressed the role of CD4 T cells in B16-OVA-bearing mice treated with
rapalogs. Although CD4 T cell depletion alone induced a delay of tumor growth, we showed
that temsirolimus treatment induced a drastic inhibition of B16-OVA growth in mice lacking
CD4 T cells (P < 0.001) (Fig. 5A). Similarly, CD4 T cell depletion significantly increased
everolimus efficacy in B16-OVA-bearing mice (Fig. 5B and C). Next, we found in B16OVA-bearing mice treated with temsirolimus a higher number of IFN-g-secreting OVAspecific CD8 T cells in the absence of CD4 T cells as compared to non-depleted mice or CD4
T cell depletion alone (Fig. 5D and E). However, the CD4 T cells depletion did not modify
mTORi efficacy in BALB/C mice bearing RENCA or 4T1 tumor (Supplementary Fig. S6).
Thus, CD4 T cells depletion highly potentiated the efficacy of mTORi by promoting potent
anti-OVA CD8 T cell responses.
The presence of FoxP3+ Treg cells in vivo altered the antitumor efficacy of temsirolimus
via the inhibition of tumor-specific CD8 T cells
We first assessed whether everolimus or temsirolimus could promote Tregs expansion in B16OVA-bearing mice. We showed that both everolimus and temsirolimus treatments increased
the Treg/tumor size ratio in tumor-bearing mice (Fig. 5F and G). To study the role exerted by
Tregs during mTORi treatment we used DEREG mice, which allow to selectively deplete Tregs
after injection of human diphtheria toxin (23). B16-OVA-bearing DEREG mice were treated
with temsirolimus and then received the diphtheria toxin when the effect of temsirolimus was
observed. The Tregs depletion was effective post diphtheria toxin administration and was
controlled during the experiments (Fig. 6A). While temsirolimus treatment delayed B16-OVA
tumor growth in DEREG mice, we showed that a potent tumor regression occurred in mice
treated with temsirolimus followed by diphtheria toxin injection (Fig. 6B). This inversion of
tumor growth rate occurred after 30 days corresponding to the time of Tregs elimination in vivo
after diphtheria toxin (Fig. 6C). In addition, Tregs depletion significantly increased the survival
87
of mice treated with temsirolimus (Fig. 6D). Similar results were found in a second model by
using anti-CD25 mAb (clone PC61.5) (27) to deplete Tregs in FoxP3-eGFP mice prior
everolimus treatment (Supplementary Fig. S7). Furthermore, in temsirolimus-treated DEREG
mice, the Tregs ablation induced a higher expansion of spontaneous OVA-specific CD8 T cells
(Fig. 6E). These anti-OVA CD8 T cells efficiently produced IFN-g indicating their in vivo
functionality (Fig. 6F). Collectively these results demonstrated that FoxP3+ Tregs removal
during everolimus or temsirolimus treatment greatly promoted tumor regression by a
mechanism involving robust antitumor CD8 T cells activation.
Tregs depleting agents are a promising therapeutic option to improve mTORi antitumor
efficacy
Based on these results, there is a rational to combine rapalogs with therapeutic agents that
deplete Treg cells or block their suppressive functions. Here, we evaluated two approaches to
target Tregs in vivo using sunitinib or CCR4 antagonist combined with everolimus or
temsirolimus respectively. As shown in Fig. 7A and B, although the tumor size was less
important in mice treated by sequential combination of everolimus following by sunitinib, the
difference observed was not significant. However, we showed in mice treated with everolimus
plus sunitinib a lower Tregs percentage and Tregs/tumor size ratio than in mice receiving
everolimus as monotherapy (Fig. 7C and D). In line with the high level of CCR4 expression
found on mTORi-exposed Tregs (Fig. 3B), we used the CCR4 antagonist, a competitive class
of Treg inhibitor in combination with temsirolimus (24). As depicted in Fig. 7E and F,
concomitant injections of temsirolimus and CCR4 antagonist efficiently delayed the B16OVA tumor growth and increased mice survival compared to temsirolimus alone.
Furthermore, in mice treated with the bitherapy, a significant decrease of T regs associated with
a higher number of anti-OVA CD8 T cells within the tumor were shown (Fig. 7G and H).
Altogether, these results strongly support the interest in combining therapeutics targeting T regs
with mTORi treatment in cancer.
88
DISCUSSION
In this study we first reported an everolimus-mediated antitumor immune modulation in a
cohort of mRCC patients. By using a dynamic immunomonitoring, we found a high
expansion of circulating FoxP3+ Tregs in patients following everolimus treatment. This increase
of Tregs started mainly two months after the beginning of everolimus treatment and remained
high in most patients compared to baseline (21/23 patients). Very few studies have
investigated the impact of Tregs in cancer patients treated with mTORi. A preliminary study by
Finke and colleagues reported a significant increase of FoxP3+ Tregs in seven mRCC patients
treated with temsirolimus (28). One previous study in mRCC patients treated with mTORi did
not show Tregs modulation but Tregs were monitored only once at one month after the
beginning of treatment (29). However in a recent study evaluating everolimus in metastatic
castration-resistant prostate cancer patients, an increase of Tregs was observed in the majority
of patients (30).
Numerous evidences show the implication of mTOR pathways in the generation of memory
CD8 T cells (14–16,31) and this also supports the recent use of mTORi to improve antiviral
and anticancer vaccines (32,33). In this cohort, no difference was observed in
CD45RO+CD127+CD62L+ central memory CD8 T cells pool (34) after everolimus treatment
(not shown). Although the memory phenotype subset of the anti-TERT Th1 cells was not
investigated, our results clearly indicate that everolimus stimulated and sustained preexisting
antitumor CD4+ Th1 responses. A shift toward Th1 polarization (IFN-g) upon everolimus
treatment has been previously shown in the aforementioned studies in mRCC patients but it
was in a non-tumor antigen-specific setting (29). We used, highly promiscuous HLA-DRrestricted peptides derived from TERT to monitor antitumor Th1 responses regardless the
HLA restriction (19,21,35). RCC is considered as an immunogenic tumor and has been shown
to respond to immunotherapies (36). Therefore, search for the stimulation of both CD4 and
CD8 T cell responses against other tumor antigens expressed on RCC such as Survivin, G250
and MUC1 during mTORi therapy would strengthen our results (37).
To investigate the relationship between the immune modulation and clinical outcome during
everolimus treatment, we used an approach based on the early (at second month) variation
rate of both Tregs and anti-TERT Th1 responses. We showed that patients belonging to
immune group 3 (where Tregs decreased significantly and the anti-TERT Th1 cells greatly
increased) showed a significantly longer PFS (13.2 months) than patients belonging to
89
immune group 2 (where immune modulation was insignificant) or group 1 (with an increase
or decrease of both Tregs and anti-TERT Th1) (8 and 4.1 months respectively, P =0.02). This
early Tregs decrease occurred in patients belonging to group 3 and did not seem to be the
consequence of a lymphopenia or a decrease in the total CD4 T cells count induced by
everolimus treatment. Most patients (11/21) in this study belonged in the immune group 2,
where a balance between Tregs and anti-TERT Th1 cells was observed. This suggests that the
anticancer non-immunologic effects of everolimus would prevail in these patients.
Conversely, in the immune group 1, the occurring of an early immunosuppression induced by
Tregs increase upon everolimus treatment could negatively impact on patient survival.
It has been shown that a variation of mTOR kinase activity can regulate Treg homeostasis
(38,39), which could explain this Treg modulation. Although both Tregs and anti-TERT Th1
cells modulation were not directly influenced by everolimus blood concentration (EBC), there
is a difference between the three immune groups. Indeed, the mean of EBC in the group 1
with the shorter PFS (4.1 months) was higher (19.6 µg/L) than in groups 2 (9 µg/L) and 3
(10.02 µg/L). This observation was also in agreement with our previous reports in mRCC
patients treated with everolimus and could differentially impact on mTOR activity (19,22).
Despite the low number of patients, the median PFS in the immune group 3 is better than that
reported in the phase III studies which is similar to PFS in immune groups 1 and 2 (4,5). Thus
the early immune modulation mediated by everolimus toward Tregs decrease and increase of
antitumor Th1 response may positively impact on clinical outcome. However our results
deserve further confirmation in a larger cohort of mRCC patients.
At the time of disease progression upon everolimus treatment, the majority of mRCC patients
totally lost the spontaneous anti-TERT Th1 response in favor to a marked increase of
circulating Tregs. In line with this, we found that Tregs exposed in vitro to everolimus or
temsirolimus strongly inhibited T cell proliferation and also decreased the production of Th1
cytokines. Similar results have been found with rapamycin-exposed Tregs in organ transplant
patients (12,40). In addition, the phenotypic analysis suggested that Tregs in mRCC patients
after everolimus treatment were natural Treg cells (Helios+) and proliferated in vivo (Ki67hi)
(25,41). Consistent with the contact-dependant mechanisms of suppression characterizing
nTreg (25), we demonstrated that the mTORi-exposed Treg inhibitory capacity was impaired
when Tregs were separated from T cells. Various subpopulations of CD4 T cells regulate tumor
immunity. In contrast to Th1 cells (18) the presence of FoxP3+ Tregs drives to an
90
immunosuppressive tumor microenvironment (42) and this has been correlated with poor
prognosis in RCC (43). Thus, the sustained increase of Tregs during everolimus treatment
overpasses the advantage of antitumor Th1 immunity in mRCC patients.
To dissect more extensively the relationship of adaptive antitumor T cells and Tregs during
anticancer rapalog treatments, we also performed in vivo T cells depletion experiments in
various mouse tumor models. We found that the impact of T cells on mTORi efficacy is based
on the immunogenicity of the tumors. Hence, in poorly immunogenic tumors such as renal
carcinoma RENCA and the mammary carcinoma 4T1, the in vivo depletion of CD8 and/or
CD4 T cells did not modify the tumor growths and consequently had no impact on mTORi
efficacy in these models. Conversely, in an immunogenic tumor model such as murine
melanoma B16-F10 expressing the ovalbumin protein (B16-OVA), we observed that mTORi
effect was greatly reduced in the absence of T cells in vivo. In contrast to CD8 T cells, the in
vivo removal of CD4 T cells in B16-OVA-bearing mice strongly increased the antitumor
effect of rapalogs. Then we focused attention on the role of Tregs in vivo during anticancer
rapalogs treatment. Like in mRCC patients, we showed that mTORi increased Tregs in B16OVA-bearing mice. The negative impact of Tregs on mTORi efficacy was confirmed by using
two different strategies to deplete Tregs in vivo. In DEREG mouse model (23), the temporally
depletion of Tregs during temsirolimus treatment led to the stop of tumor growth in most mice
and this was associated with a drastic induction of anti-OVA CD8 T cells. This strategy of
Tregs depletion in DEREG mice may preserve the positive role of Tregs in the priming of high
avidity memory CD8 T cells as recently reported (44).
Similar effect of CD4 T cells and Tregs depletion on temsirolimus efficacy was recently
reported by Wang et al. (45). However, the RENCA cell line used in this study was rendered
more immunogenic by transfection with CA9 used as tumor antigen supporting our
observation that mTORi efficacy is sculpted by the intrinsic immunogenicity of the tumor.
Furthermore, the tumor-specific T cells were induced by vaccination or provided from
adoptive transfer. In contrast, we evaluated the naturally occurring anti-OVA CD8 T cells
without any active or adoptive immunotherapy and this, in our point of view, is more
physiologically relevant. Altogether we demonstrated that during mTORi therapy, Tregs
exerted a potent inhibitory effect on host antitumor CD8 T cell responses and consequently
led to tumor progression.
91
Based on these observations, there is a rational to combine rapalogs with therapeutic agents
that deplete Treg cells or block their suppressive functions. Multiple approaches have been
developed to inhibit Tregs in cancer (46). Sunitinib, an antiangiogenic drug used in mRCC
treatment has been shown to induce a decrease of Tregs both in murine models and in patients
(47–49). We showed that sequential combination of everolimus with sunitinib could be
synergistic by promoting more Tregs decrease than with individual drugs. We also observed a
strong antitumor efficacy of the concomitant combination of temsirolimus with an antagonist
of CCR4, a receptor for chemokines CCL17 and CCL22 preferentially expressed by Tregs (50).
This association also promoted both Tregs decrease and robust anti-OVA CD8 T cell responses
in mice.
In conclusion, this study describes for the first time the implication of antitumor T cell
immunity in the clinical effectiveness of mTORi. Our results suggest that the modulation of
host antitumor T cell immunity mediated by mTORi treatments occurs in three phases. Firstly,
mTORi treatment can promote a decrease of Tregs associated with strong stimulation of
antitumor Th1 responses that positively increase the treatment efficacy. This is followed by an
immune equilibrium phase where antitumor Th1 response and Tregs variations are
insignificant. During this phase, mTORi efficacy mainly relies on its non-antitumor immune
effects. Finally, a prolonged treatment can lead to an advantage for suppressive Tregs which
overcome antitumor Th1 response and then negatively impact on treatment effectiveness.
Thus, there is a strong rational for combining mTORi with Treg blockade strategies to shift the
balance toward protective antitumor Th1 immunity.
92
REFERENCES
1.
Laplante M, Sabatini DM. mTOR signaling in growth control and disease.
Cell. 2012;149:274–93.
2.
Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell
growth. Nature. 2006;441:424–30.
3.
Porta C, Paglino C, Mosca A. Targeting PI3K/Akt/mTOR Signaling in Cancer.
Front Oncol. 2014;4:64.
4.
Hudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A, et al.
Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med.
2007;356:2271–81.
5.
Motzer RJ, Escudier B, Oudard S, Hutson TE, Porta C, Bracarda S, et al.
Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised,
placebo-controlled phase III trial. Lancet. 2008;372:449–56.
6.
Yao JC, Shah MH, Ito T, Bohas CL, Wolin EM, Van Cutsem E, et al.
Everolimus for advanced pancreatic neuroendocrine tumors. N Engl J Med. 2011;364:514–23.
7.
Baselga J, Campone M, Piccart M, Burris HA, Rugo HS, Sahmoud T, et al.
Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer. N Engl J
Med. 2012;366:520–9.
8.
Chi H. Regulation and function of mTOR signalling in T cell fate decisions.
Nat Rev Immunol. 2012;12:325–38.
9.
Delgoffe GM, Pollizzi KN, Waickman AT, Heikamp E, Meyers DJ, Horton
MR, et al. The kinase mTOR regulates the differentiation of helper T cells through the
selective activation of signaling by mTORC1 and mTORC2. Nat Immunol. 2011;12:295–303.
10.
Delgoffe GM, Kole TP, Zheng Y, Zarek PE, Matthews KL, Xiao B, et al. The
mTOR kinase differentially regulates effector and regulatory T cell lineage commitment.
Immunity. 2009;30:832–44.
11.
Battaglia M, Stabilini A, Roncarolo M-G. Rapamycin selectively expands
CD4+CD25+FoxP3+ regulatory T cells. Blood. 2005;105:4743–8.
12.
Noris M, Casiraghi F, Todeschini M, Cravedi P, Cugini D, Monteferrante G, et
al. Regulatory T cells and T cell depletion: role of immunosuppressive drugs. J Am Soc
Nephrol JASN. 2007;18:1007–18.
13.
Jung J-W, Overgaard NH, Burke MT, Isbel N, Frazer IH, Simpson F, et al.
Does the nature of residual immune function explain the differential risk of non-melanoma
skin cancer development in immunosuppressed organ transplant recipients? Int J Cancer.
2015;
14.
Araki K, Turner AP, Shaffer VO, Gangappa S, Keller SA, Bachmann MF, et
al. mTOR regulates memory CD8 T-cell differentiation. Nature. 2009;460:108–12.
15.
Li Q, Rao RR, Araki K, Pollizzi K, Odunsi K, Powell JD, et al. A central role
for mTOR kinase in homeostatic proliferation induced CD8+ T cell memory and tumor
immunity. Immunity. 2011;34:541–53.
16.
Rao RR, Li Q, Odunsi K, Shrikant PA. The mTOR kinase determines effector
versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet
and Eomesodermin. Immunity. 2010;32:67–78.
17.
Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating
immunity’s roles in cancer suppression and promotion. Science. 2011;331:1565–70.
18.
Fridman WH, Pagès F, Sautès-Fridman C, Galon J. The immune contexture in
human tumours: impact on clinical outcome. Nat Rev Cancer. 2012;12:298–306.
93
19.
Thiery-Vuillemin A, Laheurte C, Mansi L, Royer B, Pivot X, Borg C, et al.
Immunomodulatory effects of everolimus in a long responsive patient with metastatic renal
cell carcinoma. J Immunother. 2014;37:51–4.
20.
Heng DYC, Xie W, Regan MM, Warren MA, Golshayan AR, Sahi C, et al.
Prognostic factors for overall survival in patients with metastatic renal cell carcinoma treated
with vascular endothelial growth factor-targeted agents: results from a large, multicenter
study. J Clin Oncol. 2009;27:5794–9.
21.
Godet Y, Fabre E, Dosset M, Lamuraglia M, Levionnois E, Ravel P, et al.
Analysis of spontaneous tumor-specific CD4 T-cell immunity in lung cancer using
promiscuous HLA-DR telomerase-derived epitopes: potential synergistic effect with
chemotherapy response. Clin Cancer Res. 2012;18:2943–53.
22.
Thiery-Vuillemin A, Mouillet G, Nguyen Tan Hon T, Montcuquet P, Maurina
T, Almotlak H, et al. Impact of everolimus blood concentration on its anti-cancer activity in
patients with metastatic renal cell carcinoma. Cancer Chemother Pharmacol. 2014;73:999–
1007.
23.
Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G, et al.
Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease. J Exp Med.
2007;204:57–63.
24.
Pere H, Montier Y, Bayry J, Quintin-Colonna F, Merillon N, Dransart E, et al.
A CCR4 antagonist combined with vaccines induces antigen-specific CD8+ T cells and tumor
immunity against self antigens. Blood. 2011;118:4853–62.
25.
Bilate AM, Lafaille JJ. Induced CD4+Foxp3+ regulatory T cells in immune
tolerance. Annu Rev Immunol. 2012;30:733–58.
26.
Fan Y, Liu Z, Fang X, Ge Z, Ge N, Jia Y, et al. Differential expression of fulllength telomerase reverse transcriptase mRNA and telomerase activity between normal and
malignant renal tissues. Clin Cancer Res. 2005;11:4331–7.
27.
Oldenhove G, de Heusch M, Urbain-Vansanten G, Urbain J, Maliszewski C,
Leo O, et al. CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign
antigens induced by mature dendritic cells in vivo. J Exp Med. 2003;198:259–66.
28.
Salas RN, Ireland JL, Ko JS, Elson P, Garcia JA, Wood L, et al. Immune cell
changes in the peripheral blood of metastatic renal cell carcinoma (mRCC) patients (pts)
treated with sunitinib or temsirolimus. ASCO Meet Abstr. 2008;26:5099.
29.
Kobayashi M, Kubo T, Komatsu K, Fujisaki A, Terauchi F, Natsui S, et al.
Changes in peripheral blood immune cells: their prognostic significance in metastatic renal
cell carcinoma patients treated with molecular targeted therapy. Med Oncol. 2013;30:556.
30.
Templeton AJ, Dutoit V, Cathomas R, Rothermundt C, Bärtschi D, Dröge C, et
al. Phase 2 trial of single-agent everolimus in chemotherapy-naive patients with castrationresistant prostate cancer (SAKK 08/08). Eur Urol. 2013;64:150–8.
31.
Pollizzi KN, Patel CH, Sun I-H, Oh M-H, Waickman AT, Wen J, et al.
mTORC1 and mTORC2 selectively regulate CD8+ T cell differentiation. J Clin Invest.
2015;125:2090–108.
32.
Li Q, Rao R, Vazzana J, Goedegebuure P, Odunsi K, Gillanders W, et al.
Regulating mammalian target of rapamycin to tune vaccination-induced CD8(+) T cell
responses for tumor immunity. J Immunol. 2012;188:3080–7.
33.
Mannick JB, Del Giudice G, Lattanzi M, Valiante NM, Praestgaard J, Huang
B, et al. mTOR inhibition improves immune function in the elderly. Sci Transl Med.
2014;6:268ra179.
34.
Farber DL, Yudanin NA, Restifo NP. Human memory T cells: generation,
compartmentalization and homeostasis. Nat Rev Immunol. 2014;14:24–35.
94
35.
Dosset M, Godet Y, Vauchy C, Beziaud L, Lone YC, Sedlik C, et al. Universal
cancer peptide-based therapeutic vaccine breaks tolerance against telomerase and eradicates
established tumor. Clin Cancer Res. 2012;18:6284–95.
36.
Inman BA, Harrison MR, George DJ. Novel immunotherapeutic strategies in
development for renal cell carcinoma. Eur Urol. 2013;63:881–9.
37.
Vieweg J, Jackson A. Antigenic targets for renal cell carcinoma
immunotherapy. Expert Opin Biol Ther. 2004;4:1791–801.
38.
Zeng H, Yang K, Cloer C, Neale G, Vogel P, Chi H. mTORC1 couples
immune signals and metabolic programming to establish T(reg)-cell function. Nature.
2013;499:485–90.
39.
Procaccini C, De Rosa V, Galgani M, Abanni L, Calì G, Porcellini A, et al. An
oscillatory switch in mTOR kinase activity sets regulatory T cell responsiveness. Immunity.
2010;33:929–41.
40.
Bocian K, Borysowski J, Wierzbicki P, Wyzgal J, Klosowska D, Bialoszewska
A, et al. Rapamycin, unlike cyclosporine A, enhances suppressive functions of in vitroinduced CD4+CD25+ Tregs. Nephrol Dial Transplant. 2010;25:710–7.
41.
Elkord E, Sharma S, Burt DJ, Hawkins RE. Expanded subpopulation of
FoxP3+ T regulatory cells in renal cell carcinoma co-express Helios, indicating they could be
derived from natural but not induced Tregs. Clin Immunol. 2011;140:218–22.
42.
Tanchot C, Terme M, Pere H, Tran T, Benhamouda N, Strioga M, et al.
Tumor-infiltrating regulatory T cells: phenotype, role, mechanism of expansion in situ and
clinical significance. Cancer Microenviron. 2013;6:147–57.
43.
Liotta F, Gacci M, Frosali F, Querci V, Vittori G, Lapini A, et al. Frequency of
regulatory T cells in peripheral blood and in tumour-infiltrating lymphocytes correlates with
poor prognosis in renal cell carcinoma. BJU Int. 2011;107:1500–6.
44.
Pace L, Tempez A, Arnold-Schrauf C, Lemaitre F, Bousso P, Fetler L, et al.
Regulatory T cells increase the avidity of primary CD8+ T cell responses and promote
memory. Science. 2012;338:532–6.
45.
Wang Y, Sparwasser T, Figlin R, Kim HL. Foxp3+ T cells inhibit antitumor
immune memory modulated by mTOR inhibition. Cancer Res. 2014;74:2217–28.
46.
Pere H, Tanchot C, Bayry J, Terme M, Taieb J, Badoual C, et al.
Comprehensive analysis of current approaches to inhibit regulatory T cells in cancer.
Oncoimmunology. 2012;1:326–33.
47.
Adotevi O, Pere H, Ravel P, Haicheur N, Badoual C, Merillon N, et al. A
decrease of regulatory T cells correlates with overall survival after sunitinib-based
antiangiogenic therapy in metastatic renal cancer patients. J Immunother. 2010;33:991–8.
48.
Finke JH, Rini B, Ireland J, Rayman P, Richmond A, Golshayan A, et al.
Sunitinib reverses type-1 immune suppression and decreases T-regulatory cells in renal cell
carcinoma patients. Clin Cancer Res. 2008;14:6674–82.
49.
Terme M, Pernot S, Marcheteau E, Sandoval F, Benhamouda N, Colussi O, et
al. VEGFA-VEGFR pathway blockade inhibits tumor-induced regulatory T-cell proliferation
in colorectal cancer. Cancer Res. 2013;73:539–49.
50.
Iellem A, Mariani M, Lang R, Recalde H, Panina-Bordignon P, Sinigaglia F, et
al. Unique chemotactic response profile and specific expression of chemokine receptors
CCR4 and CCR8 by CD4(+)CD25(+) regulatory T cells. J Exp Med. 2001;194:847–53.
95
B.
20
250
10.0
15
10
5
150
100
50
CD25
0
e
n
li
³
e
B
B
a
a
s
s
e
M
li
n
M
e
2
6
0
C.
D.
Isotype ct
Baseline
FoxP3
M2
HELIOS
M4
ICOS
M6
CTLA-4
***
200
2
8.0
1.8
FoxP3
3.1
M6
³6
M4
M
M2
FoxP3+ Treg
absolute count (106/L)
Baseline
% FoxP3+ Treg/CD4+
***
M
A.
Baseline
F o xP 3
M6
H E L IO S
Ki67
**
IC O S
Count
C T L A -4
K I6 7
*
0
Fluorescence
F.
15000
250
**
IFN- !spots/105 cells
(17/23)
80
60
(11/23)
40
20
200
Medium
150
100
TERT
44
50
147
83
Baseline
0
M2
M4
ro
e
n
e
v
e
e
s
r
a
e
ft
A
A
ft
B
e
B
r
a
s
e
e
v
li
e
li
n
ro
e
0
H.
10
150
4
2
0
M
³
k
2
2
#
W
Patient identifiant
2
0
2
3
2
1
0
2
2
2
#
#
9
1
#
#
6
8
7
1
1
#
1
#
#
4
5
1
3
1
1
#
#
1
#
0
2
1
1
#
#
8
9
#
#
#
1
7
#
6
#
4
3
2
5
#
#
#
#
#
1
0
50
M
2
100
³
4
6
EBC!#!10.3!"g/L
2
6
8
EBC < 10.3 "g/L
k
8
IFN- !spots/105 cells
% FoxP3+ Treg/CD4+
10
W
G.
Everolimus Blood Concentration
weighted to posologie (µg/L)
10000
MFI among FoxP3+ Treg
100
% patients with anti-TERT
Th1 response
E.
5000
Time of everolimus exposure
Figure 1. Everolimus immune-mediated modulation in mRCC patients. FoxP3+ Treg cells
and spontaneous anti-TERT Th1 cells were monitored at baseline and every two months by
using flow cytometry and IFN-g-ELISPOT respectively (n=23). (A) Representative plots of
Tregs from one patient are shown. (B) Tregs evolution upon everolimus: (left) percentage, and
(right) absolute number. (C) Representative Tregs phenotype analysis from one patient is
shown (D) MFI of Tregs markers, (E) percentage of patients with spontaneous anti-TERT Th1
response, (F) (left) number of IFN-g-producing anti-TERT Th1 cells and (right) representative
IFN-g spots wells from one patient are shown. (G) Everolimus through blood concentration
weighted to posologie. (H) Correlation between everolimus blood concentration and (left)
Tregs or (right) IFN-g-producing anti-TERT Th1 cells at week 2 and month 2 after treatment.
Values shown correspond to means +/- SEM. *P<0.05, **P<0.01, ***P<0.001(Student t test)
96
A.
Immune group 1 (!pos)
0.8
DTreg
Immune group 2 (!null)
Immune group 3 (!neg)
0.6
!
0.4
0.2
Danti-TERT Th1
-1.5
-1
-0.5
0.5
1
1.5
-0.2
-0.4
-0.6
-0.8
B.
1
Immune group 1: PFS 4.1 months IC 95% (3.09-5.18)
Immune group 2: PFS 8 months IC 95% (5.95-10.05)
0.8
Immune group 3: PFS 13.2 months IC 95% (0-27.05)
Survival
(Log Rank test, P =0.02)
0.6
0.4
0.2
0
0
10
C.
20
PFS (months)
30
40
D.
10
Tregs
Anti-TERT Th1
300
Progression
Relative % of Tregs
Stop (toxicity)
5
0
-5
Relative IFN-g spots number
Ongoing
200
100
0
-1 0 0
-2 0 0
Figure 2. Impact of immune modulation on patients’ survival. Patients with mRCC treated
with everolimus (n=21) are classified into three immune groups according to their early
(between baseline and two months) variation rate of Treg and anti-TERT Th1 response (see
supplementary materials section) (A) Patients’ distribution in each group is shown. Symbols
represent individual patient. The group 1 ($pos) shows the patients whose variation rate is
significantly positive, the group 2 ($neg) shows the patients whose variation rate tends towards
0 and in the third group ($neg) variation rate is significantly negative. (B) Kaplan–Meier
curves for progression-free survival. (C) Tregs and (D) anti-TERT Th1 cells variations until
disease progression were shown.
97
B.
CD25
FoxP3
ICOS
CTLA-4
GITR
Temsiro
Evero
w/o trt
Day 0
Control isotype
**
****
****
8
6
4
GP-96
LAG-3
PD-1
CD39
CCR4
Count
2
o
ir
Fluorescence
+
+
te
e
m
v
s
e
tr
y
a
/o
w
D
ro
t
0
0
% FoxP3+ Treg/CD4+
A.
C.
D.
Day 10
SortedTregs
CD25
79.9
**
40
% Inhibition
10 days
PBMC
**
***
50
CFSE+
CD3/CD28
stimulated T
cells
+
FoxP3
mTORi
**
30
20
10
Anti-CD3/28
400
200
20
o
t
ir
tr
ro
s
e
/o
m
v
te
e
w
g
T
g
re
g
+
T
re
/2
y
3
a
g
re
re
T
T
+
+
ti
n
a
500
100
50
e
s
n
ra
/T
ti
o
T
n
+
a
w
ir
s
m
3
D
te
-C
s
n
s
m
te
m
g
te
+
T
re
g
re
T
+
o
8
/2
ll
e
w
ir
s
o
s
+
ir
T
a
n
re
/T
g
ra
te
-C
ti
n
ra
o
/T
m
3
D
w
s
s
e
T
ir
s
m
e
T
o
8
/2
ll
e
o
t
ir
tr
m
/o
w
ll
0
0
g
10
1000
re
20
IFN-g
150
IFN-g (µg/mL)
30
0
+ Treg temsiro
1:1
Transwell
IL-2
1500
ir
*
**
IL-2 (pg/mL)
% Inhibition
+ Treg temsiro
1:1
D
D
-C
te
g
H.
40
Anti-CD3/28
0
8
o
ir
e
s
m
v
e
re
T
+
T
T
re
re
g
g
w
D
g
re
T
+
+
94.61
Count
ro
t
tr
0
a
/o
y
/2
3
D
-C
n
ti
+
a
G.
CFSE
40
0
8
F.
IFN-g
60
0
+ Treg temsiro
1:1
CFSE
92.84
*
600
44.2
*
80
IL-2
800
IFN-g (µg/mL)
+ Treg evero
1:1
Count
*
*
1000
+
E.
43.1
71.65
o
s
m
te
+
+ Treg w/o trt
1:1
IL-2 (pg/mL)
61.6
ir
ro
e
v
e
+
w
D
/o
a
y
tr
0
t
0
Figure 3. Temsirolimus or everolimus-cultured Tregs present high suppressive functions.
PBMC from healthy donors were cultured in presence or not of either everolimus (100
ng/mL/day) or temsirolimus (500 ng/mL/3days) during 10 days. (A) Mean percentages of
FoxP3+ Tregs in each culture condition are shown. (B) Representative Tregs phenotype analysis.
(C) Analysis of CFSE dilution in activated CD3+ T cells co-cultured with Tregs. Results from a
representative donor are shown. (D) Percentage of inhibition of T cell proliferation by Tregs
(n=10). (E) IL-2 and IFN-g productions measured in the supernatant of co-cultures by ELISA
(n=4). (F) Analysis of CFSE dilution in CD3+ T co-cultured with Tregs in transwell assay.
Results from one representative donor are shown. (G) Percentage of inhibition of T cell
proliferation by Tregs in transwell assay (n=3). (H) IL-2 and IFN-g production in transwell
measured by ELISA (n=2). Values correspond to means +/- SEM. *P<0.05, **P<0.01,
***P<0.001, ****P<0.0001 (Student t test)
98
A.
B.
Control
Temsirolimus + rat IgG2b
GK1.5 + 2.43 mAb
Temsirolimus
+ GK1.5 + 2.43 mAb
200
150
300
Tumor size (mm2)
Tumor size (mm2)
250
*
100
50
200
100
0
10
Tumor size (mm2)
10
15
20
25
Days post tumor graft
Temsiro
+ rat IgG2b
aCD8
(2.43 mAb)
Control
*
0
15
20
25
30
Days post tumor graft
C.
Control
Rapamycin + rat IgG2b
2.43 + GK1.5 mAb
Rapamycin
+ 2.43 + GK1.5 mAb
Temsiro
+ aCD8 (2.43 mAb)
200
200
200
200
150
150
150
150
100
100
100
100
50
50
50
50
0
0
0
10
20
30
0
0
10
20
30
30
0
0
10
20
30
0
10
20
30
Days post tumor graft
D.
E.
Tumor size (mm2)
150
350
*
100
50
0
Tumor size (mm2)
Control
Temsiro + rat IgG2b
2.43 mAb
Temsiro
+ 2.43 mAb
200
Control
Evero + rat IgG2b
2.43 mAb
Evero
+ 2.43 mAb
300
250
200
*
150
100
50
0
10
15
20
25
Days post tumor graft
30
10
15
20
25
30
Days post tumor graft
Figure 4. In vivo T cells depletion decreases mTORi efficacy. B16-OVA-bearing C57BL/6
(B6) mice (n=5/group) depleted with both anti-CD4 (GK1.5) plus anti-CD8 (2.43) mAbs
injection were treated or not with mTORi. Control mice received solvent and isotype control
mAb. Comparison of tumor growth rate in mice treated by temsirolimus (A) or with
rapamycin (B) is shown. B16-OVA-bearing B6 mice (n=5/group) depleted with anti-CD8
(2.43) mAb injection were treated with mTORi. (C and D) Tumor growth and growth rate in
mice treated with temsirolimus. (E) Tumor growth rate in mice treated or not with
everolimus. The symbols represent the evolution of mean +/- SEM tumor size for each group
and the lines are the exponential regression model fitting the mean tumor size. Results
represent three (A, C to E) and two (B) independent experiments. *P<0.05.
99
Tumor size (mm2)
Temsiro
+ rat IgG2b
aCD4
(GK1.5 mAb)
Control
250
250
250
250
200
200
200
200
150
150
150
150
100
100
100
100
50
50
50
50
0
0
0
5
10
15
20
25
30
0
0
5
10
15
20
25
30
Control
Tems. + rat IgG2b
GK1.5 mAb
Temsiro
+ GK1.5 mAb
250
Temsiro
+ aCD4 (GK1.5 mAb)
Tumor size (mm2)
A.
200
150
100
50
***
0
0
5
10
15
20
25
30
0
5
10
15
20
25
0
30
10
Days post tumor graft
250
100
200
150
100
50
*
0
10
15
20
25
30
Days post tumor graft
**
75
50
25
0
0
35
15 15
20
25
30
D.
0.2
GK1.5 mAb
2.2
Temsiro
+ rat IgG2b
0.3
Temsiro +
GK1.5 mAb
40
45
50
3.2
E.
*
4
% OVA-specific
CD8 T cells
****
****
3
2
1
2
G
m e d iu m
S L 8 p e p t id e
125
50
25
b
#1
#3
#1
#2
1
t
#1
#2
#3
T e m s ir o
+ r a t Ig G 2 b
#1
#2
#3
T e m s ir o
+ G K 1 .5 m A b
s
.
m
s
+
.
G
G K 1 .5 m A b
T
e
T
m
e
#3
.5
#2
C o n tr o l
K
K
+
150
0
m
l
b
o
A
tr
m
Ig
n
1
.5
o
C
G
b
0
OVA257-264Kb dextramer
ra
CD8
Control
35
Days post tumor graft
A
Tumor size (mm2)
C.
Control
Evero + rat IgG2b
GK1.5 mAb
Everolimus
+ GK1.5 mAb
300
IFN- !spots
/105 CD8+ cells
350
% mice survival
B.
15
20
25
30
35
Days post tumor graft
o
o
ro
e
v
s
tr
n
o
C
E
o
ir
o
tr
n
m
T
e
o
C
0 .0
l
0 .0 0
0 .1
ir
0 .0 5
0 .2
s
0 .1 0
0 .3
m
0 .1 5
*
0 .4
e
Treg/Tumor size ratio
*
0 .2 0
ro
14.3
e
15.0
Tumor
*
l
CD25
11.3
Spleen
Everolimus
v
FoxP3
7.8
Temsirolimus
E
Control
Treg/Tumor size ratio
Naive
T
G.
F.
Figure 5. Increase of mTORi efficacy in mice lacking CD4 T cells in vivo. B16OVA-bearing B6 mice (n=5/group) depleted with anti-CD4 (GK1.5) mAb injection were
treated or not with mTORi. (A) Tumor growth and growth rate in mice treated or not by
temsirolimus. (B) Tumor growth rate in mice treated or not with everolimus. (C) Kaplan–
Meier curves for survival of mice treated with everolimus (**P<0.01, log-rank test) (D)
OVA257–264 Kb-dextramer ex vivo staining in spleen at day 30. (left) representative dot plots
from one representative mouse. (right) percentage of OVA257–264-specific CD8+ T cells. (E)
Functional analysis of OVA257–264-specific CD8+ T cells measured ex vivo in the spleen by
IFN-g-ELISPOT at day 30. FoxP3+ Tregs staining in tumor-bearing mice treated or not with
mTORi at day 25, (F) representative dot plots, (G) Treg/tumor size ratio (left) in the spleen and
(right) in the tumor. Results represent at least three independent experiments. Values shown
correspond to means +/- SEM. *P<0.05, **P<0.01, ***P<0.001 (Student t test).
100
A.
+ Diphtheria
toxin
B16-OVA
Day
25
DEREG mice
27
8.4
FoxP3
Temsirolimus
13
0
34
Diphtheria toxin
0.9
CD25
B.
Tumor size (mm2)
Control
Diphtheria toxin
Temsirolimus
+ Diphtheria toxin
Temsirolimus
400
400
400
400
300
300
300
300
200
200
200
200
100
100
100
100
0
0
0
10
20
30
40
0
0
10
20
30
40
0
0
10
20
30
40
0
10
20
30
40
Days post tumor graft
Tumor size (mm2)
450
D.
Control
100
Temsirolimus
Diphtheria toxin
Temsirolimus
+ Diphtheria toxin
300
%
u r v iv a l
m i c e ssurvival
% mice
C.
150
75
**
50
25
**
0
0
0
10 15
Days
20
25
30
35
40
15 15
20
25
30
35
40
D a y s p o s t tu m o r g r a f t
Days
post tumor graft
45
Diphtheria toxin
E.
0 .4
0 .2
*
6
4
2
0
50
x
h
s
ip
m
d
e
T
+
ir
#1
#2
#3
#1
#2
#3
#1
#2
#3
#4
to
o
ir
x
to
h
ip
o
th
h
s
ip
m
D
S L 8 p e p t id e
200
100
0
+
e
D
T
ri
e
a
m
to
s
x
ir
in
o
0 .0
OVA257-264Kb dextramer
IFN- !spots
/105 CD8+ cells
0 .6
8
m e d iu m
250
D ip h to x
Tem s.
T e m s ir o
+ D ip h to x
o
1.3
0 .8
10
ir
0.3
300
*
1 .0
T
e
T
m
e
s
0.4
F.
Tumor
Spleen
Temsirolimus
+ Diphtheria toxin
% OVA-specific
CD8 T cells
Temsirolimus
% OVA-specific
CD8 T cells
CD8
Diphtheria
toxin
Figure 6. FoxP3+ Tregs removal during mTORi treatment promotes protective antitumor
CD8 T cell immunity. DEREG mice (n= 4/group) were grafted with B16-OVA and then
treated or not with temsirolimus. (A) Diphtheria toxin injections (80µg/kg) and example of
Tregs depletion at sacrifice are indicated. (B) Tumor growth and (C) comparison of tumor
growth rate. The regression model was not applicable for the group treated by temsirolimus +
diphtheria toxin over the 30th day. (D) Kaplan–Meier survival curves (**P<0.01, log-rank
test). (E) OVA257–264 Kb-dextramer ex vivo staining in spleen and among TIL at day 35: (left)
Representative spleen dot plots from one representative mouse. (right) Percentage of OVA257–
+
+
264-specific CD8 T cells. (F) Functional analysis of OVA257–264-specific CD8 T cells
measured ex vivo in the spleen by IFN-g-ELISPOT at day 35. Experiments were reproduced
three times. Values shown correspond to means +/- SEM. *P<0.05, **P<0.01. (Student t test).
101
B.
Control
Everolimus
Sunitinib
Everolimus
+ Sunitinib
600
500
400
300
200
800
600
400
200
ib
in
it
n
u
S
S
+
0 .0 0
Control
250
Temsirolimus
CCR4 antagonist
Temsirolimus
+ CCR4 antagonist
200
in
ib
ro
ro
e
e
v
v
u
S
+
S
+
F.
300
100
%%mice
s u r v iv a l
m i c e survival
150
*
100
50
*
75
50
25
0
0
1 51 5
0
30
35
0 .0
o
ir
ta
4
e
m
a
n
s
s
m
m
T
e
+
s
C
C
R
4
R
C
C
+
0 .5
a
s
m
e
T
n
a
4
R
C
C
ir
is
tr
n
o
n
o
g
ta
C
CD25
o
t
0
1 .0
T
5
1 .5
o
10
*
2 .0
ir
15
l
10.5
H.
**
20
o
FoxP3
18.7
Temsirolimus
+ CCR4 anta
% FoxP3+ Treg/CD4+
Temsirolimus
25
Days post tumor graft
Days post tumor graft
G.
20
D a y s p o s t tu m o r g r a f t
30
e
25
T
20
ta
15
n
0 10 10
% OVA-specific CD8 TIL
Tumor size (mm2)
E.
it
it
o
C
u
o
ti
n
n
tr
ib
in
it
v
E
u
S
ro
ro
e
e
in
E
n
l
o
tr
n
o
n
u
S
C
0 .0 1
l
0
CD25
0 .0 2
E
5
0 .0 3
E
10
*
0 .0 4
n
15
0 .0 5
ib
*
20
v
FoxP3
25
ib
10.1
15.8
Treg/Tumor size ratio
D.
Everolimus
+ Sunitinib
Everolimus
ro
e
e
E
u
C
v
v
E
it
30
Sunitinib
% FoxP3+ Treg/CD4+
Everolimus
C.
in
o
25
n
20
o
12 15
Days
it
0
n
tr
0
ro
ib
0
100
l
Tumor size (mm2)
700
Tumor size (mm2)
At Day 29
A.
Figure 7. Sunitinib and CCR4 antagonist decrease Tregs and improve the antitumor
efficacy of mTORi. B16-OVA-bearing B6 mice were either treated sequentially with
everolimus following by sunitinib (from day 21) (40mg/kg/day, gavage) or with each drug as
monotherapy. (A) Tumor growth and (B) mean of tumor size at day 29 are shown. (C)
FoxP3+ Tregs staining at day 30. (left) Representative dot plots and (right) percentage of Tregs
in spleen. (D) Treg/tumor size ratio in spleen. B16-OVA-bearing mice were concomitantly or
individually treated with temsirolimus and CCR4 antagonist (1.5µg/mice, i.p). (E) Tumor
growth. (F) Kaplan–Meier survival curves (**P<0.05, log-rank test). (G) FoxP3+ Tregs
staining in spleen at day 25. (left) Representative dot plots and (right) percentage of Tregs (H)
OVA257–264-specific CD8+ TIL detected ex vivo by dextramer staining at day 25. n=5
mice/group were used and experiments were reproduced two times. Values shown correspond
to means +/- SEM. *P <0.05, ** P<0.01. (Student t test).
102
Supplementary Material and methods
Everolimus pharmacokinetic assessment
Pharmacokinetic assessments were regularly performed during the follow-up. Everolimus
trough blood concentration was measured using a validated liquid chromatography coupled
with tandem mass spectrometry as recently reported (22).
Flow cytometry
Tregs were analyzed by flow cytometry. PBMC were stained with surface antibodies, fixed and
permeabilized using Fixation/permeabilization buffer from eBioscience (Paris, France), and
then stained with intracellular antibodies according to the manufacturer’s protocol. The
following monoclonal antibodies with fluorescent conjugates were used: Anti-CD3 (clone
UCHT1) and anti-CD4 (SFCI12T4D11) both obtained from Beckman Coulter (Villepinte,
France); anti-CD25 (M-A251), anti-GITR (eBioAITR), anti-PD-1 (MIH4) anti-CTLA-4
(BNI3) and anti-Ki67 (B56) were obtained from BD Bioscience (Le Pont de Claix, France);
anti-CD127 (ebioRDR5), anti-Lag-3 (3DS223H) and anti-Helios (22F6) were obtained from
eBioscience; and anti-FoxP3 (259D), anti-CCR4 (L291H4), anti-ICOS (C398.4A) and antiCD39 (A1) were obtained from Biolegend (Ozyme, Saint-Quentin-en-Yvelines, France). In
mice experiments, splenocytes or TIL were stained with the following monoclonal antibodies
with fluorescent conjugates: anti-CD3 (17A2), anti-CD4 (GK1.5) and anti-CD25 (3C7) were
obtained from Biolegend and anti-FoxP3 (FJK-16s) was obtained from eBioscience. Samples
were acquired on a Facs Canto II (BD Biosciences) and analyzed with the Diva or FlowJo
softwares.
Measurement of mTORC1 and mTORC2 activities
To measure mTORC1 activity by flow cytometry, lymphocytes cultured in presence of mTOR
inhibitors were stimulated with anti-CD3 and CD28 antibodies (BD Biosciences) then fixed,
permeabilized using the Cytofix/Phosflow permeabilization buffer (BD Biosciences) and
stained with p-S6 antibodies (Cell Signaling Technology, Danvers, USA). The mTORC2
activity was measured by Western Blotting using the following antibodies: Akt
103
phosphorylated at Ser473 (D9E) (Cell Signaling Technology) and "-actin (AC-15; Sigma).
Antibody dilutions were 1:1,000 (p-Akt antibody) and 1: 1,000,000 ("-actin antibody).
Tregs suppressive assay
The immunosuppressive functions of Tregs were evaluated in a CFSE-labelled T cell
proliferation assay. Briefly, 5.105 fresh allogenic T cells from healthy donors labelled with
CellTrace 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen,
Eugene, OR) were co-cultured for 3 days at 1:1 ratio with sorted Tregs in the presence of
coated anti-CD3 (2.5 µg/mL) and anti-CD28 (5 µg/mL) antibodies (BD Bioscience). T cell
proliferation was assessed by flow cytometry. Percent inhibition was calculated using the
following formula: (Percent of T cell proliferation in suppressed condition – percent of T cell
proliferation in unsuppressed condition) / percent of T cell proliferation in unsuppressed
condition × 100. Cytokine production (IL-2 and IFN-g) was measured in the supernatants of
co-cultures by ELISA (Diaclone). Proliferation suppression assays were also performed using
transwell columns (Merck Millipore, Billerica, USA) to separate 3.105 Tregs (top chambers)
from 3.105 allogenic T cells (bottom chambers) in the presence of soluble anti-CD3 (5
µg/mL) and anti-CD28 (5 µg/mL) antibodies (BD Bioscience).
104
CD3
CD25
Count
CD127
CD3
CD4
FSC
SSC
SSC
Treg
FoxP3
Supplementary Figure 1. Treg gating strategy. Representative flow cytometry plots showing
the gating strategy used to identify CD3+CD4+CD25hiCD127loFoxP3+ regulatory CD4 T cells
(Treg).
105
A.
B.
CD3
800
NS
400
200
ro
A
ft
e
B
r
a
e
s
v
e
e
e
li
n
ro
e
v
e
r
e
ft
A
ft
e
B
r
A
r
e
s
e
e
v
li
e
n
ro
n
li
e
s
a
B
ft
e
v
e
e
0
A
ro
e
n
li
e
s
CD8
600
0
a
CD4
*
e
1000
1000
a
2000
NS
B
Absolute cell count (×106 /L)
Absolute lymphocytes count (×106 /L)
3000
Supplementary Figure 2. Total lymphocytes and CD3+CD4+/CD8+ T cells counts after
everolimus treatment. (A) Absolute total lymphocytes count at baseline and after everolimus
treatment (n=23). (B) Absolute count of CD3+, CD4+ and CD8+ T cells at baseline and after
everolimus treatment. Values correspond to means +/- SEM. *P<0.05 (Student t test)
106
A.
B.
1
1
PFS 10.97 months IC 95% (5.93-22.93)
OS 26.6 months IC 95% (15.07-nr)
0.8
Survival
Survival
0.8
0.6
0.4
0.6
0.4
0.2
0.2
0
0
PFS months
OS months
Supplementary Figure 3. Median PFS and OS of mRCC patients treated by everolimus.
(A) Kaplan–Meier curves for progression-free survival (n=23). (B) Kaplan–Meier curves for
overall survival (log-rank test).
107
A.
B.
Anti-TERT Th1 response
Antiviral T cell responses
1000
IFN- !spots/105 cells
**
300
200
800
600
400
100
200
0
n
s
s
re
g
ro
P
ft
e
B
r
a
e
s
v
e
e
li
io
ro
e
n
n
io
s
s
re
g
ro
P
A
ft
e
B
r
a
e
s
v
e
e
li
n
ro
e
0
A
IFN- !spots/105 cells
400
Supplementary Figure 4. Anti-TERT versus anti-viral T cell responses in mRCC
patients treated by everolimus. The anti-TERT Th1 response and antiviral T cell responses
were monitored at baseline after treatment and at progression (n=23) by using IFN-gELISPOT. A mixture of peptides derived from virus influenza (Flu), Epstein barr virus
(EBV), cytomegalovirus (CMV) was used to evaluate antiviral response (PA-CEF-001, CTL,
Germany). (A) number of IFN-g-producing anti-TERT Th1 cells (B) number of IFN-gproducing antiviral T cells. Horizontal bars represent mean of IFN-g spots +/-SEM. **P<0.01
(Student t test)
108
A.
B.
Everolimus Temsirolimus
mTORi
Ac_active
Count
Ac_active
Untreated
iso_active
Isotype
S6P
Akt
P-Akt
(Ser475)
Actin
Supplementary Figure 5. mTORC1 and mTORC2 kinase activities. PBMC from healthy
donors were cultured 24h in presence of everolimus (100 ng/mL) or temsirolimus (500
ng/mL) then stimulated for 30min with anti-CD3 (5 µg/mL) and anti-CD28 (5 µg/mL) and
assessed for (A) pS6 (mTORC1) by phospho-flow cytometry and (B) pAkt Ser473
(mTORC2) by westernblotting.
109
A.
RENCA
4T1
150
Control
Temsiro + rat IgG2b
GK1.5 + 2.43 mAb
Temsiro
+ GK1.5 + 2.43 mAb
200
Tumor size (mm2)
Tumor size (mm2)
300
100
0
Control
Temsiro + rat IgG2b
GK1.5 + 2.43 mAb
Temsiro
+ GK1.5 + 2.43 mAb
100
50
0
5
B.
10
15
20
Days post tumor graft
25
5
RENCA
Tumor size (mm2)
Tumor size (mm2)
100
50
0
10
C.
Control
Temsiro + rat IgG2b
2.43 mAb
Temsiro
+ 2.43 mAb
100
50
0
10
30
RENCA
100
Tumor size (mm2)
Control
Temsiro + rat IgG2b
GK1.5 mAb
Temsiro
+ GK1.5 mAb
150
15
20
Days post tumor graft
25
4T1
250
200
Tumor size (mm2)
15
20
25
Days post tumor graft
25
4T1
150
Control
Temsiro + rat IgG2b
2.43 mAb
Temsiro
+ 2.43 mAb
150
10
15
20
Days post tumor graft
50
Control
Temsiro + rat IgG2b
GK1.5 mAb
Temsiro
+ GK1.5 mAb
200
150
100
50
0
0
10
15
20
Days post tumor graft
25
20
25
30
35
Days post tumor graft
40
Supplementary Figure 6. T cells depletion in RENCA or 4T1 tumor-bearing mice
treated by mTORi. Balb/c mice (n=5/group) were depleted or not with (A) both anti-CD4
(GK1.5) and anti-CD8 (2.43) mAbs or (B) anti-CD8 (2.43) mAbs only or (C) anti-CD4
(GK1.5) only, and then grafted with (left) RENCA (5.105 cells, sc) or (right) 4T1 (1.105 cells,
sc). Tumor-bearing mice were treated or not with temsirolimus (2mg/kg/3days, ip). The
symbols represent the evolution of mean +/- SEM tumor size for each group and the lines are
the exponential regression model fitting the mean tumor size. Tumor growth rate between
groups was compared. Results represent at least two independent experiments.
110
*
20
10
s
l
0
u
E
v
e
C
ro
o
li
n
m
tr
CD4
30
o
26.1
FoxP3
15.9
% FoxP3+ Treg/CD4+
A.
B.
C.
Control
Evero + rat IgG1
PC61.5 mAb
Everolimus
+ PC61.5 mAb
250
200
100
s u r v iv a l
m i c e survival
% %mice
Tumor size (mm2)
300
150
100
50
*
*
75
50
25
0
0
0
1 51 5
20
25
30
35
D a y s p o s t tu m o r g r a f t
10
15
20
Days post tumor graft
25
Days post tumor graft
Supplementary Figure 7. Effect of anti-CD25 mAb treatment on everolimus efficacy. (A)
FoxP3-eGFP mice were treated with everolimus (0.65 mg/kg/day, gavage) during 21 days.
Representative dot plots and percentage of Tregs in the spleen at day 21. (B) FoxP3-eGFP mice
(n=5/group) were depleted or not with anti-CD25 (PC61.5) mAb and then grafted with B16OVA tumor (2.105 cells). Tumor-bearing mice, were treated or not with everolimus
(0.65mg/kg/day, gavage). The symbols represent the evolution of mean +/- SEM tumor size
for each group and the lines are the exponential regression model fitting the mean tumor size.
Tumor growth rate between groups was compared. (C) Kaplan–Meier survival curves (logrank test). Values shown correspond to means +/- SEM. Results represent two independent
experiments. *P<0.05 (Student t test)
111
Patient
Category
Risk
#1
#2
#3
#4
#5
#6
#7
#8
#9
#10
#11
#12
#13
#14
#15
#16
#17
#18
#19
#20
#21
#22
#23
High
Int
High
High
Int
High
Low
Low
Int
High
Int
High
High
Int
Low
Int
Int
Low
High
Int
Low
Low
High
Mean Everolimus
Blood
PFS
Response
Concentration
(months)
(µg/L)
53.7 (40-79)
SD
5.57
15 (8.4-18)
SD
27.57
8.8 (6-8.8)
PD
3.10
20.4 (17.4-26.5)
SD
4.87
8 (7.6-10.2)
SD
8.00
7.5 (7.5-10.5)
PD
4.13
15.6 (9.4-21.7)
SD
13.27
11.2 (7.4-23.6)
PR
6.47
3.9 (3.6-4.2)
SD
12.83
15.6 (13.4-17.2)
PR
3.03
7.4 (4.7-8.3)
SD
19.03
6.8 (3.5-10.8)
SD
7.53
28.7 (25.6-39)
PD
3.27
28.8 (18.8-38.9)
SD
5.93
6.6 (3.8-13.5)
SD
32.67
8.4 (7.7-15.5)
SD
13.13
6.2 (5.5-7)
SD
8.37
10.4 (4.7-18)
PR
17.80
20.3 (13.4-20.7)
SD
8.87
14.7 (7.5-15.3)
PD
3.53
12.2 (3.7-12.8)
PD
4.00
4 (3.8-4.3)
SD
10.97
13.4 (9.7-18.8)
SD
6.37
OS
(months)
Immune
group
28.70
33.17
8.00
15.70
15.80
15.30
35.60
7.57
15.43
5.23
18.90
10.03
22.60
12.03
32.87
35.73
5.43
18.50
9.53
15.07
5.20
10.97
6.37
NA
3
2
1
2
1
3
2
3
NA
2
2
1
1
3
2
2
2
1
2
1
2
2
Supplementary Table1. Patients’ characteristics. Category risk of renal carcinoma was
assessed with Heng et al. category risk classification (20). Response indicates the best
response during everolimus treatment. The immune groups 1, 2 and 3 were described in
supplementary materials and methods section. SD indicates stable disease, PR partial
response, PD progression disease, PFS Progression Free Survival, OS Overall Survival.
112
ARTICLE 2: A CCR4 antagonist combined with mTOR inhibitors improves
vaccination-induced antitumor memory CD8 T cell responses
Manuscrit en cours de preparation
Dans des modèles pré-cliniques, la rapamycine est apparue comme un outil très
intéressant en association avec des vaccins pour générer des réponses T CD8 mémoires antitumorales efficaces. Néanmoins, les seuls mTORi administrés chez les patients atteints de
cancer sont le temsirolimus et l’évérolimus.
Au cours de cette étude, nous avons évalué la combinaison des mTORi avec un vaccin
composé de la sous-unité B de la toxine Shiga associée à l’ovalbumine (Adotevi et al., 2007).
Nous avons montré que le temsirolimus améliore l’efficacité anti-tumorale du vaccin chez des
souris greffées avec la tumeur B16-OVA, par des mécanismes impliquant l’augmentation du
taux de LT CD8 spécifiques d’OVA dans la rate et dans la tumeur des souris.
Le traitement par évérolimus ou temsirolimus durant la phase d’expansion et de
contraction de la réponse immunitaire induite par STxB-OVA augmente le taux de LT CD8
centraux mémoires et précurseurs mémoires (CD62L+CD127+ et CD127+KLRG1lo)
spécifiques d’OVA. L'effet observé était dose-dépendant. De plus, le traitement par
temsirolimus augmente la production d’IFN-g et la dégranulation par les LT CD8 anti-OVA
après restimulation par un peptide dérivé de l'ovalbumine. Le temsirolimus améliore
également les réponses induites par des vaccinations peptidiques ciblant les LT CD4 ou LT
CD8.
Enfin, la combinaison avec un agent bloquant les Treg a été évaluée et nos résultats ont
montré que l'antagoniste du CCR4 augmente l’efficacité de la combinaison du temsirolimus
avec STxB-OVA, par des mécanismes impliquant la diminution du taux de Treg et
l’augmentation des LT CD8 anti-OVA dans la rate et la tumeur des souris greffées avec B16OVA.
En conclusion, ces travaux montrent que les mTORi anti-cancer potentialisent
l'efficacité anti-tumorale d'une vaccination thérapeutique. Ces travaux soulignent également
l'intérêt potentiel de combiner un agent bloquant les Treg à cette association thérapeutique.
113
ABSTRACT
Inhibition of the mammalian Target Of Rapamycin (mTOR) with rapamycin has emerged as
an interesting tool for generating CD8 antitumor responses induced by therapeutic vaccines.
Although the mTOR inhibitors (mTORi) everolimus and temsirolimus are used as anticancer
drugs, their contribution to the efficacy of therapeutic vaccine has not been investigated. Here
we demonstrated that temsirolimus enhanced the antitumor efficacy of STxB-OVA vaccine,
by promoting OVA-specific CD8 T cells and more particularly functional central memory and
precursor memory specific CD8 T cells. As mTORi were also shown to promote high
immunosuppressive FoxP3+ regulatory CD4 T cells (Tregs), we showed that blocking Tregs by
the CCR4 antagonist enhanced the mTORi-mediated antitumor efficacy of the therapeutic
vaccine. Altogether our results demonstrated the immunostimulatory properties of anticancer
mTORi on therapeutic vaccines and prompt the association of mTORi with immunotherapies.
114
INTRODUCTION
The mammalian Target of Rapamycin (mTOR) is a conserved serine/threonine kinase
downstream of the PI3K/AKT pathway which plays a central role in immunity as a
fundamental determinant of T lymphocytes homeostasis and functional fates (Chi, 2012).
Indeed, mTOR was shown to be a key regulator of memory CD8 T cell differentiation (Araki
et al., 2009; Rao et al., 2010) and these observations prompted the combination of rapamycin
with anticancer immunotherapies (Amiel et al., 2012; Diken et al., 2013; Li et al., 2012).
However, a recent study showed that rapamycin treatment was detrimental to the antitumor
CD8 T cell responses induced by the detoxified CyaA derived from Bordetella pertussis and
carrying the E7 protein from HPV-16 in a TC-1 mouse model of cervical cancer (Chaoul et
al., 2015a).
Most cancers are characterized by the activation of mTOR which is also involved in the
regulation of cell growth, metabolism and apoptosis (Laplante and Sabatini, 2012). The
rapamycin analogs everolimus and temsirolimus are now being used in clinical settings for
their anti-proliferative properties on tumor cells (Porta et al., 2014). We previously
demonstrated that anti-cancer mTORi treatments promote natural antitumor responses in
cancer patients and murine models (Beziaud et al. submitted).
mTOR is known to be a fundamental determinant for the differentiation and functional
regulation of CD4 T cells subset (Chi, 2012). The lack of mTOR in naïve CD4 T cells has
been shown to promote preferentially FoxP3+ regulatory T cells (Tregs) to the detriment of Th1,
Th2 or Th17 polarization (Delgoffe et al., 2009, 2011). In line with this, rapamycin is
prescribed to organ transplant patients in order to promote Tregs induction and create an
immunosuppressive environment required to prevent graft rejection (Battaglia et al., 2005;
Noris et al., 2007; Sabbatini et al., 2015). We recently reported that mTORi treatments in
cancer patients induce an increase of Tregs. mTORi treatment leads to highly
immunosuppressive Tregs which overcome the antitumor response and negatively impact on
the treatment effectiveness, suggesting that its association with therapeutics targeting Tregs
would benefit patients (Beziaud et al 2015 submitted).
CCR4 is a receptor preferentially expressed by human Tregs compared to conventional T cells
which promotes Tregs recruitment to tumor site in response to CCL17 and CCL22 chemokines
(Curiel et al., 2004). CCR4 antagonist, an emergent class of Treg inhibitors, has been shown to
block Treg recruitment mediated by CCL17 and CCL22 and is currently under clinical trials
115
(Bayry et al., 2014). Tartour et al. have developed a vaccine candidate based on the
association of the B subunit of Shiga toxin (STxB) with the ovalbumine or E7 protein and
able to establish protective CD8 T cell memory against tumor (Adotevi et al., 2007; Vingert et
al., 2006). Moreover, STxB-OVA antitumor efficacy is improved when associated with the
CCR4 antagonist by inducing a stronger effector CD8 T cell response and a higher inhibition
of tumor growth (Pere et al., 2011).
Rapamycin has emerged as a very interesting tool for generating CD8 antitumor responses
induced by therapeutic vaccines (Amiel et al., 2012; Diken et al., 2013; Li et al., 2012).
However, the clinical development of rapamycin as an anticancer agent was hampered
because of its unfavorable pharmacokinetics and thus the only mTORi administered to
patients with cancer are temsirolimus and everolimus (Faivre et al., 2006). In the present
study, we demonstrated that temsirolimus enhanced the antitumor efficacy of STxB-OVA
vaccine, by promoting OVA-specific CD8 T cells and more particularly functional central
memory and precursor memory specific CD8 T cells. We observed that blocking Tregs by the
CCR4 antagonist enhanced the mTORi-mediated antitumor efficacy of the therapeutic
vaccine.
MATERIALS AND METHODS
Mice
Female C57BL/6NCrl (B6) mice, 6-8 weeks old, were purchased from Charles River
laboratories (L’Arbresle, France) and the HLA-DRB1*0101/HLA-A*0201-transgenic mice
(A2/DR1 mice) have been previously described (Pajot et al., 2004) and were purchased at the
“Cryopréservation, Distribution, Typage et Archiva animal”. Mice were housed under
pathogen-free conditions. All experiments were carried out according to the good laboratory
practices defined by the animal experimentation rules in France.
Chemical reagents
Temsirolimus was provided by the Pharmacy unit of the University Hospital of Besançon and
everolimus was kindly obtained from Novartis (Basel, Switzerland). The CCR4 antagonist
(AF399/420/18 025) was provided by Dr. Bayry (INSERM U872, Paris, France). Anti-CD8
116
(2.43) antibody and rat IgG2b isotype control were purchased from BioXcell (West Lebanon,
NH).
Vaccines
STxB-OVA was kindly provided by Pr Tartour (INSERM U970). STxB-OVA was obtained
by chemical coupling, as previously described (Haicheur et al., 2003). Briefly, OVA was first
activated via amino groups on lysine side chains using the heterobifunctional cross-linker mmaleimidobenzoyl-N-hydroxysulfosuccinimide ester (Pierce). Activated OVA was then
reacted with STxB-Cys, and the reaction product was purified by gel filtration and
immunoaffinity chromatography.
OVA-derived peptide (OVA257-264: SIINFEKL) and UCP2 peptide (TERT578–592:
KSVWSKLQSIGIRQH) were obtained from Proimmune. The invariant natural killer T-cell
ligand aGalCer (KRN7000) was purchased from Funakoshi. Montanide was provided by
Seppic.
Tetramer staining and IFN-g-ELISPOT
The OVA-specific CD8 T cells were analyzed by using a synthetic OVA-derived peptide SL8
(OVA257–264: SIINFEKL). Ex vivo tetramer staining was conducted as previously described
(Adotevi et al., 2007). Cells were stained with phycoerythrin (PE)-conjugated SL8 Kb
tetramer (Beckman) and then analyzed by flow cytometry. Functionality of OVA-specific
CD8 T cells was analyzed by IFN-g-ELISPOT as previously described (Pere et al., 2011).
Briefly, spleen or tumor isolated cells were incubated at 1 or 2.105 cells per well (in triplicate)
in IFN-g-ELISPOT in presence of SL8 peptide. Plates were incubated for 16 to 18 hours at
37°C, and spots were revealed following the manufacturer’s instruction (Diaclone, Besançon,
France). Spot-forming cells were counted using the “C.T.L. Immunospot” system (Cellular
Technology Ltd.).
Flow Cytometry
The OVA-specific CD8 T cells phenotype were analyzed by flow cytometry. After tetramer
staining, cells were stained with the following monoclonal antibodies with fluorescent
conjugates: anti-CD3 (17A2), anti-CD8 (53-6.7) and anti-CD127 (A7R34) were obtained
from Biolegend; anti-CD62L (MEL-14) and anti-KLRG1 (2F1) were obtained from
eBioscience (Paris, France). For Treg analysis, cells were stained with surface antibodies, fixed
117
and permeabilized using Fixation/permeabilization buffer from eBioscience (Paris, France),
and then stained with intracellular antibodies according to the manufacturer’s protocol. The
following monoclonal antibodies with fluorescent conjugates were used: anti-CD3 (14A2)
CD4 (GK1.5), and anti-CD25 (3C7) were obtained from Biolegend and anti-FoxP3 (FJK-16s)
was obtained from eBioscience. Samples were acquired on a Facs Canto II (BD Biosciences)
and analyzed with Diva software.
Tumor challenge
The melanoma-B16F10 cells transfected with ovalbumin (B16-OVA) was kindly provided by
Pr. Tartour (INSERM U970, Paris, France). C57BL/6NCrl mice were subcutaneously injected
with 2.105 B16-OVA cells in 100µL of saline buffer in the abdominal flank. Tumor growth
was monitored every 2 or 3 days using a caliper and mice were euthanized when tumor mass
reached an area bigger than 300 mm2. For tumor infiltrating lymphocytes (TIL) analysis,
tumors were recovered and treated with DNAse, hyaluronidase and collagenase (SigmaAldrich) before cell suspension analysis by flow cytometry.
Therapeutic vaccination
Mice were immunized twice (day 0 and day 14) subcutaneously (s.c.) with SL8 or UCP
peptides (100µg and 150µg, respectively) emulsified with montanide (v/v), or once intraperitoneally (i.p.) with STxB-OVA (20µg) in combination with aGalCer (2µg). Mice were
then treated either with 2 or 4.5mg/kg of temsirolimus (i.p) every three days or with
everolimus administrated orally everyday by gavage at 0.65 mg/kg. The mTORi were used at
concentrations based on the study of their pharmacokinetic in mRCC patients. Mice from
control groups were injected with the solvent used to dissolve drugs. The CCR4 antagonist
was injected (i.p.) at 1.5µg per mice. To study the implication of CD8 T cells on the antitumor
effect of mTORi, mice were injected (i.p.) every 2 weeks with 200 µg of monoclonal
depleting antibodies (mAb). Anti-CD8 (2.43) antibody and rat IgG2b isotype control were
purchased from BioXcell (West Lebanon, NH). Depletion efficiency was regularly checked in
the blood.
Statistical analysis
Data from histograms are presented as means +/- Standard Error of the Mean (SEM).
Statistical comparison between groups was based on Student t test using Prism 6 GraphPad
118
Software. Mouse survival was estimated using the Kaplan-Meier method and the log-rank
test. P values less than 0.05 (*) were considered significant.
RESULTS
Synergistic antitumor efficacy of mTORi plus STxB-OVA therapeutic vaccine by
promoting antitumor CD8 T cell responses
To evaluate the impact of mTORi treatment on tumor growth and tumor-specific memory
CD8 T cells, B6 mice were grafted with the murine melanoma B16 expressing ovalbumin
protein (B16-OVA) and then vaccinated with STxB-OVA alone or in combination with
temsirolimus (Fig. 1A). First, we showed that temsirolimus or STxB-OVA vaccination alone
treatment delayed tumor growth and increased mice survival (Fig. 1, B to D). More
importantly, we showed that when administrated in combination with STxB-OVA vaccine,
temsirolimus treatment increased the therapeutic effect of STxB-OVA vaccination by
inducing a stronger inhibition of tumor growth (Fig. 1, B and C) and by increasing mice
survival (Fig. 1D). These data clearly showed that temsirolimus treatment enhanced vaccineinduced tumor regression.
To analyze the impact of CD8 T cells on the antitumor efficacy of STxB-OVA plus
temsirolimus, CD8 T cells were depleted from B16-OVA-bearing mice by the injection of
anti-CD8 mAb (clone 2.43) prior STxB-OVA vaccination and temsirolimus treatment. We
showed that CD8 T cells depletion abrogated the efficacy of combining temsirolimus with the
antitumor vaccine (Fig. 2A and B). This observation was reinforced by the increased
frequency of OVA257-264-specific CD8 T cells in the spleen of mice treated with the
combination of mTORi and vaccination (Fig. 2C). We also showed the ability of these antiOVA257-264 CD8 T cells to efficiently produce IFN-g in response to OVA257-264 peptide
stimulation (Fig. 2D). Similar results were observed within the tumor, with an increase of
IFN-g producing OVA257-264-specific CD8 TILs frequency (Fig. 2E and F). Although Tregs
were increased in mice treated with temsirolimus (Fig. 2G), the OVA-specific CD8 T cells /
Tregs ratio was significantly increased in vaccine plus temsirolimus treated mice (fig 2H).
Thus, these results demonstrate that temsirolimus treatment after a therapeutic vaccination
greatly improves its antitumor efficacy by a mechanism involving antitumor CD8 T cell
activation, amplification and tumor recruitment.
119
Temsirolimus or everolimus promotes anti-OVA CD8 T cells induced by STxB-OVA
vaccination
We previously showed that temsirolimus treatment on B16-OVA-bearing mice can modulate
OVA-specific CD8 T cells (Beziaud et al, submitted). To evaluate the impact of temsirolimus
and everolimus treatment on vaccine-induced memory CD8 T cells in a non-tumor context,
mice were vaccinated with STxB-OVA alone or in combination with temsirolimus treatment
(Fig. 3A). Despite the absence of temsirolimus impact on the percent of OVA-specific CD8 T
cells found in the spleen (Fig. 3B), we showed that OVA-specific memory CD8 T cells
generated in the presence of temsirolimus expressed higher levels of CD127, CD62L, and a
lower level of KLRG1 compared to control mice (Fig. 3B). Thus, temsirolimus treatment
stimulate the memory differentiation program resulting in a higher number of OVA-specific
CD8 T cells with the phenotypic characteristics (CD127+KLRG1- and CD127+CD62L+) of
highly functional memory cells (Fig. 3C). The administration of temsirolimus during either
the expansion or the contraction phase of the immune response induced by STxB-OVA
vaccine also modulated the memory markers on OVA-specific CD8 T cells (Supplementary
Fig. S1). In addition, temsirolimus treatment enhanced the expansion of antigen-specific
memory T cells after injection of peptide-based vaccines targeting either CD4 (UCP peptides)
or CD8 (SL8 peptide) T cells (Supplementary Fig. S2).
We then used a high dose of temsirolimus (4.5mg/kg) or everolimus (0.65mg/kg/day). We
showed that high dose of mTORi treatment drastically increased the percent of OVA-specific
CD8 T cells found in the spleen 24 days post vaccination (Fig. 3D). This high dose
temsirolimus treatment increased significantly the number of OVA-specific CD8 T cells with
the phenotypic characteristics of highly functional memory T cells (Fig. 3, E and F). These
OVA-specific CD8 T cells were able to degranulate (Fig. 3G) and to produce IFN-g (Fig. 3H)
in response to OVA257-264. Collectively, these results demonstrated that mTORi greatly
enhance vaccine-induced specific T cells with a high functional memory phenotype.
A CCR4 antagonist suppresses Tregs and increases the efficacy of STxB-OVA vaccine
plus temsirolimus
We have previously shown that mTORi treatment promotes the expansion of
immunosuppressive Tregs in cancer patients (Beziaud et al, submitted). So there is a strong
rational to combine therapeutic vaccination plus temsirolimus with depleting Tregs agents.
120
Here, we evaluated in B16OVA-bearing mice the combination of STxB-OVA vaccine plus
temsirolimus with a CCR4 antagonist. First, compared to STxB-OVA vaccine alone the
CCR4 antagonist combined to STxB-OVA vaccine slightly delayed tumor growth and
increased mice survival (Fig. 4, A to C). As depicted in Fig. 4A and B, concomitant injection
of temsirolimus plus CCR4 antagonist after STxB-OVA vaccination efficiently delayed the
B16-OVA tumor growth and improved mice survival (Fig. 4C). These data showed that
CCR4 antagonist enhanced the temsirolimus plus vaccine-induced tumor regression.
The analysis of OVA-specific CD8 T cells in mice treated with STxB-OVA combined to
temsirolimus and CCR4 antagonist revealed a small increase in the spleen (Fig. 5A) and a
more pronounced increase in the tumor (Fig. 5B). Furthermore, in mice treated with this tritherapy, the increase of tumor-specific CD8 T cells was associated with a decrease of Tregs
both in the spleen (Fig. 5C) and in the tumor (Fig. 5D). Consequently, the OVA-specific CD8
T cells / Tregs ratio was significantly increased in both spleen and tumor (Fig. 5E). In addition,
we also showed the ability of these anti-OVA CD8 T cells derived from the spleen to
efficiently produce IFN-g in response to OVA257-264 (Fig. 5F). Altogether, these results show
that blocking Tregs with a CCR4 antagonist enhanced the benefit of combining mTORi with an
antitumor therapeutic vaccine, and support the interest in combining therapeutics targeting
Tregs, mTORi treatment and active immunotherapy in cancer.
DISCUSSION
In addition to their antitumor activities some chemotherapeutics impact the immune system,
consequently
strategies
using
different
combinations
between
chemotherapy
and
immunotherapy are being evaluated. Recent studies showed that inhibiting mTOR with
rapamycin under selective conditions enhances the memory CD8 T cell differentiation and
response to vaccines in mouse models of chronic infections and tumors (Araki et al., 2009; Li
et al., 2012; Rao et al., 2010). Araki et al. demonstrated that rapamycin treatment in mice
during the expansion or contraction phase of an immune response induced by an infection
with lymphocytic choriomeningitis virus (LCMV) enhanced LCMV-specific memory CD8 T
cell differentiation. However, clinical development of rapamycin as an anticancer agent was
hampered by unfavorable pharmacokinetic properties (Faivre et al., 2006), and consequently
spurred the development of rapamycin analogues with improved pharmacokinetic properties.
121
Nowadays, temsirolimus and everolimus are two mTOR inhibitors administrated to cancer
patients. We recently showed that treatment with these rapalogs can sustain natural tumorspecific T cell responses (Beziaud et al, submitted). Therefore, here we first analyzed whether
temsirolimus could enhance the effect of a therapeutic anticancer vaccination. To address this
question, B16-OVA bearing mice were vaccinated with a vaccine based on the association of
the B subunit of Shiga toxin and ovalbumin protein, and then treated with temsirolimus. We
showed that temsirolimus greatly improves the antitumor efficacy of the therapeutic
vaccination by inducing a decrease of tumor growth kinetic. By depleting CD8 T cells and
monitoring the OVA-specific CD8 T cells, antitumor activity of the combined treatment was
shown to be dependent on CD8 T cells, confirming Wang’s observations (Wang et al., 2011,
2014). More interestingly, we showed that temsirolimus improves the antitumor efficacy of a
therapeutic vaccination, by a mechanism involving the promotion of tumor-specific CD8 T
cell responses in tumor. Conversely, Leclerc et al. showed recently an inhibitory property of
rapamycin on the generation of CD8 T cell responses and antitumor efficacy after vaccination
with CyaA-E7 vaccine derived from Bordetella pertussis and relatively similar to our vaccine
model (Chaoul et al., 2015b). Furthermore they didn’t observe any increase of CD8 T cells
and their phenotypic analysis wasn’t performed on tumor-specific CD8 T cells. In addition,
we didn’t observe an implication of CD8 T cells on the antitumor efficacy of temsirolimus in
the tumor growth of TC1 celle line model they used in their study (data not shown).
Then to assess more specifically the direct impact of mTORi treatment on the vaccineinduced specific memory CD8 T cells, we vaccinated mice with STxB-OVA vaccine which
was described to induce specifically strong CD8 T cell immune responses (Adotevi et al.,
2007). Mice were then treated with temsirolimus using different treatment regiments
(expansion; contraction; or both expansion and contraction phase of the vaccine-induced
immune response). As we previously showed that mTORi treatment on tumor-bearing mice
can directly sustain tumor-specific CD8 T cells without vaccination, this experiment was
performed in a tumor-free condition. Although temsirolimus treatment didn’t impact on the
frequency of OVA-specific CD8 T cells, we demonstrated that it promoted the expansion of
OVA-specific CD8 T cells with a phenotype of highly functional memory T cells marked by
the increase of CD127+, CD62L+ and a KLRG1low expression. We also confirmed the impact
of temsirolimus in other vaccine models by using a peptide-based vaccine, targeting either
CD8 or CD4 T cell responses.
122
The dose of mTOR inhibition with rapamycin in transplant patients is much lower than the
mTOR inhibition with rapalogs in cancer patients. Studies conducted in murine models to
promote memory CD8 T cells used rapamycin at 75µg/kg/day at a dose representative of the
drug dosing in transplant patients (2-5mg daily), and a higher dose (600µg/kg/day) was
demonstrated to be detrimental for the differentiation of memory CD8 T cells (Araki et al.,
2009; Chaoul et al., 2015b). The dose and length of administration of rapalogs in cancer
patients depend on the cancer type, as for example temsirolimus is administrated at 175mg for
three weeks to treat mantle cell lymphoma compared to 25mg weekly in mRCC patients. In
contrast to publications about rapamycin treatment in mice, in our study or in Wang et al., the
dose of temsirolimus (based on the pharmacokinetic of the drug in mRCC patients) was
equivalent to the high dose of rapamycin in murine studies and did not alter the CD8
responses. We showed that a higher dose of mTORi strongly promoted the expansion of
OVA-specific CD8 T cells with the phenotype of high functional memory CD8 T cells.
Moreover, this OVA-specific CD8 T cells induced during temsirolimus treatment showed
higher functional properties with stronger degranulation and production of IFN-g than OVAspecific CD8 T cells induced without temsirolimus. Similarly to Araki et al. who used
rapamycin, we showed that temsirolimus treatment during both the expansion and contraction
phase of the immune response induced by a vaccine is more efficient than in the expansion or
contraction phase (Araki et al., 2009). In addition, everolimus the second mTORi
administrated orally to cancer patient also showed its potential to improve memory CD8 T
cell formation.
mTORi are used as immunosuppressive drugs in organ transplant patient to prevent graft
rejection, and are well-known to induced Treg cells. We previously showed that mTORi
treatment in cancer patients leads to the expansion of high immunosuppressive Tregs and tumor
progression. In murine models, we and others also previously demonstrated that depletion of
Tregs improved the antitumor efficacy of mTORi, and prompt the association of mTORi with
immunotherapies targeting Treg (Wang et al., 2014) (Beziaud et al, submitted). Moreover,
blocking specifically Tregs rather than total CD4 T cells as suggested by Wang et al. would
decrease the risk for opportunistic infection in cancer patients. Additionally, we previously
reported the importance of tumor-specific Th1 CD4 cells promotion by everolimus in the
clinical benefits of patients. Here, we observed an increase of Tregs in mice vaccinated in
presence of temsirolimus. Nonetheless, temsirolimus expanded efficiently tumor-specific
CD8 T cells and the OVA-specific CD8 T cells / Tregs ratio was greatly increased after
123
temsirolimus treatment. Considering our previous observations showing that the Treg blocking
agent CCR4 antagonist improves the antitumor efficacy of both temsirolimus (Beziaud et al,
submitted) or STxB-OVA vaccine (Pere et al., 2011), we assessed the potential of the tritherapy combination. We observed that blocking Tregs with the CCR4 antagonist considerably
improved the antitumor efficacy of the combination of temsirolimus plus therapeutic
vaccination.
We previously suggested that prolonged treatment with mTORi could contribute to tumor
immune escape by promoting Tregs (Beziaud et al, submitted). Here we proposed that
combining mTORi treatment with therapeutic vaccination and a Tregs blockade strategy could
shift the balance toward antitumor immunity, and bring the patient back in a phase of disease
control, by strongly boost the antitumor responses induced by the therapeutic vaccine, while
removing the harmful impact of mTORi-expanded Tregs.
REFERENCES
Adotevi, O., Vingert, B., Freyburger, L., Shrikant, P., Lone, Y.-C., Quintin-Colonna,
F., Haicheur, N., Amessou, M., Herbelin, A., Langlade-Demoyen, P., et al. (2007). B subunit
of Shiga toxin-based vaccines synergize with alpha-galactosylceramide to break tolerance
against self antigen and elicit antiviral immunity. J. Immunol. Baltim. Md 1950 179, 3371–
3379.
Amiel, E., Everts, B., Freitas, T.C., King, I.L., Curtis, J.D., Pearce, E.L., and Pearce,
E.J. (2012). Inhibition of Mechanistic Target of Rapamycin Promotes Dendritic Cell
Activation and Enhances Therapeutic Autologous Vaccination in Mice. J. Immunol. 189,
2151–2158.
Araki, K., Turner, A.P., Shaffer, V.O., Gangappa, S., Keller, S.A., Bachmann, M.F.,
Larsen, C.P., and Ahmed, R. (2009). mTOR regulates memory CD8 T-cell differentiation.
Nature 460, 108–112.
Battaglia, M., Stabilini, A., and Roncarolo, M.-G. (2005). Rapamycin selectively
expands CD4+CD25+FoxP3+ regulatory T cells. Blood 105, 4743–4748.
Bayry, J., Tartour, E., and Tough, D.F. (2014). Targeting CCR4 as an emerging
strategy for cancer therapy and vaccines. Trends Pharmacol. Sci. 35, 163–165.
Chaoul, N., Fayolle, C., Desrues, B., Oberkampf, M., Tang, A., Ladant, D., and
Leclerc, C. (2015a). Rapamycin Impairs Antitumor CD8+ T-cell Responses and VaccineInduced Tumor Eradication. Cancer Res. 75, 3279–3291.
124
Chaoul, N., Fayolle, C., Desrues, B., Oberkampf, M., Tang, A., Ladant, D., and
Leclerc, C. (2015b). Rapamycin Impairs Antitumor CD8+ T-cell Responses and VaccineInduced Tumor Eradication. Cancer Res. 75, 3279–3291.
Chi, H. (2012). Regulation and function of mTOR signalling in T cell fate decisions.
Nat. Rev. Immunol. 12, 325–338.
Curiel, T.J., Coukos, G., Zou, L., Alvarez, X., Cheng, P., Mottram, P., EvdemonHogan, M., Conejo-Garcia, J.R., Zhang, L., Burow, M., et al. (2004). Specific recruitment of
regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced
survival. Nat. Med. 10, 942–949.
Delgoffe, G.M., Kole, T.P., Zheng, Y., Zarek, P.E., Matthews, K.L., Xiao, B., Worley,
P.F., Kozma, S.C., and Powell, J.D. (2009). The mTOR kinase differentially regulates effector
and regulatory T cell lineage commitment. Immunity 30, 832–844.
Delgoffe, G.M., Pollizzi, K.N., Waickman, A.T., Heikamp, E., Meyers, D.J., Horton,
M.R., Xiao, B., Worley, P.F., and Powell, J.D. (2011). The kinase mTOR regulates the
differentiation of helper T cells through the selective activation of signaling by mTORC1 and
mTORC2. Nat. Immunol. 12, 295–303.
Diken, M., Kreiter, S., Vascotto, F., Selmi, A., Attig, S., Diekmann, J., Huber, C.,
Türeci, Ö., and Sahin, U. (2013). mTOR inhibition improves antitumor effects of vaccination
with antigen-encoding RNA. Cancer Immunol. Res. 1, 386–392.
Faivre, S., Kroemer, G., and Raymond, E. (2006). Current development of mTOR
inhibitors as anticancer agents. Nat. Rev. Drug Discov. 5, 671–688.
Haicheur, N., Benchetrit, F., Amessou, M., Leclerc, C., Falguières, T., Fayolle, C.,
Bismuth, E., Fridman, W.H., Johannes, L., and Tartour, E. (2003). The B subunit of Shiga
toxin coupled to fullǦsize antigenic protein elicits humoral and cellǦmediated immune
responses associated with a Th1Ǧdominant polarization. Int. Immunol. 15, 1161–1171.
Laplante, M., and Sabatini, D.M. (2012). mTOR signaling in growth control and
disease. Cell 149, 274–293.
Li, Q., Rao, R., Vazzana, J., Goedegebuure, P., Odunsi, K., Gillanders, W., and
Shrikant, P.A. (2012). Regulating mammalian target of rapamycin to tune vaccinationinduced CD8(+) T cell responses for tumor immunity. J. Immunol. Baltim. Md 1950 188,
3080–3087.
Noris, M., Casiraghi, F., Todeschini, M., Cravedi, P., Cugini, D., Monteferrante, G.,
Aiello, S., Cassis, L., Gotti, E., Gaspari, F., et al. (2007). Regulatory T Cells and T Cell
Depletion: Role of Immunosuppressive Drugs. J. Am. Soc. Nephrol. 18, 1007–1018.
Pajot, A., Michel, M.-L., Fazilleau, N., Pancré, V., Auriault, C., Ojcius, D.M.,
Lemonnier, F.A., and Lone, Y.-C. (2004). A mouse model of human adaptive immune
functions: HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice. Eur. J.
Immunol. 34, 3060–3069.
125
Pere, H., Montier, Y., Bayry, J., Quintin-Colonna, F., Merillon, N., Dransart, E.,
Badoual, C., Gey, A., Ravel, P., Marcheteau, E., et al. (2011). A CCR4 antagonist combined
with vaccines induces antigen-specific CD8+ T cells and tumor immunity against self
antigens. Blood 118, 4853–4862.
Porta, C., Paglino, C., and Mosca, A. (2014). Targeting PI3K/Akt/mTOR Signaling in
Cancer. Front. Oncol. 4.
Rao, R.R., Li, Q., Odunsi, K., and Shrikant, P.A. (2010). The mTOR kinase
determines effector versus memory CD8+ T cell fate by regulating the expression of
transcription factors T-bet and Eomesodermin. Immunity 32, 67–78.
Sabbatini, M., Ruggiero, G., Palatucci, A.T., Rubino, V., Federico, S., Giovazzino, A.,
Apicella, L., Santopaolo, M., Matarese, G., Galgani, M., et al. (2015). Oscillatory mTOR
inhibition and Treg increase in kidney transplantation. Clin. Exp. Immunol.
Vingert, B., Adotevi, O., Patin, D., Jung, S., Shrikant, P., Freyburger, L., Eppolito, C.,
Sapoznikov, A., Amessou, M., Quintin-Colonna, F., et al. (2006). The Shiga toxin B-subunit
targets antigen in vivo to dendritic cells and elicits anti-tumor immunity. Eur. J. Immunol. 36,
1124–1135.
Wang, Y., Wang, X.-Y., Subjeck, J.R., Shrikant, P.A., and Kim, H.L. (2011).
Temsirolimus, an mTOR inhibitor, enhances anti-tumour effects of heat shock protein cancer
vaccines. Br. J. Cancer 104, 643–652.
Wang, Y., Sparwasser, T., Figlin, R., and Kim, H.L. (2014). Foxp3+ T cells inhibit
antitumor immune memory modulated by mTOR inhibition. Cancer Res. 74, 2217–2228.
126
A.
STxB-OVA + aGalCer
Day 0 14
B16-OVA
bearing mice
Tumor size (mm2)
B.
17
20
26
PBS
Temsirolimus
Temsirolimus
STxB-OVA +
Temsirolimus
STxB-OVA
400
400
400
400
300
300
300
300
200
200
200
200
100
100
100
100
0
10
15
20
25
0
10
30
15
20
25
30
0
10
15
20
25
30
0
10
15
20
25
30
Days post tumor graft
D.
400
PBS
350
T e m s ir o lim u s
100
S T x B -O V A
300
% mice survival
Tumor size
(mm2)
C.
S T x B - O V A + T e m s ir o lim u s
250
200
150
*
100
50
*
75
50
25
0
0
0
10
10
15
20
25
30
Days post tumor graft
35
0 1515
20
25
30
35
Days post tumor graft
Figure 1. Temsirolimus enhances the anti-tumor efficacy of STxB-OVA vaccination. (A)
Experimental scheme. C57Bl/6 mice (n=5/group) were grafted with B16-OVA and then
vaccinated with STxB-OVA at day 14. Temsirolimus treatment started 3 days after. (B)
Tumor growth per mice in each group. (C) Tumor growth per group (D) Kaplan–Meier curves
for survival of mice. Results represent 2 independent experiments. Values shown correspond
to means +/- SEM. *P<0.05
127
A.
400
B.
PBS
100
S T x B - O V A + T e m s ir o lim u s
300
% mice survival
Tumor size (mm2)
S T x B -O V A + T e m s ir o lim u s + a C D 8
200
100
75
50
25
*
0
0
0 10 10
15
20
25
0 15 15
20
25
Days post tumor graft
30
Days post tumor graft
D.
STxB-OVA +
Temsirolimus
8.06
5
m e d iu m
SL8
150
100
4
IF N g s p o t s n u m b e r
10
f o r 5 .1 0
% O V A - s p e c if ic
*
s p le n o c y t e s
200
15
CD8
STxB-OVA
4.74
C D 8 T c e lls
C.
30
50
0
0
+
S T x B -O VA +
u
s
T e m s ir o lim u s
T
e
m
S
s
T
ir
x
o
B
li
-O
m
V
-O
B
x
T
S
S T x B -O VA
A
V
A
OVA257-264/Kb tetramer
F.
STxB-OVA +
Temsirolimus
44.68
20
10
0
400
200
0
+
s
u
T
e
m
S
s
T
ir
x
o
li
-O
B
S T x B -O VA
+ T e m s ir o lim u s
m
V
-O
B
x
T
S
S T x B -O VA
A
V
A
OVA257-264/Kb tetramer
M e d iu m
S L 8 p e p t id e
T IL s
5
30
IF N g s p o t s /1 0
CD8
23.30
600
*
40
% O V A - s p e c if ic
STxB-OVA
C D 8 T c e lls
E.
G.
H.
*
0.4
*
40
0.3
30
0.2
20
0.0
+
A
A
u
li
o
B
s
T
ir
x
T
T
e
m
S
S
m
V
-O
B
x
li
o
ir
s
m
e
T
-O
u
m
V
-O
B
x
T
V
s
A
V
-O
B
x
T
S
S
s
0.1
0
+
10
A
C D 4 T c e lls
% o f T re g a m o n g
50
OVA-specific CD8 T cells
/ Treg ratio
Figure 2. Temsirolimus-induced STxB-OVA antitumor efficacy is dependent on OVA-specific
CD8 T cells. B16-OVA-bearing C57BL/6 (B6) mice (n=5/group) were depleted with anti-CD8 (200
µg every 2 weeks) and treated with STxB-OVA and temsirolimus (A) Tumor growth (B) Kaplan–
Meier curves for survival of mice. (C) OVA257–264 Kb-tetramer ex vivo staining in spleen. (left)
representative dot plots (right) Percentage of OVA257–264-specific CD8+ T cells. (D) Functional
analysis of OVA257–264-specific CD8+ T cells measured in the spleen by IFN-g-ELISPOT. (E) OVA257–
b
264 K -tetramer ex vivo staining in tumor. (left) representative dot plots from (right) percentage of
OVA257–264-specific CD8+ T cells. (F) Functional analysis of OVA257–264-specific CD8+ TILs measured
ex vivo by IFN-g-ELISPOT. (G) Percentage of Tregs and (H) OVA-specific CD8 T cells / Tregs ratio in
mice. Results represent 2 independent experiments. Values shown correspond to means +/- SEM.
*P<0.05.
128
Figure 3. Anti-cancer mTORi treatment induces efficient OVA-specific memory CD8 T
cells. (A) Protocol scheme: C57Bl/6 mice (n=5/group) were vaccinated with STxB-OVA
alone or in combination with temsirolimus treatment (2mg/kg) during both expansion and
contraction phase of the immune response induced by STxB-OVA. (B) ex vivo OVA257–264
Kb-tetramer staining (left) and OVA-specific CD8 T cells phenotype analysis (right) in spleen.
Representative dot plots and histograms (C) Phenotype analysis of KLRG1-CD127+ and
CD62L+CD127+ OVA-specific CD8 T cells. Representative dot plots (left) and percentage
(right). C57Bl/6 mice (n=5/group) were vaccinated with STxB-OVA in combination with
high temsirolimus treatment (4.5mg/kg) or everolimus (0.65mg/kg/day) during both
expansion and contraction phase of the immune response induced by STxB-OVA (D) (left) ex
vivo OVA257–264 Kb-tetramer representative dot plots (right) percentage of OVA257–264-specific
CD8+ T cells. (E) Phenotype analysis of OVA257–264-specific CD8+ T cells. Representative
histograms. (F) Phenotype analysis of KLRG1-CD127+ and CD62L+CD127+ OVA-specific
CD8 T cells. (G) CD107a staining. representative dot plot (left) and percentage (right). (H) ex
vivo functional analysis of OVA257–264-specific CD8+ T cells measured in the spleen by IFN-gELISPOT Results represent at 2 independent experiments. Values shown correspond to means
+/- SEM. *P<0.05, **P<0.01, ***P<0.001.
129
A.
STxB-OVA +
Temsirolimus
Tumor size (mm2)
STxB-OVA
STxB-OVA +
Temsirolimus +
CCR4 antagonist
STxB-OVA +
CCR4 antagonist
300
300
300
300
200
200
200
200
100
100
100
100
0
0
0
10
20
30
0
0
10
20
30
0
0
10
20
30
0
10
20
30
Days post tumor graft
B.
500
C.
S T x B -O V A
100
S T x B - O V A + T e m s ir o lim u s
400
S T x B - O V A + T e m s ir o lim u s
+ C C R 4 a n t a g o n is t
% mice survival
Tumor size (mm2)
S T x B - O V A + C C R 4 a n t a g o n is t
300
200
100
*
75
50
25
0
0
0
15 15
20
25
30
35
0
1 51 5
20
25
30
35
Days post tumor graft
Days post tumor graft
Figure 4. A CCR4 antagonist improves anti-tumor efficacy of STxB-OVA vaccine plus
temsirolimus. B16-OVA-bearing C57BL/6 (B6) mice (n=5/group) were vaccinated with
STxB-OVA and then treated with temsirolimus, CCR4 antagonist or combination of both. (A)
Tumor growth per mice in each group (B) Tumor growth per group. (C) Kaplan–Meier
curves for survival of mice. Results represent 2 independent experiments. Values shown
correspond to means +/- SEM. *P<0.05.
130
Tumor
B.
C D 8 T c e lls
4.68
1.0
15.22
0.5
4
V
a
A
n
+
ta
+
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OVA257-264/Kb dextramer
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4
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li
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+
0.0
OVA257-264/Kb dextramer
20
% O V A - s p e c if ic
*
CD8
CD8
% O V A - s p e c if ic
1.47
0.39
1.5
STxB-OVA +
STxB-OVA + Temsirolimus +
Temsirolimus CCR4 antagonist
m
STxB-OVA +
STxB-OVA + Temsirolimus +
Temsirolimus CCR4 antagonist
C D 8 T c e lls
Spleen
A.
ta
a
A
n
+
+
m
-O
li
C
B
s
S
+
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C
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s
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40
B
5
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10
32.28
% o f T re g a m o n g
50
44.78
m
C D 4 T c e lls
% o f T re g a m o n g
FOXP3
9.21
4
*
15
18.69
Tumor
STxB-OVA +
STxB-OVA + Temsirolimus +
Temsirolimus CCR4 antagonist
R
D.
Spleen
STxB-OVA +
STxB-OVA + Temsirolimus +
Temsirolimus CCR4 antagonist
FOXP3
C.
OVA-specific CD8 T cells
/ Treg ratio
*
1.0
*
0.8
0.15
0.6
0.10
0.4
0.05
s p le n o c y t e s
Tumor
m e d iu m
S L 8 p e p t id e
60
40
5
0.20
80
IF N g s p o t s n u m b e r
Spleen
F.
f o r 2 .1 0
E.
20
0.2
0
0.00
n
+
ta
+
S T x B -O VA
+ T e m s ir o lim u s
+ C C R 4 a n t a g o n is t
+
T
C
x
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B
R
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4
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a
A
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4
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li
B
B
o
x
ir
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S
T
s
S
m
e
T
S T x B -O VA
ta
+
0.0
Figure 5. A CCR4 antagonist enhances temsirolimus-mediated anti-OVA CD8 T cells
after vaccination and decreases Tregs. B16-OVA-bearing C57BL/6 (B6) mice (n=5/group) were
vaccinated with STxB-OVA and then treated with temsirolimus, CCR4 antagonist or combination of both. ex
vivo OVA257–264 Kb-tetramer staining in spleen (left) representative dot plots and (right)
percentage of OVA257–264-specific CD8+ T cells (A) in the spleen and (B) tumor. Tregs
phenotype analysis (left) and mean percentages of FoxP3+ Tregs (right) (C) in the spleen and
(D) tumor. (E) OVA-specific CD8 T cells / Tregs ratio in spleen and tumor. (F) Functional
analysis of OVA257–264-specific CD8+ T cells measured ex vivo in the spleen by IFN-gELISPOT. Results represent 2 independent experiments. Values shown correspond to means
+/- SEM. *P<0.05.
131
A.
Vaccine
Expansion:
Day 0
Temsirolimus
3 7
24
STxB-OVA (20µg) + aGalCer (2µg) i.p.
Contraction:
Day 0
Expansion +
Contraction: Day 0
3 7
12 16
24
12 16
24
B.
Temsirolimus (2mg/kg)
CD127
KLRG1
CD62L
7.45
10.99
17.10
3.62
8.98
28.09
30.75
9.28
7.02
17.79
21.51
11.85
6.14
46.43
26.83
28.76
PBS
Expansion
CD8
Expansion +
Contraction
Count
Contraction
OVA257-264/Kb tetramer
C.
C D 127+
K LR G 1-
PBS
C D 127+
E x p a n s io n
C D 62L+
C o n t r a c t io n
0
4
0
3
0
2
0
1
0
Exp + C ont
% a m o n g O V A - s p e c if ic C D 8 T c e lls
Supplementary Figure 1.
(A) Protocol scheme: C57Bl/6 mice (n=5/group) were vaccinated with STxB-OVA alone or
in combination with temsirolimus treatment (2mg/kg) during either expansion or contraction
or both phases of the immune response induced by STxB-OVA B) ex vivo OVA257–264 Kbtetramer staining (left) and OVA-specific CD8 T cells phenotype analysis (right) in spleen.
Representative dot plots and histograms (C) Phenotype analysis of KLRG1-CD127+ and
CD62L+CD127+ OVA-specific CD8 T cells. Representative dot plots (left) and percentage
(right). Results represent 2 independent experiments. Values shown correspond to means +/SEM.
132
Day 0
Temsirolimus
18 21 26 28 33
14
B.
SL8 peptide
+ Temsirolimus
SL8 peptide
1.34
0.62
OVA257-264/Kb
tetramer
a. SL8 peptide (100µg) s.c.
b. UCP2 peptide (150µg) s.c.
Temsirolimus (2mg/kg)
C.
1.5
*
C D 8 T c e lls
Vaccine
% O V A - s p e c if ic
A.
1.0
0.5
s
u
p
+
T
e
S
m
L
s
8
ir
o
p
li
e
e
p
8
L
S
CD8
m
p
ti
ti
d
d
e
e
0.0
D.
s p le n o c y t e s
m e d iu m
U C P 2 p e p t id e
20
5
f o r 2 .1 0
IF N - g s p o t s n u m b e r
30
10
0
#1
#2
#3
U C P 2 p e p tid e
#1
#2
#3
U C P 2 p e p tid e
+ T e m s ir o lim u s
Supplementary Figure 2.
(A) Protocol scheme: C57Bl/6 mice (n=5/group) were immunized twice (day 0 and day 14)
subcutaneously (s.c.) with SL8 and HLA-A2/DR1 Tg mice (n=5/group) were immunized
twice with UCP peptides emulsified with montanide (v/v), alone or in combination with
temsirolimus treatment. (B) OVA257–264 Kb-tetramer ex vivo staining in spleen (left)
representative dot plots from one representative mouse. (C) Percentage of OVA257–264-specific
CD8+ T cells. (D) Functional analysis of UCP2 specific CD4 T cells measured ex vivo in the
spleen by IFN-g-ELISPOT. Results represent 2 independent experiments. Values shown
correspond to means +/- SEM. *P<0.05.
133
134
DISCUSSION
135
136
La rapamycine et ses analogues l'évérolimus et le temsirolimus sont des inhibiteurs de
la voie de signalisation mTOR. Ils sont couramment prescrits dans plusieurs indications
cliniques, incluant la prévention du rejet de greffe et le traitement du cancer (Groth et al.,
1999; Laplante and Sabatini, 2012). Or ces deux indications d'administration des mTORi
apparaissent en contradiction l'une avec l'autre, l'immunosuppression et la tolérance
immunitaire nécessaires à prévenir du rejet de greffe étant délétères pour la réponse antitumorale (Gaumann et al., 2008). L'un des obstacles majeurs à l'efficacité à long terme de la
transplantation d'un organe est ainsi le risque de développer un cancer associé à
l’immunosuppression. En effet, l’incidence des cancers est plus élevée chez les patients
transplantés par rapport à la population générale. Les données épidémiologiques révèlent que
la durée et l'intensité d'exposition à un traitement immunosuppresseur sont clairement liées au
risque de développer un cancer post-transplantation (Dantal et al., 1998; Gutierrez-Dalmau
and Campistol, 2007). De manière surprenante, ce risque varie en fonction du traitement. En
comparaison aux autres immunosuppresseurs (tacrolimus, cyclosporine…), la rapamycine a
montré une diminution de l'incidence des cancers parmi les patients transplantés (Alberú et
al., 2011; Campistol et al., 2006; Euvrard et al., 2012; Vajdic CM et al., 2006). Ces
observations
suggèrent
que
la
rapamycine,
développée
pour
ses
propriétés
immunosuppressives, présenterait également des propriétés préventives contre le cancer (Law,
2005). Néanmoins, la rapamycine est prescrite en combinaison ou en substitution des
immunosuppresseurs conventionnels, empêchant l'étude de son rôle potentiel sur les réponses
immunitaires anti-tumorales chez les patients transplantés. Il n’est ainsi pas clairement
déterminé si la diminution de l’incidence des cancers chez les patients transplantés est
principalement due à la rapamycine elle-même ou plutôt au retrait des immunosuppresseurs
conventionnels. Cependant, une augmentation du pourcentage des LT CD4 effecteurs
mémoires et une diminution des LT CD4 naïfs ont été observées chez des patients
transplantés traités par la rapamycine comparé à la cyclosporine (Brouard et al., 2010).
Contrairement à la rapamycine, l’évérolimus et le temsirolimus sont prescrits à une
forte dose dans le traitement du cancer du rein métastatique respectivement à 10mg par jour et
25mg par semaine, contre 2mg par jour pour la rapamycine après une transplantation de rein
(Porta et al., 2014). Cette dose plus forte de rapalogues est administrée pour obtenir un effet
anti-prolifératif direct sur les cellules tumorales. Mais l’impact de ces traitements sur les
réponses immunitaires (induction d’une immunosuppression ou d’une réponse immunitaire
anti-tumorale) chez les patients atteints de cancer n’a pas été évalué.
137
Au cours de ce travail, nous avons analysé deux types de réponses T CD4 (Treg et Th1)
au sein d'une cohorte de 23 patients atteints de cancer rénal métastatique (mRCC) traités par
évérolimus. Les Treg et la réponse Th1 spécifique de tumeur ont été mesurés dans le sang
avant le début du traitement, puis tous les deux mois jusqu'à l'arrêt du traitement.
Expansion progressive et persistante des Treg chez des patients atteints de mRCC traités par
évérolimus
Une des observations claires de cette étude est l’augmentation progressive du taux de
Treg FoxP3+ circulants chez la majorité des patients atteints de mRCC traités par évérolimus.
Cette expansion apparaît deux mois après le début du traitement et se maintient à un taux
élevé à la fin de l'étude chez la plupart des patients (91%). Ces résultats montrent que
l'inhibition de mTOR par l'évérolimus chez des patients atteints de cancer rénal s’accompagne
d’une augmentation des Treg similairement à ce qui a été historiquement décrit en
transplantation avec la rapamycine (Battaglia et al., 2005; Hendrikx et al., 2009; Sabbatini et
al., 2015). Nous avons observé que la durée du traitement par évérolimus est corrélée à
l'augmentation des Treg chez les patients de notre cohorte, de manière similaire à l'effet tempsdépendant retrouvé avec la rapamycine (Strauss et al., 2007, 2009).
Peu d’études ont investigué l’évolution des Treg chez des patients atteints de cancer et
sous traitement par des inhibiteurs de mTOR. Une première étude chez 23 patients atteints de
cancer du rein et traités par évérolimus ou temsirolimus n’avait pas observé d’augmentation
de Treg. Cependant contrairement à notre étude, l’analyse n'a été réalisée qu’une fois à un
temps précoce (un mois) après le début du traitement ce qui peut expliquer ce résultat
(Kobayashi et al., 2013). En effet, dans notre cohorte, l’augmentation des Treg était souvent
observée à partir du 2ème mois après le début du traitement, puis nettement après 4 mois chez
la majorité des patients. Récemment, une étude pilote dans le cancer de la prostate avait
rapporté des résultats similaires aux notres à savoir une augmentation du taux de Treg après
l'administration d’évérolimus. Cette augmentation de Treg était également différée dans le
temps comme celle observée dans notre étude (Templeton et al., 2013).
Plus récemment a été décrit un rôle majeur de la voie mTOR dans la différenciation
des cellules myléoïdes suppressives (MDSC). Ainsi une inhibition de mTOR par la
rapamycine au cours d'une transplantation cardiaque dans un modèle murin a permis
l’expansion de MDSC renforcant ainsi l’immunosuppression nécessaire à la prévention du
rejet de greffe (Nakamura et al., 2015). Ces cellules appartenant à l'immunité innée sont
138
importantes dans la régulation de la réponse anti-tumorale (Vesely et al., 2011). En effet, une
forte infiltration tumorale de MDSC est souvent associée à un mauvais pronostic dans les
cancers y compris dans le RCC (Marigo et al., 2008; Rabinovich et al., 2007). Le recrutement
des MDSC favorise la néo-angiogenèse nécessaire à la croissance tumorale (Murdoch et al.,
2008). Ainsi le traitement par sunitinib, un antigiogénique administré chez les patients atteints
de cancer du rein, peut réduire l’accumulation des MDSC (Ko et al., 2009). L'activation de la
voie mTOR étant impliquée dans l’angiogenèse tumorale, il serait donc intéressant d'étudier
cette sous-population cellulaire chez les patients atteints de mRCC traités par mTORi.
Stimulation de réponses Th1 anti-TERT après traitement par évérolimus
La réponse spontanée Th1 anti-tumorale a été évaluée chez les patients par la détection
de leur LT CD4 Th1 circulants spécifiques de peptides dérivés de la télomérase (TERT).
TERT est une enzyme qui permet le maintien de la longueur des télomères dans les cellules
en division et son expression est le mécanisme principal développé par les cellules tumorales
pour échapper à la mort cellulaire dépendante des télomères (Martínez and Blasco, 2011).
Ainsi, la télomérase est surexprimée dans plus de 90% des cancers humains dont le cancer du
rein (Fan et al., 2005; Hiyama and Hiyama, 2003). Ces propriétés ont fait de TERT un
prototype d’antigène tumoral de type universel (Cheever et al., 2009). Les précédents travaux
de notre équipe ont permis l’identification de nouveaux peptides immunogènes dérivés de
TERT appelés UCP (Universal Cancer Peptide), capables de se lier à un grand nombre
d'allèles HLA-DR, permettant ainsi de cibler la majorité de la population (Godet et al., 2012).
Les LT CD4 anti-UCP produisent principalement de l'IFN-g et du TNF-a caractéristiques
d’une polarisation Th1. Des réponses naturelles T CD4 spécifiques des UCP ont été détectées
chez des patients atteints de différents types de cancers, dont le cancer du rein, confirmant le
caractère universel de ces peptides (Adotévi et al., 2013; Dosset et al., 2012). Notre équipe a
montré chez des patients atteints de cancer du poumon métastatique que la présence de
réponses Th1 anti-UCP chez les patients ayant répondu à la chimiothérapie était corrêlée à
une meilleure survie suggérant une synergie entre efficacité de la chimiothérapie et
l’immunité Th1 anti-TERT (Dosset et al., 2013; Godet et al., 2012). L’ensemble de ces
données souligne ainsi l'intérêt d’utiliser les peptides UCP comme biomarqueurs de la réponse
Th1 anti-tumorale.
En utilisant la technologie UCP, notre équipe avait rapporté une importante
modulation de la réponse spontanée Th1 anti-TERT chez une patiente atteinte de mRCC
traitée par évérolimus (Thiery-Vuillemin et al., 2014a). Au cours de cette présente étude, nous
139
avons observé la présence de réponses spontanées Th1 anti-TERT chez 48% des patients
avant traitement, et cette fréquence a atteint 74% après traitement par évérolimus, montrant
l'apparition d'une réponse immunitaire anti-tumorale chez les patients parallèlement à leur
traitement par évérolimus. De plus, l’amplitude des réponses Th1 anti-TERT augmente
significativement après traitement par évérolimus chez la majorité des patients. Ces résultats
ont clairement montré que l’inhibition de mTOR chez des patients atteints de cancer du rein
s’accompagne d’une augmentation de la fréquence et de la qualité des réponses Th1 antiTERT. Kobayashi et al. avaient observé une augmentation des réponses immunitaires de type
Th1 après traitement par mTORi chez des patients atteints de mRCC mais d’une manière non
spécifique de tumeur. En effet les LT des patients avaient été stimulés par la
PMA/ionomycine (Kobayashi et al., 2013). Notre travail est ainsi à notre connaissance la
première démonstration d’une expansion de la réponse CD4 Th1 spécifique de tumeur chez
des patients atteints de cancer et traités par un mTORi.
Plusieurs mécanismes pourraient expliquer cette stimulation de LT CD4 spécifiques de
tumeur après blocage de la voie mTOR. La première est l’impact direct de la voie mTOR sur
la différenciation des LT mémoires, qui est un processus bien décrit (Araki et al., 2009;
Pollizzi et al., 2015; Rao et al., 2010). Plusieurs sous-populations de LT mémoires ont été
décrites, telles que les TCM et les TEM (Sallusto et al., 1999). Récemment, l'équipe de N.
Restifo a caractérisé une sous-population de LT mémoires ayant des propriétés de
renouvellement importantes, les TSCM. Ces TSCM possèdent en outre des propriétés antitumorales supérieures aux autres LT mémoires (Gattinoni et al., 2011). Nos résultats ont
montré une amplification significative des LT CD4 spécifiques de TERT chez la majorité des
patients traités par évérolimus. Bien que nous n’ayons pas analysé le phénotype de ces
lymphocytes, ces données suggèrent qu'il s'agisse de LT CD4 mémoires car pré-existants chez
les patients. L’utilisation de tétramères HLA-DR spécifiques des UCP ou la détection de
cytokine (IFN-g) intracellulaire permettrait d’étudier le phénoype mémoire des LT CD4 Th1
anti-TERT. Un autre mécanisme pouvant expliquer l'activation de cette réponse Th1 antiTERT serait via une meilleure présentation antigénique aux LT CD4 suite au traitement par
évérolimus. Premièrement, l'effet cytotoxique de l'évérolimus sur la tumeur pourrait libérer la
protéine TERT des cellules nécrotiques et favoriser sa présentation aux LT CD4. Dans ce
contexte, notre équipe a étudié les mécanismes de présentation de la protéine TERT par les
molécules de CMH II. Nous avons montré que la présentation de TERT par les CMH II utilise
à la fois les voies cytosoliques et endo-lysosomales. De plus, nous avons mis en évidence un
140
rôle majeur des protéoglycanes à héparane sulfate (HSPG) présents à la surface des DC dans
l'internalisation et l’apprêtement de TERT par les molécules de CMH II (Galaine et al.,
manuscrit en préparation). Deuxièment, il a été décrit que l'inhibition de la voie mTOR dans
les DC favorise la présentation de peptides antigéniques aux LT. Ce mécanisme est soit direct,
en prolongeant la durée de vie des DC et l'expression de molécules de co-stimulation, soit
indirect en activant l'autophagie, processus connu pour optimiser la présentation de peptides
sur les molécules de CMH II (Amiel et al., 2012; Lee et al., 2010a). L’analyse de l’activation
des DC issues de patients avant et après le début du traitement par évérolimus, et de leur
capacité d’apprêtement à l’aide de la protéine TERT permettrait d’étudier l’implication des
mTORi sur l’activation des réponses Th1 anti-tumorales.
L'étude des réponses T CD8 centrales mémoires (CD45RO+CD127+CD62L+) a été
réalisée chez les patients de notre cohorte. Nos travaux n'ont pas montré d'augmentation de
cette population chez les patients suite au traitement par évérolimus. Une augmentation des
réponses T CD8 mémoires anti-virales (dirigées contre un pool de peptides dérivés du CMV,
EBV, influenza…) a été observée chez les patients après traitement. Néanmoins l'étude de la
réponse T CD8 spécifique d'antigènes de tumeur devra être réalisée par la suite pour attester
d’un impact du traitement par évérolimus sur les réponses T CD8 mémoires anti-tumorales.
En plus de peptides dérivés de la télomérase, d’autres antigènes de tumeurs exprimés dans le
cancer du rein pourront être évalués, tels que la survivine ou MUC1 (Vieweg and Jackson,
2004). Des études dans le cancer du rein ont montré que la présence de TIL T CD8 mémoires
est corrêlée à un mauvais pronostic (Giraldo et al., 2015; Nakano et al., 2001). Une des
explications serait la forte expression de récepteurs inhibiteurs (PD-1, LAG-3, PD-L1 et PDL2) dans le microenvironnement (Giraldo et al., 2015). De même, une diminution de la chaine
zeta du CD3 des TIL T CD8 dans le RCC est associée à un mauvais pronostic (Gati et al.,
2001). Néanmoins, ces observations ont été effectuées chez des patients n'ayant
majoritairement pas reçu de traitement ou alors par immunothérapie (IL-2/IFN-a) pour
quelques-uns (Giraldo et al., 2015). Il serait ainsi intéressant d'étudier la capacité des
inhibiteurs de la voie mTOR à moduler l'environnement immunitaire intra-tumoral chez les
patients. L'étude des réponses T CD8 spécifiques des tumeurs a été abordée dans nos
expériences chez la souris que nous détaillerons par la suite dans cette discussion.
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La part de l’immunité dans l’efficacité clinique de l’évérolimus
Grâce à leurs propriétés anti-prolifératives, l'évérolimus ou le temsirolimus sont
préscrit comme traitement dans le cancer du rein métastatique (Hudes et al., 2007; Motzer et
al., 2008). La médiane de la survie sans progression (PFS) calculée au sein notre cohorte de
patients est de 10,97 mois. Celle-ci est supérieure à la médiane de la PFS (4 mois) décrite
dans l'essai de phase III ayant abouti à l'autorisation de mise sur le marché de l'évérolimus
pour le traitement du mRCC (Motzer et al., 2008). Cependant l'implication du système
immunitaire dans l'efficacité clinique de ces mTORi n'est pas connue. Nous avons observé
une augmentation concomitante des Treg et des Th1 spécifiques de tumeur chez les patients
atteints de cancer du rein métastatique après traitement par évérolimus. Afin d’évaluer si cette
modulation immunitaire observée pouvait influencer la survie des patients, nous avons utilisé
un modèle mathématique prenant en compte le taux de variation au cours du temps des deux
paramètres immunitaires que sont les Treg et Th1 anti-TERT. Ce modèle a été retenu car prise
individuellement, la modulation des deux populations de LT CD4 n'avait aucune influence
significative sur la survie des patients.
En utilisant ce modèle, nous avons montré que les patients traités par évérolimus
pouvaient être classés en trois groupes immunologiques. De manière intéressante, les patients
qui présentaient une diminution précoce (à deux mois) des Treg associée à l'expansion des Th1
anti-TERT avaient une PFS significativement plus longue, de 13,2 mois. Ces résultats
suggèrent qu'une baisse précoce des cellules immunosuppressives (Treg) associée à une
stimulation des effecteurs Th1 anti-tumoraux survenant au cours du traitement agirait en
synergie avec l'effet anti-prolifératif de l'évérolimus et par conséquent augmenterait son
efficacité clinique. Dans les deux autres groupes immunologiques, le taux de variation des
Treg ou des Th1 anti-TERT était soit insignifiant (situation d'équilibre) soit les deux
paramètres évoluaient dans le même sens (augmentation ou diminution simultanée des Treg et
des Th1 anti-TERT). La majorité des patients (n=11) appartenaient au groupe dit "équilibre",
où le taux de variation des Treg ou des Th1 anti-TERT était proche de zéro, et présentaient une
PFS de 8 mois. Ainsi, en absence de modulation immunitaire favorable, l'efficacité clinique
de l'évérolimus reposerait principalement sur son effet cytotoxique direct sur la tumeur. La
médiane de la PFS dans le dernier groupe immunologique était la plus faible, de 4,1 mois. Les
patients de ce groupe présentaient un profil immunosuppressif avec une augmentation des
Treg, sans expansion des cellules Th1 anti-TERT, suggérant que la survenue précoce d'un
environnement immunosuppresseur suite au traitement par évérolimus serait délétère sur le
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plan clinique. Cette observation était confortée par l'augmentation inexorable des Treg sous
traitement par évérolimus et la perte totale des réponses Th1 anti-TERT chez la majorité des
patients au moment de la progression de la maladie. En résumé, ces résultats montrent pour la
première fois qu'une modulation précoce de la réponse immunitaire anti-tumorale induite par
les mTORi contribuerait à l'efficacité du traitement. Ces résultats confirment également notre
hypothèse de départ, suggérant que l'expansion des Treg après traitement par évérolimus
engendrerait une immunosuppression favorable à la progression du cancer.
Plusieurs données de la littérature ont rapporté que les sous-populations
lymphocytaires T CD4 helper infiltrant la tumeur ont des rôles pronostics cliniques différents
dans les cancers. Ainsi, la présence de Treg est corrélée à un mauvais pronostic, y compris
dans le cancer du rein (Griffiths et al., 2007; Liotta et al., 2011). Au contraire, une forte
densité de Th1 infiltrant les tumeurs est associée à un bon marqueur pronostic dans plusieurs
cancers, dont le cancer du rein (Fridman et al., 2012; Kondo et al., 2006). Par conséquent,
suite au blocage thérapeutique de mTOR, la formation d'un environnement immunitaire
favorable au profit d'une réponse Th1 contribuerait à l'efficacité clinique du médicament. Cet
impact immunologique de l'évérolimus sur son efficacité clinique n'a pas été observé sur la
survie globale en raison du faible nombre de patients. Ainsi, ces résultats bien que très
prometteurs méritent d'être confirmés au sein d'une plus grande cohorte de patients.
Everolimus et temsirolimus augmentent les fonctions des Treg in vitro
Nous avons montré qu'un traitement prolongé avec évérolimus est corrêlé à une
augmentation des Treg associée à une perte des réponses Th1 anti-TERT chez les patients
atteints de mRCC. Ces résultats suggèrent une inhibition des réponses Th1 anti-tumorales par
les Treg induits suite à l'inhibition de mTOR. En effet, des études chez les patients transplantés
ont montré que le traitement par la rapamycine augmente la capacité immunosuppressive des
Treg (Bocian et al., 2010; Strauss et al., 2007). Différents mécanismes de suppression utilisés
par les Treg ont été décrits: une suppression par la production de cytokines inhibitrices (TGFb, IL-10, IL-35), par une lyse cellulaire directe (perforine/granzyme), par une perturbation
métabolique (privation en IL-2 ou formation d'adénosine à partir de l'ATP du
microenvironnement via les ectonucléoisdases CD39-CD73), ou par une modulation des DC
(via les récepteurs inhibiteurs CTLA-4 ou LAG3) (Schmitt and Williams, 2013; Vignali et al.,
2008).
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Pour étudier les mécanismes de suppression dans notre contexte, nous avons dans un
premier temps analysé le phénotype fonctionnel des Treg chez les patients atteints de mRCC
avant et après traitement par évérolimus. En plus du marqueur FoxP3, les Treg retrouvés chez
les patients traités par évérolimus exprimaient les récepteurs impliqués dans la fonction
immunosuppressive tels que CTLA-4 et ICOS (Fife and Bluestone, 2008; Herman et al.,
2004). De plus, ces cellules étaient Ki67+ suggérant une prolifération in vivo. Par ailleurs,
nous avons également montré l’expression du facteur de transcription Helios sur ces Treg. Nos
résultats, qui restent à confirmer, suggèrent que ces Treg sont de type "naturel" (nTreg)
d’origine thymique par opposition aux Treg "induits" (iTreg) issus du pool périphérique de LT
CD4 (Adeegbe and Nishikawa, 2013). En effet, Helios est un facteur de transcription de la
famille Ikaros, exprimé dans les Treg thymiques, mais pas sur les iTreg (Thornton et al., 2010).
Malgré que son expression dans les LT puisse être transitoirement induite durant l'activation
(Akimova et al., 2011), il est souvent considéré comme un marqueur de nTreg, les iTreg
générés in vitro ou in vivo perdant l'expression d'Helios (Elkord et al., 2011).
Dans un second temps, nous avons exploré les mécanismes de suppression des Treg
traités par temsirolimus ou évérolimus grâce à des expériences in vitro à partir de cellules
issues de donneurs sains. Des expériences d'inhibition de la prolifération de LT allogéniques
en présence de Treg préalablement exposés au temsirolimus ou à l'évérolimus ont ainsi été
réalisées. Nous avons mis en évidence une augmentation des propriétés immunosuppressives
des Treg exposés à temsirolimus ou évérolimus. En effet, les Treg exposés in vitro à ces deux
mTORi inhibent plus efficacement la prolifération de LT allogéniques et la production d'IFNg, par rapport à des Treg contrôles. De plus, cette inhibition de la prolifération allogénique
s'accompagne d'une diminution de la production de cytokines telles que l'IL-2, le TGF-b1 et
l'IL-10. Ainsi, une hypothèse serait que la forte capacité suppressive des T reg pourrait
s'expliquer par la déprivation en IL-2 suite à une capture excessive par les Treg exprimant
fortement CD25, la chaîne a du récepteur à l'IL-2. La diminution de la production des deux
cytokines inhibitrices TGF-b1 et IL-10 parait paradoxale mais pourrait s'expliquer par une
consommation plus forte pendant les expériences de co-culture avec les LT allogéniques. En
plus de l'expression de CTLA-4 et ICOS également observée sur les Treg des patients, nous
avons montré que les Treg cultivés in vitro en présence des mTORi expriment GITR ou CD39,
des molécules impliquées dans leurs fonctions suppressives (Bastid et al., 2015; Shimizu et
al., 2002). Nous avons par la suite observé que l'inhibition de la prolifération allogénique
exercée par les Treg exposés aux mTORi était complétement abolie lorsque ces Treg étaient
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séparés des LT allogéniques dans des expériences de transwell. Cette inhibition contactdépendante suggère fortement une implication des récepteurs inhibiteurs, mais renforce
également l'origine "naturelle" de ces Treg. Il est en effet admis que les nTreg agissent
préférentiellement par des mécanismes contact-dépendants, par opposition aux iTreg supposés
agir plutôt de manière cytokine-dépendante (Bilate and Lafaille, 2012). Afin d'étudier
l'implication des récepteurs inhibiteurs, nous avons réalisé des expériences de co-culture en
présence d'anticorps bloquant GITR, ICOS, CTLA-4 et CD39. Nos résultats préliminaires
n'ont cependant pas permis de démontrer l'implication de ces molécules dans les propriétés
suppressives des Treg exposés aux mTORi. De même, le blocage des récepteurs du TGF-b1
(par du SD208, un inhibiteur spécifique) n'a pas montré de rôle potentiel du TGF-b1 dans le
processus d'inhibition.
Pour élucider les mécanismes pouvant expliquer le fort pouvoir inhibiteur des Treg
exposés aux mTORi, nous avons prévu de réaliser une analyse RNA-seq des différents
niveaux d’expression des ARNm entre les Treg exposés ou non aux mTORi, afin d'identifier
les protéines potentiellement impliquées dans les propriétés immunosuppressives de ces Treg.
Ces expériences permettront une meilleure compréhension des mécanismes suppressifs acquis
par les Treg exposés aux mTORi.
Les Treg limitent l’efficacité des mTORi chez la souris
Par la suite, nous avons utilisé des modèles in vivo afin d’étudier dans des conditions
physiologiques les relations entre la réponse Treg et l’efficacité anti-tumorale des mTORi.
Pour cela, nous avons travaillé avec le modèle tumoral B16-OVA, un mélanome murin
transfecté avec la proteine ovalbumine, nous permettant de suivre spécifiquement les réponses
T anti-tumorales naturelles. Les doses de temsirolimus (2mg/kg/3jours) et d'évérolimus
(0,65mg/kg/jour) ont été déterminées à partir des données de pharmacocinétique obtenues
chez les patients (Thiery-Vuillemin et al., 2014b). A ces doses, nous avons observé une
diminution de la vitesse de croissance tumorale chez les souris, mais jamais de régression
complète.
L'analyse du taux de Treg chez les souris greffées par une tumeur et traitées par
évérolimus ou temsirolimus a montré que le traitement augmente significativement le ratio
Treg/taille tumorale. L'un des mécanismes utilisés par les tumeurs pour échapper à la réponse
immunitaire étant le recrutement de Treg (Nishikawa and Sakaguchi, 2010), il était alors
difficile de distinguer le taux de Treg élevé provenant des tumeurs non traitées par mTORi
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(tumeur de grande taille) du taux de Treg élevé provenant du traitement par mTORi (tumeur de
petite taille). Nous avons alors calculé ce ratio Treg/taille tumorale, qui augmente après
traitement par mTORi renforçant nos observations faites chez les patients atteints de cancer
du rein.
Pour évaluer l'implication des Treg pendant les traitements par mTORi nous avons
réalisé des expériences de déplétion in vivo de LT. Dans un premier temps, la déplétion en LT
CD4 préalablement à la greffe de la tumeur a montré une importante amélioration de
l'efficacité anti-tumorale des mTORi. En effet, la croissance tumorale était diminuée et la
survie augmentée lorsque les souris étaient traitées par les mTORi en absence des LT CD4,
comparé aux autres groupes. L'absence des Treg par la déplétion en cellules CD25+ chez les
souris traitées par évérolimus conduit à des observations similaires. Ces observations
suggèrent ainsi un rôle délétère des Treg dans l'efficacité anti-tumorale des mTORi confirmant
nos hypothèses de l'impact néfaste d'un environnement immunosuppressif sous traitement par
évérolimus ou temsirolimus. L’étude plus précise de l’implication des Treg durant le traitement
par mTORi a été effectuée à l’aide du modèle de souris DEREG. Ces souris possèdent le
récepteur à la toxine diphtérique humaine sous le contrôle du promoteur FoxP3, et l'injection
de cette toxine permet de dépléter spécifiquement les Treg durant le traitement par mTORi
(Lahl et al., 2007). Ce modèle est plus pertinent pour éliminer les Treg par rapport à
l’utilisation d’un anticorps monoclonal anti-CD25 qui risque de toucher également les LT
activés si l’injection n’est pas effectuée chez des souris naïves. De plus, la déplétion tardive
des Treg permet de préserver leur rôle positif dans l'initiation des réponses T CD8 mémoires de
haute avidité (Pace et al., 2012). Par l'utilisation de ce modèle, nous avons pu dépléter les Treg
une fois que le traitement anti-mTOR eut montré un début de ralentissement de la croissance
tumorale. Nous avons alors observé que l'élimination des Treg a considérablement renforcé
l'efficacité anti-tumorale du temsirolimus, confirmant le role délétère des Treg, à la fois sur la
croissance tumorale et la survie des souris induites par les anti-mTOR.
Nos expériences in vivo dans le modèle tumoral B16-OVA nous ont permis d’évaluer
plus précisément l’implication et la modulation des réponses T CD8 anti-tumorales après
traitement par mTORi. Nous avons ainsi montré que l’absence des LT CD4 durant le
traitement par mTORi permet la génération d’une réponse T CD8 spécifique d'OVA. L’étude
plus précise de l’implication des Treg grace à leur déplétion montre leur rôle délétère dans la
génération d’une réponse T CD8 naturelle anti-OVA. L'étude des réponses Th1 anti-OVA n'a
pas été réalisée dans ce modèle mais serait interessante pour confirmer nos observations chez
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les patients. Ainsi, ces résultats suggèrent que l'efficacité anti-tumorale accrue des mTORi en
absence des Treg serait due à la levée de l’inhibition des réponses T CD8 spécifiques de la
tumeur induites par les mTORi. Ces résultats confirment notre hypothèse que les mTORi
induisent une réponse immunitaire T anti-tumorale chez les patients, mais que cette réponse
est inhibée par les Treg exposés aux mTORi.
Le rôle inhibiteur des Treg pendant le traitement par mTORi avait également été
rapporté par Wang et al. chez la souris (Wang et al., 2014). Par l'utilisation du modèle de
tumeur RENCA (tumeur du rein) transfectée avec l'antigène CA9, ils ont montré que le
traitement par temsirolimus était plus efficace en absence de LT CD4 pour ralentir la
croissance tumorale, confirmant nos observations avec le modèle B16 transfecté avec OVA.
Cependant, contrairement à notre étude, cette équipe n'a pas étudié la réponse immunitaire
anti-tumorale naturelle induite après traitement par mTORi. Par l'utilisation d'anticorps
déplétant les LT CD4 ou l'utilisation du modèle DEREG, ils ont montré le rôle délétère des
Treg sur la réponse T CD8 spécifique induite par transfert adoptif et immunisation, de plus
dans un contexte non tumoral. Notre étude est à notre connaissance la première démonstration
que le traitement par mTORi puisse induire une réponse immunitaire anti-tumorale naturelle.
En résumé, nos expériences in vivo dans le modèle B16-OVA nous ont permis
d'étudier plus précisément l'implication des Treg dans l'efficacité anti-tumorale des mTORi
Nos travaux suggèrent ainsi que les mTORi anti-cancer agissent non seulement directement
sur la tumeur, mais génèrent également une réponse immunitaire anti-tumorale, qui est
néanmoins inhibée par les Treg exposés à ces traitements.
Combinaison de mTORi avec des agents bloquant les Treg
Nous avons montré le rôle délétère des Treg pendant le traitement par les mTORi. Ces
données constituent un rationnel solide pour combiner les anti-mTOR avec des agents
thérapeutiques permettant de dépléter les Treg. De multiples approches sont actuellement
développées pour inhiber les Treg dans le cancer (Ménétrier-Caux et al., 2012). Il existe ainsi
des stratégies pour éliminer les Treg, telles que l’utilisation d’un anti-CD25 (Rech et al., 2012)
ou l’utilisation de la denileukine diftitox, une protéine recombinante conjuguant l’ADN de la
toxine diphtérique avec celui de l’IL-2 permettant de cibler les cellules exprimant fortement
CD25 (Morse et al., 2008). Des stratégies permettant d’inhiber les fonctions suppressives des
Treg sont également en développement. Par exemple, sont testés des anticorps bloquant
CTLA-4, ICOS ou GITR (Faget et al., 2012; Ko et al., 2005; Peggs et al., 2009). Nous avons
dans un premier temps évalué dans le modèle B16-OVA la combinaison séquentielle
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d'évérolimus avec le sunitinib. Le sunitinib est un inhibiteur de récepteurs tyrosine kinase
impliqués dans l’angiogenèse (VEGF-R, PDGF-R, FGF-R…). Il est prescrit dans le cancer du
rein métastatique avec un risque pronostique favorable ou intermédiaire comme traitement de
première intention (Motzer et al., 2007; Rini et al., 2009). Il a été montré que le traitement par
sunitinib entraine une immunomodulaion. Ainsi, ce traitement induit une baisse des Treg
circulants et infiltrant la tumeur chez des patients atteints de cancer du rein métastatique
(Adotevi et al., 2010; Finke et al., 2008; Terme et al., 2013). Nous avons montré que les
souris traitées par la combinaison séquentielle d’évérolimus suivie de sunitinib présentaient
un ralentissement modeste de la croissance tumorale, mais toutefois supérieur à l’effet de
l’évérolimus en monothérapie. En effet, les courbes de croissance tumorale sous évérolimus
versus évérolimus+sunitinib ont commencé à se séparer cinq jours après l’addition du
sunitinib. Cependant, l’effet anti-tumoral de la combinaison était non significatif, ce qui
pourrait s’expliquer par l’introduction tardive du sunitinib au moment où la taille des tumeurs
B16-OVA était importante (100mm2). Nous pensons ainsi qu’une introduction plus précoce
du sunitinib pourrait augmenter l’efficacité anti-tumorale de cette combinaison. L’analyse des
Treg dans les différents groupes thérapeutiques a montré une réduction significative chez les
souris traitées par la combinaison comparée à la monothérapie avec évérolimus. Bien que
nous n’ayons pas pu évaluer les réponses T CD8 spécifiques d’OVA, ces résultats montrent
que l’effet de la combinaison évérolimus+sunitinib implique une modulation de l’immunité
caractérisée par la diminution des Treg ce qui confirme également les propriétés
immunomodulatrices du sunitinib. Il serait ainsi intéressant d’évaluer cette combinaison
séquentielle en clinique, par exemple après deux mois de traitement par évérolimus au
moment où débute l’expansion des Treg chez les patients. Des essais cliniques de
combinaisons de mTORi avec des anti-angiogéniques, notamment le bévacizumab (un
anticorps anti-VEGF) ont été menés dans le cancer du rein métastatique et dans les tumeurs
neuro-endocrines, une autre indication des mTORi (Bergsland, 2015). Cependant, les résultats
attendus ont été décevants, souvent dus à des effets secondaires sévères associés à ce type de
combinaison thérapeutique (Négrier et al., 2011; Rini et al., 2014). A noter que dans la
majorité des études, l’inhibiteur de mTOR et l’anti-angiogénique étaient administrés de façon
concomitante. Plus récemment, un essai de phase I-II (patients en cours de recrutement) est
actuellement en train d'évaluer le potentiel de combiner l'évérolimus avec du
cyclophosphamide, chez des patients atteints de mRCC (Huijts et al., 2011). La capacité du
cyclophosphamide à dépléter les Treg est largement documentée (Zitvogel et al., 2008). Il a été
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en effet montré qu’une administration continue à faible dose (« dose métronomique ») de
cyclophosphamide, un agent alkylant déjà administré comme chimiothérapie dans les
lymphomes, certaines leucémies et tumeurs solides, permet d’induire une déplétion des Treg
et limiter la toxicité associée à l’utilisation de fortes doses (Ghiringhelli et al., 2006).
Nous avons également testé une seconde approche de combinaison du temsirolimus
avec un antagoniste du CCR4 (AF399/420/18 025). Nous avions observé dans nos
expériences in vitro une expression de CCR4 par les Treg. CCR4 est un récepteur exprimé
préférentiellement par les Treg comparé aux LT conventionnels, qui permet leur migration vers
la tumeur produisant les chimiokines CCL22 ou CCL17, ligands du CCR4. L’expression du
CCR4 est également associée à une forte activité suppressive des Treg (Faget et al., 2011;
Hirahara et al., 2006; Iellem et al., 2001). Une stratégie de contrôle de la fonction des Treg est
de cibler les chimiokines ou les récepteurs de chimiokines impliqués dans leur migration.
Ainsi, dans des modèles pré-cliniques, l’injection d’anticorps monoclonaux spécifiques de
CCL22 réduit significativement la migration des Treg dans des tumeurs ovariennes (Curiel et
al., 2004). Au cours de ce travail, nous avons ainsi étudié la combinaison du temsirolimus et
d'une molécule antagoniste de CCR4 qui a montré son efficacité à empêcher le recrutement
des Treg en perturbant l’interaction de CCL22/CCL17 à leur récepteur. Cet antagoniste
possède une faible demi-vie (24h) (Bayry et al., 2008) empêchant des complications autoimmunes, ce qui l’avantage par rapport à d’autres traitements ciblant les Treg (anti-CD25, antiOX40, anti-GITR…) qui ont une demi-vie plus longue (2-3 semaines) (Colombo and
Piconese, 2007). Au cours de ce travail, nous avons montré l’efficacité synergique du
traitement concomitant du temsirolimus + antagoniste du CCR4 chez des souris porteuses de
la tumeur B16-OVA. L’analyse des réponses immunitaires a montré une diminution
significative des Treg et l’induction de fortes réponses T CD8 anti-OVA chez les souris traitées
par la combinaison. L’ensemble de ces résultats confirment les observations chez les souris
déplétées en Treg par des anticorps monoclonaux ou chez les souris transgéniques DEREG.
Ainsi, nous avons mis en évidence l'intérêt de combiner les mTORi anti-cancer avec
des thérapies ciblant les Treg afin de bloquer le profil immunosuppressif induit par ces mTORi
tout en conservant leur effet bénéfique sur la réponse T anti-tumorale. Les patients atteints de
cancer et traités par mTORi pourraient ainsi bénéficier d'un traitement combiné afin
d'empêcher la balance immunitaire de pencher vers les Treg.
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Combinaison de mTORi avec une vaccination thérapeutique anti-tumorale
Nos résultats chez les patients atteints de mRCC traités par évérolimus ont montré que
la présence d'une réponse immunitaire anti-tumorale précoce est corrêlée au bénéfice clinique
du traitement. Nous avons retrouvé dans nos expériences murines l'implication des LT dans
l’efficacité anti-tumorale des mTORi. En effet, dans des modèles tumoraux murins faiblement
immunogènes tels que RENCA (cancer du rein) et 4T1 (cancer mammaire), indications des
traitements par mTORi chez l’homme, nous avons montré que la cinétique de croissance
tumorale est identique en présence ou en absence de LT. Ainsi dans ces modèles, les
déplétions de LT n’impactent pas sur l’efficacité anti-tumorale des mTORi, qui repose
entièrement sur leurs capacités anti-prolifératives. Par contre, tels que nous l’avons
précedemment décrit, l’utilisation d’un modèle tumoral murin très immunogène tel que B16OVA, a montré que l’efficacité des mTORi est modulée en fonction de la présence ou non des
populations de LT. Cette observation est confirmée dans les travaux de Wang et al., qui ont
démontré un impact des déplétions T sur l’efficacité du temsirolimus, mais dans un modèle
RENCA transfecté avec CA9. Nous avons ainsi montré l’importance de l’immunogénicité de
la tumeur pour l’implication des LT dans l’efficacité anti-tumorale des mTORi.
Le RCC est une tumeur très immunogène, et beaucoup d'immunothérapies sont
actuellement en essais cliniques (Inman et al., 2013). En effet, des études cliniques sont en
cours pour évaluer la possible synergie entre des vaccins thérapeutiques et les traitements
anti-angiogéniques conventionnels (Combe et al., 2015; Figlin, 2013). Dans des modèles précliniques murins, le sunitinib crée un microenvironnement favorable en déplétant les MDSC
et agit en synergie avec un vaccin anti-tumoral en induisant l'expansion de LT CD8
spécifiques de la tumeur (Draghiciu et al., 2015). Au cours de ce travail, nous avons évalué la
combinaison entre l’évérolimus ou le temsirolimus et une vaccination thérapeutique antitumorale. Pour cela, nous avons travaillé avec un modèle de vaccin composé de l’association
de la sous-unité B de la toxine Shiga avec la protéine ovalbumine (STxB-OVA) (Adotevi et
al., 2007). Nous avons ainsi montré que le temsirolimus améliore l’efficacité anti-tumorale du
vaccin STxB-OVA. La déplétion en LT CD8 et le suivi des réponses T CD8 spécifiques de la
tumeur B16-OVA montrent l’importance de la promotion de ces cellules dans l’efficacité de
la double thérapie. Une forte présence de LT CD8 anti-OVA est ainsi mesurée dans la rate et
la tumeur lorsque les souris sont traitées par temsirolimus. Ces observations confirment les
travaux de Wang et al. utilisant également le temsirolimus en combinaison avec différents
types de vaccins (Wang et al., 2011d, 2014). En addition, nous avons étudié le phénotype des
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LT CD8 spécifiques induits par l’immunisation. Nos travaux ont montré que l’administration
de mTORi anti-cancer durant la phase d’expansion et de contraction de la réponse
immunitaire induites par le vaccin STxB-OVA augmente le taux de réponses T CD8
spécifiques d’OVA avec un phénotype de LT mémoires très fonctionnels. Ces LT CD8
générés après traitement par mTORi ont une expression augmentée de CD62L, du CD127 et
diminuée de KLRG1, qui caractérise les LT CD8 centraux mémoires (CD62L+CD127+) et
précurseurs mémoires (CD127+KLRG1lo, qui résistent à l’apoptose durant la phase de
contraction immunitaire pour se différencier en cellules mémoires). De plus, ces LT CD8 antiOVA ont une plus forte dégranulation et production d’IFN-g par rapport aux LT CD8 induis
en absence de mTORi. Ces résultats confirment les travaux d'Araki et al. qui montraient le
rôle de la voie mTOR dans la génération de LT CD8 mémoires efficaces par l'administration
de rapamycine chez des souris infectées par le virus de la chorioméningite lymphocytaire
(LCMV) (Araki et al., 2009). Suite à ces travaux pionniers d'Araki et al., la rapamycine est
apparue comme un outil très intéressant pour générer des réponses T CD8 mémoires antitumorales efficaces induites par divers vaccins thérapeutiques (Amiel et al., 2012; Diken et
al., 2013; Li et al., 2012). A partir de ces observations, des essais cliniques de phase I sont
actuellement en cours pour évaluer la combinaison de la rapamycine avec des vaccins antitumoraux ciblant l’antigène du groupe cancer-testis NY-ESO-1, dans le cancer de l’ovaire
(NCT01536054) et dans plusieurs tumeurs solides (NCT01522820). Néanmoins, le
développement clinique de la rapamycine en tant qu’agent anti-cancer a été arrêté en raison de
ses propriétés pharmacocinétiques défavorables et donc les seuls mTORi administrés chez les
patients atteints de cancer sont le temsirolimus et l’évérolimus (Faivre et al., 2006), renforçant
l’idée d’utiliser ces mTORi plutôt que la rapamycine en combinaison avec des vaccinations
anti-tumorales.
Nos travaux ont ainsi montré que les mTORi anti-cancer peuvent agir pour
potentialiser l’efficacité d’une vaccination anti-tumorale. En addition, leur effet synergique
cytotoxique direct sur la tumeur et leur utilisation déjà autorisée chez les patients atteints de
cancer contrairement à la rapamycine en font des candidats très intéressants pour la clinique.
Cependant, il est à noter que le potentiel clinique des mTORi doit être soigneusement étudié,
dues à leurs propriétés immunosuppressives via la promotion des Treg, pour maximiser leur
efficacité thérapeutique. A la vue de nos précédents résultats, bloquer les Treg semble ainsi
être une stratégie intéressante pour améliorer la combinaison entre une vaccination
thérapeutique et une inhibition de mTOR. Pour cela, nous avons travaillé avec l'antagoniste
151
du CCR4, dont nous avions précédemment montré l'efficacité en combinaison avec le
temsirolimus, et qui a de plus montré un impact sur le bénéfice anti-tumoral du vaccin STxBOVA (Pere et al., 2011). Nous avons ainsi montré que le ciblage des Treg par l'antagoniste du
CCR4 contribue à l'amélioration de l’efficacité anti-tumorale de la combinaison entre STxBOVA et le temsirolimus. En effet, la croissance tumorale est ralentie, et le taux de LT CD8
spécifiques de la tumeur est plus élevé suite à la triple combinaison comparé aux autres
groupes thérapeutiques. Cette stratégie de déplétion semble potentiellement plus prometteuse
que celle proposée par Wang et al. qui suggèrent une association du temsirolimus à un antiCD4 (Wang et al., 2014). En addition d'un risque plus élevé d'infections opportunistes chez
les patients, une déplétion des LT CD4 bloquerait les réponses Th1 anti-tumorales dont nous
avions précédemment montré l'importance chez les patients atteints de mRCC traité par
évérolimus. Enfin, les LT CD4 jouent un rôle crucial dans la génération des LT CD8
mémoires (Shedlock and Shen, 2003; Tanchot and Rocha, 2003). Ainsi, nous avons mis en
évidence l’intérêt potentiel de combiner le temsirolimus ou l’évérolimus avec une vaccination
thérapeutique anti-tumorale. De plus, combiner cette stratégie avec une thérapie ciblant les
Treg permettrait de bloquer le profil immunosuppressif induits par les mTORi et de conserver
le potentiel adjuvant des mTORi sur les réponses anti-tumorales induites par vaccination.
152
CONCLUSION ET PERSPECTIVES
153
154
En conclusion, ce travail a montré pour la première fois une implication de la réponse
immunitaire T anti-tumorale sur le bénéfice clinique des inhibiteurs de mTOR. Nous
suggérons que les traitements par mTORi moduleraient la réponse T anti-tumorale du patient
atteint de cancer en trois phases, miroir des trois phases de l’immunoedition (Dunn et al.,
2004). Notre hypothèse est que : Premièrement, le traitement par mTORi induit une
diminution du taux de Treg parallèlement à une stimulation de la réponse T anti-tumorale, qui
est favorable à l’efficacité du traitement, marquée par une meilleure survie sans progression
du patient. Une phase d’équilibre immunitaire suit, dans laquelle les variations entre les
réponses immunitaires anti- et pro-tumorales se compensent. L’efficacité du traitement à ce
stade reposerait principalement sur son effet cytotoxique direct sur la tumeur. Finalement, un
traitement prolongé conduit à un avantage des Treg très immunosuppresseurs, qui inhibent les
réponses immunitaires anti-tumorales induites par mTORi, contribuant ainsi négativement à
l’efficacité du traitement (Figure 20).
mTORi-mediated antitumor immune modulation
Treg
Th1 Th1
Th1
Treg
Treg
Treg
Th1Th 1
Th1Th 1
Treg
Treg
Treg
Treg
Th1Th 1
Anti TERT Th1 cells
Treg
Anti-TERT
Th1 cells
Tregs
Elimination
Equilibrium
Anti-TERT Th1 cells
Tregs
Escape
Treg
Treg
Treg
Treg Treg
Treg
Th 1
ThTh1
1
Th1
Th1
Th1 Th1
Th1
Th1
Th1 Th 1
ThTh1
1
?
Th 1
ThTh1
1
Treg
Treg
Treg
Treg
Tumor cells
Normal cells
Time of everolimus exposure
Figure 20: Modèle suggéré de la modulation des réponses immunitaire anti-tumorale médiée par les
mTORi
L’administration de mTORi chez des patients atteints de cancer induit des taux de
réponses variables, et favorise généralement une stabilisation de la maladie plutôt qu’une
régression tumorale, et qui finalement abouti à une progression (Coppin et al., 2008; Hudes et
al., 2007; Motzer et al., 2008). L’hypothèse communément suggérée est l’apparition d’un
rétrocontrôle, et d’une résistance aux mTORi suite à une sur-activation de la voie mTOR
155
(O’Reilly et al., 2006). Nos travaux suggèrent ainsi que l’expansion de Treg plus
immunosuppressifs sous traitement par mTORi contribuerait fortement à cet échappement.
En perspectives cliniques de ce travail, nous suggérons que les Treg et les LT antitumoraux pourraient s’avérer intéressants en tant que biomarqueurs immunologiques
prédictifs de l’efficacité thérapeutique des mTORi. Le suivi régulier de ces populations
lymphocytaires chez les patients permettrait d’évaluer l’efficacité du traitement et de l’adapter
chez ces patients en fonction. L’induction d’un profil anti-tumoral avec une balance penchant
vers l’activation des LT anti-tumoraux conduirait à une poursuite du traitement. Au contraire,
lorsque la balance penche vers un profil immunosuppressif dû à l’expansion des Treg nous
suggérons d’arrêter l’administration des mTORi, avant la survenue de la progression clinique.
Les patients atteints de cancer et traités par mTORi pourraient potentiellement
bénéficier d'un traitement combiné avec des thérapies bloquant les Treg afin d'empêcher la
balance immunitaire de pencher vers l'immunosuppression. Les patients premièrement sous
traitement par mTORi pourraient basculer vers un traitement ciblant les Treg lorsque le taux de
Treg augmenterait (à partir du deuxième mois). Cela permettrait ainsi de conserver le bénéfice
de la réponse immunitaire T anti-tumorale induite par les mTORi.
De plus, réactiver les réponses immunitaires après un échappement tumoral à
l’immunosurveillance par le ciblage des mécanismes immunosuppressifs est une nouvelle
dynamique dans la thérapie anti-cancer. La disparition des réponses Th1 anti-tumorales
observée chez les patients traités par évérolimus dans notre étude pourrait potentiellement être
liée à une plus forte expression de molécules telles que PD-1 ou PD-L1, et suggère une
combinaison avec des anti-PD-1 ou anti-PD-L1. Une combinaison des mTORi anti-cancer
avec des vaccins thérapeutiques s'averrerait également interessante de par leur effet
cytotoxique direct sur les cancers et leur effet immuno-modulateur.
156
BIBLIOGRAPHIE
157
158
Aagaard-Tillery, K.M., and Jelinek, D.F. (1994). Inhibition of human B lymphocyte
cell cycle progression and differentiation by rapamycin. Cell. Immunol. 156, 493–507.
Adeegbe, D.O., and Nishikawa, H.M. (2013). Natural and induced T regulatory cells
in cancer. Immunol. Toler. 4, 190.
Adotevi, O., Vingert, B., Freyburger, L., Shrikant, P., Lone, Y.-C., Quintin-Colonna,
F., Haicheur, N., Amessou, M., Herbelin, A., Langlade-Demoyen, P., et al. (2007). B subunit
of Shiga toxin-based vaccines synergize with alpha-galactosylceramide to break tolerance
against self antigen and elicit antiviral immunity. J. Immunol. Baltim. Md 1950 179, 3371–
3379.
Adotevi, O., Pere, H., Ravel, P., Haicheur, N., Badoual, C., Merillon, N., Medioni, J.,
Peyrard, S., Roncelin, S., Verkarre, V., et al. (2010). A decrease of regulatory T cells
correlates with overall survival after sunitinib-based antiangiogenic therapy in metastatic
renal cancer patients. J. Immunother. Hagerstown Md 1997 33, 991–998.
Adotévi, O., Dosset, M., Galaine, J., Beziaud, L., Godet, Y., and Borg, C. (2013).
Targeting antitumor CD4 helper T cells with universal tumor-reactive helper peptides derived
from telomerase for cancer vaccine. Hum. Vaccines Immunother. 9, 1073–1077.
Aggarwal, S., Ghilardi, N., Xie, M.-H., de Sauvage, F.J., and Gurney, A.L. (2003).
Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production
of interleukin-17. J. Biol. Chem. 278, 1910–1914.
Akimova, T., Beier, U.H., Wang, L., Levine, M.H., and Hancock, W.W. (2011).
Helios Expression Is a Marker of T Cell Activation and Proliferation. PLoS ONE 6, e24226.
Alberú, J., Pascoe, M.D., Campistol, J.M., Schena, F.P., Rial, M.D.C., Polinsky, M.,
Neylan, J.F., Korth-Bradley, J., Goldberg-Alberts, R., Maller, E.S., et al. (2011). Lower
malignancy rates in renal allograft recipients converted to sirolimus-based, calcineurin
inhibitor-free immunotherapy: 24-month results from the CONVERT trial. Transplantation
92, 303–310.
Ali, A.K., Nandagopal, N., and Lee, S.-H. (2015). IL-15-PI3K-AKT-mTOR: A
Critical Pathway in the Life Journey of Natural Killer Cells. Front. Immunol. 6, 355.
Alvarez Arias, D.A., Kim, H.-J., Zhou, P., Holderried, T.A.W., Wang, X., Dranoff, G.,
and Cantor, H. (2014). Disruption of CD8+ Treg activity results in expansion of T follicular
helper cells and enhanced antitumor immunity. Cancer Immunol. Res. 2, 207–216.
Amiel, E., Everts, B., Freitas, T.C., King, I.L., Curtis, J.D., Pearce, E.L., and Pearce,
E.J. (2012). Inhibition of Mechanistic Target of Rapamycin Promotes Dendritic Cell
Activation and Enhances Therapeutic Autologous Vaccination in Mice. J. Immunol. 189,
2151–2158.
Araki, K., Turner, A.P., Shaffer, V.O., Gangappa, S., Keller, S.A., Bachmann, M.F.,
Larsen, C.P., and Ahmed, R. (2009). mTOR regulates memory CD8 T-cell differentiation.
Nature 460, 108–112.
Asma, G., Amal, G., Raja, M., Amine, D., Mohammed, C., and Amel, B.A.E. (2015).
Comparison of circulating and intratumoral regulatory T cells in patients with renal cell
carcinoma. Tumour Biol. J. Int. Soc. Oncodevelopmental Biol. Med. 36, 3727–3734.
Bastid, J., Regairaz, A., Bonnefoy, N., Déjou, C., Giustiniani, J., Laheurte, C.,
Cochaud, S., Laprevotte, E., Funck-Brentano, E., Hemon, P., et al. (2015). Inhibition of CD39
Enzymatic Function at the Surface of Tumor Cells Alleviates Their Immunosuppressive
Activity. Cancer Immunol. Res. 3, 254–265.
Basu, S., Golovina, T., Mikheeva, T., June, C.H., and Riley, J.L. (2008). Cutting edge:
Foxp3-mediated induction of pim 2 allows human T regulatory cells to preferentially expand
in rapamycin. J. Immunol. Baltim. Md 1950 180, 5794–5798.
Battaglia, M., Stabilini, A., and Roncarolo, M.-G. (2005). Rapamycin selectively
expands CD4+CD25+FoxP3+ regulatory T cells. Blood 105, 4743–4748.
159
Battaglia, M., Stabilini, A., Migliavacca, B., Horejs-Hoeck, J., Kaupper, T., and
Roncarolo,
M.-G.
(2006).
Rapamycin
promotes
expansion
of
functional
CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients.
J. Immunol. Baltim. Md 1950 177, 8338–8347.
Bayry, J., Tchilian, E.Z., Davies, M.N., Forbes, E.K., Draper, S.J., Kaveri, S.V., Hill,
A.V.S., Kazatchkine, M.D., Beverley, P.C.L., Flower, D.R., et al. (2008). In silico identified
CCR4 antagonists target regulatory T cells and exert adjuvant activity in vaccination. Proc.
Natl. Acad. Sci. 105, 10221–10226.
Benhamron, S., and Tirosh, B. (2011). Direct activation of mTOR in B lymphocytes
confers impairment in B-cell maturation andloss of marginal zone B cells. Eur. J. Immunol.
41, 2390–2396.
Benjamin, D., Colombi, M., Moroni, C., and Hall, M.N. (2011). Rapamycin passes the
torch: a new generation of mTOR inhibitors. Nat. Rev. Drug Discov. 10, 868–880.
Bergsland, E.K. (2015). Combined mammalian target of rapamycin and vascular
endothelial growth factor pathway inhibition in pancreatic neuroendocrine tumors: more than
the sum of its parts? J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 33, 1523–1526.
Bilate, A.M., and Lafaille, J.J. (2012). Induced CD4+Foxp3+ regulatory T cells in
immune tolerance. Annu. Rev. Immunol. 30, 733–758.
Blanchet, F.P., Moris, A., Nikolic, D.S., Lehmann, M., Cardinaud, S., Stalder, R.,
Garcia, E., Dinkins, C., Leuba, F., Wu, L., et al. (2010). Human Immunodeficiency Virus-1
Inhibition of Immunoamphisomes in Dendritic Cells Impairs Early Innate and Adaptive
Immune Responses. Immunity 32, 654–669.
Bocian, K., Borysowski, J., Wierzbicki, P., Wyzgal, J., Klosowska, D., Bialoszewska,
A., Paczek, L., Górski, A., and Korczak-Kowalska, G. (2010). Rapamycin, unlike
cyclosporine A, enhances suppressive functions of in vitro-induced CD4+CD25+ Tregs.
Nephrol. Dial. Transplant. Off. Publ. Eur. Dial. Transpl. Assoc. - Eur. Ren. Assoc. 25, 710–
717.
Bos, R., and Sherman, L.A. (2010). CD4+ T-cell help in the tumor milieu is required
for recruitment and cytolytic function of CD8+ T lymphocytes. Cancer Res. 70, 8368–8377.
Bourgeois, C., Veiga-Fernandes, H., Joret, A.-M., Rocha, B., and Tanchot, C. (2002a).
CD8 lethargy in the absence of CD4 help. Eur. J. Immunol. 32, 2199–2207.
Bourgeois, C., Rocha, B., and Tanchot, C. (2002b). A role for CD40 expression on
CD8+ T cells in the generation of CD8+ T cell memory. Science 297, 2060–2063.
Brahmer, J.R., Tykodi, S.S., Chow, L.Q.M., Hwu, W.-J., Topalian, S.L., Hwu, P.,
Drake, C.G., Camacho, L.H., Kauh, J., Odunsi, K., et al. (2012). Safety and activity of antiPD-L1 antibody in patients with advanced cancer. N. Engl. J. Med. 366, 2455–2465.
van den Broeke, L.T., Daschbach, E., Thomas, E.K., Andringa, G., and Berzofsky,
J.A. (2003). Dendritic cell-induced activation of adaptive and innate antitumor immunity. J.
Immunol. Baltim. Md 1950 171, 5842–5852.
Brouard, S., Puig-Pey, I., Lozano, J.-J., Pallier, A., Braud, C., Giral, M., Guillet, M.,
Londoño, M.C., Oppenheimer, F., Campistol, J.M., et al. (2010). Comparative Transcriptional
and Phenotypic Peripheral Blood Analysis of Kidney Recipients Under Cyclosporin A or
Sirolimus Monotherapy. Am. J. Transplant. 10, 2604–2614.
Brown, E.J., Albers, M.W., Shin, T.B., Ichikawa, K., Keith, C.T., Lane, W.S., and
Schreiber, S.L. (1994). A mammalian protein targeted by G1-arresting rapamycin-receptor
complex. Nature 369, 756–758.
Brugarolas, J., Lei, K., Hurley, R.L., Manning, B.D., Reiling, J.H., Hafen, E., Witters,
L.A., Ellisen, L.W., and Kaelin, W.G. (2004). Regulation of mTOR function in response to
hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 18, 2893–
2904.
160
Bruns, C.J., Koehl, G.E., Guba, M., Yezhelyev, M., Steinbauer, M., Seeliger, H.,
Schwend, A., Hoehn, A., Jauch, K.-W., and Geissler, E.K. (2004). Rapamycin-induced
endothelial cell death and tumor vessel thrombosis potentiate cytotoxic therapy against
pancreatic cancer. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 10, 2109–2119.
Budanov, A.V., and Karin, M. (2008). p53 target genes sestrin1 and sestrin2 connect
genotoxic stress and mTOR signaling. Cell 134, 451–460.
Burnet, M. (1957). Cancer; a biological approach. I. The processes of control. Br.
Med. J. 1, 779–786.
Campistol, J.M., Eris, J., Oberbauer, R., Friend, P., Hutchison, B., Morales, J.M.,
Claesson, K., Stallone, G., Russ, G., Rostaing, L., et al. (2006). Sirolimus Therapy after Early
Cyclosporine Withdrawal Reduces the Risk for Cancer in Adult Renal Transplantation. J. Am.
Soc. Nephrol. 17, 581–589.
Cantley, L.C., and Neel, B.G. (1999). New insights into tumor suppression: PTEN
suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc.
Natl. Acad. Sci. U. S. A. 96, 4240–4245.
Cao, W., Manicassamy, S., Tang, H., Kasturi, S.P., Pirani, A., Murthy, N., and
Pulendran, B. (2008). Toll-like receptor–mediated induction of type I interferon in
plasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6K
pathway. Nat. Immunol. 9, 1157–1164.
Carlson, C.M., Endrizzi, B.T., Wu, J., Ding, X., Weinreich, M.A., Walsh, E.R., Wani,
M.A., Lingrel, J.B., Hogquist, K.A., and Jameson, S.C. (2006). Kruppel-like factor 2 regulates
thymocyte and T-cell migration. Nature 442, 299–302.
Carmeliet, P., and Jain, R.K. (2000). Angiogenesis in cancer and other diseases.
Nature 407, 249–257.
Cederbom, L., Hall, H., and Ivars, F. (2000). CD4+CD25+ regulatory T cells downregulate co-stimulatory molecules on antigen-presenting cells. Eur. J. Immunol. 30, 1538–
1543.
Chalmin, F., Mignot, G., Bruchard, M., Chevriaux, A., Végran, F., Hichami, A.,
Ladoire, S., Derangère, V., Vincent, J., Masson, D., et al. (2012). Stat3 and Gfi-1 transcription
factors control Th17 cell immunosuppressive activity via the regulation of ectonucleotidase
expression. Immunity 36, 362–373.
Cheever, M.A., Allison, J.P., Ferris, A.S., Finn, O.J., Hastings, B.M., Hecht, T.T.,
Mellman, I., Prindiville, S.A., Viner, J.L., Weiner, L.M., et al. (2009). The Prioritization of
Cancer Antigens: A National Cancer Institute Pilot Project for the Acceleration of
Translational Research. Clin. Cancer Res. 15, 5323–5337.
Choo, A.Y., Yoon, S.-O., Kim, S.G., Roux, P.P., and Blenis, J. (2008). Rapamycin
differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA
translation. Proc. Natl. Acad. Sci. 105, 17414–17419.
Cichowski, K., and Jacks, T. (2001). NF1 tumor suppressor gene function: narrowing
the GAP. Cell 104, 593–604.
Cloughesy, T.F., Yoshimoto, K., Nghiemphu, P., Brown, K., Dang, J., Zhu, S., Hsueh,
T., Chen, Y., Wang, W., Youngkin, D., et al. (2008). Antitumor activity of rapamycin in a
Phase I trial for patients with recurrent PTEN-deficient glioblastoma. PLoS Med. 5, e8.
Codogno, P., and Meijer, A.J. (2005). Autophagy and signaling: their role in cell
survival and cell death. Cell Death Differ. 12 Suppl 2, 1509–1518.
Coenen, J.J.A., Koenen, H.J.P.M., van Rijssen, E., Hilbrands, L.B., and Joosten, I.
(2006). Rapamycin, and not cyclosporin A, preserves the highly suppressive CD27+ subset of
human CD4+CD25+ regulatory T cells. Blood 107, 1018–1023.
161
Collison, L.W., Workman, C.J., Kuo, T.T., Boyd, K., Wang, Y., Vignali, K.M., Cross,
R., Sehy, D., Blumberg, R.S., and Vignali, D.A.A. (2007). The inhibitory cytokine IL-35
contributes to regulatory T-cell function. Nature 450, 566–569.
Colombetti, S., Basso, V., Mueller, D.L., and Mondino, A. (2006). Prolonged
TCR/CD28 engagement drives IL-2-independent T cell clonal expansion through signaling
mediated by the mammalian target of rapamycin. J. Immunol. Baltim. Md 1950 176, 2730–
2738.
Colombo, M.P., and Piconese, S. (2007). Regulatory T-cell inhibition versus
depletion: the right choice in cancer immunotherapy. Nat. Rev. Cancer 7, 880–887.
Combe, P., de Guillebon, E., Thibault, C., Granier, C., Tartour, E., and Oudard, S.
(2015). Trial Watch: Therapeutic vaccines in metastatic renal cell carcinoma.
Oncoimmunology 4, e1001236.
Copp, J., Manning, G., and Hunter, T. (2009). TORC-specific phosphorylation of
mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR
signaling complex 2. Cancer Res. 69, 1821–1827.
Coppin, C., Le, L., Porzsolt, F., and Wilt, T. (2008). Targeted therapy for advanced
renal cell carcinoma. Cochrane Database Syst. Rev. CD006017.
Coppola, D., Nebozhyn, M., Khalil, F., Dai, H., Yeatman, T., Loboda, A., and Mulé,
J.J. (2011). Unique ectopic lymph node-like structures present in human primary colorectal
carcinoma are identified by immune gene array profiling. Am. J. Pathol. 179, 37–45.
Cunningham, J.T., Rodgers, J.T., Arlow, D.H., Vazquez, F., Mootha, V.K., and
Puigserver, P. (2007). mTOR controls mitochondrial oxidative function through a YY1-PGC1alpha transcriptional complex. Nature 450, 736–740.
Curiel, T.J., Coukos, G., Zou, L., Alvarez, X., Cheng, P., Mottram, P., EvdemonHogan, M., Conejo-Garcia, J.R., Zhang, L., Burow, M., et al. (2004). Specific recruitment of
regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced
survival. Nat. Med. 10, 942–949.
Daniel, C., Wennhold, K., Kim, H.-J., and Boehmer, H. von (2010). Enhancement of
antigen-specific Treg vaccination in vivo. Proc. Natl. Acad. Sci. 107, 16246–16251.
Dantal, J., Hourmant, M., Cantarovich, D., Giral, M., Blancho, G., Dreno, B., and
Soulillou, J.-P. (1998). Effect of long-term immunosuppression in kidney-graft recipients on
cancer incidence: randomised comparison of two cyclosporin regimens. The Lancet 351, 623–
628.
Daurkin, I., Eruslanov, E., Stoffs, T., Perrin, G.Q., Algood, C., Gilbert, S.M., Rosser,
C.J., Su, L.-M., Vieweg, J., and Kusmartsev, S. (2011). Tumor-associated macrophages
mediate immunosuppression in the renal cancer microenvironment by activating the 15lipoxygenase-2 pathway. Cancer Res. 71, 6400–6409.
Deaglio, S., Dwyer, K.M., Gao, W., Friedman, D., Usheva, A., Erat, A., Chen, J.-F.,
Enjyoji, K., Linden, J., Oukka, M., et al. (2007). Adenosine generation catalyzed by CD39
and CD73 expressed on regulatory T cells mediates immune suppression. J. Exp. Med. 204,
1257–1265.
Delgoffe, G.M., Kole, T.P., Zheng, Y., Zarek, P.E., Matthews, K.L., Xiao, B., Worley,
P.F., Kozma, S.C., and Powell, J.D. (2009). The mTOR kinase differentially regulates effector
and regulatory T cell lineage commitment. Immunity 30, 832–844.
Delgoffe, G.M., Pollizzi, K.N., Waickman, A.T., Heikamp, E., Meyers, D.J., Horton,
M.R., Xiao, B., Worley, P.F., and Powell, J.D. (2011). The kinase mTOR regulates the
differentiation of helper T cells through the selective activation of signaling by mTORC1 and
mTORC2. Nat. Immunol. 12, 295–303.
De Monte, L., Reni, M., Tassi, E., Clavenna, D., Papa, I., Recalde, H., Braga, M., Di
Carlo, V., Doglioni, C., and Protti, M.P. (2011). Intratumor T helper type 2 cell infiltrate
162
correlates with cancer-associated fibroblast thymic stromal lymphopoietin production and
reduced survival in pancreatic cancer. J. Exp. Med. 208, 469–478.
Dighe, A.S., Richards, E., Old, L.J., and Schreiber, R.D. (1994). Enhanced in vivo
growth and resistance to rejection of tumor cells expressing dominant negative IFN gamma
receptors. Immunity 1, 447–456.
Diken, M., Kreiter, S., Vascotto, F., Selmi, A., Attig, S., Diekmann, J., Huber, C.,
Türeci, Ö., and Sahin, U. (2013). mTOR inhibition improves antitumor effects of vaccination
with antigen-encoding RNA. Cancer Immunol. Res. 1, 386–392.
Ding, X., Du, H., Yoder, M.C., and Yan, C. (2014). Critical role of the mTOR
pathway in development and function of myeloid-derived suppressor cells in lal-/- mice. Am.
J. Pathol. 184, 397–408.
Ding, X., Wu, L., Yan, C., and Du, H. (2015). Establishment of lal-/- myeloid lineage
cell line that resembles myeloid-derived suppressive cells. PloS One 10, e0121001.
Donahue, A.C., and Fruman, D.A. (2007). Distinct signaling mechanisms activate the
target of rapamycin in response to different B-cell stimuli. Eur. J. Immunol. 37, 2923–2936.
Donnelly, R.P., Loftus, R.M., Keating, S.E., Liou, K.T., Biron, C.A., Gardiner, C.M.,
and Finlay, D.K. (2014). mTORC1-dependent metabolic reprogramming is a prerequisite for
NK cell effector function. J. Immunol. Baltim. Md 1950 193, 4477–4484.
Dorrello, N.V., Peschiaroli, A., Guardavaccaro, D., Colburn, N.H., Sherman, N.E., and
Pagano, M. (2006). S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein
translation and cell growth. Science 314, 467–471.
Dosset, M., Godet, Y., Vauchy, C., Beziaud, L., Lone, Y.C., Sedlik, C., Liard, C.,
Levionnois, E., Clerc, B., Sandoval, F., et al. (2012). Universal cancer peptide-based
therapeutic vaccine breaks tolerance against telomerase and eradicates established tumor.
Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 18, 6284–6295.
Dosset, M., Vauchy, C., Beziaud, L., Adotevi, O., and Godet, Y. (2013). Universal
tumor-reactive helper peptides from telomerase as new tools for anticancer vaccination.
Oncoimmunology 2, e23430.
Douros, J., and Suffness, M. (1981). New antitumor substances of natural origin.
Cancer Treat. Rev. 8, 63–87.
Dowling, R.J.O., Topisirovic, I., Alain, T., Bidinosti, M., Fonseca, B.D., Petroulakis,
E., Wang, X., Larsson, O., Selvaraj, A., Liu, Y., et al. (2010). mTORC1-mediated cell
proliferation, but not cell growth, controlled by the 4E-BPs. Science 328, 1172–1176.
Draghiciu, O., Nijman, H.W., Hoogeboom, B.N., Meijerhof, T., and Daemen, T.
(2015). Sunitinib depletes myeloid-derived suppressor cells and synergizes with a cancer
vaccine to enhance antigen-specific immune responses and tumor eradication.
Oncoimmunology 4, e989764.
Dunker, K., Schlaf, G., Bukur, J., Altermann, W.W., Handke, D., and Seliger, B.
(2008). Expression and regulation of non-classical HLA-G in renal cell carcinoma. Tissue
Antigens 72, 137–148.
Dunn, G.P., Old, L.J., and Schreiber, R.D. (2004). The three Es of cancer
immunoediting. Annu. Rev. Immunol. 22, 329–360.
Dutcher, J.P., and Nanus, D. (2011). Long-term survival of patients with sarcomatoid
renal cell cancer treated with chemotherapy. Med. Oncol. Northwood Lond. Engl. 28, 1530–
1533.
Düvel, K., Yecies, J.L., Menon, S., Raman, P., Lipovsky, A.I., Souza, A.L.,
Triantafellow, E., Ma, Q., Gorski, R., Cleaver, S., et al. (2010). Activation of a metabolic
gene regulatory network downstream of mTOR complex 1. Mol. Cell 39, 171–183.
Eckl, J., Buchner, A., Prinz, P.U., Riesenberg, R., Siegert, S.I., Kammerer, R., Nelson,
P.J., and Noessner, E. (2012). Transcript signature predicts tissue NK cell content and defines
163
renal cell carcinoma subgroups independent of TNM staging. J. Mol. Med. Berl. Ger. 90, 55–
66.
Elhilali, M.M., Gleave, M., Fradet, Y., Davis, I., Venner, P., Saad, F., Klotz, L.,
Moore, R., Ernst, S., and Paton, V. (2000). Placebo-associated remissions in a multicentre,
randomized, double-blind trial of interferon gamma-1b for the treatment of metastatic renal
cell carcinoma. The Canadian Urologic Oncology Group. BJU Int. 86, 613–618.
Elkord, E., Sharma, S., Burt, D.J., and Hawkins, R.E. (2011). Expanded subpopulation
of FoxP3+ T regulatory cells in renal cell carcinoma co-express Helios, indicating they could
be derived from natural but not induced Tregs. Clin. Immunol. Orlando Fla 140, 218–222.
Euvrard, S., Morelon, E., Rostaing, L., Goffin, E., Brocard, A., Tromme, I., Broeders,
N., del Marmol, V., Chatelet, V., Dompmartin, A., et al. (2012). Sirolimus and Secondary
Skin-Cancer Prevention in Kidney Transplantation. N. Engl. J. Med. 367, 329–339.
Facciabene, A., Peng, X., Hagemann, I.S., Balint, K., Barchetti, A., Wang, L.-P.,
Gimotty, P.A., Gilks, C.B., Lal, P., Zhang, L., et al. (2011). Tumour hypoxia promotes
tolerance and angiogenesis via CCL28 and Treg cells. Nature 475, 226–230.
Faget, J., Biota, C., Bachelot, T., Gobert, M., Treilleux, I., Goutagny, N., Durand, I.,
Léon-Goddard, S., Blay, J.Y., Caux, C., et al. (2011). Early Detection of Tumor Cells by
Innate Immune Cells Leads to Treg Recruitment through CCL22 Production by Tumor Cells.
Cancer Res. 71, 6143–6152.
Faget, J., Bendriss-Vermare, N., Gobert, M., Durand, I., Olive, D., Biota, C., Bachelot,
T., Treilleux, I., Goddard-Leon, S., Lavergne, E., et al. (2012). ICOS-Ligand Expression on
Plasmacytoid Dendritic Cells Supports Breast Cancer Progression by Promoting the
Accumulation of Immunosuppressive CD4+ T Cells. Cancer Res. 72, 6130–6141.
Faivre, S., Kroemer, G., and Raymond, E. (2006). Current development of mTOR
inhibitors as anticancer agents. Nat. Rev. Drug Discov. 5, 671–688.
Fan, Y., Liu, Z., Fang, X., Ge, Z., Ge, N., Jia, Y., Sun, P., Lou, F., Björkholm, M.,
Gruber, A., et al. (2005). Differential expression of full-length telomerase reverse
transcriptase mRNA and telomerase activity between normal and malignant renal tissues.
Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 11, 4331–4337.
Fang, Y., Park, I.-H., Wu, A.-L., Du, G., Huang, P., Frohman, M.A., Walker, S.J.,
Brown, H.A., and Chen, J. (2003). PLD1 regulates mTOR signaling and mediates Cdc42
activation of S6K1. Curr. Biol. CB 13, 2037–2044.
Feng, Z., Zhang, H., Levine, A.J., and Jin, S. (2005). The coordinate regulation of the
p53 and mTOR pathways in cells. Proc. Natl. Acad. Sci. U. S. A. 102, 8204–8209.
Fife, B.T., and Bluestone, J.A. (2008). Control of peripheral T-cell tolerance and
autoimmunity via the CTLA-4 and PD-1 pathways. Immunol. Rev. 224, 166–182.
Figlin, R.A. (2013). A novel personalized vaccine approach in combination with
targeted therapy in advanced renal cell carcinoma. Immunotherapy 6, 261–268.
Finke, J., Ko, J., Rini, B., Rayman, P., Ireland, J., and Cohen, P. (2011). MDSC as a
mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy. Int.
Immunopharmacol. 11, 856–861.
Finke, J.H., Rini, B., Ireland, J., Rayman, P., Richmond, A., Golshayan, A., Wood, L.,
Elson, P., Garcia, J., Dreicer, R., et al. (2008). Sunitinib reverses type-1 immune suppression
and decreases T-regulatory cells in renal cell carcinoma patients. Clin. Cancer Res. Off. J.
Am. Assoc. Cancer Res. 14, 6674–6682.
Finlay, D., and Cantrell, D. (2010). Phosphoinositide 3-kinase and the mammalian
target of rapamycin pathways control T cell migration. Ann. N. Y. Acad. Sci. 1183, 149–157.
Foley, J.E., Jung, U., Miera, A., Borenstein, T., Mariotti, J., Eckhaus, M., Bierer, B.E.,
and Fowler, D.H. (2005). Ex vivo rapamycin generates donor Th2 cells that potently inhibit
164
graft-versus-host disease and graft-versus-tumor effects via an IL-4-dependent mechanism. J.
Immunol. Baltim. Md 1950 175, 5732–5743.
Francisco, L.M., Salinas, V.H., Brown, K.E., Vanguri, V.K., Freeman, G.J., Kuchroo,
V.K., and Sharpe, A.H. (2009). PD-L1 regulates the development, maintenance, and function
of induced regulatory T cells. J. Exp. Med. 206, 3015–3029.
Frauwirth, K.A., Riley, J.L., Harris, M.H., Parry, R.V., Rathmell, J.C., Plas, D.R.,
Elstrom, R.L., June, C.H., and Thompson, C.B. (2002). The CD28 signaling pathway
regulates glucose metabolism. Immunity 16, 769–777.
Fridman, W.H., Pagès, F., Sautès-Fridman, C., and Galon, J. (2012). The immune
contexture in human tumours: impact on clinical outcome. Nat. Rev. Cancer 12, 298–306.
Fyfe, G., Fisher, R.I., Rosenberg, S.A., Sznol, M., Parkinson, D.R., and Louie, A.C.
(1995). Results of treatment of 255 patients with metastatic renal cell carcinoma who received
high-dose recombinant interleukin-2 therapy. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 13,
688–696.
Gabrilovich, D.I., and Nagaraj, S. (2009). Myeloid-derived suppressor cells as
regulators of the immune system. Nat. Rev. Immunol. 9, 162–174.
Ganley, I.G., Lam, D.H., Wang, J., Ding, X., Chen, S., and Jiang, X. (2009).
ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy. J.
Biol. Chem. 284, 12297–12305.
Garín, M.I., Chu, C.-C., Golshayan, D., Cernuda-Morollón, E., Wait, R., and Lechler,
R.I. (2007). Galectin-1: a key effector of regulation mediated by CD4+CD25+ T cells. Blood
109, 2058–2065.
Gati, A., Guerra, N., Giron-Michel, J., Azzarone, B., Angevin, E., Moretta, A.,
Chouaib, S., and Caignard, A. (2001). Tumor cells regulate the lytic activity of tumor-specific
cytotoxic t lymphocytes by modulating the inhibitory natural killer receptor function. Cancer
Res. 61, 3240–3244.
Gattinoni, L., Lugli, E., Ji, Y., Pos, Z., Paulos, C.M., Quigley, M.F., Almeida, J.R.,
Gostick, E., Yu, Z., Carpenito, C., et al. (2011). A human memory T cell subset with stem
cell-like properties. Nat. Med. 17, 1290–1297.
Gaumann, A., Schlitt, H.J., and Geissler, E.K. (2008). Immunosuppression and tumor
development in organ transplant recipients: the emerging dualistic role of rapamycin. Transpl.
Int. 21, 207–217.
Gerriets, V.A., and Rathmell, J.C. (2012). Metabolic pathways in T cell fate and
function. Trends Immunol. 33, 168–173.
Ghiringhelli, F., Puig, P.E., Roux, S., Parcellier, A., Schmitt, E., Solary, E., Kroemer,
G., Martin, F., Chauffert, B., and Zitvogel, L. (2005). Tumor cells convert immature myeloid
dendritic cells into TGF-"–secreting cells inducing CD4+CD25+ regulatory T cell
proliferation. J. Exp. Med. 202, 919–929.
Ghiringhelli, F., Menard, C., Puig, P.E., Ladoire, S., Roux, S., Martin, F., Solary, E.,
Cesne, A.L., Zitvogel, L., and Chauffert, B. (2006). Metronomic cyclophosphamide regimen
selectively depletes CD4+CD25+ regulatory T cells and restores T and NK effector functions
in end stage cancer patients. Cancer Immunol. Immunother. 56, 641–648.
Giraldo, N.A., Becht, E., Pagès, F., Skliris, G., Verkarre, V., Vano, Y., Mejean, A.,
Saint-Aubert, N., Lacroix, L., Natario, I., et al. (2015). Orchestration and Prognostic
Significance of Immune Checkpoints in the Microenvironment of Primary and Metastatic
Renal Cell Cancer. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res.
Gleave, M.E., Elhilali, M., Fradet, Y., Davis, I., Venner, P., Saad, F., Klotz, L.H.,
Moore, M.J., Paton, V., and Bajamonde, A. (1998). Interferon gamma-1b compared with
placebo in metastatic renal-cell carcinoma. Canadian Urologic Oncology Group. N. Engl. J.
Med. 338, 1265–1271.
165
Gobert, M., Treilleux, I., Bendriss-Vermare, N., Bachelot, T., Goddard-Leon, S., Arfi,
V., Biota, C., Doffin, A.C., Durand, I., Olive, D., et al. (2009). Regulatory T Cells Recruited
through CCL22/CCR4 Are Selectively Activated in Lymphoid Infiltrates Surrounding
Primary Breast Tumors and Lead to an Adverse Clinical Outcome. Cancer Res. 69, 2000–
2009.
Godet, Y., Fabre, E., Dosset, M., Lamuraglia, M., Levionnois, E., Ravel, P.,
Benhamouda, N., Cazes, A., Le Pimpec-Barthes, F., Gaugler, B., et al. (2012). Analysis of
spontaneous tumor-specific CD4 T-cell immunity in lung cancer using promiscuous HLA-DR
telomerase-derived epitopes: potential synergistic effect with chemotherapy response. Clin.
Cancer Res. Off. J. Am. Assoc. Cancer Res. 18, 2943–2953.
Gondek, D.C., Lu, L.-F., Quezada, S.A., Sakaguchi, S., and Noelle, R.J. (2005).
Cutting Edge: Contact-Mediated Suppression by CD4+CD25+ Regulatory Cells Involves a
Granzyme B-Dependent, Perforin-Independent Mechanism. J. Immunol. 174, 1783–1786.
Gounaris, E., Blatner, N.R., Dennis, K., Magnusson, F., Gurish, M.F., Strom, T.B.,
Beckhove, P., Gounari, F., and Khazaie, K. (2009). T-Regulatory Cells Shift from a
Protective Anti-Inflammatory to a Cancer-Promoting Proinflammatory Phenotype in
Polyposis. Cancer Res. 69, 5490–5497.
Griffiths, R.W., Elkord, E., Gilham, D.E., Ramani, V., Clarke, N., Stern, P.L., and
Hawkins, R.E. (2007). Frequency of regulatory T cells in renal cell carcinoma patients and
investigation of correlation with survival. Cancer Immunol. Immunother. CII 56, 1743–1753.
Grossman, W.J., Verbsky, J.W., Tollefsen, B.L., Kemper, C., Atkinson, J.P., and Ley,
T.J. (2004). Differential expression of granzymes A and B in human cytotoxic lymphocyte
subsets and T regulatory cells. Blood 104, 2840–2848.
Groth, C.G., Bäckman, L., Morales, J.M., Calne, R., Kreis, H., Lang, P., Touraine,
J.L., Claesson, K., Campistol, J.M., Durand, D., et al. (1999). Sirolimus (rapamycin)-based
therapy in human renal transplantation: similar efficacy and different toxicity compared with
cyclosporine. Sirolimus European Renal Transplant Study Group. Transplantation 67, 1036–
1042.
Guba, M., von Breitenbuch, P., Steinbauer, M., Koehl, G., Flegel, S., Hornung, M.,
Bruns, C.J., Zuelke, C., Farkas, S., Anthuber, M., et al. (2002). Rapamycin inhibits primary
and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth
factor. Nat. Med. 8, 128–135.
Gupta, K., Miller, J.D., Li, J.Z., Russell, M.W., and Charbonneau, C. (2008).
Epidemiologic and socioeconomic burden of metastatic renal cell carcinoma (mRCC): A
literature review. Cancer Treat. Rev. 34, 193–205.
Gutierrez-Dalmau, A., and Campistol, J.M. (2007). Immunosuppressive therapy and
malignancy in organ transplant recipients: a systematic review. Drugs 67, 1167–1198.
Gu-Trantien, C., Loi, S., Garaud, S., Equeter, C., Libin, M., de Wind, A., Ravoet, M.,
Le Buanec, H., Sibille, C., Manfouo-Foutsop, G., et al. (2013). CD4+ follicular helper T cell
infiltration predicts breast cancer survival. J. Clin. Invest. 123, 2873–2892.
Gwinn, D.M., Shackelford, D.B., Egan, D.F., Mihaylova, M.M., Mery, A., Vasquez,
D.S., Turk, B.E., and Shaw, R.J. (2008). AMPK phosphorylation of raptor mediates a
metabolic checkpoint. Mol. Cell 30, 214–226.
Hahn, S., Gehri, R., and Erb, P. (1995). Mechanism and biological significance of
CD4-mediated cytotoxicity. Immunol. Rev. 146, 57–79.
Haidinger, M., Poglitsch, M., Geyeregger, R., Kasturi, S., Zeyda, M., Zlabinger, G.J.,
Pulendran, B., Hörl, W.H., Säemann, M.D., and Weichhart, T. (2010). A Versatile Role of
Mammalian Target of Rapamycin in Human Dendritic Cell Function and Differentiation. J.
Immunol. 185, 3919–3931.
166
Halloran, P.F. (2004). Immunosuppressive drugs for kidney transplantation. N. Engl.
J. Med. 351, 2715–2729.
Hanahan, D., and Weinberg, R.A. (2000). The hallmarks of cancer. Cell 100, 57–70.
Hanahan, D., and Weinberg, R.A. (2011). Hallmarks of cancer: the next generation.
Cell 144, 646–674.
Hara, K., Yonezawa, K., Weng, Q.P., Kozlowski, M.T., Belham, C., and Avruch, J.
(1998). Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a
common effector mechanism. J. Biol. Chem. 273, 14484–14494.
Hara, K., Maruki, Y., Long, X., Yoshino, K., Oshiro, N., Hidayat, S., Tokunaga, C.,
Avruch, J., and Yonezawa, K. (2002). Raptor, a binding partner of target of rapamycin
(TOR), mediates TOR action. Cell 110, 177–189.
Harrington, L.E., Hatton, R.D., Mangan, P.R., Turner, H., Murphy, T.L., Murphy,
K.M., and Weaver, C.T. (2005). Interleukin 17-producing CD4+ effector T cells develop via a
lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. 6, 1123–1132.
Haxhinasto, S., Mathis, D., and Benoist, C. (2008). The AKT-mTOR axis regulates de
novo differentiation of CD4+Foxp3+ cells. J. Exp. Med. 205, 565–574.
Heitman, J., Movva, N.R., and Hall, M.N. (1991). Targets for cell cycle arrest by the
immunosuppressant rapamycin in yeast. Science 253, 905–909.
Hendrikx, T.K., van Gurp, E.A.F.J., Sewgobind, V.D.K.D., Mol, W.M., Schoordijk,
W., Klepper, M., Velthuis, J.H.L., Geel, A., Ijzermans, J.N.M., Weimar, W., et al. (2009).
Generation of donor-specific regulatory T-cell function in kidney transplant patients.
Transplantation 87, 376–383.
Heng, D.Y.C., Xie, W., Regan, M.M., Warren, M.A., Golshayan, A.R., Sahi, C., Eigl,
B.J., Ruether, J.D., Cheng, T., North, S., et al. (2009). Prognostic factors for overall survival
in patients with metastatic renal cell carcinoma treated with vascular endothelial growth
factor-targeted agents: results from a large, multicenter study. J. Clin. Oncol. Off. J. Am. Soc.
Clin. Oncol. 27, 5794–5799.
Herman, A.E., Freeman, G.J., Mathis, D., and Benoist, C. (2004). CD4+CD25+ T
Regulatory Cells Dependent on ICOS Promote Regulation of Effector Cells in the Prediabetic
Lesion. J. Exp. Med. 199, 1479–1489.
Hirahara, K., Liu, L., Clark, R.A., Yamanaka, K., Fuhlbrigge, R.C., and Kupper, T.S.
(2006). The majority of human peripheral blood CD4+CD25highFoxp3+ regulatory T cells
bear functional skin-homing receptors. J. Immunol. Baltim. Md 1950 177, 4488–4494.
Hiyama, E., and Hiyama, K. (2003). Telomerase as tumor marker. Cancer Lett. 194,
221–233.
Hodi, F.S., O’Day, S.J., McDermott, D.F., Weber, R.W., Sosman, J.A., Haanen, J.B.,
Gonzalez, R., Robert, C., Schadendorf, D., Hassel, J.C., et al. (2010). Improved survival with
ipilimumab in patients with metastatic melanoma. N. Engl. J. Med. 363, 711–723.
Holz, M.K. (2012). The role of S6K1 in ER-positive breast cancer. Cell Cycle
Georget. Tex 11, 3159–3165.
Hori, S., Nomura, T., and Sakaguchi, S. (2003). Control of regulatory T cell
development by the transcription factor Foxp3. Science 299, 1057–1061.
Hosoi, H., Dilling, M.B., Shikata, T., Liu, L.N., Shu, L., Ashmun, R.A., Germain,
G.S., Abraham, R.T., and Houghton, P.J. (1999). Rapamycin causes poorly reversible
inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma
cells. Cancer Res. 59, 886–894.
Hsieh, A.C., Costa, M., Zollo, O., Davis, C., Feldman, M.E., Testa, J.R., Meyuhas, O.,
Shokat, K.M., and Ruggero, D. (2010). Genetic dissection of the oncogenic mTOR pathway
reveals druggable addiction to translational control via 4EBP-eIF4E. Cancer Cell 17, 249–
261.
167
Huang, S., Apasov, S., Koshiba, M., and Sitkovsky, M. (1997). Role of A2a
Extracellular Adenosine Receptor-Mediated Signaling in Adenosine-Mediated Inhibition of
T-Cell Activation and Expansion. Blood 90, 1600–1610.
Hudes, G., Carducci, M., Tomczak, P., Dutcher, J., Figlin, R., Kapoor, A.,
Staroslawska, E., Sosman, J., McDermott, D., Bodrogi, I., et al. (2007). Temsirolimus,
interferon alfa, or both for advanced renal-cell carcinoma. N. Engl. J. Med. 356, 2271–2281.
Huijts, C.M., Santegoets, S.J., van den Eertwegh, A.J., Pijpers, L.S., Haanen, J.B., de
Gruijl, T.D., Verheul, H.M., and van der Vliet, H.J. (2011). Phase I-II study of everolimus and
low-dose oral cyclophosphamide in patients with metastatic renal cell cancer. BMC Cancer
11, 505.
Hung, K., Hayashi, R., Lafond-Walker, A., Lowenstein, C., Pardoll, D., and Levitsky,
H. (1998). The central role of CD4(+) T cells in the antitumor immune response. J. Exp. Med.
188, 2357–2368.
Iellem, A., Mariani, M., Lang, R., Recalde, H., Panina-Bordignon, P., Sinigaglia, F.,
and D’Ambrosio, D. (2001). Unique chemotactic response profile and specific expression of
chemokine receptors CCR4 and CCR8 by CD4(+)CD25(+) regulatory T cells. J. Exp. Med.
194, 847–853.
Inman, B.A., Harrison, M.R., and George, D.J. (2013). Novel immunotherapeutic
strategies in development for renal cell carcinoma. Eur. Urol. 63, 881–889.
Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K.-L. (2002). TSC2 is phosphorylated and
inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4, 648–657.
Inoki, K., Li, Y., Xu, T., and Guan, K.-L. (2003). Rheb GTPase is a direct target of
TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 17, 1829–1834.
Inoki, K., Ouyang, H., Zhu, T., Lindvall, C., Wang, Y., Zhang, X., Yang, Q., Bennett,
C., Harada, Y., Stankunas, K., et al. (2006). TSC2 integrates Wnt and energy signals via a
coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 126, 955–968.
Intlekofer, A.M., Takemoto, N., Wherry, E.J., Longworth, S.A., Northrup, J.T.,
Palanivel, V.R., Mullen, A.C., Gasink, C.R., Kaech, S.M., Miller, J.D., et al. (2005). Effector
and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat. Immunol. 6, 1236–
1244.
Jacinto, E., Loewith, R., Schmidt, A., Lin, S., Rüegg, M.A., Hall, A., and Hall, M.N.
(2004). Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin
insensitive. Nat. Cell Biol. 6, 1122–1128.
Jacinto, E., Facchinetti, V., Liu, D., Soto, N., Wei, S., Jung, S.Y., Huang, Q., Qin, J.,
and Su, B. (2006). SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt
phosphorylation and substrate specificity. Cell 127, 125–137.
Jagannath, C., Lindsey, D.R., Dhandayuthapani, S., Xu, Y., Hunter, R.L., and Eissa,
N.T. (2009). Autophagy enhances the efficacy of BCG vaccine by increasing peptide
presentation in mouse dendritic cells. Nat. Med. 15, 267–276.
Janzen, N.K., Kim, H.L., Figlin, R.A., and Belldegrun, A.S. (2003). Surveillance after
radical or partial nephrectomy for localized renal cell carcinoma and management of recurrent
disease. Urol. Clin. North Am. 30, 843–852.
Jhun, B.S., Oh, Y.T., Lee, J.Y., Kong, Y., Yoon, K.-S., Kim, S.S., Baik, H.H., Ha, J.,
and Kang, I. (2005). AICAR suppresses IL-2 expression through inhibition of GSK-3
phosphorylation and NF-AT activation in Jurkat T cells. Biochem. Biophys. Res. Commun.
332, 339–346.
Jung, J.-W., Overgaard, N.H., Burke, M.T., Isbel, N., Frazer, I.H., Simpson, F., and
Wells, J.W. (2015). Does the nature of residual immune function explain the differential risk
of non-melanoma skin cancer development in immunosuppressed organ transplant recipients?
Int. J. Cancer J. Int. Cancer.
168
Jung, U., Foley, J.E., Erdmann, A.A., Toda, Y., Borenstein, T., Mariotti, J., and
Fowler, D.H. (2006). Ex vivo rapamycin generates Th1/Tc1 or Th2/Tc2 Effector T cells with
enhanced in vivo function and differential sensitivity to post-transplant rapamycin therapy.
Biol. Blood Marrow Transplant. J. Am. Soc. Blood Marrow Transplant. 12, 905–918.
Kaech, S.M., and Cui, W. (2012). Transcriptional control of effector and memory
CD8+ T cell differentiation. Nat. Rev. Immunol. 12, 749–761.
Kaizuka, T., Hara, T., Oshiro, N., Kikkawa, U., Yonezawa, K., Takehana, K., Iemura,
S.-I., Natsume, T., and Mizushima, N. (2010). Tti1 and Tel2 are critical factors in mammalian
target of rapamycin complex assembly. J. Biol. Chem. 285, 20109–20116.
Kantidakis, T., Ramsbottom, B.A., Birch, J.L., Dowding, S.N., and White, R.J. (2010).
mTOR associates with TFIIIC, is found at tRNA and 5S rRNA genes, and targets their
repressor Maf1. Proc. Natl. Acad. Sci. U. S. A. 107, 11823–11828.
Katzman, S.D., O’Gorman, W.E., Villarino, A.V., Gallo, E., Friedman, R.S.,
Krummel, M.F., Nolan, G.P., and Abbas, A.K. (2010). Duration of antigen receptor signaling
determines T-cell tolerance or activation. Proc. Natl. Acad. Sci. U. S. A. 107, 18085–18090.
Keir, M.E., Butte, M.J., Freeman, G.J., and Sharpe, A.H. (2008). PD-1 and its ligands
in tolerance and immunity. Annu. Rev. Immunol. 26, 677–704.
Kellersch, B., and Brocker, T. (2013). Langerhans cell homeostasis in mice is
dependent on mTORC1 but not mTORC2 function. Blood 121, 298–307.
Kennedy, R., and Celis, E. (2008). Multiple roles for CD4+ T cells in anti-tumor
immune responses. Immunol. Rev. 222, 129–144.
Kerdiles, Y.M., Beisner, D.R., Tinoco, R., Dejean, A.S., Castrillon, D.H., DePinho,
R.A., and Hedrick, S.M. (2009). Foxo1 links homing and survival of naive T cells by
regulating L-selectin, CCR7 and interleukin 7 receptor. Nat. Immunol. 10, 176–184.
Kim, B.-S., Kim, I.-K., Park, Y.-J., Kim, Y.-S., Kim, Y.-J., Chang, W.-S., Lee, Y.-S.,
Kweon, M.-N., Chung, Y., and Kang, C.-Y. (2010). Conversion of Th2 memory cells into
Foxp3+ regulatory T cells suppressing Th2-mediated allergic asthma. Proc. Natl. Acad. Sci.
U. S. A. 107, 8742–8747.
Kim, D.-H., Sarbassov, D.D., Ali, S.M., King, J.E., Latek, R.R., Erdjument-Bromage,
H., Tempst, P., and Sabatini, D.M. (2002). mTOR interacts with raptor to form a nutrientsensitive complex that signals to the cell growth machinery. Cell 110, 163–175.
Kim, D.-H., Sarbassov, D.D., Ali, S.M., Latek, R.R., Guntur, K.V.P., ErdjumentBromage, H., Tempst, P., and Sabatini, D.M. (2003). GbetaL, a positive regulator of the
rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and
mTOR. Mol. Cell 11, 895–904.
Klapper, J.A., Downey, S.G., Smith, F.O., Yang, J.C., Hughes, M.S., Kammula, U.S.,
Sherry, R.M., Royal, R.E., Steinberg, S.M., and Rosenberg, S. (2008). High-dose interleukin2 for the treatment of metastatic renal cell carcinoma%: a retrospective analysis of response and
survival in patients treated in the surgery branch at the National Cancer Institute between
1986 and 2006. Cancer 113, 293–301.
Ko, J.S., Zea, A.H., Rini, B.I., Ireland, J.L., Elson, P., Cohen, P., Golshayan, A.,
Rayman, P.A., Wood, L., Garcia, J., et al. (2009). Sunitinib mediates reversal of myeloidderived suppressor cell accumulation in renal cell carcinoma patients. Clin. Cancer Res. Off.
J. Am. Assoc. Cancer Res. 15, 2148–2157.
Ko, K., Yamazaki, S., Nakamura, K., Nishioka, T., Hirota, K., Yamaguchi, T.,
Shimizu, J., Nomura, T., Chiba, T., and Sakaguchi, S. (2005). Treatment of advanced tumors
with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+
regulatory T cells. J. Exp. Med. 202, 885–891.
Kobayashi, M., Kubo, T., Komatsu, K., Fujisaki, A., Terauchi, F., Natsui, S., Nukui,
A., Kurokawa, S., and Morita, T. (2013). Changes in peripheral blood immune cells: their
169
prognostic significance in metastatic renal cell carcinoma patients treated with molecular
targeted therapy. Med. Oncol. Northwood Lond. Engl. 30, 556.
Kondo, T., Nakazawa, H., Ito, F., Hashimoto, Y., Osaka, Y., Futatsuyama, K., Toma,
H., and Tanabe, K. (2006). Favorable prognosis of renal cell carcinoma with increased
expression of chemokines associated with a Th1-type immune response. Cancer Sci. 97, 780–
786.
Kopf, H., de la Rosa, G.M., Howard, O.M.Z., and Chen, X. (2007). Rapamycin
inhibits differentiation of Th17 cells and promotes generation of FoxP3+ T regulatory cells.
Int. Immunopharmacol. 7, 1819–1824.
Krishna, S., Yang, J., Wang, H., Qiu, Y., and Zhong, X.-P. (2014). Role of Tumor
Suppressor TSC1 in Regulating Antigen-Specific Primary and Memory CD8 T Cell
Responses to Bacterial Infection. Infect. Immun. 82, 3045–3057.
Kryczek, I., Banerjee, M., Cheng, P., Vatan, L., Szeliga, W., Wei, S., Huang, E.,
Finlayson, E., Simeone, D., Welling, T.H., et al. (2009). Phenotype, distribution, generation,
and functional and clinical relevance of Th17 cells in the human tumor environments. Blood
114, 1141–1149.
Kubach, J., Lutter, P., Bopp, T., Stoll, S., Becker, C., Huter, E., Richter, C.,
Weingarten, P., Warger, T., Knop, J., et al. (2007). Human CD4+CD25+ regulatory T cells:
proteome analysis identifies galectin-10 as a novel marker essential for their anergy and
suppressive function. Blood 110, 1550–1558.
Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N.R., and Hall,
M.N. (1993). Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase
homolog required for G1 progression. Cell 73, 585–596.
Laar, L. van de, Buitenhuis, M., Wensveen, F.M., Janssen, H.L.A., Coffer, P.J., and
Woltman, A.M. (2010). Human CD34-Derived Myeloid Dendritic Cell Development
Requires Intact Phosphatidylinositol 3-Kinase–Protein Kinase B–Mammalian Target of
Rapamycin Signaling. J. Immunol. 184, 6600–6611.
Laar, L. van de, Bosch, A. van den, Boonstra, A., Binda, R.S., Buitenhuis, M.,
Janssen, H.L.A., Coffer, P.J., and Woltman, A.M. (2012). PI3K-PKB hyperactivation
augments human plasmacytoid dendritic cell development and function. Blood 120, 4982–
4991.
Lahl, K., Loddenkemper, C., Drouin, C., Freyer, J., Arnason, J., Eberl, G., Hamann,
A., Wagner, H., Huehn, J., and Sparwasser, T. (2007). Selective depletion of Foxp3+
regulatory T cells induces a scurfy-like disease. J. Exp. Med. 204, 57–63.
Lam, J.S., Bergman, J., Breda, A., and Schulam, P.G. (2008). Importance of surgical
margins in the management of renal cell carcinoma. Nat. Rev. Urol. 5, 308–317.
Laplante, M., and Sabatini, D.M. (2009). An emerging role of mTOR in lipid
biosynthesis. Curr. Biol. CB 19, R1046–R1052.
Laplante, M., and Sabatini, D.M. (2012). mTOR signaling in growth control and
disease. Cell 149, 274–293.
Law, B.K. (2005). Rapamycin: an anti-cancer immunosuppressant? Crit. Rev. Oncol.
Hematol. 56, 47–60.
Lee, D.-F., Kuo, H.-P., Chen, C.-T., Hsu, J.-M., Chou, C.-K., Wei, Y., Sun, H.-L., Li,
L.-Y., Ping, B., Huang, W.-C., et al. (2007). IKK beta suppression of TSC1 links
inflammation and tumor angiogenesis via the mTOR pathway. Cell 130, 440–455.
Lee, H.K., Mattei, L.M., Steinberg, B.E., Alberts, P., Lee, Y.H., Chervonsky, A.,
Mizushima, N., Grinstein, S., and Iwasaki, A. (2010a). In Vivo Requirement for Atg5 in
Antigen Presentation by Dendritic Cells. Immunity 32, 227–239.
Lee, K., Gudapati, P., Dragovic, S., Spencer, C., Joyce, S., Killeen, N., Magnuson,
M.A., and Boothby, M. (2010b). Mammalian target of rapamycin protein complex 2 regulates
170
differentiation of Th1 and Th2 cell subsets via distinct signaling pathways. Immunity 32,
743–753.
Li, C., Liu, B., Dai, Z., and Tao, Y. (2011a). Knockdown of VEGF receptor-1
(VEGFR-1) impairs macrophage infiltration, angiogenesis and growth of clear cell renal cell
carcinoma (CRCC). Cancer Biol. Ther. 12, 872–880.
Li, M.O., Sanjabi, S., and Flavell, R.A. (2006). Transforming Growth Factor-"
Controls Development, Homeostasis, and Tolerance of T Cells by Regulatory T CellDependent and -Independent Mechanisms. Immunity 25, 455–471.
Li, Q., Rao, R.R., Araki, K., Pollizzi, K., Odunsi, K., Powell, J.D., and Shrikant, P.A.
(2011b). A central role for mTOR kinase in homeostatic proliferation induced CD8+ T cell
memory and tumor immunity. Immunity 34, 541–553.
Li, Q., Rao, R., Vazzana, J., Goedegebuure, P., Odunsi, K., Gillanders, W., and
Shrikant, P.A. (2012). Regulating mammalian target of rapamycin to tune vaccinationinduced CD8(+) T cell responses for tumor immunity. J. Immunol. Baltim. Md 1950 188,
3080–3087.
Liang, B., Workman, C., Lee, J., Chew, C., Dale, B.M., Colonna, L., Flores, M., Li,
N., Schweighoffer, E., Greenberg, S., et al. (2008). Regulatory T Cells Inhibit Dendritic Cells
by Lymphocyte Activation Gene-3 Engagement of MHC Class II. J. Immunol. 180, 5916–
5926.
Liotta, F., Gacci, M., Frosali, F., Querci, V., Vittori, G., Lapini, A., Santarlasci, V.,
Serni, S., Cosmi, L., Maggi, L., et al. (2011). Frequency of regulatory T cells in peripheral
blood and in tumour-infiltrating lymphocytes correlates with poor prognosis in renal cell
carcinoma. BJU Int. 107, 1500–1506.
Liu, G., Burns, S., Huang, G., Boyd, K., Proia, R.L., Flavell, R.A., and Chi, H. (2009).
The receptor S1P1 overrides regulatory T cell-mediated immune suppression through AktmTOR. Nat. Immunol. 10, 769–777.
Liu, G., Yang, K., Burns, S., Shrestha, S., and Chi, H. (2010). The S1P(1)-mTOR axis
directs the reciprocal differentiation of T(H)1 and T(reg) cells. Nat. Immunol. 11, 1047–1056.
Ljungberg, B., Cowan, N.C., Hanbury, D.C., Hora, M., Kuczyk, M.A., Merseburger,
A.S., Patard, J.-J., Mulders, P.F.A., Sinescu, I.C., and European Association of Urology
Guideline Group (2010). EAU guidelines on renal cell carcinoma: the 2010 update. Eur. Urol.
58, 398–406.
Lu, L., Qian, X.F., Rao, J.H., Wang, X.H., Zheng, S.G., and Zhang, F. (2010).
Rapamycin promotes the expansion of CD4(+) Foxp3(+) regulatory T cells after liver
transplantation. Transplant. Proc. 42, 1755–1757.
Lu, Y., Hong, S., Li, H., Park, J., Hong, B., Wang, L., Zheng, Y., Liu, Z., Xu, J., He,
J., et al. (2012). Th9 cells promote antitumor immune responses in vivo. J. Clin. Invest. 122,
4160–4171.
Ma, X.M., and Blenis, J. (2009). Molecular mechanisms of mTOR-mediated
translational control. Nat. Rev. Mol. Cell Biol. 10, 307–318.
Ma, A., Koka, R., and Burkett, P. (2006). Diverse functions of IL-2, IL-15, and IL-7 in
lymphoid homeostasis. Annu. Rev. Immunol. 24, 657–679.
Makki, K., Taront, S., Molendi-Coste, O., Bouchaert, E., Neve, B., Eury, E., Lobbens,
S., Labalette, M., Duez, H., Staels, B., et al. (2014). Beneficial metabolic effects of rapamycin
are associated with enhanced regulatory cells in diet-induced obese mice. PloS One 9,
e92684.
Mandala, S., Hajdu, R., Bergstrom, J., Quackenbush, E., Xie, J., Milligan, J.,
Thornton, R., Shei, G.-J., Card, D., Keohane, C., et al. (2002). Alteration of Lymphocyte
Trafficking by Sphingosine-1-Phosphate Receptor Agonists. Science 296, 346–349.
171
Marçais, A., Cherfils-Vicini, J., Viant, C., Degouve, S., Viel, S., Fenis, A., Rabilloud,
J., Mayol, K., Tavares, A., Bienvenu, J., et al. (2014). The metabolic checkpoint kinase
mTOR is essential for IL-15 signaling during the development and activation of NK cells.
Nat. Immunol. 15, 749–757.
Marigo, I., Dolcetti, L., Serafini, P., Zanovello, P., and Bronte, V. (2008). Tumorinduced tolerance and immune suppression by myeloid derived suppressor cells. Immunol.
Rev. 222, 162–179.
Mariotti, J., Foley, J., Jung, U., Borenstein, T., Kantardzic, N., Han, S., Hanson, J.T.,
Wong, E., Buxhoeveden, N., Trepel, J.B., et al. (2008). Ex vivo rapamycin generates
apoptosis-resistant donor Th2 cells that persist in vivo and prevent hemopoietic stem cell graft
rejection. J. Immunol. Baltim. Md 1950 180, 89–105.
Markel, G., Cohen-Sinai, T., Besser, M.J., Oved, K., Itzhaki, O., Seidman, R.,
Fridman, E., Treves, A.J., Keisari, Y., Dotan, Z., et al. (2009). Preclinical evaluation of
adoptive cell therapy for patients with metastatic renal cell carcinoma. Anticancer Res. 29,
145–154.
Martínez, P., and Blasco, M.A. (2011). Telomeric and extra-telomeric roles for
telomerase and the telomere-binding proteins. Nat. Rev. Cancer 11, 161–176.
Martin-Orozco, N., Muranski, P., Chung, Y., Yang, X.O., Yamazaki, T., Lu, S., Hwu,
P., Restifo, N.P., Overwijk, W.W., and Dong, C. (2009). T helper 17 cells promote cytotoxic
T cell activation in tumor immunity. Immunity 31, 787–798.
Marzo, A.L., Kinnear, B.F., Lake, R.A., Frelinger, J.J., Collins, E.J., Robinson, B.W.,
and Scott, B. (2000). Tumor-specific CD4+ T cells have a major “post-licensing” role in CTL
mediated anti-tumor immunity. J. Immunol. Baltim. Md 1950 165, 6047–6055.
Mayer, C., Zhao, J., Yuan, X., and Grummt, I. (2004). mTOR-dependent activation of
the transcription factor TIF-IA links rRNA synthesis to nutrient availability. Genes Dev. 18,
423–434.
McDermott, D.F., Regan, M.M., Clark, J.I., Flaherty, L.E., Weiss, G.R., Logan, T.F.,
Kirkwood, J.M., Gordon, M.S., Sosman, J.A., Ernstoff, M.S., et al. (2005). Randomized phase
III trial of high-dose interleukin-2 versus subcutaneous interleukin-2 and interferon in patients
with metastatic renal cell carcinoma. J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 23, 133–
141.
Mellor, A.L., and Munn, D.H. (2004). Ido expression by dendritic cells: tolerance and
tryptophan catabolism. Nat. Rev. Immunol. 4, 762–774.
Menendez, J.A., and Lupu, R. (2007). Fatty acid synthase and the lipogenic phenotype
in cancer pathogenesis. Nat. Rev. Cancer 7, 763–777.
Ménétrier-Caux, C., Curiel, T., Faget, J., Manuel, M., Caux, C., and Zou, W. (2012).
Targeting regulatory T cells. Target. Oncol. 7, 15–28.
Menon, S., Yecies, J.L., Zhang, H.H., Howell, J.J., Nicholatos, J., Harputlugil, E.,
Bronson, R.T., Kwiatkowski, D.J., and Manning, B.D. (2012). Chronic activation of mTOR
complex 1 is sufficient to cause hepatocellular carcinoma in mice. Sci. Signal. 5, ra24.
Merkenschlager, M., and von Boehmer, H. (2010). PI3 kinase signalling blocks Foxp3
expression by sequestering Foxo factors. J. Exp. Med. 207, 1347–1350.
Merlo, A., Casalini, P., Carcangiu, M.L., Malventano, C., Triulzi, T., Mènard, S.,
Tagliabue, E., and Balsari, A. (2009). FOXP3 Expression and Overall Survival in Breast
Cancer. J. Clin. Oncol. 27, 1746–1752.
Modiano, J.F., Johnson, L.D.S., and Bellgrau, D. (2008). Negative regulators in
homeostasis of naïve peripheral T cells. Immunol. Res. 41, 137–153.
Morse, M.A., Hobeika, A.C., Osada, T., Serra, D., Niedzwiecki, D., Lyerly, H.K., and
Clay, T.M. (2008). Depletion of human regulatory T cells specifically enhances antigenspecific immune responses to cancer vaccines. Blood 112, 610–618.
172
Mosmann, T.R., Cherwinski, H., Bond, M.W., Giedlin, M.A., and Coffman, R.L.
(1986). Two types of murine helper T cell clone. I. Definition according to profiles of
lymphokine activities and secreted proteins. J. Immunol. Baltim. Md 1950 136, 2348–2357.
Motzer, R.J., Hutson, T.E., Tomczak, P., Michaelson, M.D., Bukowski, R.M., Rixe,
O., Oudard, S., Negrier, S., Szczylik, C., Kim, S.T., et al. (2007). Sunitinib versus interferon
alfa in metastatic renal-cell carcinoma. N. Engl. J. Med. 356, 115–124.
Motzer, R.J., Escudier, B., Oudard, S., Hutson, T.E., Porta, C., Bracarda, S.,
Grünwald, V., Thompson, J.A., Figlin, R.A., Hollaender, N., et al. (2008). Efficacy of
everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled
phase III trial. Lancet 372, 449–456.
Motzer, R.J., Rini, B.I., McDermott, D.F., Redman, B.G., Kuzel, T.M., Harrison,
M.R., Vaishampayan, U.N., Drabkin, H.A., George, S., Logan, T.F., et al. (2015). Nivolumab
for Metastatic Renal Cell Carcinoma: Results of a Randomized Phase II Trial. J. Clin. Oncol.
Off. J. Am. Soc. Clin. Oncol. 33, 1430–1437.
Muranski, P., and Restifo, N.P. (2009). Does IL-17 promote tumor growth? Blood
114, 231–232.
Murdoch, C., Muthana, M., Coffelt, S.B., and Lewis, C.E. (2008). The role of myeloid
cells in the promotion of tumour angiogenesis. Nat. Rev. Cancer 8, 618–631.
Nakamura, K., Kitani, A., and Strober, W. (2001). Cell Contact–Dependent
Immunosuppression by Cd4+Cd25+Regulatory T Cells Is Mediated by Cell Surface–Bound
Transforming Growth Factor ". J. Exp. Med. 194, 629–644.
Nakamura, T., Nakao, T., Yoshimura, N., and Ashihara, E. (2015). Rapamycin
Prolongs Cardiac Allograft Survival in a Mouse Model by Inducing Myeloid-Derived
Suppressor Cells. Am. J. Transplant. Off. J. Am. Soc. Transplant. Am. Soc. Transpl. Surg.
Nakano, O., Sato, M., Naito, Y., Suzuki, K., Orikasa, S., Aizawa, M., Suzuki, Y.,
Shintaku, I., Nagura, H., and Ohtani, H. (2001). Proliferative Activity of Intratumoral CD8+
T-Lymphocytes As a Prognostic Factor in Human Renal Cell Carcinoma Clinicopathologic
Demonstration of Antitumor Immunity. Cancer Res. 61, 5132–5136.
Namba, R., Young, L.J.T., Abbey, C.K., Kim, L., Damonte, P., Borowsky, A.D., Qi,
J., Tepper, C.G., MacLeod, C.L., Cardiff, R.D., et al. (2006). Rapamycin inhibits growth of
premalignant and malignant mammary lesions in a mouse model of ductal carcinoma in situ.
Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 12, 2613–2621.
Nanus, D.M., Garino, A., Milowsky, M.I., Larkin, M., and Dutcher, J.P. (2004).
Active chemotherapy for sarcomatoid and rapidly progressing renal cell carcinoma. Cancer
101, 1545–1551.
Négrier, S., Gravis, G., Pérol, D., Chevreau, C., Delva, R., Bay, J.-O., Blanc, E.,
Ferlay, C., Geoffrois, L., Rolland, F., et al. (2011). Temsirolimus and bevacizumab, or
sunitinib, or interferon alfa and bevacizumab for patients with advanced renal cell carcinoma
(TORAVA): a randomised phase 2 trial. Lancet Oncol. 12, 673–680.
Nickerson, M.L., Jaeger, E., Shi, Y., Durocher, J.A., Mahurkar, S., Zaridze, D.,
Matveev, V., Janout, V., Kollarova, H., Bencko, V., et al. (2008). Improved identification of
von Hippel-Lindau gene alterations in clear cell renal tumors. Clin. Cancer Res. Off. J. Am.
Assoc. Cancer Res. 14, 4726–4734.
Nishikawa, H., and Sakaguchi, S. (2010). Regulatory T cells in tumor immunity. Int. J.
Cancer 127, 759–767.
Noessner, E., Brech, D., Mendler, A.N., Masouris, I., Schlenker, R., and Prinz, P.U.
(2012). Intratumoral alterations of dendritic-cell differentiation and CD8+ T-cell anergy are
immune escape mechanisms of clear cell renal cell carcinoma. OncoImmunology 1, 1451–
1453.
173
Noris, M., Casiraghi, F., Todeschini, M., Cravedi, P., Cugini, D., Monteferrante, G.,
Aiello, S., Cassis, L., Gotti, E., Gaspari, F., et al. (2007). Regulatory T cells and T cell
depletion: role of immunosuppressive drugs. J. Am. Soc. Nephrol. JASN 18, 1007–1018.
Nourse, J., Firpo, E., Flanagan, W.M., Coats, S., Polyak, K., Lee, M.H., Massague, J.,
Crabtree, G.R., and Roberts, J.M. (1994). Interleukin-2-mediated elimination of the p27Kip1
cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 372, 570–573.
Ochi, A., Nguyen, A.H., Bedrosian, A.S., Mushlin, H.M., Zarbakhsh, S., Barilla, R.,
Zambirinis, C.P., Fallon, N.C., Rehman, A., Pylayeva-Gupta, Y., et al. (2012). MyD88
inhibition amplifies dendritic cell capacity to promote pancreatic carcinogenesis via Th2 cells.
J. Exp. Med. 209, 1671–1687.
Ohtani, M., Nagai, S., Kondo, S., Mizuno, S., Nakamura, K., Tanabe, M., Takeuchi,
T., Matsuda, S., and Koyasu, S. (2008). Mammalian target of rapamycin and glycogen
synthase kinase 3 differentially regulate lipopolysaccharide-induced interleukin-12 production
in dendritic cells. Blood 112, 635–643.
O’Reilly, K.E., Rojo, F., She, Q.-B., Solit, D., Mills, G.B., Smith, D., Lane, H.,
Hofmann, F., Hicklin, D.J., Ludwig, D.L., et al. (2006). mTOR Inhibition Induces Upstream
Receptor Tyrosine Kinase Signaling and Activates Akt. Cancer Res. 66, 1500–1508.
O’Shea, J.J., and Paul, W.E. (2010). Mechanisms underlying lineage commitment and
plasticity of helper CD4+ T cells. Science 327, 1098–1102.
Ostrand-Rosenberg, S. (2005). CD4+ T lymphocytes: a critical component of
antitumor immunity. Cancer Invest. 23, 413–419.
Oudard, S., Banu, E., Vieillefond, A., Fournier, L., Priou, F., Medioni, J., Banu, A.,
Duclos, B., Rolland, F., Escudier, B., et al. (2007). Prospective multicenter phase II study of
gemcitabine plus platinum salt for metastatic collecting duct carcinoma: results of a GETUG
(Groupe d’Etudes des Tumeurs Uro-Génitales) study. J. Urol. 177, 1698–1702.
Ouyang, W., Beckett, O., Ma, Q., Paik, J., DePinho, R.A., and Li, M.O. (2010). Foxo
proteins cooperatively control the differentiation of Foxp3+ regulatory T cells. Nat. Immunol.
11, 618–627.
Pace, L., Tempez, A., Arnold-Schrauf, C., Lemaitre, F., Bousso, P., Fetler, L.,
Sparwasser, T., and Amigorena, S. (2012). Regulatory T cells increase the avidity of primary
CD8+ T cell responses and promote memory. Science 338, 532–536.
Pallotta, M.T., Orabona, C., Volpi, C., Vacca, C., Belladonna, M.L., Bianchi, R.,
Servillo, G., Brunacci, C., Calvitti, M., Bicciato, S., et al. (2011). Indoleamine 2,3dioxygenase is a signaling protein in long-term tolerance by dendritic cells. Nat. Immunol. 12,
870–878.
Pandiyan, P., Zheng, L., Ishihara, S., Reed, J., and Lenardo, M.J. (2007).
CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation–mediated apoptosis of
effector CD4+ T cells. Nat. Immunol. 8, 1353–1362.
Park, Y., Jin, H.-S., Lopez, J., Elly, C., Kim, G., Murai, M., Kronenberg, M., and Liu,
Y.-C. (2013). TSC1 regulates the balance between effector and regulatory T cells. J. Clin.
Invest. 123, 5165–5178.
Pearce, E.L. (2010). Metabolism in T cell activation and differentiation. Curr. Opin.
Immunol. 22, 314–320.
Pearce, E.L., Walsh, M.C., Cejas, P.J., Harms, G.M., Shen, H., Wang, L.-S., Jones,
R.G., and Choi, Y. (2009). Enhancing CD8 T-cell memory by modulating fatty acid
metabolism. Nature 460, 103–107.
Pearce, L.R., Huang, X., Boudeau, J., Paw&owski, R., Wullschleger, S., Deak, M.,
Ibrahim, A.F.M., Gourlay, R., Magnuson, M.A., and Alessi, D.R. (2007). Identification of
Protor as a novel Rictor-binding component of mTOR complex-2. Biochem. J. 405, 513–522.
174
Peggs, K.S., Quezada, S.A., Chambers, C.A., Korman, A.J., and Allison, J.P. (2009).
Blockade of CTLA-4 on both effector and regulatory T cell compartments contributes to the
antitumor activity of anti–CTLA-4 antibodies. J. Exp. Med. 206, 1717–1725.
Pere, H., Montier, Y., Bayry, J., Quintin-Colonna, F., Merillon, N., Dransart, E.,
Badoual, C., Gey, A., Ravel, P., Marcheteau, E., et al. (2011). A CCR4 antagonist combined
with vaccines induces antigen-specific CD8+ T cells and tumor immunity against self
antigens. Blood 118, 4853–4862.
Peterson, T.R., Laplante, M., Thoreen, C.C., Sancak, Y., Kang, S.A., Kuehl, W.M.,
Gray, N.S., and Sabatini, D.M. (2009). DEPTOR is an mTOR inhibitor frequently
overexpressed in multiple myeloma cells and required for their survival. Cell 137, 873–886.
Peterson, T.R., Sengupta, S.S., Harris, T.E., Carmack, A.E., Kang, S.A., Balderas, E.,
Guertin, D.A., Madden, K.L., Carpenter, A.E., Finck, B.N., et al. (2011). mTOR complex 1
regulates lipin 1 localization to control the SREBP pathway. Cell 146, 408–420.
Pollizzi, K.N., Patel, C.H., Sun, I.-H., Oh, M.-H., Waickman, A.T., Wen, J., Delgoffe,
G.M., and Powell, J.D. (2015). mTORC1 and mTORC2 selectively regulate CD8+ T cell
differentiation. J. Clin. Invest. 125, 2090–2108.
Porta, C., Paglino, C., and Mosca, A. (2014). Targeting PI3K/Akt/mTOR Signaling in
Cancer. Front. Oncol. 4, 64.
Potter, C.J., Pedraza, L.G., and Xu, T. (2002). Akt regulates growth by directly
phosphorylating Tsc2. Nat. Cell Biol. 4, 658–665.
Procaccini, C., De Rosa, V., Galgani, M., Abanni, L., Calì, G., Porcellini, A., Carbone,
F., Fontana, S., Horvath, T.L., La Cava, A., et al. (2010). An oscillatory switch in mTOR
kinase activity sets regulatory T cell responsiveness. Immunity 33, 929–941.
Purwar, R., Schlapbach, C., Xiao, S., Kang, H.S., Elyaman, W., Jiang, X., Jetten,
A.M., Khoury, S.J., Fuhlbrigge, R.C., Kuchroo, V.K., et al. (2012). Robust tumor immunity to
melanoma mediated by interleukin-9-producing T cells. Nat. Med. 18, 1248–1253.
Qian, X., Wang, K., Wang, X., Zheng, S.G., and Lu, L. (2011). Generation of human
regulatory T cells de novo with suppressive function prevent xenogeneic graft versus host
disease. Int. Immunopharmacol. 11, 630–637.
Qin, Z., and Blankenstein, T. (2000). CD4+ T cell--mediated tumor rejection involves
inhibition of angiogenesis that is dependent on IFN gamma receptor expression by
nonhematopoietic cells. Immunity 12, 677–686.
Qin, Z., Richter, G., Schüler, T., Ibe, S., Cao, X., and Blankenstein, T. (1998). B cells
inhibit induction of T cell-dependent tumor immunity. Nat. Med. 4, 627–630.
Rabinovich, G.A., Gabrilovich, D., and Sotomayor, E.M. (2007). Immunosuppressive
strategies that are mediated by tumor cells. Annu. Rev. Immunol. 25, 267–296.
Rao, R.R., Li, Q., Odunsi, K., and Shrikant, P.A. (2010). The mTOR kinase
determines effector versus memory CD8+ T cell fate by regulating the expression of
transcription factors T-bet and Eomesodermin. Immunity 32, 67–78.
Rao, R.R., Li, Q., Bupp, M.R.G., and Shrikant, P.A. (2012). Transcription Factor
Foxo1 Represses T-bet-Mediated Effector Functions and Promotes Memory CD8+ T Cell
Differentiation. Immunity 36, 374–387.
Raught, B., Peiretti, F., Gingras, A.-C., Livingstone, M., Shahbazian, D., Mayeur,
G.L., Polakiewicz, R.D., Sonenberg, N., and Hershey, J.W.B. (2004). Phosphorylation of
eucaryotic translation initiation factor 4B Ser422 is modulated by S6 kinases. EMBO J. 23,
1761–1769.
Rebecca, V.W., Massaro, R.R., Fedorenko, I.V., Sondak, V.K., Anderson, A.R.A.,
Kim, E., Amaravadi, R.K., Maria-Engler, S.S., Messina, J.L., Gibney, G.T., et al. (2014).
Inhibition of autophagy enhances the effects of the AKT inhibitor MK-2206 when combined
175
with paclitaxel and carboplatin in BRAF wild-type melanoma. Pigment Cell Melanoma Res.
27, 465–478.
Rech, A.J., Mick, R., Martin, S., Recio, A., Aqui, N.A., Powell, D.J., Colligon, T.A.,
Trosko, J.A., Leinbach, L.I., Pletcher, C.H., et al. (2012). CD25 blockade depletes and
selectively reprograms regulatory T cells in concert with immunotherapy in cancer patients.
Sci. Transl. Med. 4, 134ra62.
Redjimi, N., Raffin, C., Raimbaud, I., Pignon, P., Matsuzaki, J., Odunsi, K., Valmori,
D., and Ayyoub, M. (2012). CXCR3+ T Regulatory Cells Selectively Accumulate in Human
Ovarian Carcinomas to Limit Type I Immunity. Cancer Res. 72, 4351–4360.
Ridge, J.P., Di Rosa, F., and Matzinger, P. (1998). A conditioned dendritic cell can be
a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393, 474–478.
Rini, B.I., Campbell, S.C., and Escudier, B. (2009). Renal cell carcinoma. Lancet
Lond. Engl. 373, 1119–1132.
Rini, B.I., Bellmunt, J., Clancy, J., Wang, K., Niethammer, A.G., Hariharan, S., and
Escudier, B. (2014). Randomized phase III trial of temsirolimus and bevacizumab versus
interferon alfa and bevacizumab in metastatic renal cell carcinoma: INTORACT trial. J. Clin.
Oncol. Off. J. Am. Soc. Clin. Oncol. 32, 752–759.
Rivet, J., Mourah, S., Murata, H., Mounier, N., Pisonero, H., Mongiat-Artus, P.,
Teillac, P., Calvo, F., Janin, A., and Dosquet, C. (2008). VEGF and VEGFR-1 are
coexpressed by epithelial and stromal cells of renal cell carcinoma. Cancer 112, 433–442.
Rubtsov, Y.P., Rasmussen, J.P., Chi, E.Y., Fontenot, J., Castelli, L., Ye, X., Treuting,
P., Siewe, L., Roers, A., Henderson Jr., W.R., et al. (2008). Regulatory T Cell-Derived
Interleukin-10 Limits Inflammation at Environmental Interfaces. Immunity 28, 546–558.
Sabatini, D.M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S.H.
(1994). RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent
fashion and is homologous to yeast TORs. Cell 78, 35–43.
Sabbatini, M., Ruggiero, G., Palatucci, A.T., Rubino, V., Federico, S., Giovazzino, A.,
Apicella, L., Santopaolo, M., Matarese, G., Galgani, M., et al. (2015). Oscillatory mTOR
inhibition and Treg increase in kidney transplantation. Clin. Exp. Immunol.
Sabers, C.J., Martin, M.M., Brunn, G.J., Williams, J.M., Dumont, F.J., Wiederrecht,
G., and Abraham, R.T. (1995). Isolation of a protein target of the FKBP12-rapamycin
complex in mammalian cells. J. Biol. Chem. 270, 815–822.
Sakaguchi, S. (2004). Naturally Arising CD4+ Regulatory T Cells for Immunologic
Self-Tolerance and Negative Control of Immune Responses. Annu. Rev. Immunol. 22, 531–
562.
Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., and Toda, M. (1995). Immunologic
self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25).
Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J.
Immunol. Baltim. Md 1950 155, 1151–1164.
Sakata, A., Kuwahara, K., Ohmura, T., Inui, S., and Sakaguchi, N. (1999).
Involvement of a rapamycin-sensitive pathway in CD40-mediated activation of murine B cells
in vitro. Immunol. Lett. 68, 301–309.
Salama, P., Phillips, M., Grieu, F., Morris, M., Zeps, N., Joseph, D., Platell, C., and
Iacopetta, B. (2009). Tumor-Infiltrating FOXP3+ T Regulatory Cells Show Strong Prognostic
Significance in Colorectal Cancer. J. Clin. Oncol. 27, 186–192.
Sallusto, F., Lenig, D., Förster, R., Lipp, M., and Lanzavecchia, A. (1999). Two
subsets of memory T lymphocytes with distinct homing potentials and effector functions.
Nature 401, 708–712.
Salmond, R.J., Emery, J., Okkenhaug, K., and Zamoyska, R. (2009). MAPK,
phosphatidylinositol 3-kinase, and mammalian target of rapamycin pathways converge at the
176
level of ribosomal protein S6 phosphorylation to control metabolic signaling in CD8 T cells.
J. Immunol. Baltim. Md 1950 183, 7388–7397.
Sancak, Y., Peterson, T.R., Shaul, Y.D., Lindquist, R.A., Thoreen, C.C., Bar-Peled, L.,
and Sabatini, D.M. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to
mTORC1. Science 320, 1496–1501.
Sarbassov, D.D., Ali, S.M., Kim, D.-H., Guertin, D.A., Latek, R.R., ErdjumentBromage, H., Tempst, P., and Sabatini, D.M. (2004). Rictor, a novel binding partner of
mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the
cytoskeleton. Curr. Biol. CB 14, 1296–1302.
Sarbassov, D.D., Guertin, D.A., Ali, S.M., and Sabatini, D.M. (2005). Phosphorylation
and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101.
Sarbassov, D.D., Ali, S.M., Sengupta, S., Sheen, J.-H., Hsu, P.P., Bagley, A.F.,
Markhard, A.L., and Sabatini, D.M. (2006). Prolonged rapamycin treatment inhibits
mTORC2 assembly and Akt/PKB. Mol. Cell 22, 159–168.
Sarris, M., Andersen, K.G., Randow, F., Mayr, L., and Betz, A.G. (2008). Neuropilin1 Expression on Regulatory T Cells Enhances Their Interactions with Dendritic Cells during
Antigen Recognition. Immunity 28, 402–413.
Sathaliyawala, T., O’Gorman, W.E., Greter, M., Bogunovic, M., Konjufca, V., Hou,
Z.E., Nolan, G.P., Miller, M.J., Merad, M., and Reizis, B. (2010). Mammalian Target of
Rapamycin Controls Dendritic Cell Development Downstream of Flt3 Ligand Signaling.
Immunity 33, 597–606.
Sauer, S., Bruno, L., Hertweck, A., Finlay, D., Leleu, M., Spivakov, M., Knight, Z.A.,
Cobb, B.S., Cantrell, D., O’Connor, E., et al. (2008). T cell receptor signaling controls Foxp3
expression via PI3K, Akt, and mTOR. Proc. Natl. Acad. Sci. U. S. A. 105, 7797–7802.
Schmitt, E., and Bopp, T. (2012). Amazing IL-9: revealing a new function for an “old”
cytokine. J. Clin. Invest. 122, 3857–3859.
Schmitt, E.G., and Williams, C.B. (2013). Generation and function of induced
regulatory T cells. Immunol. Toler. 4, 152.
Schreiber, T.H., Wolf, D., Bodero, M., and Podack, E. (2012). Tumor antigen specific
iTreg accumulate in the tumor microenvironment and suppress therapeutic vaccination.
Oncoimmunology 1, 642–648.
Schultz, E.S., Lethé, B., Cambiaso, C.L., Van Snick, J., Chaux, P., Corthals, J.,
Heirman, C., Thielemans, K., Boon, T., and van der Bruggen, P. (2000). A MAGE-A3
peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T
lymphocytes. Cancer Res. 60, 6272–6275.
Shafer-Weaver, K.A., Watkins, S.K., Anderson, M.J., Draper, L.J., Malyguine, A.,
Alvord, W.G., Greenberg, N.M., and Hurwitz, A.A. (2009). Immunity to murine prostatic
tumors: continuous provision of T-cell help prevents CD8 T-cell tolerance and activates
tumor-infiltrating dendritic cells. Cancer Res. 69, 6256–6264.
Shankaran, V., Ikeda, H., Bruce, A.T., White, J.M., Swanson, P.E., Old, L.J., and
Schreiber, R.D. (2001). IFNgamma and lymphocytes prevent primary tumour development
and shape tumour immunogenicity. Nature 410, 1107–1111.
Shaw, R.J., and Cantley, L.C. (2006). Ras, PI(3)K and mTOR signalling controls
tumour cell growth. Nature 441, 424–430.
Shedlock, D.J., and Shen, H. (2003). Requirement for CD4 T Cell Help in Generating
Functional CD8 T Cell Memory. Science 300, 337–339.
Shimizu, J., Yamazaki, S., Takahashi, T., Ishida, Y., and Sakaguchi, S. (2002).
Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological selftolerance. Nat. Immunol. 3, 135–142.
177
Shin, H.-J., Baker, J., Leveson-Gower, D.B., Smith, A.T., Sega, E.I., and Negrin, R.S.
(2011). Rapamycin and IL-2 reduce lethal acute graft-versus-host disease associated with
increased expansion of donor type CD4+CD25+Foxp3+ regulatory T cells. Blood 118, 2342–
2350.
Shrestha, S., Yang, K., Wei, J., Karmaus, P.W.F., Neale, G., and Chi, H. (2014). Tsc1
promotes the differentiation of memory CD8+ T cells via orchestrating the transcriptional and
metabolic programs. Proc. Natl. Acad. Sci. U. S. A. 111, 14858–14863.
Sinclair, L.V., Finlay, D., Feijoo, C., Cornish, G.H., Gray, A., Ager, A., Okkenhaug,
K., Hagenbeek, T.J., Spits, H., and Cantrell, D.A. (2008). Phosphatidylinositol-3-OH kinase
and nutrient-sensing mTOR pathways control T lymphocyte trafficking. Nat. Immunol. 9,
513–521.
Sittig, S.P., Køllgaard, T., Grønbæk, K., Idorn, M., Hennenlotter, J., Stenzl, A.,
Gouttefangeas, C., and Thor Straten, P. (2013). Clonal expansion of renal cell carcinomainfiltrating T lymphocytes. Oncoimmunology 2, e26014.
van Slegtenhorst, M., de Hoogt, R., Hermans, C., Nellist, M., Janssen, B., Verhoef, S.,
Lindhout, D., van den Ouweland, A., Halley, D., Young, J., et al. (1997). Identification of the
tuberous sclerosis gene TSC1 on chromosome 9q34. Science 277, 805–808.
So, T., Choi, H., and Croft, M. (2011). OX40 complexes with phosphoinositide 3kinase and protein kinase B (PKB) to augment TCR-dependent PKB signaling. J. Immunol.
Baltim. Md 1950 186, 3547–3555.
Steinbrink, K., Wölfl, M., Jonuleit, H., Knop, J., and Enk, A.H. (1997). Induction of
tolerance by IL-10-treated dendritic cells. J. Immunol. 159, 4772–4780.
Strauss, L., Whiteside, T.L., Knights, A., Bergmann, C., Knuth, A., and Zippelius, A.
(2007). Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T
cells cultured with rapamycin. J. Immunol. Baltim. Md 1950 178, 320–329.
Strauss, L., Czystowska, M., Szajnik, M., Mandapathil, M., and Whiteside, T.L.
(2009). Differential responses of human regulatory T cells (Treg) and effector T cells to
rapamycin. PloS One 4, e5994.
Surh, C.D., and Sprent, J. (2008). Homeostasis of naive and memory T cells.
Immunity 29, 848–862.
Tanchot, C., and Rocha, B. (2003). CD8 and B cell memory: same strategy, same
signals. Nat. Immunol. 4, 431–432.
Taqueti, V.R., Grabie, N., Colvin, R., Pang, H., Jarolim, P., Luster, A.D., Glimcher,
L.H., and Lichtman, A.H. (2006). T-bet Controls Pathogenicity of CTLs in the Heart by
Separable Effects on Migration and Effector Activity. J. Immunol. 177, 5890–5901.
Tartour, E., Fossiez, F., Joyeux, I., Galinha, A., Gey, A., Claret, E., Sastre-Garau, X.,
Couturier, J., Mosseri, V., Vives, V., et al. (1999). Interleukin 17, a T-cell-derived cytokine,
promotes tumorigenicity of human cervical tumors in nude mice. Cancer Res. 59, 3698–3704.
Tatsumi, T., Kierstead, L.S., Ranieri, E., Gesualdo, L., Schena, F.P., Finke, J.H.,
Bukowski, R.M., Mueller-Berghaus, J., Kirkwood, J.M., Kwok, W.W., et al. (2002). Diseaseassociated bias in T helper type 1 (Th1)/Th2 CD4(+) T cell responses against MAGE-6 in
HLA-DRB10401(+) patients with renal cell carcinoma or melanoma. J. Exp. Med. 196, 619–
628.
Tee, A.R., Fingar, D.C., Manning, B.D., Kwiatkowski, D.J., Cantley, L.C., and Blenis,
J. (2002). Tuberous sclerosis complex-1 and -2 gene products function together to inhibit
mammalian target of rapamycin (mTOR)-mediated downstream signaling. Proc. Natl. Acad.
Sci. U. S. A. 99, 13571–13576.
Templeton, A.J., Dutoit, V., Cathomas, R., Rothermundt, C., Bärtschi, D., Dröge, C.,
Gautschi, O., Borner, M., Fechter, E., Stenner, F., et al. (2013). Phase 2 trial of single-agent
178
everolimus in chemotherapy-naive patients with castration-resistant prostate cancer (SAKK
08/08). Eur. Urol. 64, 150–158.
Tepper, R.I., Pattengale, P.K., and Leder, P. (1989). Murine interleukin-4 displays
potent anti-tumor activity in vivo. Cell 57, 503–512.
Tepper, R.I., Coffman, R.L., and Leder, P. (1992). An eosinophil-dependent
mechanism for the antitumor effect of interleukin-4. Science 257, 548–551.
Terme, M., Pernot, S., Marcheteau, E., Sandoval, F., Benhamouda, N., Colussi, O.,
Dubreuil, O., Carpentier, A.F., Tartour, E., and Taieb, J. (2013). VEGFA-VEGFR pathway
blockade inhibits tumor-induced regulatory T-cell proliferation in colorectal cancer. Cancer
Res. 73, 539–549.
Thiery-Vuillemin, A., Laheurte, C., Mansi, L., Royer, B., Pivot, X., Borg, C., and
Adotevi, O. (2014a). Immunomodulatory effects of everolimus in a long responsive patient
with metastatic renal cell carcinoma. J. Immunother. Hagerstown Md 1997 37, 51–54.
Thiery-Vuillemin, A., Mouillet, G., Nguyen Tan Hon, T., Montcuquet, P., Maurina,
T., Almotlak, H., Stein, U., Montange, D., Foubert, A., Nerich, V., et al. (2014b). Impact of
everolimus blood concentration on its anti-cancer activity in patients with metastatic renal cell
carcinoma. Cancer Chemother. Pharmacol. 73, 999–1007.
Thompson, R.H., Kuntz, S.M., Leibovich, B.C., Dong, H., Lohse, C.M., Webster,
W.S., Sengupta, S., Frank, I., Parker, A.S., Zincke, H., et al. (2006). Tumor B7-H1 is
associated with poor prognosis in renal cell carcinoma patients with long-term follow-up.
Cancer Res. 66, 3381–3385.
Thompson, R.H., Dong, H., Lohse, C.M., Leibovich, B.C., Blute, M.L., Cheville, J.C.,
and Kwon, E.D. (2007). PD-1 is expressed by tumor-infiltrating immune cells and is
associated with poor outcome for patients with renal cell carcinoma. Clin. Cancer Res. Off. J.
Am. Assoc. Cancer Res. 13, 1757–1761.
Thornton, A.M., Donovan, E.E., Piccirillo, C.A., and Shevach, E.M. (2004). Cutting
edge: IL-2 is critically required for the in vitro activation of CD4+CD25+ T cell suppressor
function. J. Immunol. Baltim. Md 1950 172, 6519–6523.
Thornton, A.M., Korty, P.E., Tran, D.Q., Wohlfert, E.A., Murray, P.E., Belkaid, Y.,
and Shevach, E.M. (2010). Expression of Helios, an Ikaros Transcription Factor Family
Member, Differentiates Thymic-Derived from Peripherally Induced Foxp3+ T Regulatory
Cells. J. Immunol. 184, 3433–3441.
Toge, H., Inagaki, T., Kojimoto, Y., Shinka, T., and Hara, I. (2009). Angiogenesis in
renal cell carcinoma: the role of tumor-associated macrophages. Int. J. Urol. Off. J. Jpn. Urol.
Assoc. 16, 801–807.
Tone, Y., Furuuchi, K., Kojima, Y., Tykocinski, M.L., Greene, M.I., and Tone, M.
(2008). Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer. Nat.
Immunol. 9, 194–202.
Turner, A.P., Shaffer, V.O., Araki, K., Martens, C., Turner, P.L., Gangappa, S., Ford,
M.L., Ahmed, R., Kirk, A.D., and Larsen, C.P. (2011). Sirolimus Enhances the Magnitude
and Quality of Viral-Specific CD8+ T-Cell Responses to Vaccinia Virus Vaccination in
Rhesus Macaques. Am. J. Transplant. 11, 613–618.
Turner, M.S., Kane, L.P., and Morel, P.A. (2009). Dominant role of antigen dose in
CD4+Foxp3+ regulatory T cell induction and expansion. J. Immunol. Baltim. Md 1950 183,
4895–4903.
Turnquist, H.R., Cardinal, J., Macedo, C., Rosborough, B.R., Sumpter, T.L., Geller,
D.A., Metes, D., and Thomson, A.W. (2010). mTOR and GSK-3 shape the CD4+ T-cell
stimulatory and differentiation capacity of myeloid DCs after exposure to LPS. Blood 115,
4758–4769.
179
Tzankov, A., Meier, C., Hirschmann, P., Went, P., Pileri, S.A., and Dirnhofer, S.
(2008). Correlation of high numbers of intratumoral FOXP3+ regulatory T cells with
improved survival in germinal center-like diffuse large B-cell lymphoma, follicular
lymphoma and classical Hodgkin’s lymphoma. Haematologica 93, 193–200.
Vacchelli, E., Aranda, F., Obrist, F., Eggermont, A., Galon, J., Cremer, I., Zitvogel, L.,
Kroemer, G., and Galluzzi, L. (2014). Trial watch: Immunostimulatory cytokines in cancer
therapy. Oncoimmunology 3, e29030.
Vajdic CM, McDonald SP, McCredie ME, and et al (2006). CAncer incidence before
and after kidney transplantation. JAMA 296, 2823–2831.
Vallotton, L., Hadaya, K., Venetz, J.-P., Buehler, L.H., Ciuffreda, D., Nseir, G.,
Codarri, L., Villard, J., Pantaleo, G., and Pascual, M. (2011). Monitoring of
CD4+CD25highIL-7R*high activated T cells in kidney transplant recipients. Clin. J. Am.
Soc. Nephrol. CJASN 6, 2025–2033.
Vander Haar, E., Lee, S.-I., Bandhakavi, S., Griffin, T.J., and Kim, D.-H. (2007).
Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell Biol. 9,
316–323.
Végran, F., Berger, H., Boidot, R., Mignot, G., Bruchard, M., Dosset, M., Chalmin, F.,
Rébé, C., Dérangère, V., Ryffel, B., et al. (2014). The transcription factor IRF1 dictates the
IL-21-dependent anticancer functions of TH9 cells. Nat. Immunol. 15, 758–766.
Vesely, M.D., Kershaw, M.H., Schreiber, R.D., and Smyth, M.J. (2011). Natural
innate and adaptive immunity to cancer. Annu. Rev. Immunol. 29, 235–271.
Vézina, C., Kudelski, A., and Sehgal, S.N. (1975). Rapamycin (AY-22,989), a new
antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active
principle. J. Antibiot. (Tokyo) 28, 721–726.
Vieweg, J., and Jackson, A. (2004). Antigenic targets for renal cell carcinoma
immunotherapy. Expert Opin. Biol. Ther. 4, 1791–1801.
Vignali, D.A.A., Collison, L.W., and Workman, C.J. (2008). How regulatory T cells
work. Nat. Rev. Immunol. 8, 523–532.
de Visser, K.E., Korets, L.V., and Coussens, L.M. (2005). De novo carcinogenesis
promoted by chronic inflammation is B lymphocyte dependent. Cancer Cell 7, 411–423.
Vivier, E., Tomasello, E., Baratin, M., Walzer, T., and Ugolini, S. (2008). Functions of
natural killer cells. Nat. Immunol. 9, 503–510.
Walker, M.R., Kasprowicz, D.J., Gersuk, V.H., Benard, A., Van Landeghen, M.,
Buckner, J.H., and Ziegler, S.F. (2003). Induction of FoxP3 and acquisition of T regulatory
activity by stimulated human CD4+CD25- T cells. J. Clin. Invest. 112, 1437–1443.
Wang, B.T., Ducker, G.S., Barczak, A.J., Barbeau, R., Erle, D.J., and Shokat, K.M.
(2011a). The mammalian target of rapamycin regulates cholesterol biosynthetic gene
expression and exhibits a rapamycin-resistant transcriptional profile. Proc. Natl. Acad. Sci. U.
S. A. 108, 15201–15206.
Wang, G.-Y., Yang, Y., Li, H., Zhang, J., Li, M.-R., Zhang, Q., and Chen, G.-H.
(2012a). Rapamycin combined with donor immature dendritic cells promotes liver allograft
survival in association with CD4(+) CD25(+) Foxp3(+) regulatory T cell expansion. Hepatol.
Res. Off. J. Jpn. Soc. Hepatol. 42, 192–202.
Wang, L., Harris, T.E., Roth, R.A., and Lawrence, J.C. (2007). PRAS40 regulates
mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding. J. Biol.
Chem. 282, 20036–20044.
Wang, L., Yi, T., Kortylewski, M., Pardoll, D.M., Zeng, D., and Yu, H. (2009). IL-17
can promote tumor growth through an IL-6-Stat3 signaling pathway. J. Exp. Med. 206, 1457–
1464.
180
Wang, Q.J., Hanada, K.-I., Robbins, P.F., Li, Y.F., and Yang, J.C. (2012b). Distinctive
features of the differentiated phenotype and infiltration of tumor-reactive lymphocytes in clear
cell renal cell carcinoma. Cancer Res. 72, 6119–6129.
Wang, Y., Camirand, G., Lin, Y., Froicu, M., Deng, S., Shlomchik, W.D., Lakkis,
F.G., and Rothstein, D.M. (2011b). Regulatory T cells require mammalian target of
rapamycin signaling to maintain both homeostasis and alloantigen-driven proliferation in
lymphocyte-replete mice. J. Immunol. Baltim. Md 1950 186, 2809–2818.
Wang, Y., Wang, X.-Y., Subjeck, J.R., Shrikant, P.A., and Kim, H.L. (2011c).
Temsirolimus, an mTOR inhibitor, enhances anti-tumour effects of heat shock protein cancer
vaccines. Br. J. Cancer 104, 643–652.
Wang, Y., Wang, X.-Y., Subjeck, J.R., Shrikant, P.A., and Kim, H.L. (2011d).
Temsirolimus, an mTOR inhibitor, enhances anti-tumour effects of heat shock protein cancer
vaccines. Br. J. Cancer 104, 643–652.
Wang, Y., Sparwasser, T., Figlin, R., and Kim, H.L. (2014). Foxp3+ T cells inhibit
antitumor immune memory modulated by mTOR inhibition. Cancer Res. 74, 2217–2228.
Weichhart, T., Costantino, G., Poglitsch, M., Rosner, M., Zeyda, M., Stuhlmeier,
K.M., Kolbe, T., Stulnig, T.M., Hörl, W.H., Hengstschläger, M., et al. (2008). The TSCmTOR Signaling Pathway Regulates the Innate Inflammatory Response. Immunity 29, 565–
577.
Wherry, E.J., Teichgräber, V., Becker, T.C., Masopust, D., Kaech, S.M., Antia, R.,
von Andrian, U.H., and Ahmed, R. (2003). Lineage relationship and protective immunity of
memory CD8 T cell subsets. Nat. Immunol. 4, 225–234.
Wilke, C.M., Kryczek, I., Wei, S., Zhao, E., Wu, K., Wang, G., and Zou, W. (2011).
Th17 cells in cancer: help or hindrance? Carcinogenesis 32, 643–649.
Williams, M.A., and Bevan, M.J. (2007). Effector and memory CTL differentiation.
Annu. Rev. Immunol. 25, 171–192.
Wofford, J.A., Wieman, H.L., Jacobs, S.R., Zhao, Y., and Rathmell, J.C. (2008). IL-7
promotes Glut1 trafficking and glucose uptake via STAT5-mediated activation of Akt to
support T-cell survival. Blood 111, 2101–2111.
Wu, Q., Kiguchi, K., Kawamoto, T., Ajiki, T., Traag, J., Carbajal, S., Ruffino, L.,
Thames, H., Wistuba, I., Thomas, M., et al. (2007). Therapeutic effect of rapamycin on
gallbladder cancer in a transgenic mouse model. Cancer Res. 67, 3794–3800.
Wu, Q., Liu, Y., Chen, C., Ikenoue, T., Qiao, Y., Li, C.-S., Li, W., Guan, K.-L., Liu,
Y., and Zheng, P. (2011). The tuberous sclerosis complex-mammalian target of rapamycin
pathway maintains the quiescence and survival of naive T cells. J. Immunol. Baltim. Md 1950
187, 1106–1112.
Yang, Z., and Klionsky, D.J. (2010). Eaten alive: a history of macroautophagy. Nat.
Cell Biol. 12, 814–822.
Yang, K., Neale, G., Green, D.R., He, W., and Chi, H. (2011). The tumor suppressor
Tsc1 enforces quiescence of naive T cells to promote immune homeostasis and function. Nat.
Immunol. 12, 888–897.
Yokosuka, T., Kobayashi, W., Takamatsu, M., Sakata-Sogawa, K., Zeng, H.,
Hashimoto-Tane, A., Yagita, H., Tokunaga, M., and Saito, T. (2010). Spatiotemporal Basis of
CTLA-4 Costimulatory Molecule-Mediated Negative Regulation of T Cell Activation.
Immunity 33, 326–339.
Yu, C.-R., Mahdi, R.M., Ebong, S., Vistica, B.P., Gery, I., and Egwuagu, C.E. (2003).
Suppressor of cytokine signaling 3 regulates proliferation and activation of T-helper cells. J.
Biol. Chem. 278, 29752–29759.
Yuan, T.L., and Cantley, L.C. (2008). PI3K pathway alterations in cancer: variations
on a theme. Oncogene 27, 5497–5510.
181
Zeiser, R., Leveson-Gower, D.B., Zambricki, E.A., Kambham, N., Beilhack, A., Loh,
J., Hou, J.-Z., and Negrin, R.S. (2008). Differential impact of mammalian target of rapamycin
inhibition on CD4+CD25+Foxp3+ regulatory T cells compared with conventional CD4+ T
cells. Blood 111, 453–462.
Zeng, H., Yang, K., Cloer, C., Neale, G., Vogel, P., and Chi, H. (2013). mTORC1
couples immune signals and metabolic programming to establish T(reg)-cell function. Nature
499, 485–490.
Zhang, S., Readinger, J.A., DuBois, W., Janka-Junttila, M., Robinson, R., Pruitt, M.,
Bliskovsky, V., Wu, J.Z., Sakakibara, K., Patel, J., et al. (2011). Constitutive reductions in
mTOR alter cell size, immune cell development, and antibody production. Blood 117, 1228–
1238.
Zhang, W., Zhang, D., Shen, M., Liu, Y., Tian, Y., Thomson, A.W., Lee, W.P.A., and
Zheng, X.X. (2010a). Combined administration of a mutant TGF-beta1/Fc and rapamycin
promotes induction of regulatory T cells and islet allograft tolerance. J. Immunol. Baltim. Md
1950 185, 4750–4759.
Zhang, Y.-L., Li, J., Mo, H.-Y., Qiu, F., Zheng, L.-M., Qian, C.-N., and Zeng, Y.-X.
(2010b). Different subsets of tumor infiltrating lymphocytes correlate with NPC progression
in different ways. Mol. Cancer 9, 4.
Zhao, T., Du, H., Ding, X., Walls, K., and Yan, C. (2015). Activation of mTOR
pathway in myeloid-derived suppressor cells stimulates cancer cell proliferation and
metastasis in lal|[minus]|/|[minus]| mice. Oncogene 34, 1938–1948.
Zheng, Y., Delgoffe, G.M., Meyer, C.F., Chan, W., and Powell, J.D. (2009). Anergic
T cells are metabolically anergic. J. Immunol. Baltim. Md 1950 183, 6095–6101.
Zhu, J., Yamane, H., and Paul, W.E. (2010). Differentiation of effector CD4 T cell
populations (*). Annu. Rev. Immunol. 28, 445–489.
Zinzalla, V., Stracka, D., Oppliger, W., and Hall, M.N. (2011). Activation of
mTORC2 by association with the ribosome. Cell 144, 757–768.
Zitvogel, L., Apetoh, L., Ghiringhelli, F., André, F., Tesniere, A., and Kroemer, G.
(2008). The anticancer immune response: indispensable for therapeutic success? J. Clin.
Invest. 118, 1991–2001.
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ANNEXES
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COMMENTARY
COMMENTARY
Human Vaccines & Immunotherapeutics 9:5, 1073–1077; May 2013; © 2013 Landes Bioscience
Olivier Adotévi,1,2,3,4,* Magalie Dosset,1,2,3 Jeanne Galaine,1,2,3 Laurent Beziaud,1,2,3 Yann Godet1,2,3 and Christophe Borg1,2,3,4
1
INSERM; Unité Mixte de Recherche 1098; Besançon, France; 2Etablissement Français du Sang de Bourgogne Franche-Comté; UMR1098; Besançon cedex,
France; 3Université de Franche-Comté; UMR1098; SFR IBCT; Besançon, France; 4CHRU de Besançon; Service d’Oncologie; Besançon, France
C
Downloaded by [Inserm Disc Ist] at 14:53 29 November 2015
urrent cancer immunotherapies
predominantly rely on CD8 + T
cells to fight against tumors. However
accumulative evidence showed that proinflammatory CD4 + helper T cells are
critical determinants of effective antitumor immunity. The utilization of
universal tumor-reactive helper peptides
from telomerase represents a powerful
approach to the fully use of CD4 + T cellbased immunotherapy.
Critical Roles of CD4+ Helper T
Cells in Antitumor Immunity
Keywords: CD4 T cell, helper peptide,
telomerase, cancer vaccine
Submitted: 01/04/13
Accepted: 01/13/13
http://dx.doi.org/10.4161/hv.23587
*Correspondence to: Olivier Adotévi;
Email: [email protected]
www.landesbioscience.com
The cancer immunoediting hypothesis
indicates that tumor cells could be immunogenic and that the adaptive immune
system is involved in the active elimination and selection of tumor cells.1 Among
adaptive immune cells involved in antitumor responses, CD8 + T cells (CTL)
have been considered as the main protagonists because they exhibit a direct
cytotoxic activity toward cancer cells.
However, recent advances on the fields
indicate that different subpopulations of
CD4 + T helper (TH) lymphocytes regulate
the antitumor response.2 Among them,
tumor-reactive CD4 + T helper 1 cells
(TH1), which produce IFN-γ, TNF-α
and IL-2, play a critical role in the orchestration of cell-mediated immunity against
tumors.3 The concept of CD4 + T-cell help
initially emerged from studies showing
that successful generation of antitumor
CTL depends on the presence of CD4 +
T cells. Hence, adoptive cell transfer with
CD4 + TH cells induces tumor protection
or regression, whereas depletion of CD4 +
Human Vaccines & Immunotherapeutics
T cells inhibits vaccine-induced protective
immunity.4,5
One generally accepted model implies
that CD4 + T cells are necessary to license
dendritic cells (DC) for efficient CD8 +
T-cell priming through the interaction of
costimulatory receptors such as CD40CD40L.6,7 TH1 cells also promote NK
cells and macrophages (M1) activation in
vivo.2,8 They have also been shown to contribute to the inhibition of tumor angiogenesis via an IFN-γ and TSP-1 dependent
pathway.9,10 Accordingly, in human, high
density of tumor-infiltrating TH1 cells has
been shown as a good prognostic marker
in several cancers.11 The expression of the
TH1 specific transcription factor Tbet in
tumor infiltrating lymphocytes predicts
survival of breast cancer patients treated
with traztuzumab and chemotherapy.12
All above emphasize the growing interest
to specifically target tumor-reactive CD4
TH1 cells for cancer immunotherapy.
Use of Universal CD4 Helper
Peptides from Telomerase
as a Relevant Tool to Target
CD4 T Cells in Vivo
Because CTLs have been shown as the
most powerful effector cells, most previous cancer vaccines targeted MHC
class I-restricted peptides derived from
tumor antigens to stimulate anticancer CTL responses.13 In the meanwhile;
CD4 + TH cells have gained interest in
antitumor immunity and immunotherapy. As a result, increasing attention has
focused on identifying MHC class II
epitopes from tumor antigens to actively
1073
©2013 Landes Bioscience. Do not distribute.
Targeting antitumor CD4 helper T cells with universal tumor-reactive
helper peptides derived from telomerase for cancer vaccine
1074
UCP-specific CD4 + T cells fulfill helper
features necessary to generate potent cellular antitumor responses. Indeed, the
addition of UCP as helper peptide drastically increased tumor-specific CD8 +
T cell responses. We showed that UCPbased vaccine was associated with high
antitumor CTL avidity and memory, two
critical functions for tumor eradication.
Furthermore, the magnitude and quality
of the CD8 + T cell responses were closely
correlated with the number of IFN-γ and
IL-2-secreting UCP-specific CD4 + T cells
in vivo. The induction of DC activation
represents one major helper mechanism
used by CD4 + TH1 cells to sustain antigen presentation and to provide costimulatory signals to CTL. This is referred as
the “ménage à trois” model.23 Fully DC
activation was also found increased in
vivo following UCP vaccination and in
vitro after coculture of immature DC with
UCP-specific CD4 + T cells. The upregulation of activation markers such as CD86
and MHC class II on DC depends on
both IFN-γ and GM-CSF secretion and
CD40L expression by UCP-specific CD4 +
T cells (Fig. 1). Finally, by using a model
of transplantable mouse melanoma (B16HLA-A2),24 we showed that the addition
of UCP as helper peptide was required for
effective protection against tumor growth
in a therapeutic peptide vaccine using
the HLA-A2+ self/TERT CTL peptides
(pY988 or pY572).25,26 Collectively our
results provide a robust method to comprehensively analyze tumor-derived helper
peptides and support that the stimulation
of tumor-reactive CD4 TH1 cells is a powerful method to improve the efficacy of
cancer vaccines.
Tumor-Reactive Helper
Peptides are More Effective
for Intratumoral Recruitment
of Effector CD8+ T Cells
A critical consideration for vaccination
is the nature of the helper peptide used.
One approach to induction of CD4 + T cell
help is to use of xenogenic or non-tumor
antigens that stimulate recall responses
or nonspecific help. The synthetic helper
peptide PADRE derived from keyhole
limpet hemocyanin (KLH), and the tetanus toxoid-derived helper peptide are
Human Vaccines & Immunotherapeutics
commonly used in anticancer vaccines.27-30
Although, tumor-specific CTL responses
appear to be increased by co-administration of non-tumor helper peptide, the
clinical benefit of this strategy has not
been clearly established, as exemplified in
a recent study in melanoma. In this report,
patients were vaccinated with MHC class
I tumor-derived peptides in conjunction with either helper peptides derived
from melanoma antigens or the tetanusderived helper peptide. Although higher
CD8 + T cell responses were induced in
the arm with tetanus helper peptide than
in melanoma helper peptides one, the clinical impact was quite similar in the two
groups.31 One explanation could be related
to the inefficiency of non-tumor specific
CD4 + T cells to guide effector CD8 + T cells
within the tumor as recently demonstrated
by Sherman L and colleagues.32,33 Indeed,
CD8 + effector T-cell recruitment within
the tumor was enhanced by tumor-specific
CD4 + T cells and this effect was promoted
by IFN-γ-dependent production of chemokines such as CXCL9 and CXCL10. In
addition, the production of IL-2 by tumor
resident CD4 + T cells enhanced CD8 +
T-cell proliferation and function.34
In our study, the use of UCP as helper
peptide during therapeutic vaccine promoted tumor infiltration by tumor-specific CD8 + T cells which explain the best
tumor control observed in mice (Fig. 1).22
In a previous related study, Gross and
colleagues reported that vaccination
with the same self/TERT CTL peptides
(pY572 and pY988) induced tumor protection only when coupled with a CD4 +
helper peptide derived from the hepatitis
B virus.35 However around 25% of mice
vaccinated prophylactically achieved
tumor protection compared with 60%
in our therapeutic vaccine study using
TERT-derived UCP. Thus we speculate
that the difference observed in the two
studies could be related to the specificity
of the helper signal delivered by CD4 + T
cells. This new role of CD4 + helper T cells
on CD8 T attraction to the site of attack
emerges as a new general mechanism and
has also been reported by Nakanishi et
al. in the case of infected mucosa.36 Thus
only tumor-reactive CD4 + T cells are
effectively able to induce a better homing
of killer cells at the tumor site.
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©2013 Landes Bioscience. Do not distribute.
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target antitumor CD4 + T cells in vivo.14,15
However, the use of tumor-reactive MHC
class II helper peptide should require particular caution to prevent the induction of
detrimental immune response as different
subpopulations of CD4 + T cells are known
to regulate host immune responses.16
For instance, TH2 and regulatory T cell
are frequently associated with an inhibitory environment within the tumor17 and
the role of TH17 cells in the antitumor
response is still controversial and seems to
depend on the type of cancer.11,18
In a recent study, we used an optimized
reverse immunology approach to identify four novel MHC class II-restricted
peptides derived from human telomerase
reverse transcriptase (TERT).19 TERT
maintains telomere length in dividing
cells and its expression is the predominant
mechanism developed by malignant cells
to escape telomere-dependent cell death.20
Telomerase activity has been observed in
the vast majority of cancers and emerges
as a clinically relevant tumor antigen for
immunotherapy.21 These novel TERTderived peptides referred as “Universal
Cancer Peptides” (UCP), effectively bind
to most commonly found HLA-DR molecules which increase their applicability in
a large number of cancer patients.19 This
promiscuous binding capacity of UCP
circumvents one major limit of the clinical use of tumor-derived helper peptides
that only bind few MHC class II alleles.14
UCP-specific CD4 + T-cell repertoire is
present in human and naturally occurring
CD4 + T-cells responses against UCP were
detected in patients with various types of
cancers and these cells mainly produce
IFN-γ and TNF-α revealing their TH1
polarization.
In a second study, UCP were use to
actively target CD4 + T cells in a preclinical tumor model and the helper properties
of UCP-specific CD4 + T cells were systematically analyzed.22 Using the HLAA2/HLA-DR1 transgenic mouse model,
we showed that UCP vaccinations induce
high avidity and tumor-specific CD4 + TH1
polarized responses. The UCP-specific
CD4 + T cells produced high amount of
IFN-γ and IL-2 and but no IL-4, IL-5,
IL-10 and IL-17. Co-immunization of
MHC class I restricted tumor-derived peptides with or without UCP showed that
,QWHUSOD\%HWZHHQ7(576SHFLÀF
CD4+ T Immune Responses
and Cytotoxic Chemotherapy:
An Emerging Synergistic
Antitumor Effects
In a report by Godet and colleagues,
we used the universal characteristic of
UCP to monitor the UCP-specific CD4 +
T-cell responses in metastatic non-small
cell lung carcinoma (NSCLC) patients
treated by platinum-based doublet chemotherapy. Naturally occurring UCPspecific TH1 CD4 responses were found
in 38% of these patients prior chemotherapy. We observed that the presence of
this response prior treatment significantly
www.landesbioscience.com
increases the survival of chemotherapy
responding patients (median overall survival: 13.2 vs 10 mo, p = 0.034).19 Of
note, antiviral T-cell responses measured
at the same time in the two groups of
patients were similar and had no effect
on survival. On other hand, patients with
progressive disease after first line chemotherapy do not benefit from UCP-specific
immune responses. These results strongly
suggested the interplay between UCPspecific CD4 TH1-cell immunity and chemotherapy efficacy.
Original work from Laurence Zitvogel
and Guido Kroemer highlighted the capacity of several anticancer agents including
classical chemotherapeutics, and targeted
Human Vaccines & Immunotherapeutics
compounds stimulate tumor-specific
immune responses either by inducing the
immunogenic death of tumor cells or by
engaging immune effector mechanisms.37
Such an immunogenic cell death relies on
the coordinated emission of specific signals from dying cancer cells and their perception by the host immune system. The
molecular mechanisms imply the early
exposition of calreticulin, ERP57 and the
MRP on the cell surface together with
the secretion of ATP.38 In line with these
observations, our data suggest that the
tumor cell lysis induced by chemotherapy
promotes an immunological milieu and
the release of TERT, which is taken up by
antigen presenting cells that subsequently
1075
©2013 Landes Bioscience. Do not distribute.
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Figure 1. Antitumor immune effects of novel universal CD4 helper peptides derived from telomerase (UCP). UCP-based antitumor vaccines favor DC
activation which provides costimulatory signals to antitumor CTL, enhancing their quantity, quality and recruitment at the tumor site. Concomittantly
chemotherapy can act by promoting immunogenic cell death and antitumor T cell activation.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were
disclosed.
12.
Acknowledgments
The authors wish to acknowledge funding
received from Ligue Contre le Cancer, and
Association pour la Recherche contre le
Cancer, France and grant from the province of Franche Comté, France.
1076
13.
14.
Vesely MD, Kershaw MH, Schreiber RD, Smyth
MJ. Natural innate and adaptive immunity to
cancer. Annu Rev Immunol 2011; 29:235-71;
PMID :21219185;
http://dx.doi.org/10.1146/
annurev-immunol-031210-101324.
Kennedy R, Celis E. Multiple roles for CD4+ T cells
in anti-tumor immune responses. Immunol Rev
2008; 222:129-44; PMID:18363998; http://dx.doi.
org/10.1111/j.1600-065X.2008.00616.x.
Pardoll DM, Topalian SL. The role of CD4+ T
cell responses in antitumor immunity. Curr Opin
Immunol 1998; 10:588-94; PMID:9794842; http://
dx.doi.org/10.1016/S0952-7915(98)80228-8.
Fayolle C, Deriaud E, Leclerc C. In vivo induction of cytotoxic T cell response by a free synthetic
peptide requires CD4+ T cell help. J Immunol 1991;
147:4069-73; PMID:1684372.
Hunder NN, Wallen H, Cao J, Hendricks DW,
Reilly JZ, Rodmyre R, et al. Treatment of metastatic melanoma with autologous CD4+ T cells
against NY-ESO-1. N Engl J Med 2008; 358:2698703; PMID:18565862; http://dx.doi.org/10.1056/
NEJMoa0800251.
Bennett SR, Carbone FR, Karamalis F, Flavell RA,
Miller JF, Heath WR. Help for cytotoxic-T-cell
responses is mediated by CD40 signalling. Nature
1998; 393:478-80; PMID:9624004; http://dx.doi.
org/10.1038/30996.
Smith CM, Wilson NS, Waithman J, Villadangos
JA, Carbone FR, Heath WR, et al. Cognate CD4(+)
T cell licensing of dendritic cells in CD8(+) T
cell immunity. Nat Immunol 2004; 5:1143-8;
PMID:15475958; http://dx.doi.org/10.1038/ni1129.
Fehniger TA, Cooper MA, Nuovo GJ, Cella M,
Facchetti F, Colonna M, et al. CD56bright natural
killer cells are present in human lymph nodes and
are activated by T cell-derived IL-2: a potential new
link between adaptive and innate immunity. Blood
2003; 101:3052-7; PMID:12480696; http://dx.doi.
org/10.1182/blood-2002-09-2876.
Qin Z, Blankenstein T. CD4+ T cell--mediated
tumor rejection involves inhibition of angiogenesis
that is dependent on IFN gamma receptor expression
by nonhematopoietic cells. Immunity 2000; 12:67786; PMID:10894167; http://dx.doi.org/10.1016/
S1074-7613(00)80218-6.
Rakhra K, Bachireddy P, Zabuawala T, Zeiser R,
Xu L, Kopelman A, et al. CD4(+) T cells contribute to the remodeling of the microenvironment
required for sustained tumor regression upon oncogene inactivation. Cancer Cell 2010; 18:485-98;
PMID:21035406 ;
http://dx.doi.org/10.1016/j.
ccr.2010.10.002.
Fridman WH, Pagès F, Sautès-Fridman C, Galon J.
The immune contexture in human tumours: impact
on clinical outcome. Nat Rev Cancer 2012; 12:298306; PMID:22419253; http://dx.doi.org/10.1038/
nrc3245.
Ladoire S, Arnould L, Mignot G, Apetoh L, Rébé
C, Martin F, et al. T-bet expression in intratumoral
lymphoid structures after neoadjuvant trastuzumab
plus docetaxel for HER2-overexpressing breast carcinoma predicts survival. Br J Cancer 2011; 105:36671; PMID:21750556; http://dx.doi.org/10.1038/
bjc.2011.261.
Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines. Nat
Med 2004; 10:909-15; PMID:15340416; http://
dx.doi.org/10.1038/nm1100.
Kobayashi H, Celis E. Peptide epitope identification
for tumor-reactive CD4 T cells. Curr Opin Immunol
2008; 20:221-7; PMID:18499419; http://dx.doi.
org/10.1016/j.coi.2008.04.011.
Human Vaccines & Immunotherapeutics
15. Knutson KL, Disis ML. Tumor antigen-specific T
helper cells in cancer immunity and immunotherapy. Cancer Immunol Immunother 2005; 54:7218; PMID:16010587; http://dx.doi.org/10.1007/
s00262-004-0653-2.
16. Zhu J, Paul WE. CD4 T cells: fates, functions, and
faults. Blood 2008; 112:1557-69; PMID:18725574;
http://dx.doi.org/10.1182/blood-2008-05-078154.
17. De Monte L, Reni M, Tassi E, Clavenna D, Papa
I, Recalde H, et al. Intratumor T helper type 2
cell infiltrate correlates with cancer-associated fibroblast thymic stromal lymphopoietin production and
reduced survival in pancreatic cancer. J Exp Med
2011; 208:469-78; PMID:21339327; http://dx.doi.
org/10.1084/jem.20101876.
18. Tosolini M, Kirilovsky A, Mlecnik B, Fredriksen
T, Mauger S, Bindea G, et al. Clinical impact
of different classes of infiltrating T cytotoxic and
helper cells (Th1, th2, treg, th17) in patients with
colorectal cancer. Cancer Res 2011; 71:1263-71;
PMID:21303976; http://dx.doi.org/10.1158/00085472.CAN-10-2907.
19. Godet Y, Fabre E, Dosset M, Lamuraglia M,
Levionnois E, Ravel P, et al. Analysis of spontaneous
tumor-specific CD4 T-cell immunity in lung cancer using promiscuous HLA-DR telomerase-derived
epitopes: potential synergistic effect with chemotherapy response. Clin Cancer Res 2012; 18:2943-53;
PMID:22407833; http://dx.doi.org/10.1158/10780432.CCR-11-3185.
20. Martínez P, Blasco MA. Telomeric and extratelomeric roles for telomerase and the telomerebinding proteins. Nat Rev Cancer 2011; 11:16176; PMID:21346783; http://dx.doi.org/10.1038/
nrc3025.
21. Harley CB. Telomerase and cancer therapeutics.
Nat Rev Cancer 2008; 8:167-79; PMID:18256617;
http://dx.doi.org/10.1038/nrc2275.
22. Dosset M, Godet Y, Vauchy C, Beziaud L, Lone YC,
Sedlik C, et al. Universal cancer peptide-based therapeutic vaccine breaks tolerance against telomerase
and eradicates established tumor. Clin Cancer Res
2012; 18:6284-95; PMID:23032748; http://dx.doi.
org/10.1158/1078-0432.CCR-12-0896.
23. Ridge JP, Di Rosa F, Matzinger P. A conditioned dendritic cell can be a temporal bridge between a CD4+
T-helper and a T-killer cell. Nature 1998; 393:474-8;
PMID:9624003; http://dx.doi.org/10.1038/30989.
24. Adotévi O, Mollier K, Neuveut C, Dosset M, Ravel
P, Fridman WH, et al. Targeting human telomerase reverse transcriptase with recombinant lentivector is highly effective to stimulate antitumor CD8
T-cell immunity in vivo. Blood 2010; 115:3025-32;
PMID:20130242; http://dx.doi.org/10.1182/blood2009-11-253641.
25. Hernandez J, Garcia-Pons F, Lone YC, Firat
H, Schmidt JD, Langlade-Demoyen P, et al.
Identification of a human telomerase reverse transcriptase peptide of low affinity for HLA A2.1
that induces cytotoxic T lymphocytes and mediates lysis of tumor cells. Proc Natl Acad Sci U S A
2002; 99:12275-80; PMID:12218171; http://dx.doi.
org/10.1073/pnas.182418399.
26. Scardino A, Gross DA, Alves P, Schultze JL, GraffDubois S, Faure O, et al. HER-2/neu and hTERT
cryptic epitopes as novel targets for broad spectrum
tumor immunotherapy. J Immunol 2002; 168:59006; PMID:12023395.
27. del Guercio MF, Alexander J, Kubo RT, Arrhenius
T, Maewal A, Appella E, et al. Potent immunogenic
short linear peptide constructs composed of B cell
epitopes and Pan DR T helper epitopes (PADRE) for
antibody responses in vivo. Vaccine 1997; 15:441-8;
PMID:9141216; http://dx.doi.org/10.1016/S0264410X(97)00186-2.
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amplify preexisting tumor-specific CD4 +
T cells.39 An additional postchemotherapy
monitoring of UCP-specific CD4 + T cell
responses will be performed for a complete
study of CD4 + T cell modulation.
In contrast to CD8 T cell responses,
few data are available on the mechanisms by which anticancer drugs modulate antitumor CD4 + T cell immunity.38
Although cyclophosphamide (CTX) has
been thought as the most potent CD4activating anticancer drug in several
experimental models; the mechanisms
underlying its effect are not well understood. In addition to its well-known
effect of depleting suppressor T cells,
recent data suggest a link between CD4 +
T-cell responses and an immunogenic
milieu such as proinflammatory cytokines induced by CTX.40 We and others
have shown that blocking VEGF/VEGFR
pathway with antiangiogenic drugs modulates CD4 + Foxp3 + regulatory T cells
number and functions in tumor-bearing
mice and in metastatic cancer patients.41,42
Thus, understanding how the efficiency
of conventional chemotherapy influenced
CD4 + helper T cell response is a challenging study for chemoimmunotherapy.
In conclusion, there is great interest
for cancer vaccines to stimulate tumorreactive TH1 responses by using tumorreactive CD4 helper peptides such as
TERT-derived UCP that would extend
the potential application to various types
of cancers. These results also provide a
new tool for comprehensive monitoring
of antitumor CD4 + T cell responses and
support the concept of the immunomodulation of chemotherapy efficacy in cancer
patients.
www.landesbioscience.com
35. Gross DA, Graff-Dubois S, Opolon P, Cornet S,
Alves P, Bennaceur-Griscelli A, et al. High vaccination efficiency of low-affinity epitopes in antitumor
immunotherapy. J Clin Invest 2004; 113:425-33;
PMID:14755339.
36. Nakanishi Y, Lu B, Gerard C, Iwasaki A. CD8(+)
T lymphocyte mobilization to virus-infected tissue
requires CD4(+) T-cell help. Nature 2009; 462:5103; PMID:19898495; http://dx.doi.org/10.1038/
nature08511.
37. Zitvogel L, Kepp O, Kroemer G. Immune parameters affecting the efficacy of chemotherapeutic
regimens. Nat Rev Clin Oncol 2011; 8:151-60;
PMID:21364688; http://dx.doi.org/10.1038/nrclinonc.2010.223.
38. Galluzzi L, Senovilla L, Zitvogel L, Kroemer G.
The secret ally: immunostimulation by anticancer drugs. Nat Rev Drug Discov 2012; 11:21533; PMID:22301798; http://dx.doi.org/10.1038/
nrd3626.
39. Godet Y, Dosset M, Borg C, Adotevi O. Is preexisting
antitumor CD4 T cell response indispensable for the
chemotherapy induced immune regression of cancer?
Oncoimmunology 2012; 1:1617-9; PMID:23264913;
http://dx.doi.org/10.4161/onci.21513.
Human Vaccines & Immunotherapeutics
40. Ding ZC, Zhou G. Cytotoxic chemotherapy and
CD4+ effector T cells: an emerging alliance for
durable antitumor effects. Clin Dev Immunol 2012;
2012:890178; PMID:22400040; http://dx.doi.
org/10.1155/2012/890178.
41. Terme M, Pernot S, Marcheteau E, Sandoval F,
Benhamouda N, Colussi O, et al. VEGFA-VEGF
Receptor pathway blockade inhibits tumor-induced
regulatory T cell proliferation in colorectal cancer. Cancer Res 2013; 73:539-49; PMID:23108136;
http://dx.doi.org/10.1158/0008-5472.CA N-122325.
42. Adotevi O, Pere H, Ravel P, Haicheur N, Badoual
C, Merillon N, et al. A decrease of regulatory
T cells correlates with overall survival after sunitinib-based antiangiogenic therapy in metastatic
renal cancer patients. J Immunother 2010; 33:9918; PMID:20948437; http://dx.doi.org/10.1097/
CJI.0b013e3181f4c208.
1077
©2013 Landes Bioscience. Do not distribute.
Downloaded by [Inserm Disc Ist] at 14:53 29 November 2015
28. Helling F, Zhang S, Shang A, Adluri S, Calves M,
Koganty R, et al. GM2-KLH conjugate vaccine:
increased immunogenicity in melanoma patients after
administration with immunological adjuvant QS-21.
Cancer Res 1995; 55:2783-8; PMID:7796403.
29. Scheibenbogen C, Schadendorf D, Bechrakis NE,
Nagorsen D, Hofmann U, Servetopoulou F, et al.
Effects of granulocyte-macrophage colony-stimulating factor and foreign helper protein as immunologic
adjuvants on the T-cell response to vaccination with
tyrosinase peptides. Int J Cancer 2003; 104:18894; PMID:12569574; http://dx.doi.org/10.1002/
ijc.10961.
30. Valmori D, Pessi A, Bianchi E, Corradin G. Use of
human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination. J Immunol
1992; 149:717-21; PMID:1378079.
31. Slingluff CL Jr., Petroni GR, Chianese-Bullock KA,
Smolkin ME, Ross MI, Haas NB, et al. Randomized
multicenter trial of the effects of melanoma-associated
helper peptides and cyclophosphamide on the immunogenicity of a multipeptide melanoma vaccine. J
Clin Oncol 2011; 29:2924-32; PMID:21690475;
http://dx.doi.org/10.1200/JCO.2010.33.8053.
32. Wong SB, Bos R, Sherman LA. Tumor-specific
CD4+ T cells render the tumor environment permissive for infiltration by low-avidity CD8+ T cells. J
Immunol 2008; 180:3122-31; PMID:18292535.
33. Marzo AL, Kinnear BF, Lake RA, Frelinger JJ,
Collins EJ, Robinson BW, et al. Tumor-specific
CD4+ T cells have a major “post-licensing” role in
CTL mediated anti-tumor immunity. J Immunol
2000; 165:6047-55; PMID:11086036.
34. Bos R, Sherman LA. CD4+ T-cell help in the
tumor milieu is required for recruitment and cytolytic function of CD8+ T lymphocytes. Cancer Res
2010; 70:8368-77; PMID:20940398; http://dx.doi.
org/10.1158/0008-5472.CAN-10-1322.
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AUTHOR’S VIEW
AUTHOR’S VIEW
OncoImmunology 2:3, e23430; March 2013; © 2013 Landes Bioscience
Universal tumor-reactive helper peptides
from telomerase as new tools
for anticancer vaccination
Magalie Dosset,1,2,3 Charline Vauchy,1,2,3 Laurent Beziaud,1,2,3 Olivier Adotevi1,2,3,4 and Yann Godet1,2,3,*
INSERM; Unité Mixte de Recherche 1098; Besançon, France; 2Etablissement Français du Sang de Bourgogne Franche-Comté; UMR1098; Besançon, France;
3
Université de Franche-Comté; UMR1098; SFR IBCT; Besançon, France; 4CHRU de Besançon; Service d’Oncologie; Besançon, France
1
Downloaded by [Inserm Disc Ist] at 14:55 29 November 2015
Keywords: CD4 T cell, cancer vaccine, helper peptide, immunotherapy, telomerase
Accumulating evidence demonstrates the importance of CD4+ T cells in antitumor immune responses. Identifying
promiscuous MHC Class II-binding peptides derived from relevant tumor-associated antigens that specifically target
CD4+ helper T cells in vivo represent a powerful approach to fully exploit these cells for anticancer immunotherapy.
Recent advances indicate that adaptive
immune responses play a critical role
in cancer immunosurveillance. Among
various cell types involved in adaptive
immunity, CD4 + helper T-cell subpopulations are critical for antitumor immune
response.1 In particular, tumor-reactive
CD4 + T helper 1 cells (TH1) produce
cytokines such as interferon γ (IFNγ),
tumor necrosis factor α (TNFα) and
interleukin (IL-)2, which are essential for
the induction of cell-mediated immunity
against tumors. Thus, CD4 + TH1 cells
play a key role in “helping” antigen-specific CD8 + T cells to efficiently undergo
activation and proliferation. According
to a commonly accepted model, CD4 +
T cells license dendritic cells (DCs) for
efficient CD8 + T-cell priming through
the interaction of co-stimulatory receptors such as CD40 with their ligands
(e.g., CD40L).2 Supporting the critical
role of CD4 + T cells in antitumor immunity, a high density of tumor-infiltrating
TH1 cells has been shown to constitute a
good prognostic marker in several cancers.3 Hence, there is a growing interest to
specifically stimulate CD4 + TH1 cells for
cancer immunotherapy.
We have recently described four MHC
Class II-restricted peptides derived from
human telomerase reverse transcriptase
(TERT). TERT is a prototype of universal tumor-associated antigen, as it is overexpressed by the vast majority of human
cancers and appears as an attractive target
for anticancer immunotherapy.4 These
novel peptides, which we referred to as
“Universal Cancer Peptides” (UCPs), efficiently bind various HLA-DR molecules,
increasing their likelihood to be immunogenic in a large number of patients.5 Such a
promiscuous binding capacity of UCPs circumvents one major limitation of the clinical use of tumor-derived peptides, which
only bind a few MHC Class II molecules.
In a recent report, UCPs were used
to actively target CD4 + TH1 cells in vivo
and the helper properties of UCP-specific
CD4 + T cells were systematically analyzed
in a preclinical tumor model.6 Using the
HLA-A2/HLA-DR1 transgenic mouse
model, we showed that vaccinations with
UCPs induce high-avidity specific CD4 +
TH1 responses. These UCP-specific CD4 +
T cells produce high amount of IFNγ
and IL-2 and but not IL-4, IL-5, IL-10
and IL-17. The immunization of mice
with MHC Class I-restricted tumor peptide alone or combined with UCP showed
that UCP-specific CD4 + T cells fulfill
the helper features that are necessary
to generate potent cellular antitumor
responses. Indeed, the addition of UCPs
as helper peptides drastically increased
tumor-specific cytotoxic T lymphocyte
(CTL) responses and mice survival. The
magnitude and quality of CTL responses
were closely correlated with the strength
of UCP-specific CD4 + TH1 responses.
The activation of DCs was also found to
be increased in vivo following vaccination
with UCPs and in vitro after the co-culture of immature DCs with UCP-specific
CD4 + T cells.
We demonstrated that the mechanism underpinning the upregulation of
activation markers such as CD86 and
MHC Class II on DCs involves both
the secretion of IFNγ and granulocytemacrophage colony-stimulating factor
(GM-CSF) and the expression of CD40L
by UCP-specific CD4 + T cells. Finally,
by using a model of transplantable mouse
melanoma (B16-HLA-A2 cells), we
showed that the addition of UCPs to a
MHC Class I peptide-based therapeutic
vaccination is necessary to promote tumor
regression by fostering the recruitment of
tumor-specific CD8 + effector T cells at the
tumor site. Altogether, these results indicate that the stimulation of UCP-specific
CD4 + TH1 cells may constitute a powerful
*Correspondence to: Yann Godet; Email: [email protected]
Submitted: 12/20/12; Accepted: 12/28/12
Citation: Dosset M, Vauchy C, Beziaud L, Adotevi O, Godet Y. Universal tumor-reactive helper peptides from telomerase as new tools for anticancer vaccination.
OncoImmunology 2013; 2:e23430; http://dx.doi.org/10.4161/onci.23430
www.landesbioscience.com
OncoImmunology
e23430-1
Downloaded by [Inserm Disc Ist] at 14:55 29 November 2015
Figure 1. Cellular effects of universal cancer peptides-based antitumor vaccination. CTL, cytotoxic
T lymphocyte; DC, dendritic cell; GM-CSF, granulocyte-macrophage colony-stimulating factor;
IFNγ, interferon γ; IL, interleukin; TCR, T-cell receptor; TH1; T helper 1; UCP, universal cancer peptides.
method to improve the efficiency of anticancer vaccines (Fig. 1).
During the past ten years, great interest
has been attracted by the characterization
of immunogenic helper epitopes derived
from tumor-associated antigens to actively
target CD4 + T cells.7 However, the use of
tumor-reactive CD4 + T cell-targeting peptides requires particular caution to prevent
e23430-2
the induction of detrimental immune
responses, as different subpopulations
of CD4 + TH cells are known to regulate
host antitumor immune responses. For
instance, TH2 and regulatory T cells are
frequently associated with the establishment of an immunosuppressive environment within the tumor, whereas the role of
TH17 cells is still controversial and seems
OncoImmunology
to vary, at least in part, with cancer type.1,3
Thus, only TH1 immune response have
been shown to mediate bona fide anticancer effects, providing a strong rationale to
develop anticancer vaccines that stimulate
antitumor TH1 immunity. Nevertheless,
only few tumor-reactive CD4 + T cell-targeting peptides are currently used in clinical settings, and in most cases the helper
features of these peptides had not been
evaluated before their clinical deployment. Another critical consideration is the
nature of helper peptides used for anticancer vaccination. The synthetic helper
peptide PADRE, which is derived from
keyhole limpet hemocyanin (KLH), and
the tetanus toxoid-derived helper peptide
are commonly used in anticancer vaccines
although they have never provided a real
impact on disease outcome, as exemplified in a recent study involving melanoma
patients.8 In this clinical trial, patients were
vaccinated with MHC Class I-restricted
tumor-derived peptides in conjunction with either helper peptides derived
from melanoma antigens or the tetanus
toxoid-derived helper peptide. Although
more robust CD8 + T-cell responses were
induced in patients receiving the tetanus
toxoid-derived helper peptide than in
individuals getting the melanoma-derived
helper peptide, the clinical outcome was
relatively similar in the two groups. One
possible explanation for these observations
is that helper peptides unrelated to tumor
antigens may be ineffective in guiding
effector CD8 + T cells within the tumor,
as recently demonstrated by Sherman
and colleagues.9,10 Indeed, only CD4 +
T cells specific for tumor-associated antigens are effectively able to pave the way
for the entry of CTLs within the tumor.
In view of these findings, our results provide a robust method to comprehensively
analyze tumor-derived helper peptides for
anticancer vaccines.
In conclusion, there is great interest
to stimulate antitumor TH1 responses
by using universal tumor-reactive helper
peptides such as TERT-derived UCPs, as
these may potentially be applied to several
distinct types of cancers.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were
disclosed.
Volume 2 Issue 3
References
1.
2.
3.
6.
7.
Godet Y, Fabre E, Dosset M, Lamuraglia M, Levionnois
E, Ravel P, et al. Analysis of spontaneous tumor-specific
CD4 T-cell immunity in lung cancer using promiscuous HLA-DR telomerase-derived epitopes: potential
synergistic effect with chemotherapy response. Clin
Cancer Res 2012; 18:2943-53; PMID:22407833;
http://dx.doi.org/10.1158/1078-0432.CCR-11-3185.
Dosset M, Godet Y, Vauchy C, Beziaud L, Lone
YC, Sedlik C, et al. Universal cancer peptide-based
therapeutic vaccine breaks tolerance against telomerase
and eradicates established tumor. Clin Cancer Res
2012; 18:6284-95; PMID:23032748; http://dx.doi.
org/10.1158/1078-0432.CCR-12-0896.
Kobayashi H, Celis E. Peptide epitope identification
for tumor-reactive CD4 T cells. Curr Opin Immunol
2008; 20:221-7; PMID:18499419; http://dx.doi.
org/10.1016/j.coi.2008.04.011.
8.
Slingluff CL Jr., Petroni GR, Chianese-Bullock KA,
Smolkin ME, Ross MI, Haas NB, et al. Randomized
multicenter trial of the effects of melanoma-associated
helper peptides and cyclophosphamide on the immunogenicity of a multipeptide melanoma vaccine. J Clin
Oncol 2011; 29:2924-32; PMID:21690475; http://
dx.doi.org/10.1200/JCO.2010.33.8053.
9. Bos R, Sherman LA. CD4+ T-cell help in the tumor
milieu is required for recruitment and cytolytic function of CD8+ T lymphocytes. Cancer Res 2010;
70:8368-77; PMID:20940398; http://dx.doi.
org/10.1158/0008-5472.CAN-10-1322.
10. Wong SBJ, Bos R, Sherman LA. Tumor-specific CD4+
T cells render the tumor environment permissive for
infiltration by low-avidity CD8+ T cells. J Immunol
2008; 180:3122-31; PMID:18292535.
Downloaded by [Inserm Disc Ist] at 14:55 29 November 2015
4.
Kennedy R, Celis E. Multiple roles for CD4+ T
cells in anti-tumor immune responses. Immunol Rev
2008; 222:129-44; PMID:18363998; http://dx.doi.
org/10.1111/j.1600-065X.2008.00616.x.
Ridge JP, Di Rosa F, Matzinger P. A conditioned dendritic cell can be a temporal bridge between a CD4+
T-helper and a T-killer cell. Nature 1998; 393:474-8;
PMID:9624003; http://dx.doi.org/10.1038/30989.
Fridman WH, Pagès F, Sautès-Fridman C, Galon J.
The immune contexture in human tumours: impact on
clinical outcome. Nat Rev Cancer 2012; 12:298-306;
PMID:22419253; http://dx.doi.org/10.1038/nrc3245.
Harley CB. Telomerase and cancer therapeutics. Nat
Rev Cancer 2008; 8:167-79; PMID:18256617; http://
dx.doi.org/10.1038/nrc2275.
5.
www.landesbioscience.com
OncoImmunology
e23430-3
Published OnlineFirst October 2, 2012; DOI: 10.1158/1078-0432.CCR-12-0896
Clinical
Cancer
Research
Cancer Therapy: Preclinical
Universal Cancer Peptide-Based Therapeutic Vaccine
Breaks Tolerance against Telomerase and Eradicates
Established Tumor
Magalie Dosset1,2,3, Yann Godet1,2,3, Charline Vauchy1,2,3, Laurent Beziaud1,2,3, Yu Chun Lone5,
Christine Sedlik6, Christelle Liard7, Emeline Levionnois8, Bertrand Clerc1,2,3, Federico Sandoval8,
Etienne Daguindau1,2,3, Simon Wain-Hobson7, Eric Tartour8, Pierre Langlade-Demoyen7,
!vi1,2,3,4
Christophe Borg1,2,3,4, and Olivier Adote
Abstract
Purpose: To evaluate CD4þ helper functions and antitumor effect of promiscuous universal cancer
peptides (UCP) derived from telomerase reverse transcriptase (TERT).
Experimental Design: To evaluate the widespread immunogenicity of UCPs in humans, spontaneous
T-cell responses against UCPs were measured in various types of cancers using T-cell proliferation and
ELISPOT assays. The humanized HLA-DRB1" 0101/HLA-A" 0201 transgenic mice were used to study the
CD4þ helper effects of UCPs on antitumor CTL responses. UCP-based antitumor therapeutic vaccine was
evaluated using HLA-A" 0201–positive B16 melanoma that express TERT.
Results: The presence of a high number of UCP-specific CD4þ T cells was found in the blood of patients
with various types of cancer. These UCP-specific T cells mainly produce IFN-g and TNF-a. In HLA transgenic
mice, UCP vaccinations induced high avidity CD4þ TH1 cells and activated dendritic cells that produced
interleukin-12. UCP-based vaccination breaks self-tolerance against TERT and enhances primary and
memory CTL responses. Furthermore, the use of UCP strongly improves the efficacy of therapeutic
vaccination against established B16-HLA-A" 0201 melanoma and promotes tumor infiltration by TERTspecific CD8þ T cells.
Conclusions: Our results showed that UCP-based vaccinations strongly stimulate antitumor immune
responses and could be used to design efficient immunotherapies in multiple types of cancers. Clin Cancer
Res; 18(22); 6284–95. #2012 AACR.
Introduction
The introduction of immunotherapy in the clinical
cancer practice emphasizes the role of immune responses
in cancer prognosis and has led to a growing interest to
extend this approach to several human cancers (1). Considerable knowledge has been obtained on the elements
that are relevant in antitumor immune responses, hence,
! de FrancheAuthors' Affiliations: 1INSERM, UMR1098; 2Universite
!; 4CHU Besançon, Oncologie
!; 3EFS Bourgogne Franche-Comte
Comte
5
!dicale, Besançon; INSERM U1014, Ho
^ pital Paul Brousse Ba
^timent
me
!partement de Transfert,
INSERM Lavoisier, Villejuif; 6Institut Curie, De
7
8
INSERM U932; Invectys, Institut Pasteur Biotop; and INSERM U970
! Paris Descartes, Ho
^ pital Europe
!en Georges Pompidou,
PARCC, Universite
Service d'Immunologie Biologique, (AP-HP), Paris, France
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Olivier Adotevi, INSERM, UMR1098, 1
Boulevard A Fleming BP 1937, Besancon cedex, F-25020, Besancon, France. Phone: 33-381-615-615; Fax: 33-381-615-617; E-mail:
[email protected]
doi: 10.1158/1078-0432.CCR-12-0896
#2012 American Association for Cancer Research.
6284
CD8 CTLs have been identified as the most powerful
effector cells (2). As a consequence, most previous cancer
vaccines target class I MHC-restricted peptides derived
from tumor antigens to stimulate CTL responses. However, the clinical impact of CTL peptide–based cancer
vaccines remains still modest, even if a recent gp100derived peptide vaccination was shown to increase patient
survival in melanoma (3, 4).
In the meanwhile, CD4 helper T cells have gained interest
in antitumor immunity and immunotherapy (5). The concept of CD4þ T-cell help initially emerged from studies
showing that successful generation of antitumor CTL
depends on the presence of CD4þ T cells. Adoptive cell
transfer with CD4þ T cells induces tumor protection or
regression, whereas depletion of CD4 T cells inhibits vaccine-induced protective immunity (6–8). CD4þ T cells have
been thought to play a key role in "helping" antigen-specific
CD8þ T cells to undergo efficient activation and proliferation (9). In particular, tumor-reactive CD4þ T-helper 1 cells
(TH1) produce several cytokines [such as IFN-g, TNF-a, and
interleukin-2 (IL-2)] essential for the induction of cellmediated immunity against tumors (10). One widely
Clin Cancer Res; 18(22) November 15, 2012
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Published OnlineFirst October 2, 2012; DOI: 10.1158/1078-0432.CCR-12-0896
UCP-Specific CD4 T Cells Help with Antitumor CTL Responses
Translational Relevance
The stimulation of CD4þ T-helper cell responses has
gained considerable interest for cancer immunotherapy.
This article reports a systematic analysis of CD4þ Thelper cell functions in response to universal cancer
peptides (UCP), novel promiscuous HLA-DR–restricted,
and TERT-derived peptides. Using a relevant preclinical
HLA transgenic mouse model, we showed that UCPspecific CD4þ T cells induced after vaccination fulfilled
helper features necessary to generate antitumor immune
responses. UCP-based vaccinations break self-tolerance
against TERT and greatly increase primary and memory
CTL responses. Furthermore, the use of UCPs in therapeutic vaccination eradicates established mouse melanoma by promoting massive TERT-specific CD8þ T-cell
recruitment at the tumor site. Together with the presence
of natural UCP-specific T-cell responses in many human
cancers, these results support that the stimulation of
UCP-specific CD4þ helper T cells is a powerful method
to improve the efficiency of cancer vaccines.
accepted model shows the ability of CD4þ T cells to license
dendritic cells (DC) for efficient CD8þ T-cell priming
through the interaction of costimulatory receptors (11,
12). The cytokines secreted by CD4þ TH1 cells also exert
direct antitumor and antiangiogenic effects (13). More
importantly, only tumor-reactive CD4þ T cells have been
found to ensure efficient effector CTL recruitment at the
tumor site (14). In human cancers, a high density of tumorinfiltrating CD4þ TH1 cells has been shown as a good
prognostic marker in patients with colorectal cancer emphasizing the role of these cells in cancer immunosurveillance
(15). Altogether, these results underline the growing interest in stimulating tumor-specific CD4þ TH1 cells for antitumor immunotherapy.
As a result, increasing attention has focused on identifying MHC class II epitopes from tumor antigens to actively
target antitumor CD4þ T cells in vivo (16). However, the
CD4þ helper T-cell subpopulation is known to be plastic
(17, 18). Thus, the choice of tumor-reactive CD4 epitopes
should require special caution to prevent the induction of
detrimental CD4þ T-cell responses.
Recently, we characterized potent immunogenic CD4
epitopes referred as universal cancer peptides (UCP)
derived from telomerase reverse transcriptase (TERT;
ref. 19). TERT expression has been detected in all studied
cancer forms including stem cell-like tumor cells (20, 21).
Thus TERT has emerged as a clinically relevant tumor
antigen for cancer vaccines (22). These TERT-derived UCPs
effectively bind to the most commonly found HLA-DR
alleles (19).
In the present study, we found naturally occurring
CD4þ T-cell responses against UCPs in patients with
various types of cancers. We then evaluated the potential
of UCP for active immunotherapy in a preclinical tumor
www.aacrjournals.org
model. By using the humanized HLA-DRB1" 0101/HLAA" 0201 transgenic mice, we found that UCP vaccinations
stimulate CD4þ TH1 cells that drastically improved antitumor CTL responses in vivo. Subsequently, UCP-based
therapeutic vaccine was shown to inhibit tumor growth
by mechanisms that involve CD8þ T cells.
Materials and Methods
Synthetic peptides
The 4 peptides derived from TERT called UCPs: UCP1
(TERT44–58: PAAFRALVAQCLVCV), UCP2 (TERT578–592:
KSVWSKLQSIGIRQH), UCP3 (TERT916–930: GTAFVQMPAHGLFPW), and UCP4 (TERT1041–1055: SLCYSILKAKNAGMS) have been described recently (19). The modified
(first amino acid substitution with a tyrosine) HLA-A2–
restricted pY988 (YLQVNSLQTV) and pY572 (YLFFYRKSV)
peptides derived from TERT have been described elsewhere
as high-affinity forms of their cryptic counterparts (23, 24).
The native forms of these 2 peptides are fully conserved in
human and mouse TERT (23, 24). Synthetic peptides
(>80% purity) were purchased from Activotec.
Detection of UCP-specific T-cell responses in cancer
patients
Blood was collected from patients with cancer at the
University Hospital of Besançon (Besançon, France) after
informed consent. The study was conducted in accordance
with the French laws and after approval by the local ethics
committee. Ficoll-isolated lymphocytes were analyzed by
3
H-thymidine incorporation as described previously (25).
After a short in vitro stimulation of lymphocytes with
UCPs as previously reported (19), UCP-specific immune
responses were analyzed by human ELISPOT assay (GenProbe). Concomitantly, cytokines production was measured
after a 15-hour culture with or without UCPs using DIAplex
Human Th1/Th2 kit (GenProbe) according to the manufacturer’s instructions.
Tumor cell lines and TERT expression analysis
The HLA-A2.1–positive B16F10 murine tumor cell line
(referred as B16-A2) was previously described (26). Telomerase detection in cell lines was achieved by Western blot
analysis using anti-hTERT monoclonal antibody (clone
2C4; Novus Biologicals), which cross reacts with mouse
TERT. FaDu cell line (human head and neck squamous cell
carcinoma) and murine fibroblast were used as positive and
negative controls, respectively. Telomerase activity was
assessed by TRAP-ELISA assay using the TeloTAGGG Telomerase PCR ELISAPLUS kit (Roche Diagnostics) according to
the manufacturer’s instructions.
Mouse and vaccinations
The HLA-DRB1" 0101/HLA-A" 0201-transgenic mice (A2/
DR1 mice) have been previously described (25) and were
purchased at the "Cryopr!eservation, Distribution, Typage et
Archivage animal". These mice are H-2 class I and IA class II
knockout, and their CD8 T and CD4 T cells are restricted by
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6285
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Published OnlineFirst October 2, 2012; DOI: 10.1158/1078-0432.CCR-12-0896
Dosset et al.
the sole HLA-A" 0201 and HLA-DR1" 0101 molecules,
respectively. To study the processing of UCP, 8- to 10week-old A2/DR1 mice were immunized with a pTrip-TERT
DNA (100 mg) at days 0 and 14 as previously reported (26).
In some experiments, CD4 T cells were depleted with antiCD4 monoclonal antibody treatment (clone GK1.5) before
DNA immunization. For UCP immunization, mice were
injected twice with 100 mg of each UCP emulsified in
incomplete Freund adjuvant (IFA, Sigma-Aldrich). In some
experiments, 50 mg of pY988 peptide was coinjected with
100 mg of each UCP in IFA. All peptide vaccinations were
done subcutaneously in the right abdominal flank. All
experiments were carried out according to the good laboratory practices defined by the animal experimentation rules
in France.
Pentamer staining and ELISPOT
Ex vivo pentamer staining was conducted as previously
described (26, 27). Cells were stained with phycoerythrin
(PE)-conjugated pY988 and pY572 HLA-A2.1 pentamer
(ProImmune). After cell staining, samples were analyzed
by flow cytometry on a FACS Canto II (BD Biosciences)
using Diva software. Ex vivo ELISPOT was conducted as
previously described (26, 27). Briefly, freshly ficoll-purified
lymphocytes or spleen-isolated CD8þ or CD4þ T cells from
immunized mice (T cell isolation kit, Miltenyi Biotec) were
incubated at 1 or 2 # 105 cells per well (in triplicates) in
Elispot IFN-g or inteleukin (IL)-2 plates in presence of the
relevant or control peptides. Plates were incubated for 16 to
18 hours at 37$ C, and spots were revealed following the
manufacturer’s instructions (GenProbe). Spot-forming cells
were counted using the « C.T.L. Immunospot » system
(Cellular Technology Ltd.).
Cytotoxicity assays
The in vivo CTL killing assays were conducted using CFSElabeled target cells (carboxyfluorescein-diacetate succinimidyl ester, Molecular Probes) as described previously (28).
CFSEhigh splenocytes from na€$ve mice were pulsed with
peptides at 10 mg/mL and nonpulsed CFSElow splenocytes
served as control. Equal numbers of each cell fraction (high
or low) were injected intravenously into immunized and
nonimmunized mice. After 15 hours, cells were recovered
from spleen or blood and analyzed by flow cytometry. The
specific lysis was calculated as previously described (28). In
vitro cytotoxicity assay was conducted using a standard
51
chromium-release assay as described previously (26). The
cytolytic activity of CTL from immunized mice was tested
against TERT-expressing tumor cells.
Dendritic cells generation and activation
Spleen or lymph nodes CD11cþ DCs from peptideimmunized mice were directly analyzed for costimulatory
receptor expression. In some experiments, bone marrow
cells from na€$ve mice (8.106/mL) were cultured for 6 days in
Iscove’s modified Dulbecco’s medium (IMDM; SigmaAldrich) supplemented with 10% fetal calf serum, 2
mmol/L L-glutamine (Sigma-Aldrich), 5 mmol/L sodium
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pyruvate (Gibco), and 50 mmol/L 2-mercaptoethanol
(Gibco) with 30% conditioned medium from granulocyte
macrophage colony-stimulating factor (GM-CSF)–producing NIH-3T3 (R1 medium). Isolated CD4 T cells from mice
immunized with UCP or IFA alone were then cultured for 24
hours in the presence of UCP with immature bone marrow–
derived DCs (iDC) from A2/DR1 mice. In some cases,
blocking CD40L (MR1) or IFN-g (XMG1.2) antibodies
(20mg/mL; Bio X Cell) were added to the culture. Cells were
then stained for cell surface expression of costimulatory
receptors and cytokines production.
Tumor challenge
A2/DR1 mice were subcutaneously injected with 2.105
B16-A2 cells in 100 mL of saline buffer in the abdominal
flank. At day 5, groups of mice were immunized with either
the mix of pY988 and pY572 peptides (100 mg) with or
without UCP2 (100 mg). A boost injection was done at day
17. Control mice were treated with IFA in saline buffer.
Tumor growth was monitored every 2 to 3 days using a
caliper and mice were euthanized when the tumor mass
reached an area of more than 200 mm2. The mice survival
was assessed using the Kaplan–Meier model. For tumor
infiltrative lymphocyte (TIL) analysis, tumor-bearing mice
were treated as above and 7 days after the last immunization, tumors were recovered and treated with DNAse (Sigma-Aldrich) and collagenase (Roche) before cell suspension analysis by flow cytometry, and antigen specificity of
TILs was done ex vivo by ELISPOT assay.
Statistics
Data are presented as mean % SD. Statistical comparison
between groups was based on Student t test using Prism 4
GraphPad Software. Mouse survival time was estimated
using the Kaplan–Meier method and the log-rank test.
P values less than 0.05 (" ) were considered significant.
Results
Presence of naturally occurring UCP-specific CD4þ Tcell responses in various human cancers
Recently, we found frequent occurrence of spontaneous
UCP-specific CD4þ T-cell response in patients with
advanced lung cancer (19). On the basis of the broad
expression of TERT in cancers, we sought to extend this
study in patients of different histologic origins. For this
purpose, we measured 3H-thymidine incorporation of
blood lymphocytes obtained from patients or healthy
donors directly stimulated with UCPs during 6 days. In
contrast with healthy donors, blood lymphocytes from
patients with cancer specifically proliferate upon UCP stimulation (Fig. 1A). Next, UCP-specific T cells were measured
by IFN-g ELISPOT after short-term in vitro stimulation.
Accordingly, high number of IFN-g–producing T cells
directed against UCP was found in patients as compared
with healthy donors (Fig. 1B). These responses included
T cells specific of each UCP, supporting their immunogenicity (Fig. 1C). Furthermore, the UCP-specific T cells
mainly produce TH1 cytokines but not IL-4, IL-10, or IL-17
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UCP-Specific CD4 T Cells Help with Antitumor CTL Responses
Thus, the UCP-specific T-cell repertoire is spontaneously
stimulated in various cancers such as colon, kidney, lung,
stomach, and leukemia. This also underlined the universal
nature of the promiscuous HLA-DR–restricted UCPs.
Figure 1. Analysis of spontaneous UCP-specific T-cell responses in
humans. A, blood lymphocytes from patients with cancer were directly
cultured with pool of UCPs during 5 days and specific proliferation was
3
measured by H-thymidine incorporation. Representative data from 3
healthy donors and 9 responding patients are shown. Results are
considered positive for a proliferation index more than 2. B–D,
lymphocytes were cultured in vitro with pool of UCPs for one week. B,
detection of UCP-specific T cells by IFN-g ELISPOT. Representative data
from healthy donors and 9 responding patients are shown. Columns,
mean of triplicate; bars, SD. C, T-cell responses against individual UCP
for 6 responding patients. D, detection of cytokine production by DIAplex
assay in supernatant after 15 hours of culture in the presence of UCPs.
Columns, mean cytokine levels from 3 patients; bars, SD. NSCLC, non–
small cell lung cancer; RCC, renal cell carcinoma; HNSCC, head and neck
squamous cell carcinoma; AML, acute myeloid leukemia; CRC,
colorectal carcinoma.
(Fig. 1D). This result was also confirmed by the obvious
TH1 polarization of UCP-specific CD4þ T-cell clones isolated from 1 patient with cancer (Supplementary Fig. S1).
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UCPs are endogenously processed and induce high
avidity TH1-polarized CD4þ T-cell responses in vivo
On the basis of the equivalent binding capacity of UCPs
to HLA-DRB1" 0101 molecules, we then used A2/DR1 mice
to study the in vivo immunogenicity and natural processing
of UCPs. To assess whether UCPs can be endogenously
processed from the TERT protein, we conducted immunizations with a plasmid DNA encoding the full length TERT
sequence, and the UCP-specific CD4 T-cell proliferation was
monitored by a 5-day 3H-thymidine incorporation assay. As
shown in Fig. 2A, all the UCPs stimulate proliferation of
spleen lymphocytes from DNA-immunized mice. Especially, high T-cell proliferation was measured in response to
UCP2 and 3 as compared with UCP1 or UCP4. We confirmed these results by using ex vivo IFN-g ELISPOT assay
(Fig. 2B). These data clearly indicate that UCPs are differentially processed and presented to CD4þ T cells in vivo in
the context of DRB1" 0101 restriction.
Different populations of CD4þ TH cells control the
antitumor immune responses (9), thus, we studied the
polarization of the UCP-specific CD4þ T-cell responses in
vivo. To this end, freshly isolated CD4þ T cells from UCPvaccinated mice were cultured in the presence of syngenic
iDC pulsed or not with UCP and cytokines production
was measured. In all cases, we showed that UCP-specific
CD4þ T cells produce IFN-g and IL-2, but not IL-4, IL-5,
IL-10, or IL-17, indicating that UCP immunization preferentially induces a TH1-polarized immune response in
vivo (Fig. 2C).
Next, to assess the avidity of UCP-specific CD4þ T cells,
freshly purified CD4þ T cells from UCP-immunized mice
were cultured in the presence of decreasing concentrations
of peptide and the number of specific IFN-g–producing
CD4þ T cells was measured. Results in Fig. 2D showed that
mice immunized with UCP2 or UCP3 induced high avidity–specific CD4 T cells (< 10&7 mg/mL). In comparison,
CD4þ T cells from mice vaccinated with UCP1 or UCP4
responded to 10&1 and 10&3 mg/mL of peptide concentration, respectively. In addition, low doses of UCP2 or UCP3
peptides ('1 mg) stimulated potent IFN-g þ CD4þ T cells
in vivo (Fig. 2E). Collectively, these results show that UCPs
are efficiently processed in vivo and stimulate high avidity TH
1-polarized CD4þ T cells in A2/DR1 mice.
UCP-specific CD4þ TH1 cells provide help for optimal
anti-self/TERT CD8þ T-cell responses in vivo
CD4þ T-cell helper functions are thought to be important
for the generation of potent and sustained CTL responses
(29, 30). To address this question concerning UCP-specific
CD4þ T cells, we coimmunized mice with pY988 an HLAA2þ self/TERT peptide in the presence of UCP. The pY988specific CTL response was measured ex vivo by pentamer
staining and ELISPOT assays. As shown in Fig. 3A, a higher
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frequency of functional pY988-specific CD8þ T cells was
detected in mice immunized with pY988 plus UCPs compared with pY988/IFA group. Although UCP1 vaccination
had little impact on the frequency of pY988/A2 pentamerþ
CD8 T cell-specific response, all UCPs were able to significantly increase the number of IFN-g–secreting CD8þ T cells
against TERT (Fig. 3B). The magnitude of the pY988-specific
CD8þ T cells response was strongly correlated with the
intensity of UCP-specific CD4 T-cell responses concomitantly induced in mice (Fig. 3C and D). Furthermore, these
UCPs exerted similar helper effect on the self/TERT pY572specific CTL responses in vivo (Supplementary Fig. S2).
Thus, the addition of UCPs as helper peptides efficiently
breaks immune tolerance against TERT in vivo.
We next sought out to study the impact of UCPs helper
peptides on CTL avidity and memory, 2 critical functions for
tumor eradication. To this end we focused on the UCP2 that
induces potent TH1 immune responses in vivo. In addition,
compared with a HLA-DR1–restricted viral peptides such as
Tax-derived peptide (16), UCP2 strongly enhanced CTL
responses (Supplementary Fig. S3). As shown in Fig. 4A,
freshly isolated CD8þ T cells from mice immunized with
pY988 þ UCP2 were still reactive against very low concentrations of peptide (<10&3 mg/mL). These cells also recognized the cryptic native counterpart p988 (data not shown),
underlining their high avidity. Accordingly, mice vaccinated
with pY988 þ UCP2 displayed stronger in vivo cytotoxicity
against CFSE-labeled target cells (Fig. 4B) than in pY988/
IFA group. In addition, TERT-specific CTLs from mice
immunized in the presence of UCP2 exhibit strong in vitro
cytotoxicity against TERT-expressing B16-A2 cells (Fig. 4C
and D).
Furthermore, long-lasting TERT-specific CTL response
was detected in mice coinjected with UCP2. This response
was correlated to the sustained UCP2-specific CD4þ T-cell
response in vivo (Fig. 4E). Similar helper functions of UCP2
were obtained in other tumor antigen model such as E7
from HPV-16, (Supplementary Fig. S4). By using a second
model of DNA immunization, we also showed in mice,
depleted or not of CD4þ cells, that UCP-specific CD4 T cells
are necessary for the induction of TERT-specific CD8 T cells
(Supplementary Fig. S4C). Collectively, UCP2 helper
immune responses enhance the magnitude and quality of
antitumor CTL response.
Figure 2. UCP vaccinations stimulate high avidity TH1-polarized CD4 Tcell responses. A and B, A2/DR1 mice (n ¼ 8) were immunized twice with a
DNA encoding TERT. A, proliferation of spleen lymphocytes in the
presence of UCPs. B, CD8-depleted spleen lymphocytes from DNAimmunized mice were assayed in ex vivo IFN-g ELISPOT. Columns, mean
of triplicate from 4 mice; bars, SD. C and D, mice (3–4/group) were
immunized once with each UCP in IFA. C, ten days later, spleen-isolated
CD4 T cells were cultured overnight in presence of DC loaded with UCP.
The cytokines production was measured in the supernatant by Luminex
assay. Columns, mean of cytokine levels; bars, SD. D, isolated CD4
T cells were cultured ex vivo with increasing concentrations of peptide as
indicated. IFN-g production was measured by ELISPOT. Curves,
mean responses from 3 mice; bars, SD. E, mice were vaccinated once
with low dose of UCP as indicated. UCP-specific T-cell responses were
evaluated in spleen by ex vivo IFN-g ELISPOT.
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UCP-Specific CD4 T Cells Help with Antitumor CTL Responses
UCP-specific CD4þ T cells promote DC activation
in vivo
The induction of DC activation represents one major
helper mechanism used by CD4þ TH1 cells to sustain
antigen presentation and provide costimulatory signals to
the CTLs. This is referred as the "m!enage %a trois" model (31).
To test this mechanism, we analyzed the expression of
costimulating receptors on DCs from mice immunized with
the mix of pY988 % UCP2. As shown in Fig. 5A, lymph
nodes CD11cþ DCs from UCP2-immunized mice expressed
higher level of HLA-DR molecules and slight increase of
CD86 as compared with control mice. In a second set of
experiments, CD4þ T cells isolated from UCP2/IFA or IFAinjected mice were cocultured with syngenic iDCs (Fig. 5B).
Similar increase of DC activation was found in the presence
of UCP2-specific CD4 T cells (Fig. 5C, left). In addition,
high rate of CD40Lþ CD4þ T cells were detected in UCP2immunized mice (Fig. 5C, middle) and significant amounts
of TH1-associated cytokines such as IL-12, IFN-g, and GMCSF were found in the supernatant of CD4UCP2/DC coculture (Fig. 5C, right). This DC activation could be partially
inhibited by blocking CD40L and/or IFN-g antibodies (Fig.
5D). Together, these results showed that the stimulation of
UCP2-specific CD4þ T cells shapes the phenotype and
function of DC in vivo.
UCP2 helper peptide enhances the efficacy of self/TERT
CD8 peptides vaccination against established HLAA" 0201þ B16F10 melanoma
To investigate the helper role of UCP2 in a therapeutic
vaccination protocol, we used the aggressive and poor
immunogenic B16F10-HLA-A" 0201 melanoma (B16-A2;
ref. 26). Mice were challenged with 2 # 105 B16-A2 cells
and tumor bearing mice were then vaccinated twice either
with the 2 self/TERT CTL peptides (pY572 þ pY988/IFA)
alone or in presence of the UCP2. As shown in Fig 6A, the
tumor growth reached an area of more than 200 mm2 at day
25 in the control group injected with IFA alone. In this
representative experiment, tumor regression was observed
in 1 of 8 mice vaccinated with pY572 þ pY988/IFA, whereas
2 mice achieved a delay in tumor growth. In the group
vaccinated with pY988 þ pY572/IFA combined with UCP2,
complete tumor regression was achieved in 5 of 8 mice.
Accordingly, survival analysis out on day 50 after tumor cell
injection showed that 63% of mice vaccinated in the presence of UCP2 were still alive as compared with 13% in the
group of mice injected with pY988 þ pY572/IFA (P <
0.05; Fig. 6B).
Figure 3. CD4 helper role of UCP vaccinations on the self/TERT-specific
CTL responses. Mice (3/group) were immunized either with pY988 plus
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each UCP in IFA or with pY988/IFA alone and the immune responses were
monitored 10 days later in the spleen. A, freshly isolated CD8 T cells were
þ
stained with TERT pY988/A2 pentamer. Representative flow cytometry
dot plots (top) and mean percentages of pY988/A2þ CD8 T cells (bottom)
are shown. B, ex vivo detection of anti-pY988 CD8 T cells by IFN-g
ELISPOT. C and D, simultaneous UCP-specific CD4 T-cell responses
were assessed in CD8-depleted fraction by IFN-g (C) and IL-2 (D)
ELISPOT assays. DR1-restricted Tax191-250 was used as irrelevant
peptide. Columns, mean of spots from 3 mice; bars, SD. Data are
representative of 3 independent experiments.
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Figure 4. Immunization in the
presence of UCP2 enhances the
quality of self pY988-specific CTL
responses. Mice (3–4/group) were
immunized once either with pY988
plus UCP2 (UCP2 þ pY988/IFA) or
with pY988/IFA alone. A, ten days
later, freshly isolated spleen CD8 T
cells were cultured with increasing
pY988 peptide concentration and
IFN-g–secreting CD8 T cells were
detected by ex vivo ELISPOT. B,
in vivo cytototoxic assay.
Representative flow cytometry
histograms showing lysis of CFSElabeled pY988-loaded target cells
compared with unpulsed (UP) and
the mean of in vivo percentage lysis
are shown. C, TERT expression by
Western blot (left) and activity by
TRAP-ELISA assay (right) in B16A2 melanoma cells. HT, heattreated cells and &, untreated cells.
D, cytotoxicity of T cells against
TERT-positive B16 or B16-A2
tumor cells after 5 days of in vitro
stimulation of splenocytes with
pY988. Results represent the
specific lysis (percentage) % SD in
each immunized group of mice. E,
long-term T-cell responses were
evaluated 30 days after
immunization. Frequencies of
pY988/A2 pentamerþ CD8 T cells
hi
lo
gated on CD44 CD62 cells (left)
and by IFN-g secretion assay
(middle). UCP2-specific CD4 T-cell
response measured in CD8depleted fraction by ex vivo IFN-g
ELISPOT (right).
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UCP-Specific CD4 T Cells Help with Antitumor CTL Responses
Figure 5. UCP2-specific CD4 TH1
cells activate dendritic cells. A, mice
(3/group) were immunized once
either with UCP2 þ pY988/IFA or
pY988/IFA alone. Ten days later, the
expression of activation markers
CD80, CD86, and HLA-DR were
analyzed on lymph nodes CD11cþ
DC by flow cytometry.
Representative flow cytometry
histograms (top) and the mean of
mean fluorescence intensity (MFI;
bottom) are shown. Columns, mean
of MFI; bars, SD. B–E, analysis of DC
and CD4 T cells cross talk. B, schema
of the in vitro DC-CD4 T cell
coculture. C, expression of CD86
and HLA-DR on CD11cþ DC (left).
CD40L expression on CD4 T cells
(middle). IFN-g, GM-CSF, and IL12p70 production measured by
ELISA in the supernatant (right). D,
expression of CD86 on CD11cþ DC
cocultured with CD4 T cells from
UCP2-immunized mice in presence
or not of blocking CD40L and/or IFNg antibodies. Representative flow
cytometry histograms (left) and mean
of percentage from 3 mice (right) are
shown. Data are representative of 2
independent experiments.
The density of tumor-infiltrating CD8 T cells was shown
to be critical for tumor control (32). Therefore, we analyzed
immune cell infiltration within tumor in mice treated with
the same vaccination protocols. Higher total CD3þCD8þ T-
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cell infiltration was observed in mice that received vaccine
plus UCP2 helper peptide as compared with pY988 þ
pY572/IFA group (67% vs 40%, P < 0.05; Fig. 6C). In
contrast, UCP2 vaccination did not influence CD4þ TILs,
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Figure 6. Therapeutic antitumor effect of UCP-based vaccination. Tumor-bearing mice (6–8 mice/group) were therapeutically vaccinated with peptides as
described (Materials and Methods). A, follow-up of tumor size. The numbers in parentheses indicate mice with tumor regression per group. B, survival
curves recorded until 50 days. C, tumor-bearing mice were vaccinated as above and tumor-infiltrating immune cells were analyzed by flow cytometry.
Columns, mean of percentages of cells from 4 mice; bars, SD. D, TERT-specific T cells in spleen and in tumor were analyzed by ex vivo IFN-g ELISPOT.
Columns, mean of spots from 5 mice; bars, SD. All data are representative of 3 independent experiments.
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UCP-Specific CD4 T Cells Help with Antitumor CTL Responses
natural killer, or regulatory T cells (Treg), suggesting that
UCP2-specific immunity mainly drive CTLs to the tumor
microenvironment.
In line with this observation, we detected a large number
of TERT-specific CD8þ TILs in mice that received UCP2based vaccine (Fig. 6D, bottom). TERT-specific CTL
response was also detected in spleen of mice, which is
correlated to UCP2-specific CD4 T-cell response (Fig. 6D,
top). However, UCP-specific CD4þ TILs were not detected
at the tumor site. This could be due to the low level of CD4þ
TILs or to the lack of HLA-DR expression on the B16/A2
model used. Together, our results clearly showed that
UCP2-specific CD4þ T cells exert strong helper activity on
tumor-specific CTL responses in vivo. Moreover, the addition of UCP2 influences the homing of CD8þ T cells at the
tumor site. All these data support the use of UCP for
antitumor therapeutic vaccination.
Discussion
CD4 TH1 response against tumor is gaining considerable
interest in cancer immunity. In this study, we found spontaneous TH1 CD4þ T-cell responses against recently
described TERT-derived UCP in patients with different types
of cancers. This observation underlines the great interest of
these peptides for immunotherapy. To evaluate the potential applicability of UCP for cancer vaccine, we used the
preclinical A2/DR1 mouse model. We have found that UCP
vaccination induces high avidity TH1-polarized CD4þ T
cells that greatly increase CTL responses against self/TERT
epitopes in vivo and promote potent antitumor immunity.
Different subpopulations of CD4þ TH cells regulate host
antitumor immune responses (10). Indeed, TH2 CD4þ T
cells and Tregs are often associated with an inhibitory
environment within the tumor (10, 33). The role of TH17
cells in antitumor immune response is still controversial
and seems to depend on the type of cancer (34). In contrast,
TH1 immunity has a clear positive effect in cancer cell
eradication. The CD4þ TH1 cells provide help for CTLs
through multiple interactions during the induction and
effector phases of antitumor immune responses (35, 36).
Thus, there is a strong rational to develop cancer vaccines
that stimulate antitumor TH1 immunity (5, 37). Nevertheless, in recent randomized trials, the use of melanomaassociated helper peptides paradoxically decreased CD8þ
T-cell responses to a melanoma vaccine (38). This could be
related to the plasticity of CD4þ TH cell responses (17).
Consequently, the choice of tumor-reactive CD4 helper
peptides for cancer vaccine needs to be done carefully.
On the basis of its expression profile and its role in
multiple human tumors, TERT is an attractive target for
cancer vaccination (22, 39). Schroers and colleagues have
previously described TERT-derived promiscuous HLA-DR–
restricted peptides (40, 41). However, their role on cellmediated tumor immunity was not completely addressed
neither in preclinical nor in clinical trials setting. Recently, a
cancer vaccine using a TERT-derived CD4 helper peptide
called GV1001 was able to stimulate specific CD4 T-cell
immunity. Clinical trials using GV1001 suggest an
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increased survival in patients with cancer when combined
with cytotoxic agents (42, 43). Nevertheless, GV1001 vaccine also failed to induce specific immune responses and
clinical benefit in other cancers (44). The impact of
GV1001-specific CD4þ T-cell help on antitumor CTL
responses remains to be investigated.
Here, we used a relevant mouse model to conduct a
systematic analysis of UCP-specific CD4þ T-cell help on
antitumor CTL responses in A2/DR1 mice. To this end, we
selected 2 HLA-A2þ TERT peptides called pY572 and pY988
because they are self-epitopes in mouse and also fully
conserved in human TERT (23, 24). In addition, these
peptides are already used for cancer vaccines in humans
(45, 46). We found that the presence of UCP-specific TH1
cells drastically enhances self/TERT-specific CD8þ T-cell
responses as compared with mice immunized with CD8
peptides alone. The anti-self/TERT CTL induced in UCPvaccinated mice displayed higher avidity and stronger cytotoxicity than the helper less counterpart. Furthermore, the
addition of UCP2 to the CD8 TERT peptide vaccine led to
B16-A2 tumor regression and improved the survival of
mice.
Previous studies have already shown the requirement of
CD4 help for the generation of CTL against the self/TERT
epitopes used in this study (23, 47). Gross and colleagues
reported that vaccination of HHD mice with these peptides
promote tumor protection only when they were coupled
with a helper peptide derived from the hepatitis B virus. In
this study, the vaccine was used prophylactically: approximately 25% of vaccinated mice were tumor free compared
with 60% in our therapeutic vaccine study (47). This difference could be related to the nature of the help signal
delivered by CD4 T cells. We used the tumor-reactive helper
peptide UCP2 that mediates a better homing of CD8þ TILs
than nontumor antigen–specific CD4 TH1 cells as previously reported (14, 48, 49). Indeed, we found that CD4 T cells
specific for UCP2 cross-recognized its mTERT-derived counterpart peptide p568 (differing by one amino acid; Supplementary Fig. S5). Consequently, the contribution of xenogenic response in UCP2-mediated helper effect in mice
studies seems to be weak. In agreement with previous
studies, no sign of autoimmunity has been observed in all
immunized mice suggesting the safety of TERT-based vaccination (26, 47).
Moreover, immunization with UCP2 stimulates specific CD4þ T cells secreting high levels of IL-2 and GM-CSF,
which are known to be central components for the
generation of CD8þ T-cell memory and DC licensing,
respectively (36, 50). Therefore, fully activated DCs and
sustained self/TERT CTL responses were found in A2/DR1
mice coimmunized with UCP2. Finally, we found that
spontaneous UCP-specific TH1 responses are detected in
patients with various cancers indicating the presence of a
functional UCP-specific T-cell repertoire. In our recent
study, this preexisting UCP-specific CD4þ T-cell immunity was shown to be associated with an increased overall
survival of patients with lung cancer responding to the
first-line chemotherapy (19).
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In conclusion, our study shows that the stimulation of
UCP-specific CD4 TH cells is a powerful method to improve
cancer vaccine efficacy and also highlights the interest of
TERT-derived UCPs for the monitoring of antitumor CD4þ
T-cell responses.
Disclosure of Potential Conflicts of Interest
P. Langlade-Demoyen is a member of advisory board, INVECTYS Co.
(current patent holder for UCPs). No potential conflicts of interest were
disclosed by other authors.
Authors' Contributions
Conception and design: P. Langlade-Demoyen, C. Borg, O. Adotevi
Development of methodology: M. Dosset, Y.C. Lone, E. Levionnois, B.
Clerc, P. Langlade-Demoyen
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): M. Dosset, Y. Godet, C. Vauchy, L. Beziaud, Y.C.
Lone, B. Clerc, E. Daguindau, O. Adotevi
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Dosset, Y. Godet, L. Beziaud, E.
Levionnois, F. Sandoval, P. Langlade-Demoyen, O. Adotevi
Writing, review, and/or revision of the manuscript: M. Dosset, Y. Godet,
C. Liard, S. Wain-Hobson, E. Tartour, P. Langlade-Demoyen, C. Borg,
O. Adotevi
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Sedlik, C. Liard, E. Levionnois
Study supervision: E. Tartour, P. Langlade-Demoyen, C. Borg, O. Adotevi
Acknowledgments
The authors thank Dr. Nathalie Chaput for providing B16/HLA-A" 0201
cell lines. The authors also thank Drs. E. Fabre, T. Nguyen, K. Stefano, and A.
Thierry-Vuillemin for the recruitment of patients with cancer.
Grant Support
This work was supported by Ligue Contre le Cancer, Association pour la
Recherche contre le Cancer (ARC), and BQR grant from university of Franche
Comt!e.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received March 19, 2012; revised August 20, 2012; accepted September 16,
2012; published OnlineFirst October 2, 2012.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
6294
Topalian SL, Weiner GJ, Pardoll DM. Cancer immunotherapy comes of
age. J Clin Oncol 2011;29:4828–36.
Vesely MD, Kershaw MH, Schreiber RD, Smyth MJ. Natural innate and
adaptive immunity to cancer. Annu Rev Immunol 2011;29:235–71.
Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving
beyond current vaccines. Nat Med 2004;10:909–15.
Schwartzentruber DJ, Lawson DH, Richards JM, Conry RM, Miller DM,
Treisman J, et al. gp100 peptide vaccine and interleukin-2 in patients
with advanced melanoma. N Engl J Med 2011;364:2119–27.
Knutson KL, Disis ML. Tumor antigen-specific T helper cells in cancer
immunity and immunotherapy. Cancer Immunol Immunother 2005;54:
721–8.
Antony PA, Piccirillo CA, Akpinarli A, Finkelstein SE, Speiss PJ, Surman DR, et al. CD8þ T cell immunity against a tumor/self-antigen is
augmented by CD4þ T helper cells and hindered by naturally occurring
T regulatory cells. J Immunol 2005;174:2591–601.
Fayolle C, Deriaud E, Leclerc C. In vivo induction of cytotoxic T cell
response by a free synthetic peptide requires CD4þ T cell help.
J Immunol 1991;147:4069–73.
Hunder NN, Wallen H, Cao J, Hendricks DW, Reilly JZ, Rodmyre R,
et al. Treatment of metastatic melanoma with autologous CD4þ T cells
against NY-ESO-1. N Engl J Med 2008;358:2698–703.
Pardoll DM, Topalian SL. The role of CD4þ T cell responses in
antitumor immunity. Curr Opin Immunol 1998;10:588–94.
Kennedy R, Celis E. Multiple roles for CD4þ T cells in anti-tumor
immune responses. Immunol Rev 2008;222:129–44.
Bennett SR, Carbone FR, Karamalis F, Flavell RA, Miller JF, Heath WR.
Help for cytotoxic-T-cell responses is mediated by CD40 signalling.
Nature 1998;393:478–80.
Smith CM, Wilson NS, Waithman J, Villadangos JA, Carbone FR, Heath
WR, et al. Cognate CD4(þ) T cell licensing of dendritic cells in CD8(þ) T
cell immunity. Nat Immunol 2004;5:1143–8.
Qin Z, Blankenstein T. CD4þ T cell–mediated tumor rejection involves
inhibition of angiogenesis that is dependent on IFN gamma receptor
expression by nonhematopoietic cells. Immunity 2000;12:677–86.
Bos R, Sherman LA. CD4þ T-cell help in the tumor milieu is required for
recruitment and cytolytic function of CD8þ T lymphocytes. Cancer
Res 2010;70:8368–77.
Tosolini M, Kirilovsky A, Mlecnik B, Fredriksen T, Mauger S, Bindea G,
et al. Clinical impact of different classes of infiltrating T cytotoxic and
helper cells (Th1, th2, treg, th17) in patients with colorectal cancer.
Cancer Res 2011;71:1263–71.
Kobayashi H, Celis E. Peptide epitope identification for tumor-reactive
CD4 T cells. Curr Opin Immunol 2008;20:221–7.
Clin Cancer Res; 18(22) November 15, 2012
17. Quezada SA, Peggs KS. Tumor-reactive CD4þ T cells: plasticity
beyond helper and regulatory activities. Immunotherapy 2011;3:
915–7.
18. Zhu J, Paul WE. CD4 T cells: fates, functions, and faults. Blood
2008;112:1557–69.
19. Godet Y, Fabre-Guillevin E, Dosset M, Lamuraglia M, Levionnois E,
Ravel P, et al. Analysis of spontaneous tumor-specific CD4 T cell
immunity in lung cancer using promiscuous HLA-DR telomerasederived epitopes: potential synergistic effect with chemotherapy
response. Clin Cancer Res 2012;18:2943–53.
20. Artandi SE, DePinho RA. Telomeres and telomerase in cancer. Carcinogenesis 2010;31:9–18.
21. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.
Cell 2011;144:646–74.
22. Harley CB. Telomerase and cancer therapeutics. Nat Rev Cancer
2008;8:167–79.
23. Hernandez J, Garcia-Pons F, Lone YC, Firat H, Schmidt JD, LangladeDemoyen P, et al. Identification of a human telomerase reverse transcriptase peptide of low affinity for HLA A2.1 that induces cytotoxic T
lymphocytes and mediates lysis of tumor cells. Proc Natl Acad Sci
U S A 2002;99:12275–80.
24. Scardino A, Gross DA, Alves P, Schultze JL, Graff-Dubois S, Faure O,
et al. HER-2/neu and hTERT cryptic epitopes as novel targets for broad
spectrum tumor immunotherapy. J Immunol 2002;168:5900–6.
25. Pajot A, Michel ML, Fazilleau N, Pancre V, Auriault C, Ojcius DM, et al. A
mouse model of human adaptive immune functions: HLA-A2.1-/HLADR1-transgenic H-2 class I-/class II-knockout mice. Eur J Immunol
2004;34:3060–9.
26. Adotevi O, Mollier K, Neuveut C, Dosset M, Ravel P, Fridman WH, et al.
Targeting human telomerase reverse transcriptase with recombinant
lentivector is highly effective to stimulate antitumor CD8 T-cell immunity in vivo. Blood 2010;115:3025–32.
27. Adotevi O, Mollier K, Neuveut C, Cardinaud S, Boulanger E, Mignen B,
et al. Immunogenic HLA-B" 0702-restricted epitopes derived from
human telomerase reverse transcriptase that elicit antitumor cytotoxic
T-cell responses. Clin Cancer Res 2006;12:3158–67.
28. Rusakiewicz S, Dosset M, Mollier K, Souque P, Charneau P, WainHobson S, et al. Immunogenicity of a recombinant lentiviral vector
carrying human telomerase tumor antigen in HLA-B" 0702 transgenic
mice. Vaccine 2010;28:6374–81.
29. Brandmaier AG, Leitner WW, Ha SP, Sidney J, Restifo NP, Touloukian
CE. High-avidity autoreactive CD4þ T cells induce host CTL, overcome T(regs) and mediate tumor destruction. J Immunother 2009;32:
677–88.
Clinical Cancer Research
Downloaded from clincancerres.aacrjournals.org on November 29, 2015. © 2012 American Association for Cancer Research.
Published OnlineFirst October 2, 2012; DOI: 10.1158/1078-0432.CCR-12-0896
UCP-Specific CD4 T Cells Help with Antitumor CTL Responses
30. Shedlock DJ, Shen H. Requirement for CD4 T cell help in generating
functional CD8 T cell memory. Science 2003;300:337–9.
31. Ridge JP, Di Rosa F, Matzinger P. A conditioned dendritic cell can be a
temporal bridge between a CD4þ T-helper and a T-killer cell. Nature
1998;393:474–8.
32. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, LagorcePages C, et al. Type, density, and location of immune cells within
human colorectal tumors predict clinical outcome. Science 2006;313:
1960–4.
33. Nishimura T, Iwakabe K, Sekimoto M, Ohmi Y, Yahata T, Nakui M, et al.
Distinct role of antigen-specific T helper type 1 (Th1) and Th2 cells in
tumor eradication in vivo. J Exp Med 1999;190:617–27.
34. Murugaiyan G, Saha B. Protumor vs antitumor functions of IL-17.
J Immunol 2009;183:4169–75.
35. Rocha B, Tanchot C. Towards a cellular definition of CD8þ T-cell
memory: the role of CD4þ T-cell help in CD8þ T-cell responses. Curr
Opin Immunol 2004;16:259–63.
36. Zhu J, Yamane H, Paul WE. Differentiation of effector CD4 T cell
populations (" ). Annu Rev Immunol 2010;28:445–89.
37. Fujiki F, Oka Y, Kawakatsu M, Tsuboi A, Tanaka-Harada Y, Hosen N,
et al. A clear correlation between WT1-specific Th response and
clinical response in WT1 CTL epitope vaccination. Anticancer Res
2010;30:2247–54.
38. Slingluff CL Jr, Petroni GR, Chianese-Bullock KA, Smolkin ME, Ross
MI, Haas NB, et al. Randomized multicenter trial of the effects of
melanoma-associated helper peptides and cyclophosphamide on the
immunogenicity of a multipeptide melanoma vaccine. J Clin Oncol
2011;29:2924–32.
39. Beatty GL, Vonderheide RH. Telomerase as a universal tumor antigen
for cancer vaccines. Expert Rev Vaccines 2008;7:881–7.
40. Schroers R, Huang XF, Hammer J, Zhang J, Chen SY. Identification of
HLA DR7-restricted epitopes from human telomerase reverse transcriptase recognized by CD4þ T-helper cells. Cancer Res 2002;62:
2600–5.
41. Schroers R, Shen L, Rollins L, Rooney CM, Slawin K, Sonderstrup G,
et al. Human telomerase reverse transcriptase-specific T-helper
www.aacrjournals.org
42.
43.
44.
45.
46.
47.
48.
49.
50.
responses induced by promiscuous major histocompatibility complex
class II-restricted epitopes. Clin Cancer Res 2003;9:4743–55.
Brunsvig PF, Kyte JA, Kersten C, Sundstrom S, Moller M, Nyakas M,
et al. Telomerase peptide vaccination in NSCLC: a phase II trial in stage
III patients vaccinated after chemoradiotherapy and an 8-year update
on a phase I/II trial. Clin Cancer Res 2011;17:6847–57.
Kyte JA, Gaudernack G, Dueland S, Trachsel S, Julsrud L, Aamdal S.
Telomerase peptide vaccination combined with temozolomide: a
clinical trial in stage IV melanoma patients. Clin Cancer Res 2011;17:
4568–80.
Schlapbach C, Yerly D, Daubner B, Yawalkar N, Hunger RE. Telomerase-specific GV1001 peptide vaccination fails to induce objective
tumor response in patients with cutaneous T cell lymphoma. J Dermatol Sci 2011;62:75–83.
Bolonaki I, Kotsakis A, Papadimitraki E, Aggouraki D, Konsolakis G,
Vagia A, et al. Vaccination of patients with advanced non-small-cell
lung cancer with an optimized cryptic human telomerase reverse
transcriptase peptide. J Clin Oncol 2007;25:2727–34.
Kotsakis A, Vetsika EK, Christou S, Hatzidaki D, Vardakis N, Aggouraki
D, et al. Clinical outcome of patients with various advanced cancer
types vaccinated with an optimized cryptic human telomerase reverse
transcriptase (TERT) peptide: results of an expanded phase II study.
Ann Oncol 2012;2:442–9
Gross DA, Graff-Dubois S, Opolon P, Cornet S, Alves P, BennaceurGriscelli A, et al. High vaccination efficiency of low-affinity epitopes in
antitumor immunotherapy. J Clin Invest 2004;113:425–33.
Marzo AL, Kinnear BF, Lake RA, Frelinger JJ, Collins EJ, Robinson
BW, et al. Tumor-specific CD4þ T cells have a major "post-licensing" role in CTL mediated anti-tumor immunity. J Immunol 2000;
165:6047–55.
Wong SB, Bos R, Sherman LA. Tumor-specific CD4þ T cells render the
tumor environment permissive for infiltration by low-avidity CD8þ T
cells. J Immunol 2008;180:3122–31.
Williams MA, Tyznik AJ, Bevan MJ. Interleukin-2 signals during priming
are required for secondary expansion of CD8þ memory T cells. Nature
2006;441:890–3.
Clin Cancer Res; 18(22) November 15, 2012
6295
Downloaded from clincancerres.aacrjournals.org on November 29, 2015. © 2012 American Association for Cancer Research.
Published OnlineFirst October 2, 2012; DOI: 10.1158/1078-0432.CCR-12-0896
Universal Cancer Peptide-Based Therapeutic Vaccine Breaks
Tolerance against Telomerase and Eradicates Established Tumor
Magalie Dosset, Yann Godet, Charline Vauchy, et al.
Clin Cancer Res 2012;18:6284-6295. Published OnlineFirst October 2, 2012.
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IJC
International Journal of Cancer
CD20 alternative splicing isoform generates immunogenic CD4
helper T epitopes
Charline Vauchy1,2,3, Clementine Gamonet1,2,3, Christophe Ferrand1,2,3, Etienne Daguindau4, Jeanne Galaine1,2,3,
Laurent Beziaud1,2,3, Adrien Chauchet4, Carole J. Henry Dunand5, Marina Deschamps1,2,3, Pierre Simon Rohrlich1,2,6,
Christophe Borg1,2,3,7, Olivier Adotevi1,2,3,7 and Yann Godet1,2,3
1
INSERM UMR1098, F25020 Besançon cedex, France
Universite de Franche-Comte, F25020 Besançon cedex, France
3
EFS Bourgogne Franche-Comte, F25020 Besançon cedex, France
4
Department of Hematology, University Hospital of Besançon, F25020 Besançon cedex, France
5
The Department of Medicine, Section of Rheumatology, The Knapp Center for Lupus and Immunology Research, The University of Chicago, Chicago, IL
6
Department of Pediatrics, University Hospital of Besançon, F25020 Besançon cedex, France
7
Department of Medical Oncology, University Hospital of Besançon, F25020 Besançon cedex, France
2
Tumor Immunology
Cancer-specific splice variants gain significant interest as they generate neo-antigens that could be targeted by immune cells.
CD20, a membrane antigen broadly expressed in mature B cells and in B cell lymphomas, is subject to an alternative splicing
named D393-CD20 leading to loss of membrane expression of the spliced isoform. D393-CD20 expression is detectable in
transformed B cells and upregulated in various lymphoma B cells. In this study, we show that D393-CD20 is translated in
malignant B cells and that D393-CD20 specific CD4 T cells producing IFN-c are present in B-cell lymphoma patients. Then, we
have investigated whether the 20mer D393-CD20 peptide spanning the splicing site might be targeted by the immune system
and we have shown that D393-CD20-specific CD4 Th1 clones could directly recognize malignant B cell lines and kill autologous lymphoma B cells indicating that D393-CD20-derived epitopes are naturally processed and presented on tumor cells.
Finally, D393-CD20 peptide-based vaccination induced specific CD8 and CD4 T cell responses in HLA-humanized transgenic
mice suggesting the presentation of D393-CD20 derived peptides on both HLA Class-I and -II. These findings support further
investigations on the potential use of D393-CD20 directed specific immunotherapy in B cell malignancies.
The effective discovery of clinically relevant tumor antigens
holds a fundamental role for the development of new diagnostic tools and anticancer immunotherapies. Generally,
tumor antigens are classified as unique antigens derived from
point mutations or as shared antigens. The shared antigens
are further divided into tumor-specific antigens, differentiation antigens and overexpressed antigens. The prioritization
of cancer antigens is based on criteria such as immunogenicity, specificity, oncogenicity.1 One mechanism that would
Key words: immunotherapy, B-cell lymphoma, CD20 antigen, CD4
T cells, splicing
Additional Supporting Information may be found in the online
version of this article.
Conflict of interest: The authors declare no conflict of interest.
Grant sponsor: Ligue contre le cancer, the ICB network of the
University of Franche-Comte, the Conseil Regional de FrancheComte, the Agence Nationale de la Recherche (Labex LipSTIC,
ANR-11-LABX-0021), the Fondation de France and the Etablissement Français du Sang (AO#2010)
DOI: 10.1002/ijc.29366
History: Received 22 Aug 2014; Accepted 18 Nov 2014; Online 28
Nov 2014
Correspondence to: Y. Godet, INSERM UMR1098, F25020
Besançon cedex, France, E-mail: [email protected]
C 2014 UICC
Int. J. Cancer: 137, 116–126 (2015) V
lead to such priority targets could be alternative splicing.
Alternative splicing can change the structure of mRNA by
inclusion or skipping of exons, and this may alter the function, stability or binding properties of the encoded protein.2
Aside from its role in physiological cell adaptation, alternative splicing has been shown to occur in human diseases,
including cancer.3 Particularly, the splice variants differ
between cancer and normal corresponding tissues.4,5 Cancerspecific splice variants are thus of significant interest as they
may be involved in pathogenesis and may further potentially
be used as biomarkers and generate novel targets for therapy.
Because immune recognition of antigenic epitopes is highly
specific, alternative exon splicing could provide the structural
basis for expression of novel amino-acid sequences not subject to self repertoire tolerance.
We previously identified an alternative transcript of the B
cell lineage membrane receptor CD20. This alternative transcript lacks 168 nucleotides within Exon 3 to 7 compared to
the wild-type CD20 transcript and referred thereafter as
D393-CD20.6 D393-CD20 translation gives rise to a protein
lacking the extracellular domain and the most part of the
four transmembrane-spanning domains and is, therefore,
cytoplasmic. D393-CD20 mRNA is absent from normal resting B cells but present in various malignant or transformed B
cells.6–8 Moreover, high expression of D393-CD20 protein
117
Vauchy et al.
What’s new?
Cancer-specific splice variants are generating interest as they may potentially be used as biomarkers and generate novel targets for therapy. Here, the authors investigate whether an alternative transcript of the B cell lineage membrane receptor CD20
could generate epitopes that are recognized by T lymphocytes and eligible as new targets for immunotherapy. They show that
D393-CD20 generates promiscuous HLA-DR epitopes recognized by CD4 T cells, and that naturally occurring D393-CD20specific T cells are present in B cell lymphoma patients. The findings support further studies on the modulation of D393CD20-specific T cell response by Rituximab and the development of D393-CD20-specific immunotherapies.
VIAGI), FRR (FRRMSSLELVIAGIV), RRM (RRMSSLEL
VIAGIVE) and RMS (RMSSLELVIAGIVEN) were predicted
to bind multiples HLA molecules by using SYFPETHI
(www.syfpeithi.de), NetMHCpan 2.1 (http://www.cbs.dtu.
dk/services/NetMHCIIpan/) and NetMHCII 2.2 (http://
www.cbs.dtu.dk/services/NetMHCII/) software. Synthetic
peptides (>80% purity) were purchased from ProImmune
(Oxford, UK).
Cell lines
Human cell lines FaDu (-DRB1*04), Ramos (-DRB1*12, DRB1*15), Daudi (-DRB1*07), SKW6.4 (-DRB1*13), MEC1
(-DRB1*07) and Pfeiffer were obtained from the DSMZ or
ATCC cell banks. Jijoye and Raji cell lines (Human Burkitt
Lymphoma) were provided by Diaclone (Besançon, France).
Cells were maintained in RPMI 1640 or DMEM (Lonza,
Paris, France) with 10% heat inactivated endotoxin free foetal
calf serum added (Invitrogen, Cergy-Pontoise, France). For
HLA-DR restriction analysis, FaDu cell line was treated with
IFN-g (100 UI/mL; Peprotech, Neuilly-Sur-Seine, France) for
48 hr before coculture with T cell clones at 1:1 ratio.
D393-CD20 expression in B cell lines
D393-CD20 expression was investigated by Western Blotting.
Briefly, cells were lysed with sample buffer (2% SDS in
125 mM Tris HCl, pH 6.8). Proteins were extracted from 0.2
3 107 to 1 3 107 cells and by electrophoresis on 12.5% SDSpolyacrylamide gels and transferred to PDVF membranes
(Bio-Rad, Marnes-la-Coquette, France). The blots were then
blocked for 1 hr in 6% milk before incubation with specific
antibody against human CD20 (rabbit anti-human CD20 specific to the COOH-terminal region, Spring Bioscience, Pleasanton, CA). Blotted proteins were detected and quantified on
a bioluminescence imager and BIO-1D advanced software
(Vilber-Lourmat, France) after incubating blots with a horseradish peroxidase-conjugated appropriate secondary antibody
(Beckman Coulter, Villepinte, France). The exposure time
was longer for D393-CD20 detection than for CD20 and
actin due to difference in the protein quantity.
Material and Methods
Peptide sequences
The six peptides derived from D393-CD2028–47 (KPLFRR
MSSLELVIAGIVEN), referred as KPL (KPLFRR MSSLEL
VIA), PLF (PLFRRMSSLELVIAG), LFR (LFRRMSSLEL
C 2014 UICC
Int. J. Cancer: 137, 116–126 (2015) V
In vitro generation of D393-CD20-specific CD4 T-cell
lymphocytes
Peripheral blood was obtained from clinical trial for patients
or from a blood bank for healthy patients. Informed consent
Tumor Immunology
has been found in malignant B cells of relapsed patients previously treated with the anti-CD20 therapeutic monoclonal
antibody Rituximab.6 Therefore, we reasoned that the selective expression in leukemic B cells, as well as the expression
of D393-CD20 in resistant lymphomas confers to this spliced
mRNA the potential to be a tumor associated antigen. We
thus investigated whether the D393-CD20 could generate epitopes recognized by T lymphocytes and eligible as new targets for immunotherapy.
Recent progresses on the fields of tumor immunology
highlight the critical role of CD4 T helper cells in antitumor
immunity.9 Among subsets of CD4 helper T cells (Th), Th1
lymphocytes, which mainly produce IFN-g, control cellmediated immunity against tumors. Particularly, recent evidence obtained in rodent models showed that tumor-specific
CD4 T cells are indispensable for effective homing of effector
T cells within the tumor.10,11 Then, it is of particular interest
to identify new MHC Class II-restricted peptides (helper peptides) derived from relevant tumor antigens in order to
actively target antitumor CD4 helper T cell response in vivo.
The pivotal role of cancer specific helper peptides has also
been reported in clinical trials showing effective antitumor T
cell responses and clinical benefits when therapeutic cancer
vaccines contain tumor-derived MHC Class II-restricted
peptides.12,13
In this study, we identify novel promiscuous D393-CD20derived MHC Class II restricted epitopes that bind various
HLA-DR alleles. IFN-g-producing D393-CD20 specific CD4
T cell responses were detected in blood lymphocytes from
lymphoma patients and D393-CD20 specific CD4 Th1 clones
were capable to recognize both lymphoma cell lines and
autologous lymphoma cells and to induce their apoptosis. In
a preclinical model, D393-CD20-derived-peptide immunization induces specific CD4 T cells and also primes CD8 T cell
responses against D393-CD20. Collectively, our results show
that the splicing variant D393-CD20 is immunogenic and
support the interest to stimulate or adoptively transfer D393CD20-specific T cells.
118
for functional tests and genetic analysis was obtained from
patients and healthy donors and this study was approved by
the local ethic committee of Besançon hospital. To evaluate
the presence of memory D393-CD20 specific T cell response,
peripheral blood mononuclear cells (PBMC) from cancer
patients were isolated by density centrifugation on FicollHyperpaque gradients (Sigma-Aldrich) and plated at 2.105
cells per well in a 96-well plate in RPMI 8% human serum
with 10 lmol/L of D393-CD20 20mer peptide. Recombinants
interleukin (IL) 7 (5 ng/mL; Peprotech) and IL-2 (50 UI/mL;
Novartis, Rueil-Malmaison, France) were added at Days 1
and 3, respectively. After 10 days of cell culture, the presence
of D393-CD20 specific T cells was investigated by intracellular cytokine staining after a 10 lmol/L peptide stimulation.
To evaluate the presence of a D393-CD20 specific T cell repertoire in healthy donor’s PBMC, a different protocol was
used with the adjunction of 20mer peptide at Day 7 and IL-2
at Day 10. The intracellular staining was performed at Day
14 after the first peptide stimulation.
Tumor Immunology
D393-CD20 T-cell clones isolation and B lymphoma cell
recognition
T-cell clones were isolated by limiting dilution and amplified
after stimulation by PHA in presence of irradiated allogenic
PBMCs, B-EBV cell line and 150 UI of IL-2 as previously
described.14 Functional analyses of specific CD4 T-cell clones
were done by using intracellular IFN-g staining, IFN-g
ELISA (Diaclone) and Human Ten-plex cytokines assay
(Human Th1/Th2/Inflammation Diaplex; Diaclone). Th0 cells
were isolated using the na€ıve CD4 T cell isolation kit II (Miltenyi Biotec, Paris, France). For lymphoma cell recognition, a
patient’s derived D393-CD20 CD4 T cell clone was cocultured with B-cell lines or autologous PBMC at 1:1 or 2:1
ratio respectively. Supernatants from T-cell clones and B-cell
lines coculture were collected after 18 hr for cytokines detection. Control CD4 T cells were selected using the CD4
MicroBeads Kit (Miltenyi Biotec) and amplified as T-cell
clones.
Flow cytometry analysis
For intracellular IFN-g detection, cells were stimulated for 6
to 18 hr with or without 10 lmol/L peptide, with 1 lL/mL
Golgi Plug (BD Bioscience, Le Pont de Claix, France) before
staining for flow cytometric analysis. Surface staining was
performed using FITC (fluorescein isothiocyanate)-CD4
(clone B-A1), PE-CD8 (clone B-Z31, Diaclone) and Pacific
Blue-CD3 (clone UCHT1, BD Biosciences), cells were then
fixed and permeabilized using CytoFix/Perm Buffer (BD Biosciences) before staining with APC (allophycocyanin)-IFN-g
(clone B27, BD Biosciences). Samples were acquired on a
FACS Canto II (BD Biosciences) and analyzed with the
DIVA software. In antibody blocking experiments, anti-HLADR (clone L243) and anti-HLA-DP (clone B7/21) antibodies
were used at 5 lg/mL. Chemokine receptors expression was
investigated using APC-CCR6 (clone 11A9), PE-Cy7-CXCR3
CD20 alternative splicing D393-CD20 is recognized by CD4 T cells
(clone 1C6) and PE-CCR4 (clone 1G1, BD Biosciences). Positive staining was validated on healthy donor’s PBMC (Supporting Information Fig. S1). For detection of Annexin V,
cells were stained using APC-CD20 (clone 2H7, BD Biosciences) and PE-Kappa light chain (clone TB28-2, eBioscience,
Paris, France). After transfer in binding buffer, V450Annexin V and 7-AAD (BD Biosciences) were added.
Real-time PCR analysis
RNA was extracted using RNeasy Mini kit (Qiagen, CergyPontoise, France) with RLT buffer (Qiagen) supplemented
with b-mercaptoethanol (Sigma-Aldrich). Total RNA was
subjected to reverse transcription (High Capacity RNA-tocDNA Master Mix, Applied Biosystems, Courtaboeuf, France)
and quantified by real-time quantitative PCR using primer/
probe sets listed as follows: T-bet (Hs00203436_m1), Gata3
(Hs00231122_m1),
RORcT
(Hs01076112_m1),
FoxP3
(Hs01085834_m1) (Assay On Demand, Applied Biosystems).
Real-time PCR were performed on the iCycler CFX96 realtime PCR system (Bio-Rad, France). Relative expression for
the mRNA transcripts was calculated using the DDCt method
and G6PDH mRNA transcript as reference.
Mouse and vaccinations
The HLA-DRB1*0101/HLA-A*0201-transgenic mice (A2/
DR1 mice) previously described were kindly provided by Pr
François Lemonnier. Briefly, these mice are H-2 Class I and
IA Class II knockout, and their CD8 T and CD4 T cells are
restricted by the sole HLA-A*0201 and HLA-DR1*0101 molecules, respectively.11,15 For D393-CD2028–47 (20mer) and
D393-CD2033–41 (9mer) immunization, mice were injected
twice with 100 lg of D393-CD20 20mer or 50 mg of D393CD20 9mer were emulsified in incomplete Freund adjuvant
(IFA, Sigma-Aldrich). All peptide vaccinations were done
subcutaneously at the base of the tail at Days 1 and 14. In
some experiments, CD8 T cells were depleted with anti-CD8
monoclonal antibody treatment (500 lg, clone 2.43, BioXcell,
West Lebanon, NH) before sacrifice. All protocols were performed according to the approval of the “Services
Veterinaires de la Sante et de la Protection Animale” delivered by the Ministry of Agriculture (Paris, France).
IFN-c ELISPOT assay
Briefly, splenocytes from immunized mice (2.105 per well)
were cultured in anti-mouse IFN-g monoclonal antibody precoated ELISPOT plate with D393-CD20 20mer or 9mer
(5 lmol/L) in X-VIVO medium (Invitrogen, Saint Aubin,
France) for 18 hr at 37 C. In some experiments, CD8 T cells
were selected using the CD8a MicroBeads Kit (Miltenyi Biotec) before ELISPOT assay. Cells cultured with medium alone
or phorbol-12-myristate-13-acetate (25 ng/mL; SigmaAldrich) and ionomycin (1 lg/mL; Sigma-Aldrich) were used
as negative and positive controls, respectively. The IFN-g
spots were revealed following the manufacturer’s instructions
(Diaclone). Spot-forming cells were counted using the C.T.L.
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Immunospot system (Cellular Technology). All the experiments were conducted in triplicates.
Results
D393-CD20 isoform protein expression and
immunogenicity
To determine whether the D393-CD20 protein is frequently
expressed in B cell malignancies, D393-CD20 protein expression was evaluated in various B cell lines. As shown in Figure
1a, D393-CD20 was expressed in B cell lines derived from
patients with circulating lymphoblastic, marginal zone, Burkitt, mantle or diffuse large B cell lymphomas (Fig. 1a). As
expected, we detected a signal at position 33 to 35 kDa, corresponding to the wild type CD20 protein isoforms (differentially phosphorylated),16 but also two additional bands at 15
to 17 kDa. The two bands at position 15 to 17 kDa may correspond to different phosphorylation states of the D393CD20 protein.6 To assess its expression in primary lymphoma cells, we looked for D393-CD20 protein in peripheral
blood lymphocytes of patient with circulating lymphoma
cells. D393-CD20 protein expression was detected in five
tested patient’s PBMC and was absent in healthy donor’s
PBMC (Fig. 1b). This result confirms D393-CD20 as a lymphoma associated antigen. D393-CD20 is an in frame alternative spliced isoform of CD20. Thus, their amino acids
sequences are similar excepting the lack of the CD2037–92
region (Fig. 1c). Peptides overlapping the splicing site (position 37/38) are thus specific of D393-CD20 and could be
used to stimulate lymphoma specific T cells (Fig. 1d).
To evaluate the presence of D393-CD20 specific T cell in
the human repertoire, the D393-CD2028–47 20mer-peptide
overlapping the splicing site was used to stimulate T cells.
For this purpose, peripheral blood lymphocytes of healthy
volunteers were in vitro stimulated twice using 10 mmol/L of
D393-CD2028–47 in 96 well microplates during 14 days as
described in the material and method section. The presence
of specific CD4 T cells was assessed using IFN-g intracellular
staining after peptide stimulation. D393-CD20-specific CD4
T cell responses were detected in four out of six healthy
donors (Fig. 1e) and frequencies of positive microcultures
were 14/96, 10/96, 3/96 and 8/96 for the four positive healthy
donors with specific CD4 T cell frequencies ranging from 1
to 18.5%.
We then evaluated the presence of D393-CD20 specific T
cell responses in lymphoma patients (Supporting Information
Table I). Ficoll-isolated blood lymphocytes from eight
patients were stimulated once (10 days) with the D393CD2028–47 peptide, and specific CD4 T cell responses were
measured by IFN-g intracellular staining. D393-CD20 specific
CD4 T cell responses were detected in six out of the eight
patients tested (Fig. 1f). For the six patients, frequencies of
positive microcultures were 6/96, 4/96, 1/36, 6/96, 2/96 and
9/96 with IFN-g positive CD4 T cell frequencies ranging
from 1 to 6.9%. Thus a D393-CD20 specific CD4 T cell is
present in peripheral T cell repertoire of lymphoma patients.
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119
Characterization of HLA-DR restricted D393-CD20 specific
CD4 T cells
To further characterize these responses, we derived D393CD2028–47-specific CD4 T cell clones by limiting dilution
from patients’ lymphocyte cultures as previously described
(Godet et al., 2012). We successfully derived CD4 specific
T cell clones from two patients P#1 (P1.4 and P1.17) and
P#7 (P7.10). These CD4 T cell clones were strongly reactive
in the presence of cognate peptide and showed a halfmaximal IFN-g secretion at peptide concentration ranging
from 30 ng/mL (13 nM) to 150 ng/mL (66 nM; Fig. 2a). Of
note, these clones were strictly D393-CD20 specific as they
did not recognize the CD20 peptide overlapping this
sequence (Fig. 2b).
Next, we determined the HLA Class II restriction of
D393-CD20-specific CD4 T cell clones. As shown in Figure
2c, D393-CD2028-47 peptide recognition by all these T cell
clones was inhibited in presence of HLA-DR blocking antibodies indicating their HLA-DR restriction (Fig. 2c). To further investigate the promiscuous HLA-DR binding capacity
of D393-CD2028–47 peptide and the degeneracy of T cell recognition, the reactivity of the CD4 T cell clones was evaluated against various HLA-DR-positive and D393-CD20negative loaded with the D393-CD2028–47 peptide. CD4 T
cell clones from patient P#1 (HLA-DRB1*04 homozygote)
and patient P#7 (HLA-DRB1*04 and -DRB1*11) were reactive against the HLA-DRB1*04 positive cell line FaDu loaded
with the cognate peptide (Fig. 2d).
Investigations have well-documented that the nature of
the Th epitope presented to the TCR is the first signal for
the differentiation of a na€ıve Th cell into a specific Th phenotype.17 However, D393-CD2028–47 specific CD4 T cell
clones isolated from patient P#1 exhibited a CXCR31,
CCR61 and CCR42 phenotype (Fig. 3a) and expressed both
the transcription factors T-bet and RORgT that are associated with a Th1/Th17 profile whereas the CD4 T cell clone
isolated from the patient P#7 was CXCR31, CCR62, CCR42
and expressed T-bet, that is, associated with a Th1 profile
(Fig. 3b). All these clones produced high amount of Th1
associated cytokines such as IFN-g, TNF-a and IL-2 but only
P1.4 and P1.17 produced IL-17 in accordance with their
molecular patterns (Fig. 3c).
D393-CD20 specific CD4 T cell clones recognize malignant
HLA-DR1 B cells
Natural D393-CD20-derived peptide processing was evaluated by a coculture of the CD4 T cell clones with HLADR1 D393-CD201 cell lines (Fig. 4a). P1.4 and P1.17 CD4
T cell clones were able to recognize SKW6.4 cells (HLADRB1*13) and P7.10 recognized HLA-DRB1*07 positive
Daudi cells (Fig. 4a). The HLA-DR restriction of these
responses was confirmed using HLA-DR blocking antibodies
(Fig. 4a). These results suggested that these CD4 T cell
clones were alloreactive or were able to recognize the D393-
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Vauchy et al.
CD20 alternative splicing D393-CD20 is recognized by CD4 T cells
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120
Figure 1. D393-CD20 expression and T cell specific repertoire. Western blot analysis, after denaturing acrylamide electrophoresis, with antiC-term CD20 antibody of whole-cell lysates from B-cell lines (a) and from PBMC of patients (P#) or healthy donors (HD#) (b). A signal at
position 33 to 35 kDa, corresponding to the WT-CD20 protein isoforms and two additional bands at 15 to 17 kDa corresponding to different
post-translational modifications of the D393-CD20 protein could be detected. The exposure time was longer for D393-CD20 detection than
for CD20 and actin due to difference in the protein quantity. Data are representative of at least three independent experiments. Sequence
alignment of amino-acids preceding or following splice donor and acceptor sites respectively of CD20 and D393-CD20 (c). Peptides spanning the splice site used for epitope-mapping studies (d). The three first amino-acids used to name these peptides are indicated in boldface. T cell lines were obtained from healthy donors’ PBMCs after two rounds of in vitro stimulation in microplates with 10 lg/mL of D393CD2028–47 peptide. Specific responses were assessed by CD4 and IFN-g staining. A microculture was considered as positive when (i) the
fraction of IFN-g producing T cells was twofold higher upon peptide stimulation than unstimulated, (ii) and higher than 1% of CD4 T-cell
after deducting the background. The bars represent the mean of the CD4 T cell responses in the microcultures (e). T cell lines were
obtained from PBMCs of patients with B-cell lymphomas cultured with 10 lg/mL of D393-CD2028–47 peptide during 10 days, and specific
responses in microcultures were assessed by CD4 and IFN-g staining (n 5 96 microcultures for patients 1, 2, 4, 5, 6, 7, 8 and n 5 36 microcultures for Patient 3) (f).
CD2028–47 peptide in different HLA Class II contexts as it
has been previously reported for other CD4 T cells.18–21 As
P7.10 did not recognized the HLA-DRB1*07 positive MEC-
1 cell line (Fig. 4a) and P1.4 and P1.17 did not recognized a
HLA-DRB1*13 melanoma cell line (Data not shown), the
alloreactivity of these D393-CD20 specific T cell clones was
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Vauchy et al.
Figure 2. Functional characterization of D393-CD20-specific CD4 T cell clones. D393-CD20-specific CD4 T cell clones isolated from B-cell
lymphoma patients were stimulated 6 hr with a range of D393-CD2028–47 peptide concentration and their reactivity was assessed by IFN-g
intracellular staining (a). CD4 T-cell clones were stimulated by D393-CD20 or CD20 derived peptides for 6 hr and their reactivity was
assessed by IFN-g intracellular staining (b). D393-CD20 specific T-cell clones were stimulated with D393-CD2028–47 peptide (10 lg/mL) in
presence of 5 lg/mL of anti-HLA-DR (L243) or HLA-DP (B7/21) blocking antibodies and their reactivity was assessed by IFN-g intracellular
staining (c). HLA-class-II-positive cell line FaDu was loaded with the D393-CD20 derived peptide and CD4 T cell clones reactivity was
assessed in an IFN-g ELISA (d). Data are representative of three independent experiments.
rule out. These results indicate the natural processing of
D393-CD20 on HLA-DR molecules by malignant B-cell
lines.
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To identify the minimal epitope recognized within the
specific sequence of D393-CD20, we tested the reactivity the
CD4 T cell clones against the six 15mer peptides derived
CD20 alternative splicing D393-CD20 is recognized by CD4 T cells
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122
Figure 3. Polarization of D393-CD20-specific CD4 T cell clones. CXCR3, CCR6 and CCR4 expression on resting D393-CD20 specific T cell
clones was assessed by flow cytometry (a). Quantitative reverse transcriptase–PCR analysis for master transcription factors associated with
Th1, Th2, Th17 and Treg phenotypes was performed using mRNA isolated from resting T cell clones or na€ıve T CD4 cells isolated from a
healthy donor (Th0). Mean 6 s.e.m. of gene expression levels are represented. Data are representative of two independent experiments performed in duplicate (b). Cytokines produced by D393-CD20-specific T cell clones in response to phorbol-12-myristate-13-acetate (PMA; 25
ng/mL) and Ionomycine (1 lg/mL) stimulation were assessed using a human Ten-plex cytokines assay. Data represent the mean result
from duplicate after background subtraction (c).
from the D393-CD20 specific sequence (Fig. 1d). On the one
hand, the CD4 T cell clone isolated from the patient P#7
needed the proline (Pro29) and the alanine (Ala42) meaning
that the 13mer peptide (PLFRRMSSLEVIA) would be the minimal epitope recognized (Fig. 4b). On the other hand, CD4 T
cells clones isolated from the patient P#1 required the leucine
(Leu30) and the alanine (Ala42) meaning that the 12mer peptide
(LFRRMSSLEVIA) would be the minimal epitope recognized
(Fig. 4b). Nonetheless as all of these clones recognized a series
of D393-CD20 derived peptides, we cannot formally assess that
the shortest ones will be the exact peptides naturally presented
by HLA-DR molecules on B cell lymphoma cells.
As effector CD4 T cells have also been shown to exert
direct cytotoxicity against tumor cells,22,23 we further
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Vauchy et al.
Figure 4. D393-CD20-specific CD4 T cell clones recognize malignant B cells. Reactivity of each T cell clones against HLA-Class II expressing
B-cell lines (Ratio 1/1) was assessed after 16 hr in an IFN-g ELISA. Data represent the mean result from three independent experiments (a).
CD4 T-cell clones were cultured with 10 lg/mL of the 20mer (D393-CD2028–47) or 15mer peptides (KPL, PLF, LFR, FRR, RRM, RMS) during 18
hr and IFN-g production was assessed by intracellular staining. Data represent the mean result from two independent experiments (b). The
antitumor activity of the P1.17 T cell clone derived from Patient 1 with circulating lymphoplasmacytic B cell lymphoma cells was evaluated
against unloaded autologous PBMC (Ratio 1/1) after a 72 hr coculture by annexin V staining on CD201 j1 B cells (c) and its reactivity was
assessed by human Ten-plex cytokines assay (d). Control cells are CD4 T cells sorted from Patient 1 and amplified alike P1.17 T cell clone.
Data are representative from at least three independent experiments.
evaluated the cytotoxic activity of one of the D393-CD20specific CD4 T cell clone P1.17 derived from a patient (P#1)
with an active disease with peripheral blood lymphocytes
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containing 11.6% of lymphoma B cells (Data not shown).
P1.17 was cocultured with autologous B lymphoma cells and
the cell cytotoxicity was measured by annexin-V staining. As
124
CD20 alternative splicing D393-CD20 is recognized by CD4 T cells
Tumor Immunology
Figure 5. Vaccination with D393-CD20 derived long peptide induces both CD8 and CD4 T cells priming. HLA-A2/HLA-DR1 transgenic mice
immunized with D393-CD2028–47 were CD8 T cells depleted with 500 lg of CD8 clone 2.43 and splenocytes were stimulated with D393CD20 derived peptides and their reactivity was assessed ex vivo in an IFN-g ELISPOT (a). D393-CD2033–41 (SP) specific T cell recognition
after D393-CD20 SP or D393-CD20 LP vaccination was assessed ex vivo on sorted CD8 T cells (b). D393-CD2028–47 (LP) specific T cell recognition after D393-CD20 SP or D393-CD20 LP vaccination was assessed ex vivo on CD8 T cells depleted splenocytes in an IFN-g ELISPOT (c).
Five mice per group were used in each experiment.
shown in Figure 4c, higher percent of B cell lymphoma apoptosis was found in presence of P1.17 than in presence of
autologous irrelevant control CD4 T cells (59% vs. 18.3%;
Fig. 4c). This specific recognition was also assessed by TNF-a
secretion during the coculture experiment (Fig. 4d). Together,
these results show a cytolytic activity of D393-CD20-specific
CD4 T cell clone and also support the natural processing of
D393-CD20-derived MHC Class II epitopes on B lymphoma
cells.
D393-CD20–derived peptide vaccination stimulates both
specific CD4 and CD8 T cells in HLA-A2/HLA-DR1
transgenic mice
To assess the capacity of the 20mer long peptide D393CD2028–47 (LP) to prime a specific T cell response in vivo,
we performed a peptide immunization in HLA-A2/HLA-DR1
transgenic mice and specific T cell responses were measured
by ex vivo IFN-g ELISPOT assay. As shown in Figure 5a,
specific CD4 T cell responses were induced in vivo against
the D393-CD2028–47 LP and against four of the six D393CD20 derived 15mer peptides supporting that these peptides
effectively binds to HLA-DR1 molecules. Previous studies
indicate that the use of synthetic long peptides can induce
more effective cytotoxic T cell (CTL) responses than minimal
MHC Class I restricted peptide-based vaccine.24 We then
evaluated the capacity of the D393-CD2028–47 long peptide
(LP) to prime HLA-A2-restricetd CTL response. To this end,
mice were immunized either with the D393-CD2028–47 LP or
with the HLA-A2-restricted D393-CD20 derived short peptide D393-CD2033–41 (SP). As shown in Figure 5b, higher
IFN-g producing specific-CTL were detected ex vivo after
immunization with the D393-CD2028–47 LP than with the SP.
This could be related to the help provided by the D393CD2028–47-specific CD4 T cells simultaneously induced in
vivo (Fig. 5c). Collectively, these results suggest that after
uptake of D393-CD2028–47, antigen presenting cells could
simultaneously prime D393-CD20-specific CTL and CD4 T
cell responses in vivo.
Discussion
Recent reports have shown that a cancer antigen could be
processed into different variants as the result of RNA alternative splicing.25,26 Alternative splicing can change the structure
of mRNA by inclusion or skipping of exons, and this may
alter the function, stability or binding properties of the
encoded protein.2 Cancer-specific splice variants are thus of
significant interest as they could be involved in pathogenesis
and may further potentially be used as biomarkers and generate novel targets for therapy. The new amino-acid sequences
generated in that case have previously shown to be potentially immunogenic.27,28 Moreover, some of these epitopes
produced by cancer-related alternative splicing are recognized
by specific CD8 T cells29–32 or CD4 T cells33 from cancer
patients. The presence of candidate antigens derived from
alternative splicing has however never been described in B
cell lymphomas.
In this study, we identified promiscuous HLA-DR epitopes
derived from the D393-CD20 splice variant of the MS4A1
gene. Even if CD4 T cells are not considered to recognize
MHC-II expressing cancer cells efficiently, direct recognitions
of B cell lymphomas by D393-CD20 specific CD4 T cell
clones have been observed. Particularly antitumor activity of
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D393-CD20 specific CD4 T cells has been assessed on autologous lymphoma B cells. Such direct tumor recognition by
CD4 T cells has nevertheless already been reported for many
tumor antigens and may be dependent of the epitope recognized.34,35 This is the first demonstration of the immunogenicity of D393-CD20 and of the presence of spontaneous
D393-CD20 specific CD4 T cell responses in patients with B
cell malignancies.
Different subpopulations of CD4 T helper lymphocytes
regulate the antitumor response36 and Th1 immunity has a
clear positive effect in cancer cell eradication while the role
of Th17 cells is still controversial and seems to depend on
the type of cancer. It is now clear that Th17 could convert
toward a Th1 like phenotype (See review37). For instance,
Th17 cells specific for the cancer testis antigen MAGE-A3
isolated from a patient with lung cancer were readily converted into IFN-g–secreting Th1-like effectors.38 In this
study, according to the secretion, chemokine receptors and
transcription factor expression profiles, we have isolated both
Th1 and Th1/Th17 D393-CD20 specific T cell clones and
IFN-g-secreting D393-CD20-specific T cells were detected in
B cell lymphoma patients suggesting the presence of an active
antitumor CD4 T cell response. However, even if Signal 1
influences the CD4 T cell polarization39,40 we could not
exclude the presence of D393-CD20 with other polarization
as we have not addressed the presence of Th2 or Treg polarized D393-CD20 specific T cell responses.
HLA-DR restriction of the D393-CD20 specific CD4 T
cell response was assessed by HLA-DR-blocking antibody
experiments, and interestingly HLA-DR alleles of recognized
B cell lines were not shared. These results are in line with
previous studies showing that several human T cells are able
to recognize different peptides in the context of one or several restriction elements.18–21 This result supports the promiscuous nature of D393-CD20 and of the TCRs expressed
by these CD4 T cell clones. In this study, significant frequencies of IFN-g-secreting D393-CD20-specific T cells were
detected in patients with B cell malignancies but also in
healthy donors. One explanation of the presence of D393CD20 specific responses in healthy donors could be explained
by the expression of D393-CD20 in EBV infected B cells.6
Thus, the D393-CD20 specific response could be both an
antiviral and an antitumor immune response. However, we
have not compared the presence of this response in EBV positive and in EBV negative donors.
Recent studies have shown that vaccines containing natural CTL epitopes included in long peptides were superior
to those comprising minimal CTL epitopes because of
long-lasting cross-presentation of the longer peptides.41,42
We showed that D393-CD2028–47 was superior to D393CD2033–41 to stimulate D393-CD20 specific CTLs in HLAA2/HLA-DR1 transgenic mice. However, we did not
observed spontaneous HLA-A2 restricted D393-CD20 specific CTL responses in human. Therefore, we are not able
to conclude that using D393-CD2028–47 would lead to
D393-CD20 specific CTL responses in human. Analyzing
cancer data sets which included lymphoblastic and Burkitt
lymphomas (The Alternative Splicing and Transcript
Diversity database43), Stranzl et al. have shown that alternative splicing in cancer cell is associated with the loss of
epitopes restricted by frequently expressed HLA Class I
compared to splicing in normal tissue.44 This observation
is also present here since the CD20188–196 previously
described45 HLA-A2 restricted epitope is absent from the
D393-CD20 alternative spliced protein missing the
CD2037–204 region.6
In conclusion, we have shown that the D393-CD20 splice
variant generates promiscuous HLA-DR epitopes recognized
by CD4 T cells and that naturally occurring D393-CD20 specific Th1 and Th1/Th17 antitumoral cells are present in B
cell lymphoma patients. These findings support further studies on the modulation of D393-CD20 specific T cell response
by Rituximab treatment and the development of D393-CD20
specific immunotherapies in B cell malignancies.
References
1.
2.
3.
4.
5.
6.
Cheever MA, Allison JP, Ferris AS, et al. The prioritization of cancer antigens: a national cancer
institute pilot project for the acceleration of
translational research. Clin Cancer Res 2009;15:
5323–37.
Faustino NA, Cooper TA. Pre-mRNA splicing
and human disease. Genes Dev 2003;17:419–37.
Kaida D, Schneider-Poetsch T, Yoshida M. Splicing in oncogenesis and tumor suppression. Cancer Sci 2012;103:1611–6.
Gardina PJ, Clark TA, Shimada B, et al. Alternative splicing and differential gene expression in
colon cancer detected by a whole genome exon
array. BMC Genomics 2006;7:325.
He C, Zuo Z, Chen H, et al. Genome-wide detection of testis- and testicular cancer-specific alternative splicing. Carcinogenesis 2007;28:2484–90.
Henry C, Deschamps M, Rohrlich P-S, et al.
Identification of an alternative CD20 transcript
variant in B-cell malignancies coding for a novel
protein associated to rituximab resistance. Blood
2010;115:2420–9.
7. Gamonet C, Ferrand C, Colliou N, et al. Lack of
expression of an alternative CD20 transcript variant in circulating B cells from patients with pemphigus. Exp Dermatol 2014;23:66–7.
8. Small GW, McLeod HL, Richards KL. Analysis of
innate and acquired resistance to anti-CD20 antibodies in malignant and nonmalignant B cells.
PeerJ 2013;1:e31.
9. Kennedy R, Celis E. Multiple roles for CD41 T
cells in anti-tumor immune responses. Immunol
Rev 2008;222:129–44.
10. Bos R, Sherman LA. CD41 T-cell help in the
tumor milieu is required for recruitment and
cytolytic function of CD81 T lymphocytes. Cancer Res 2010;70:8368–77.
11. Dosset M, Godet Y, Vauchy C, et al. Universal
cancer peptide-based therapeutic vaccine breaks
tolerance against telomerase and eradicates estab-
C 2014 UICC
Int. J. Cancer: 137, 116–126 (2015) V
lished tumor. Clin Cancer Res Off J Am Assoc
Cancer Res 2012;18:6284–95.
12. Aarntzen EHJG, De Vries IJM, Lesterhuis WJ,
et al. Targeting CD4(1) T-helper cells improves
the induction of antitumor responses in dendritic cell-based vaccination. Cancer Res 2013;73:
19–29.
13. Slingluff CL Jr, Lee S, Zhao F, et al. A randomized phase II trial of multiepitope vaccination
with melanoma peptides for cytotoxic T cells and
helper T cells for patients with metastatic melanoma (E1602). Clin Cancer Res Off J Am Assoc
Cancer Res 2013;19:4228–38.
14. Godet Y, Fabre E, Dosset M, Lamuraglia M, et al.
Analysis of spontaneous tumor-specific CD4 Tcell immunity in lung cancer using promiscuous
HLA-DR telomerase-derived epitopes: potential
synergistic effect with chemotherapy response.
Clin Cancer Res Off J Am Assoc Cancer Res 2012;
18:2943–53.
Tumor Immunology
125
Vauchy et al.
Tumor Immunology
126
15. Pajot A, Michel M-L, Fazilleau N, et al. A mouse
model of human adaptive immune functions:
HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/
class II-knockout mice. Eur J Immunol 2004;34:
3060–9.
16. Tedder TF, Schlossman SF. Phosphorylation of
the B1 (CD20) molecule by normal and malignant human B lymphocytes. J Biol Chem 1988;
263:10009–15.
17. Pfeiffer C, Stein J, Southwood S, et al. Altered
peptide ligands can control CD4 T lymphocyte
differentiation in vivo. J Exp Med 1995;181:1569–
74.
18. Doherty DG, Penzotti JE, Koelle DM, et al. Structural basis of specificity and degeneracy of T cell
recognition: pluriallelic restriction of T cell
responses to a peptide antigen involves both specific and promiscuous interactions between the T
cell receptor, peptide, and HLA-DR. J Immunol
Baltim Md 1950 1998;161:3527–35.
19. Mycko MP, Waldner H, Anderson DE, et al.
Cross-reactive TCR responses to self antigens
presented by different MHC class II molecules.
J Immunol Baltim Md 1950 2004;173:1689–98.
20. Hennecke J, Wiley DC. Structure of a complex of
the human alpha/beta T cell receptor (TCR)
HA1.7, influenza hemagglutinin peptide, and
major histocompatibility complex class II molecule, HLA-DR4 (DRA*0101 and DRB1*0401):
insight into TCR cross-restriction and alloreactivity. J Exp Med 2002;195:571–81.
21. Maccalli C, Li YF, El-Gamil M, et al. Identification of a Colorectal Tumor-Associated Antigen
(COA-1) Recognized by CD41 T Lymphocytes.
Cancer Res 2003;63:6735–43.
22. Guo Y, Niiya H, Azuma T, et al. Direct recognition and lysis of leukemia cells by WT1-specific
CD41 T lymphocytes in an HLA class IIrestricted manner. Blood 2005;106:1415–8.
23. Akhmetzyanova I, Zelinskyy G, Schimmer S,
et al. Tumor-specific CD41 T cells develop cytotoxic activity and eliminate virus-induced tumor
cells in the absence of regulatory T cells. Cancer
Immunol Immunother CII 2013;62:257–71.
24. Bijker MS, van den Eeden SJF, Franken KL, et al.
CD81 CTL priming by exact peptide epitopes in
incomplete Freund’s adjuvant induces a vanishing
CTL response, whereas long peptides induce sustained CTL reactivity. J Immunol Baltim Md
1950 2007;179:5033–40.
CD20 alternative splicing D393-CD20 is recognized by CD4 T cells
25. Zhang L, Vlad A, Milcarek C, Finn OJ. Human
mucin MUC1 RNA undergoes different types of
alternative splicing resulting in multiple isoforms.
Cancer Immunol Immunother CII 2013;62:423–
35.
26. Lin J, Lin L, Thomas DG, et al. Melanoma-associated antigens in esophageal adenocarcinoma:
identification of novel MAGE-A10 splice variants.
Clin Cancer Res Off J Am Assoc Cancer Res 2004;
10:5708–16.
27. Kobayashi J, Torigoe T, Hirohashi Y, et al. Comparative study on the immunogenicity between
an HLA-A24-restricted cytotoxic T-cell epitope
derived from survivin and that from its splice
variant survivin-2B in oral cancer patients.
J Transl Med 2009;7:1.
28. Volpe G, Cignetti A, Panuzzo C, et al. Alternative
BCR/ABL splice variants in Philadelphia
chromosome-positive leukemias result in novel
tumor-specific fusion proteins that may represent
potential targets for immunotherapy approaches.
Cancer Res 2007;67:5300–7.
29. Lupetti R, Pisarra P, Verrecchia A, v. Translation
of a retained intron in tyrosinase-related protein
(TRP) 2 mRNA generates a new cytotoxic T lymphocyte (CTL)-defined and shared human melanoma antigen not expressed in normal cells of
the melanocytic lineage. J Exp Med 1998;188:
1005–16.
30. Guilloux Y, Lucas S, Brichard VG, et al. A peptide recognized by human cytolytic
T lymphocytes on HLA-A2 melanomas is
encoded by an intron sequence of the
N-acetylglucosaminyltransferase V gene. J Exp
Med 1996;183:1173–83.
31. Robbins PF, El-Gamil M, Li YF, et al. The
intronic region of an incompletely spliced gp100
gene transcript encodes an epitope recognized by
melanoma-reactive tumor-infiltrating lymphocytes. J Immunol Baltim Md 1950 1997;159:
303–8.
32. Andersen RS, Andersen SR, Hjortsø MD, et al.
High frequency of T cells specific for cryptic epitopes in melanoma patients. Oncoimmunology
2013;2:e25374.
33. Slager EH, van der Minne CE, Kr€
use M, et al.
Identification of multiple HLA-DR-restricted epitopes of the tumor-associated antigen CAMEL by
CD41 Th1/Th2 lymphocytes. J Immunol Baltim
Md 1950 2004;172:5095–102.
34. Friedman KM, Prieto PA, Devillier LE, et al.
Tumor-specific CD41 melanoma tumorinfiltrating lymphocytes. J Immunother Hagerstown Md 1997 2012;35:400–8.
35. Matsuzaki J, Tsuji T, Luescher I, et al. Nonclassical antigen-processing pathways are required for
MHC class II-restricted direct tumor recognition
by NY-ESO-1-specific CD4(1) T cells. Cancer
Immunol Res 2014;2:341–50.
36. Zhu J, Paul WE. Peripheral CD41 T-cell differentiation regulated by networks of cytokines and
transcription factors. Immunol Rev 2010;238:247–
62.
37. Muranski P, Restifo NP. Essentials of Th17 cell
commitment and plasticity. Blood 2013;121:2402–
14.
38. Hama€ı A, Pignon P, Raimbaud I, et al. Human
T(H)17 immune cells specific for the tumor antigen MAGE-A3 convert to IFN-g-secreting cells
as they differentiate into effector T cells in vivo.
Cancer Res 2012;72:1059–63.
39. Cecil DL, Holt GE, Park KH, et al. Elimination of
IL-10-inducing T-helper epitopes from an
IGFBP-2 vaccine ensures potent antitumor activity. Cancer Res 2014;74:2710–8.
40. Van Panhuys N, Klauschen F, Germain RN.
T-cell-receptor-dependent signal intensity dominantly controls CD4(1) T cell polarization in
vivo. Immunity 2014;41:63–74.
41. Speetjens FM, Kuppen PJK, Welters MJP, et al.
Induction of p53-specific immunity by a p53 synthetic long peptide vaccine in patients treated for
metastatic colorectal cancer. Clin Cancer Res Off J
Am Assoc Cancer Res 2009;15:1086–95.
42. Melief CJM, van der Burg SH. Immunotherapy of
established (pre)malignant disease by synthetic
long peptide vaccines. Nat Rev Cancer 2008;8:
351–60.
43. Koscielny G, Le Texier V, Gopalakrishnan C, et al.
ASTD: The Alternative Splicing and Transcript
Diversity database. Genomics 2009;93:213–20.
44. Stranzl T, Larsen MV, Lund O, et al. The cancer
exome generated by alternative mRNA splicing
dilutes predicted HLA class I epitope density.
PloS One 2012;7:e38670.
45. Bae J, Martinson JA, Klingemann HG. Identification of CD19 and CD20 peptides for induction of
antigen-specific CTLs against B-cell malignancies.
Clin Cancer Res Off J Am Assoc Cancer Res 2005;
11:1629–38.
C 2014 UICC
Int. J. Cancer: 137, 116–126 (2015) V
Article
Synergistic CD40 signaling on APCs and
CD8 T cells drives efficient CD8 response
and memory differentiation
Sylvain Meunier,*,†,1 Laëtitia Rapetti,*,1 Laurent Beziaud,* Christiane Pontoux,*
Agnès Legrand,* and Corinne Tanchot*,†,2
*Institut National de la Santé et de la Recherche Médicale, INSERM U1020, and †INSERM U970 Paris Cardiovascular Research
Center, Université Paris Descartes, Paris, France
RECEIVED JUNE 17, 2011; REVISED NOVEMBER 9, 2011; ACCEPTED NOVEMBER 30, 2011. DOI: 10.1189/jlb.0611292
ABSTRACT
The role of CD4 help during CD8 response and memory
differentiation has been clearly demonstrated in different experimental models. However, the exact mechanisms of CD4 help remain largely unknown and preclude replacement therapy to develop. Interestingly,
studies have shown that administration of an agonist
aCD40ab can substitute CD4 help in vitro and in vivo,
whereas the targets of this antibody remain elusive. In
this study, we address the exact role of CD40 expression on APCs and CD8 T cells using aCD40ab treatment in mice. We demonstrate that aCD40 antibodies
have synergetic effects on APCs and CD8 T cells. Full
efficiency of aCD40 treatment requires CD40 expression on both populations: if one of these cell populations is CD40-deficient, the CD8 T cell response is impaired. Most importantly, direct CD40 signaling on
APCs and CD8 T cells affects CD8 T cell differentiation
differently. In our model, CD40 expression on APCs plays
an important but dispensable role on CD8 T cell expansion and effector functions during the early phase of the
immune response. Conversely, CD40 on CD8 T cells is
crucial and nonredundant for their progressive differentiation into memory cells. Altogether, these results highlight that CD40 –CD40L-dependent and independent effects of CD4 help to drive a complete CD8 T cell differentiation. J. Leukoc. Biol. 91: 859 – 869; 2012.
INTRODUCTION
The generation of memory CD8 T cells is a key factor for clinical immunotherapy. However, the mechanisms underlying ef-
Abbreviations: aCD40ab5anti-CD40 antibody, ARC5Association de la recherche contre le cancer, BM5bone marrow, CD3-«2/25CD3-«-deficient,
Ct5threshold cycle, d5dilution, hprt5hypoxanthine guanine phosphoribosyl transferase, L5ligand, LCMV5lymphocytic choriomeningitis virus,
LIP5lymphopenia-induced proliferation, TCM5central memory T cells,
Tg5transgenic, Zfy-15zinc finger protein Y-linked
The online version of this paper, found at www.jleukbio.org, includes
supplemental information.
0741-5400/12/0091-859 © Society for Leukocyte Biology
fector and memory CD8 T cell differentiation are far from being established entirely [1–3]. Elucidation of these mechanisms, leading to the generation of long-lasting and highly
efficient cells, may pave new pathways toward improved clinical therapy and vaccination.
A few years ago, we and others [4 – 8] demonstrated that the
generation of efficient memory CD8 T cells requires the presence of CD4 T cells. Vaccinations and treatments relying on
CD8 response should consequently target CD4 and CD8 T cell
populations. An alternative is to bypass the requirement for
CD4 T cells. This implies that the underlying mechanisms of
CD4 help, which are still under extensive debates, should be
dissected further [9 –11].
CD4 help on CD8 T cell responses was described initially
during CD8 T cell activation and involved CD40 –CD40L interactions, expressed, respectively, on APCs and CD4 T cells [12].
Based on these observations, a sequential model has been proposed. This model supports the hypothesis that CD4 T cells
express CD40L upon activation and activate the APCs, which
express CD40. The licensed APCs would then drive CD8 responses [12]. Additionally, stimulations by an agonist aCD40ab
have been proven to be sufficient to induce efficient CD8 responses in the absence of CD4 T cell help in vitro and in vivo
[13–16]. Such aCD40ab treatments are used in tumor [17, 18]
as well as in viral models [19] to increase CD8 T cell responsiveness. However, injection of aCD40ab may also induce sideeffects. The administration of aCD40ab in certain tumor models reduced CD8 T cell response [20] and provoked the expression of several angiogenic factors enhancing tumor growth
[21–24]. Other studies indicated that aCD40ab could profoundly suppress CD8 response to LCMV infection [25]
and failed to induce effector functions in an influenza model
[26]. Breakdowns of peripheral tolerance, inducing autoimmune diseases, have also been reported [27, 28].
Additionally to the APCs, CD8 T cells can express CD40 as
well [5, 12]. Therefore, we hypothesize that such diversity of
1. These authors contributed equally to this work.
2. Correspondence: INSERM U1020, Institut Necker, 156 Rue de Vaugirard,
75015, Paris, France. E-mail: [email protected]
Volume 91, June 2012
Journal of Leukocyte Biology 859
Mice
CD3-«2/2 mice; CD3-«2/2CD402/2 mice; Rag22/2 mice expressing a
TCR-ab Tg specific for the HY male antigens, restricted to MHC class II
IAb [31] or restricted to MHC class I Db [4]; and Rag22/2 CD402/2 HY Tg
mice [5] were bred at the Center for the Development of Advanced Experimental Techniques (Orleans, France). Experimental procedures were approved by the French University Animals Ethics Committee and conducted
according to the institutional guidelines of the European Community.
Immunization protocol
Sublethally irradiated (400 Rad) CD3-«2/2 and CD3-«2/2CD402/2 female
mice were injected with 0.5 3 106 male 1 4.5 3 106 female BM cells from
CD3-«2/2 or CD3-«2/2CD402/2 mice, respectively. BM cells have a high
capacity for cell divisions, allowing male cells to grow in host mice at early
time-points after immunization, thus mimicking infectious antigenic spread
or tumoral antigenic proliferation. Three days later, 0.5 3 106 LN CD8 HY
Tg T cells (from Rag22/2 HY Tg CD401/1 or CD402/2 female mice) were
injected alone, with an equal number of LN CD4 HY Tg T cells or with an
agonist aCD40ab (FGK45; Fig. 1A). These HY Tg T cells are specific for
male cells (expressing the HY antigen). The aCD40ab was injected at 50
mg/mouse at Days 0, 2, and 4. This administration protocol insures the
presence of aCD40ab when APCs and CD8 T cells express the CD40 molecule. The activation of APCs results in a rapid up-regulation of CD40,
whereas CD8 T cells in our in vivo experimental system express CD40 transitory, with an expression peak reached 4 days postimmunization [5]. At
different time-points after the immunization, the number of CD8 T cells
recovered from the spleen, a pool of LN, and liver was determined and referred to as number of CD8 T cells/mouse.
To perform in vivo secondary response, CD8 T cells were isolated
and purified from the spleen of the different chimeras at Day 60 of the
primary immune response. Then, 0.5 3 106 CD8 T cells were injected with
0.5 3 106 naive CD4 T cells into new chimeras immunized with the male
antigen. As control, lethargic CD8 T cells (injected without CD4 help)
were also isolated at Day 60 and injected alone into new chimeras immunized with the male antigen.
Immunofluorescence analysis
The following mAb were used: biotin-labeled anti-CD127 (IL-7Ra) and antiCD62L revealed by streptavidin-allophycocyanin; PerCP-labeled anti-CD4
and anti-CD8; PE-labeled anti-IFN-g; and FITC-labeled anti-T3.70 (anti-
860 Journal of Leukocyte Biology
Volume 91, June 2012
CD3-/- female recipients
CD40+/+ or CD40-/-
Day -3
CD3 -/- bone marrow cells
CD40+/+ or CD40-/90% female
10% male
(HY male)
B
9
8
7
6
5
4
3
2
1
0
Day
Day 0
Anti-HY CD8 + T cells
CD40+/+ or CD40-/+
anti-HY CD4 + T cells
or
anti-CD40 antibody
C
# of male cells / 10 6 cells (*104 )
MATERIALS AND METHODS
A
Day 4
Day 7
4 7
No CD8
4 7
CD8
CFSE
D
# CD8 T cells / mouse (*10 6)
effects is related to the diversity of targets. Indeed, we have
demonstrated, in our experimental model, that CD40-deficient
CD8 T cells were not able to receive CD4 T cell help and were
incapable of differentiating into memory cells [5]. CD8CD402/2
T cells exhibited major defects in secondary response and on expansion, antigenic load control, effector functions, cytokine receptor expression patterns, as well as higher sensitivity to inhibitory cytokines [29]. These and other studies highlighted the significant importance of CD40 –CD40L interactions in CD8
responses [30] and led to a reconsideration of aCD40 adjuvant
treatment.
In this study, we investigated the effect of aCD40ab treatment on CD8 immune responses compared with the provision
of CD4 help and determine the relative importance of CD40
expression on APCs and CD8 T cells. For this purpose, we
used an experimental procedure permitting the restriction of
CD40 expression on none, only one, or both of these populations.
18
16
14
12
10
8
6
4
2
0
Day
0,05
0,04
0,03
0,02
0,01
0
4
7
No Ag
4
7
Ag
Figure 1. Description and characteristic of the experimental model.
(A) Schema of the experimental strategy as described in Materials
and Methods. (B) CD3-«2/2 female mice were injected with 0.5 3 106
male 1 4.5 3 106 female BM cells from CD3-«2/2 mice. Three days
later, one-half of them was injected with 0.5 3 106 CD8 1 0.5 3 106
CD4 T cells, and one-half of them was not injected with T cells. Results show the number of male cells detected/million splenic cells recovered at Day 4 (white histograms) and Day 7 (black histograms)
from hosts injected (CD8) or not (No CD8) with T cells. Each sample
was performed in triplicate. Data show the average 6 sd of two mice/
group and are representative of one experiment. (C and D) CD3-«2/2
female mice were injected with 5 3 106 female BM cells from CD3«2/2 mice. Three days later, they were injected with 0.5 3 106 CD8 T
cells. In parallel, CD3-«2/2 female mice were injected with 0.5 3 106
male 1 4.5 3 106 female BM cells from CD3-«2/2 mice. Three days
later, they were injected with 0.5 3 106 CD8 1 0.5 3 106 CD4 T cells.
CD8 T cells were stained with CFSE before injection. (C) Results show
CFSE staining of CD8 T cells isolated from female hosts injected (thin
lines) or not (bold lines) with male BM cells at Days 4 and 7, respectively. Dotted lines represents the isotype control. (D) Results show
the absolute number of CD8 T cells recovered at Day 4 (white histograms) and Day 7 (black histograms) from female hosts injected (Ag)
or not (No Ag) with male BM cells. Data show the average 6 sd of
two mice/group and are representative of one experiment.
www.jleukbio.org
Meunier et al.
TCR-a Tg; PharMingen, San Diego, CA, USA). For some experiments, CD8
T cells were stained with CFSE before injection, as described previously
[5]. Flow cytometry was performed using a FACSCalibur cytometer and data analysis via Flow Jo software (Becton Dickinson, San Jose, CA, USA).
Antigen load
The antigen load was determined directly by quantifying the number of
male cells remaining in the spleen during the primary response, as described previously [29]. Briefly, we quantified the genomic DNA-encoded
Zfy-1 gene (present at one copy/antigenic male cells only) and hprt gene
(present at two copies on male and female cells) using real-time PCR (7900
HT, Applied Biosystems, Warrington, UK) with SYBR Green dye (Applied
Biosystems).
The number of male cells/million of total cells was calculated as follows: 2 3
2(Cthprt2CtZfy-1) 3 (dhprt/dZfy-1) 3 106.
Cytokine secretion
Splenic CD8 T cells recovered from the chimeras were purified further by
negative selection using a cocktail of mAb coated with Dynabeads (Dynal,
A.S., Oslo, Norway) recognizing B cells, macrophages, and CD4 T cells (purity .99%). Purified CD8 T cells (0.53106) were then incubated at 37°C
with 1 3 106 spleen APCs (from CD3-«2/2 female mice) and 2.5 mg antiCD3 mAb (clone 2C11, PharMingen)/well. After 2 h of incubation in the
presence of Brefeldin A (10 mg/mL, Becton Dickinson), intracellular staining was performed as described previously [5]. IFN-g and IL-2 secretions
were determined in culture medium supernatants by ELISA (R&D Systems,
Minneapolis, MN, USA) after 24 h of incubation.
Statistical analysis
Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and the two-tailed Mann-Whitney test. All mentioned differences are statistically significant: *P , 0.05, **P , 0.01,
and ***P , 0.001.
RESULTS
Experimental strategy
To characterize CD8 response and memory differentiation, we
used a well-described, noninfectious immunization system directed against the HY male antigen. Figure 1A recapitulates
the experimental strategy. CD3-«2/2 female mice were injected with male BM cells. As BM cells have a high capacity for
CD40 signaling on CD8 responses
cell divisions, it allows male cells to grow in host mice. Indeed,
the number of male cells increased constantly into host mice
if the naïve HY CD8 Tg T cell were not injected (Fig. 1B). On
the contrary, when CD8 T cells were injected, they acquired
cytotoxic function and eliminated male cells (Fig. 1B). At Day
7, the elimination was almost completed, and by Day 14, no
male cells were detected in the hosts. This experimental system relies on adoptive transfer of naïve T cells into an empty
host, which could introduce a bias as a result of LIP. However,
this particular naïve HY CD8 Tg T cell population did not suffer from LIP, as we and others have already shown [5, 32, 33].
We confirmed this in the present study. CD8 T cells injected
into CD3-«2/2 female mice, not reconstituted with male BM
cells, did not divide (Fig. 1C). Accordingly, the number of
CD8 T cells recovered at Days 4 and 7 was similar and corresponded to the homing of CD8 T cells in the spleen (10% of
the injected population; Fig. 1D). Contrary, in the presence of
male cells, CD8 T cells have already strongly divided at Day 4,
as no CFSE staining was detected at Day 7 (Fig. 1C). This correlated to a strong increase in absolute number of CD8 T cells
(Fig. 1D). Thus, in this system, stimulation with the male antigen is strictly required for in vivo proliferation and differentiation of naïve CD8 T cells.
To evaluate the relative implication of CD40 expression on
APCs and CD8 T cells, different chimeras immunized with the
male antigen have been generated. Four groups of chimeras have
been designed: CD401 chimeras, APCs and CD8 T cells were
CD401/1; CD402 chimeras, APCs and CD8 T cells were
CD402/2; CD8 CD402 chimeras, APCs were CD401/1, and CD8
T cells were CD402/2; APC CD402 chimeras, APCs were
CD402/2 , and CD8 T cells were CD401/1. These different settings allow restricting CD40 expression on APCs and/or CD8 T
cells or neither of them. Within each group, CD8 T cells were
injected alone, with CD4 T cells or with aCD40ab (Table 1).
CD40 signaling on APCs is important for CD8 T cell
expansion
We first determined the absolute number of CD8 T cells recovered from lymphoid organs in the different chimeras (Fig. 2).
TABLE 1. Description of the different groups of immunization
CD8 T cells
CD401/1
APCs
CD402/2
CD401/1
CD401 chimeras
CD8 CD402 chimeras
CD402/2
APC CD402 chimeras
CD402 chimeras
Alone
CD4 T cells
aCD40ab
Alone
CD4 T cells
aCD40ab
Different chimeras immunized with the male antigen have been generated, allowing restricted CD40 expression on different target cells. Four
different groups can thus be considered: 1) CD401 chimeras, APCs and CD8 T cells were CD401/1; 2) CD402 chimeras, APCs and CD8 T cells
were CD402/2; 3) CD8 CD402 chimeras, APCs were CD401/1, and CD8 T cells were CD402/2; 4) APC CD402 chimeras, APCs were CD402/2,
and CD8 T cells were CD401/1. In each group, CD8 T cells have been injected alone, with CD4 T cells or with aCD40ab. For all chimeras injected with CD8 T cells alone (groups 1– 4), similar results were obtained. Therefore, only the chimeras, where both APCs and CD8 T cells were
competent for CD40 (CD401 chimeras), are shown in Results.
www.jleukbio.org
Volume 91, June 2012
Journal of Leukocyte Biology 861
A
CD40+ Chimeras
20
A
CD8 only
B
CD4
15
C
aCD40
10
5
0
0
B
5
10
15
CD40Chimeras
20
25
30
25
30
25
30
10
15
20
25
Days after immunization
60
30
20
CD4
K
aCD40
L
15
# CD8 T cells / mouse (*10 6)
10
5
0
0
C
5 CD4010 Chimeras
15
20
CD8
20
E
CD4
aCD40
F
15
10
5
0
0
D
5 CD4010 Chimeras
15
20
APC
20
H
CD4
IaCD40
15
10
5
0
0
5
Figure 2. Synergic effect of CD40 pathways on CD8 T cell expansion
in lymphoid organs. Total number of CD8 T cells recovered from lymphoid organs (spleen1LN) of individual mice at different time-points
after immunization. (A) CD8 T cells recovered from CD401 chimeras.
CD8 T cells were injected alone (3) or coinjected with CD4 T cells
(n) or aCD40ab (▫). (B) CD8 T cells recovered from CD402 chimeras. CD8 T cells were coinjected with CD4 T cells (l) or aCD40ab
(L). (C) CD8 T cells recovered from CD8 CD402 chimeras. CD8 T
cells were coinjected with CD4 T cells (Œ) or aCD40ab (‚). (D) CD8
T cells recovered from APC CD402 chimeras. CD8 T cells were coinjected with CD4 T cells (F) or aCD40ab (E). Data show the average 6
sem of three mice/group and are representative of six independent
experiments.
862 Journal of Leukocyte Biology
Volume 91, June 2012
In CD401 chimeras, the expansion of CD8 T cells, injected
alone, was extremely low (Fig. 2A), as previously described for
lethargic cells [4, 5]. In the presence of CD4 T cells, the CD8
T cell expansion followed a classical pattern with a peak at Day
7 and then a contraction (up to Day 14) and resting phase
(Fig. 2A). When aCD40ab was used instead of CD4 T cells, no
differences were observed between the two groups (Fig. 2A).
Therefore, in a full CD40-competent environment, aCD40ab
can efficiently substitute CD4 help in regard to CD8 T cell expansion.
In CD402 chimeras, CD8 T cell expansion was observed in
the presence of CD4 T cells (Fig. 2B), but the number of CD8
T cells recovered at Day 7 was decreased significantly, compared with their CD401 counterparts (6.5310661.13106 vs.
13.8310662.73106; P,0.01; Supplemental Fig. 1). In CD402
chimeras injected with aCD40ab, CD8 T cell expansion was
severely impaired and similar to the lethargic population: the
aCD40ab treatment had no effect, as expected. The next step
was to determine whether CD40 agonist signals were mediated
through APCs and/or CD8 T cells.
In CD8 CD402 chimeras, the expansion of CD8CD402/2 T
cells, injected with CD4 T cells or with aCD40ab, was similar to
CD401 chimeras (Fig. 2C). In APC CD402 chimeras receiving
CD4 T cells, the expansion remained similar to CD401 chimeras as well (Fig. 2D). The number of CD8 T cells recovered at
Day 7 was reduced slightly but not statistically different compared with CD401 chimeras (Supplemental Fig. 1). Conversely, CD8 T cell expansion was severely impaired in APC
CD402 chimeras receiving aCD40ab (Fig. 2D). At Day 7, the
number of cells was fourfold reduced compared with CD401
chimeras (3.5310661.33106 vs. 13.9310662.93106; P,0.01;
Supplemental Fig. 1). Of note, the expansion of CD8 T cells
in this group was significantly higher compared with the lethargic cells (3.5310661.33106 vs. 1.2310660.253106;
P,0.05; Supplemental Fig. 1), showing that some signals were
mediated through CD40 expression by CD8 T cells.
Collectively, our data demonstrate that direct CD40 signaling on APCs is important for CD8 T cell expansion but can be
bypassed by other CD4 help signals.
To further explore the role of CD40 signaling on CD8 T
cell expansion and their following migration in the periphery,
we studied the number of CD8 T cells recovered from the
liver (Fig. 3). The number of lethargic cells remained very low
all along the primary response (Fig. 3A). On the contrary, in
CD401 chimeras injected with CD4 T cells or aCD40ab, the
number of CD8 T cells strongly increased until Day 7 (Fig.
3A). Overall, the numbers of CD8 T cells recovered upon
coinjection with CD4 T cells were similar in all studied groups
(Fig. 3A–D). This demonstrated that the deficiency of CD40
on APCs and/or CD8 did not impact T cell migration capacity
in an otherwise full CD4 help-competent environment. In CD8
CD402 chimeras injected with aCD40ab, no differences were
observed, as well compared with CD401 chimeras (Fig. 3C
and A, respectively). On the contrary, in CD402 and APC
CD402 chimeras, the numbers of CD8 T cells were reduced
dramatically and comparable with those observed for lethargic
cells. These results confirm the predominant role of CD40 expression on APCs to induce CD8 T cell expansion and conse-
www.jleukbio.org
CD40 signaling on CD8 responses
Meunier et al.
A
CD40+ Chimeras
9
CD8 only
A
BCD4
CaCD40
8
7
6
5
4
3
2
1
0
B
0
5
10
15
CD40Chimeras
20
25
30
9
CD4
K
LaCD40
8
7
6
5
quent migration to peripheral tissues. Interestingly, in CD401
and CD8 CD402 chimeras injected with aCD40ab, the contraction phase of CD8 T cells was delayed compared with their
counterpart injected with CD4 T cells, suggesting that
aCD40ab could provide more prolonged survival signals than
CD4 T cells. However, few CD8 T cells remained in the liver at
the end of the primary responses in all chimeras.
The differences in CD8 T cell expansion observed among
the groups injected with CD4 T cells were not a result of a defect in CD4 T cell expansion, as the numbers of CD4 T cells
recovered did not significantly differ for these groups in lymphoid organs (Supplemental Fig. 2) and the liver (data not
shown). No differences were found as well in the numbers of
B cells recovered from lymphoid organs and liver of different
chimeras (injected with CD4 T cells or aCD40ab; data not
shown). Thus, the administration of aCD40ab has no impact
on the survival or the toxicity toward the B cells in the settings
used.
# CD8 T cells / mouse (*10 6)
4
3
CD40 signaling on APCs is involved in the acquisition
of CD8 T cell effector functions in the early phase of
the immune responses
2
1
0
C
0
5 CD4010 Chimeras
15
20
CD8
25
30
25
30
9
ECD4
FaCD40
8
7
6
5
4
3
2
1
0
D
0
5 CD4010 Chimeras
15
20
APC
9
8
CD4
H
IaCD40
7
To assess the in vivo cytotoxic capacity of CD8 T cells, we
quantified the male antigen load by real-time PCR (Fig. 4).
The male antigen was eliminated almost completely at Day 7
and totally undetectable at Day 14 in all groups, including the
lethargic ones. This demonstrates that CD4 help is not strictly
required for the development of CD8 cytotoxic functions leading to antigen elimination. However, CD4 T cells induced a
faster kinetic of antigen elimination in CD401 chimeras, as
the number of male cells detected was, respectively, twoand threefold lower at Days 4 and 5 compared with the lethargic one (Fig. 4). No differences were observed in the presence
of aCD40ab compared with the presence of CD4 T cells, demonstrating that CD40 signaling bypassed CD4 help completely.
In contrast, the kinetic of antigen elimination in CD402 chi-
6
5
4
2
1
0
0
5
10
15
20
25
Days after immunization
60
30
Figure 3. Effect of CD40 deficiency on CD8 T cell migration. Total
number of CD8 T cells recovered from the liver of individual mice at
different time-points after immunization. (A) CD8 T cells recovered
from CD401 chimeras. CD8 T cells were injected alone (3) or coinjected with CD4 T cells (n) or aCD40ab (▫). (B) CD8 T cells recovered from CD402 chimeras. CD8 T cells were coinjected with CD4 T
cells (l) or aCD40ab (L). (C) CD8 T cells recovered from CD8
CD402 chimeras. CD8 T cells were coinjected with CD4 T cells (Œ) or
aCD40ab (‚). (D) CD8 T cells recovered from APC CD402 chimeras.
CD8 T cells were coinjected with CD4 T cells (F) or aCD40ab (E).
Data show the average 6 sem of three mice/group and are representative of six independent experiments.
www.jleukbio.org
# of male cells / 10 6 cells (*104 )
7
3
6
ns
*
ns
5
4
3
2
1
0
aCD40ab CD4 T cell -
+
+
-
+
+
-
+
+
-
+
+
-
Figure 4. Delayed antigen elimination under CD40 deficiency. Male
antigen quantification using genomic Zfy-1 DNA real-time PCR. Results show the number of male cells detected/million splenic cells.
The histograms represent the quantification of male cells in the different chimeras at Day 4 (white histograms), Day 5 (gray histograms),
and Day 7 (black histograms). Each sample was performed in triplicate. Data show the average 6 sem of three mice/group and are representative of three independent experiments.
Volume 91, June 2012
Journal of Leukocyte Biology 863
A
120
CD8 only
A
B
CD4
CaCD40
80
60
40
20
0
0
B
120
We finally evaluated the regulation of IL-7Ra and CD62L expression, which includes two important markers of the effector
CD8 T cell phase. The expected down-regulation of IL-7Ra on
CD8 T cells shortly after activation was observed in all groups
at Day 3 (data not shown). However, kinetic of its re-expression differed among them. The IL-7Ra expression on lethargic
CD8 T cells remained low at Day 7 (Fig. 6A and Supplemental
Fig. 3A). In CD401 chimeras, injected with CD4 T cells or
aCD40ab, the re-expression of IL-7Ra was observed as soon as
Day 5, and ;65% of CD8 T cells re-expressed it at Day 7. In
CD402 chimeras injected with CD4 T cells, CD8 T cells initiated IL-7Ra re-expression by Day 5, but the re-expression was
delayed slightly at Day 7 compared with CD401 chimeras
(53.4%60.4 vs. 67.463.3; P,0.05). In the presence of
aCD40ab, the IL-7Ra re-expression was strongly delayed
and comparable with the profile observed in lethargic cells. In
CD8 CD402 chimeras, injected with CD4 T cells or aCD40ab,
CD8 T cells initiated IL-7Ra re-expression by Day 5, but the
re-expression was delayed slightly at Day 7 compared with
CD401 chimeras (and similar to CD402 chimeras injected
with CD4 T cells). CD40 signaling on CD8 T cells thus contrib864 Journal of Leukocyte Biology
Volume 91, June 2012
2
4
6
8
10
12
14
10
12
14
12
14
12
14
CD40- Chimeras
100
KCD4
LaCD40
80
60
40
20
0
0
C
2
4
6
8
CD8 CD40- Chimeras
120
100
ECD4
FaCD40
80
60
40
20
0
0
D
2
4
6
8
10
APC CD40- Chimeras
120
CD40 signaling on APCs modulates the kinetics of
IL-7Ra and CD62L expression on CD8 T cells
CD40+ Chimeras
100
% of IFN- expression by CD8 T cells
meras injected with CD4 T cells or aCD40ab was similar to the
lethargic group, suggesting that CD4 help could not bypass
CD40 deficiency on the APCs and CD8 T cells. No differences
were observed between CD8 CD402 chimeras and their
CD401 counterparts. Thus, aCD40ab stimulation on the APCs
alone is sufficient to allow rapid antigen elimination by CD8 T
cells. In APC CD402 chimeras injected with CD4 T cells or
aCD40ab, the level of antigen load was similar to the lethargic
one at Day 4. However, at Day 5, only the chimera injected
with aCD40ab showed a delay in antigen elimination. Altogether, this suggests that CD4 help involves mainly CD40 signaling on APCs to allow rapid antigen elimination.
To further analyze the effect of CD40 signaling on CD8 T
cell effector functions, we investigated their ability to express
IFN-g after a short in vitro restimulation (Fig. 5). Only onethird of lethargic CD8 T cells expressed IFN-g (;35%66% at
Days 5 and 7), and they did not significantly improve their
IFN-g expression thereafter (Fig. 5A). In CD401 chimeras, no
differences were observed between CD4 T cells and aCD40ab
stimulation. Approximately 55% of CD8 T cells are able to express IFN-g as soon as Day 4, and this percentage increased
constantly thereafter (Fig. 5A). In fact, CD8 T cells isolated
from all groups of mice injected with CD4 T cells have a similar profile of IFN-g expression until Day 7, demonstrating that
CD40 signaling through APCs or CD8 T cells was not required
strictly during the effector phase (Fig. 5A–D). The production
of IFN-g by CD8 T cells in CD402 or APC CD402 chimeras
injected with aCD40ab was, however, altered and similar to the
lethargic group (Fig. 5A, B, and D), whereas its production by
CD8 T cells from CD8 CD402 chimeras was similar to that of
CD401 chimeras (Fig. 5C and A, respectively). Therefore, during the effector phase of the immune response, CD40 expression on APCs is important to induce IFN-g expression by CD8
T cells but could be bypassed by other CD4 helper signaling.
100
HCD4
IaCD40
80
60
40
20
0
0
2
4
6
8
10
Days after immunization
Figure 5. CD40 deficiency altered IFN-g expression during effector
phase. Intracellular expression of IFN-g by splenic CD8 T cells after
2 h of in vitro restimulation. (A) Percentage of CD8 T cells expressing
IFN-g from CD401 chimeras. CD8 T cells were injected alone (3) or
coinjected with CD4 T cells (n) or aCD40ab (▫). (B) Percentage of
CD8 T cells expressing IFN-g from CD402 chimeras. CD8 T cells
were coinjected with CD4 T cells (l) or aCD40ab (L). (C) Percentage of CD8 T cells expressing IFN-g from CD8 CD402 chimeras. CD8
T cells were coinjected with CD4 T cells (Œ) or aCD40ab (‚). (D)
Percentage of CD8 T cells expressing IFN-g from APC CD402 chimeras. CD8 T cells were coinjected with CD4 T cells (F) or aCD40ab
(E). Data show the average 6 sem of three mice/group and are representative of six independent experiments.
www.jleukbio.org
Meunier et al.
utes mildly to IL-7Ra re-expression. In APC CD402 chimeras,
injected with CD4 T cells, the kinetic of IL-7Ra re-expression
was identical to CD401 chimeras. In the presence of
aCD40ab, defects in receptor dynamics were comparable with
those observed in CD402 chimeras (Fig. 6A), showing that
CD40 signaling on the APCs is important. In conclusion, injection of aCD40ab is sufficient to ensure re-expression of IL-7Ra
when APCs expressed CD40. However, in the absence of CD40
expression by the APCs, CD4 T cells allow a faster IL-7Ra expression, suggesting that CD4 help may be provided in a
CD40-independent pathway.
Regarding CD62L expression, in all chimeras injected with
CD4 T cells, the expected transitory down-regulation on CD8
T cells after activation was observed at Day 5 and maintained
at Day 7 (Fig. 6B and Supplemental Fig. 3B). On the contrary,
the down-regulation of CD62L was severely delayed on lethargic cells and still not detected at Day 5. In CD401 and CD8
CD402 chimeras injected with aCD40ab, the CD62L expression profile was similar to those injected with CD4 T cells. In
contrast, in APC CD402 and CD402 chimeras injected with
aCD40ab, the CD62L expression profile mimicked that of lethargic cells (Fig. 6B). Collectively, these results demonstrate
that CD40 on APCs participates in the down-regulation of
CD62L. However, other signals from CD4 T cells can overcome this deficiency.
% of IL-7Rα expression
A 100
90
80
70
60
50
40
30
20
10
0
*
B 100
% of CD62L expression
90
80
70
60
50
40
30
20
10
0
aCD40ab CD4 T cell -
+
+
-
+
+
-
+
+
-
+
+
-
Figure 6. Effect of CD40 deficiency on IL-7Ra and CD62L expression.
Percentage of IL-7Ra 1(A) and CD62L1 (B) on gated, splenic CD8 T
cells recovered from the different chimeras at Day 5 (white histograms) and Day 7 (black histograms). Data show the average 6 sem of
three mice/group and are representative of six independent experiments.
www.jleukbio.org
CD40 signaling on CD8 responses
CD40 signaling on CD8 T cells is crucial for memory
differentiation
We studied the number and the phenotype of CD8 T cells recovered from the different chimeras at the late phase of the
primary response. CD8 T cell numbers were equivalent in all
groups except for the lethargic group and the CD402 group
injected with aCD40ab, which exhibited reduced CD8 T cell
numbers (Fig. 2 and Supplemental Fig. 4). Additionally, CD8
T cells isolated at Day 60 all expressed IL-7Ra at a higher level
than naïve cells (Supplemental Fig. 3A). CD8 T cells from all
chimeras also progressively re-expressed CD62L and at Day 60;
;90% of them were CD62Lhigh (Supplemental Fig. 3B).
Therefore, irrespective of CD4 help and/or CD40 signaling,
CD8 T cells surviving the contraction phase harbored the
same profile of IL-7Ra and CD62L expression.
However, the quantity and the phenotype of CD8 T cells did
not necessarily reflect their quality (functional properties). For
instance, CD62L and CCR7 molecules were described originally in humans to discriminate between effector memory T
cells and TCM, the later lacking effector functions [34]. Subsequent experiments in mice reported, however, that TCM cells
differed from the original description of human TCM cells in
that they retained effector functions [35]. Therefore, to assess
the quality of CD8 T cells generated at the end of the primary
response in the different settings of CD4 help, we monitored
their potential for high cytokine production and cell division
upon secondary challenge, two important hallmarks of CD8
memory T cells.
We first studied IFN-g expression by CD8 T cells at Day 60
after a short in vitro restimulation (Fig. 7A). The percentage
of lethargic CD8 T cells expressing IFN-g did not increase after the contraction phase (Fig. 7A), whereas its expression by
CD8 T cells from CD401 chimeras, injected with CD4 T cell
or aCD40ab, was strongly increased. Thirty-eight percent 6 7%
of lethargic cells compared with 81% 6 6.2% of CD8 T cells
in CD401 chimeras injected with CD4 T cells expressed it
(P,0.01). Therefore, aCD40ab stimulation can fully substitute
CD4 help to induce a high capacity of cytokine productions by
CD8 T cells. Conversely, CD8 T cells from CD402 chimeras,
injected with CD4 T cells or aCD40ab, did not improve their
capacity to express IFN-g after the contraction phase (Fig. 7A).
As the ability to maintain a high capacity of cytokine productions is a hallmark of memory T cells [1–3], these results demonstrate that CD40 signaling on APCs and/or on CD8 T cells
is involved in memory differentiation. In CD8 CD402 chimeras, injected with CD4 T cells or aCD40ab, the percentage of
CD8CD402/2 T cells expressing IFN-g did not increased after
the contraction phase (Fig. 7A). For example, in chimeras injected with CD4 T cells, 55% 6 4.7% of CD8 T cells in CD8
CD402 chimeras expressed IFN-g compared with 81% 6 6.2%
in CD401 chimeras (P,0.01). Therefore, CD40 on CD8 T
cells plays a crucial and nonredundant function in long-term
IFN-g expression. In APC CD402 chimeras, the IFN-g expression by CD8 T cells was not altered in the presence of CD4 T
cells (Fig. 7A). CD8 T cells constantly increased their capacity
to express IFN-g throughout the immune response. At Day 60,
75% 6 6.7% of cells expressed IFN-g compared with 81% 6
Volume 91, June 2012
Journal of Leukocyte Biology 865
ns
0,8
0,8
*
D3
0,6
0,6
0,4
0,4
# CD8 T cells / mouse (*10 6)
B
0,2
0,2
-B
+
+C
-
-K
+
+L
-
-E
+
+F
-
-H
+
+I
-
50
80
00
A
40
40
B
D7
K
ns E
H
*
30
30
70
60
50
30
40
20
30
20
20
IL-2 (ng.ml-1)
40
20
10
10
10
00
CD4 T cell
-
A
+
B
+
K
+
E
+
H
10
0
aCD40ab CD4 T cell -
0
+
+
-
+
6.2% in CD401 chimeras (P,0.57). Therefore, CD40 on APCs
is not involved in the high capacity of IFN-g production by
CD8 T cells. Importantly, in the presence of aCD40ab, the capacity of CD8 T cells to express IFN-g was impaired drastically
and was similar to the expression by lethargic cells (Fig. 7A).
Thus, the unique stimulation of CD40 through CD8 T cells is
not sufficient to induce memory differentiation.
To further analyze CD8 T cell memory differentiation, the
secretion of IFN-g and IL-2 by CD8 T cells isolated at Day 60
was determined after in vitro restimulation (Fig. 7B). As shown
previously [4, 5], lethargic CD8 T cells secreted very low levels
of both cytokines. In CD401 chimeras, stimulated with CD4 T
cells or aCD40ab, CD8 T cells secreted similarly high levels of
IFN-g and IL-2, showing that aCD40ab is sufficient to substitute CD4 help. Besides, CD40 signaling is important, as in
CD402 chimeras, CD8 T cells were unable to secrete high levels of IFN-g and IL-2 in the presence of CD4 T cells or
aCD40ab. In CD8 CD402 chimeras, stimulated with CD4 T
cells or aCD40ab, a three- to fourfold diminution of IFN-g
and IL-2 secretions was observed when compared with CD401
chimeras (Fig. 7B). In APC CD402 chimeras, CD8 T cells
stimulated with CD4 T cells secreted similarly high levels of
IFN-g and IL-2, whereas a fourfold decrease was measured
upon aCD40ab stimulation. These results confirm the prominent role of CD40 expression on CD8 T cells, for their differentiation into memory cells.
Finally, we performed in vivo secondary responses for all
chimeras injected with CD4 T cells and analyzed CD8 T cell
secondary expansion capacity (Fig. 7C). CD8 T cells, which
exhibited a defect in cytokine secretions (from CD402
and CD8 CD402 chimeras or lethargic CD8 T cells), have se866 Journal of Leukocyte Biology
C
ns
**
**
aCD40ab ACD4 T cell -
IFN- (ng.ml-1 )
Figure 7. CD40 deficiency altered the
memory differentiation. (A) Intracellular
expression of IFN-g by splenic CD8 T
cells at Day 60, after 2 h of in vitro restimulation. (B) IFN-g and IL-2 secretion
(ELISA) by CD8 T cells at Day 60, after
24 h of in vitro restimulation. Data show
the average 6 sem of three mice/group
and are representative of six independent
experiments. (C) Total number of CD8 T
cells recovered from the spleen and LN
of individual mice at Days 3 (D3; upper
graph) and 7 (D7; lower graph) after secondary immunization (as described in
Materials and Methods) in the presence
of CD4 T cells. The secondary response
of lethargic CD401 CD8 T cells is also
shown. Data show the average 6 sem of
three mice/group and are representative
of two independent experiments.
100
100
00
90
9090
80
8080
70
7070
60
6060
50
5050
40
4040
30
3030
20
2020
10
1010
000
% of IFN- expression
by CD8 T cells
A
Volume 91, June 2012
+
-
+
+
-
+
+
-
verely altered expansion at Days 3 and 7 upon secondary immunization compared with CD8 T cells that secreted high levels of cytokines (from CD401 and APC CD402 chimeras).
Therefore, although CD8 T cells from CD402 and CD8
CD402 persisted to a relatively high number at the end of the
primary response, they were not endowed with high cell-division capacity.
Altogether, these results demonstrate that CD40 expression
on CD8 T cells is required to enhance cytokine secretions
and cell divisions of CD8 T cells upon secondary challenge,
therefore allowing their differentiation into memory cells,
whereas CD40 expression on APCs is less involved in this process.
DISCUSSION
Among the multiple mechanisms of CD4 help discovered so
far, the CD40 –CD40L interactions play an important role in
CD8 immune responses [30]. Accordingly, researchers have
used agonist aCD40ab to increase CD8 T cell responses to bypass CD4 help in a number of immune models [30, 36]. In
several cancer models, for example, aCD40ab can induce direct apoptosis of CD40-expressing tumor cells and may prove a
promising tool in cancer treatments [37]. However, its administration may have unexpected consequences. Well-documented reviews disclose the side-effects of aCD40ab treatments
[21, 30, 38]. The administration of aCD40ab in a number of
cancer models provokes the expression of several angiogenic
factors promoting tumor growth [22–25]. Moreover, sustained
systemic treatment with aCD40ab often engenders toxicity
www.jleukbio.org
Meunier et al.
[30]. Dissecting the exact impact of aCD40ab on the various
cell targets may provide novel strategy to prevent the development of side-effects. In this study, we designed an experimental system allowing restricted expression of CD40 on APCs or
CD8 T cells, and we carefully distinguished the primary effector immune response from further differentiation into memory CD8 T cells.
In agreement with many earlier studies, we observed that
aCD40ab could efficiently substitute CD4 help during the primary response [13–16]. However, complete substitution required a full CD40-competent environment: CD40 deficiency
on APCs or CD8 T cells resulted in altered responses. Importantly, we found that the different parameters of CD8 responses (cell division, antigen clearance, effector functions,
and memory differentiation) did not follow the same requirements regarding CD40 expression (Table 2).
During the early phase of the primary response, in CD8
CD402 chimeras, the sole injection of aCD40ab is sufficient to
obtain an immune response similar to the one observed in
CD401 chimeras, as assessed by expansion, antigen elimination, IL-7Ra, CD62L, and IFN-g expression. In contrast, the
injection of aCD40ab into APC CD402 was ineffective, confirming a major role of CD40 expression on APCs during primary responses. Importantly, when APCs are CD402/2, the
injection of CD4 T cells fully bypassed CD40 deficiency, demonstrating that CD40 expression on APCs is not strictly required. Finally, in CD402 chimeras, CD4 T cells did not fully
restore the immune response, reinforcing the synergistic role
of CD40 expression on APCs and CD8 T cells. These results
suggest that some helper signals were CD40-dependent
and not redundant with other signals provided by CD4 T cells
(Table 2). This is in accordance with an earlier study describing a CD40-independent pathway of CD4 help on APC activation, as well as direct CD4 –CD8 T cell communication to deliver helper signals to CD8 T cells [39].
A different picture emerged during the late phase of immune
response, whereas CD8 T cells progressively acquired memory
properties. Studying intracellular IFN-g expression and IFN-g
and IL-2 productions after in vitro restimulation (mimicking the
secondary response), we showed the important and nonredundant role of CD40 expression by CD8 T cells on memory genera-
CD40 signaling on CD8 responses
tion (Table 2). Indeed, when CD8 T cells are CD40-deficient,
neither the stimulation of the APCs by aCD40ab nor by CD4 T
cells could overcome the defects in IFN-g and IL-2 productions
by CD8 T cells. Conversely, when APCs are CD402/2, enhanced
IFN-g and IL-2 secretions by CD8 T cells were observed in the
presence of CD4 help but not upon aCD40ab stimulation. Importantly, CD8 T cells exhibiting a default in cytokine secretions also
had a severely altered expansion upon in vivo secondary immunization compared with CD8 T cells that secreted high levels of
cytokines. These results demonstrate that CD40 expression on
CD8 T cells is fundamental to allow memory generation. However, as shown by the defects observed in the APC CD402 chimera injected with aCD40ab, CD40 expression on CD8 T cells is
not strictly sufficient for CD8 T cell differentiation. Other CD40independent signals provided by CD4 T cells are also required to
allow CD8 memory differentiation. Overall, this demonstrates that
CD8 T cells must go through different checkpoints to differentiate
into memory cells. One may hypothesis that some CD4 help signaling, mainly through the APCs, is necessary to drive efficient effector
phase. This transition to an effector stage may be a prerequisite for
further memory differentiation but may not be sufficient. Some
other CD4 help signaling, mainly through CD40 on CD8 T cells, is
necessary to fulfill complete memory differentiation.
Among the signals provided by CD4 help that are CD40-independent, other members of the TNF–TNFR are good candidates.
Numerous studies have demonstrated the important role of
TRANCE, CD70, OX40, and 41BB on CD8 expansion, survival,
and/or acquisition of effector functions [40 – 44]. Interestingly, a
report about the role of OX40 –OX40L interaction in CD4 T cell
survival reached a similar conclusion to our observations with
CD40 –CD40L interactions [45]. The authors demonstrated that
OX40 and OX40L could be expressed on activated APCs
and CD4 T cells. The deficiency of OX40L, on APCs or CD4 T
cells, induced a significant reduction in T cell proliferation [45].
Our study confirms and extents the important role of
CD40 –CD40L interactions in noninfectious immune responses. The implication of these interactions is more controversial in pathogen models [46-54]. For example, the CD8 response against LCMV infection is mostly described as CD40 –
CD40L-dependent [47, 48]. In the Listeria model, depending
on the authors, the response is CD40 –CD40L-dependent [49,
TABLE 2. Schematic view of the CD40 signaling and CD4 help
CD40 signaling
CD40 on APC
CD40 on CD8
Early phase of primary
response
Important but
redundant
Modest contribution
and redundant
Late phase of primary
response
Modest contribution
and redundant
Strictly necessary
and not redundant
CD40 on APC
and CD8
Synergistic action
and not fully
redundant
Strictly necessary
and not redundant
CD4 help signals by CD40independent signaling
Partially rescued expansion
and effectors fonctions
Could not overcome CD40
deficiency by CD8 T cells
CD40 signaling versus CD4 help signals are highlighted here. The impact of CD40 signaling during the early phase (effector phase) and the
late phase of the primary response (memory differentiation) is distinguished. It is also mentioned whether CD40 signaling is redundant, i.e., could
be replaced by other signals provided by CD4 T cells or strictly necessary. The impact of CD4 T cell help by CD40-independent signals is also
shown.
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Volume 91, June 2012
Journal of Leukocyte Biology 867
50] or independent [51, 52]. These discrepancies remain to
be elucidated but could be related to the infectious doses, the
pathogens used, or the timing and kinetics of the response.
Only few studies have dissected the role of CD40 expression
on APCs and CD8 T cells in response to pathogen infection
[52, 53]. No crucial role of CD40 expression on CD8 T cells
has been detected in these studies, neither during primary nor
secondary responses. Accordingly, in primary response, we
showed that CD40 expression on CD8 T cells was not involved.
In these infectious models, secondary responses were assessed
only for CD8 expansion at one time-point of the immune response. Similar expansions were found between CD402/2
and CD401/1 CD8 T cells in these analyses. However, in a
previous report, we made an extensive analysis of the secondary responses of CD8CD401/1 and CD8CD402/2 T cells [29].
We found that the secondary response of CD8CD402/2 T cells
was severely altered compared with CD8CD401/1 T cells. Defects in proliferation were found at early time-points, but the
most striking observation was the profound cytotoxic defaults
of CD8CD402/2 T cells [29]. Thus, discrepancies between
these and our studies may rely on the different readouts used
and the time-point considered. Interestingly, a recent study
supported a role of CD40 expression on CD8 T cells in a viral
model [54]. Upon certain viral infections, the APCs expressed
CD40L, suggesting that CD40 –CD40L interactions could be
bidirectional on APCs, CD4, and CD8 T cells. Most importantly, CD40 deficiency on CD8 T cells inhibited the killing of
the infected target cells, demonstrating that CD40 expression
on CD8 T cells could play a role in certain viral models as well
[54]. Finally, using a model of Leishmania donovani infection in
susceptible BALB/c mice, Martin’s group [55] reported that
CD401 CD8 T cells executed CD40-dependent cytotoxicity on
CD41 CD251 regulatory T cells. They demonstrated that
CD40 signaled through Ras, PI3K, and PKC, resulting in NFkB-dependent induction of the cytotoxic mediators granzyme
and perforin. These data sustained our previous study, where
we found that CD8CD402/2 T cells have severely altered perforin and granzyme B cytotoxic functions compared with
CD8CD401/1 T cells [29].
Further studies are therefore required to evaluate the role of
CD40 signaling on CD8 T cells in response to pathogen infections. With respect to the numerous mechanisms of CD4 help
described so far, it is, however, tempting to speculate that CD4
helper signals will differ depending of the kind of CD8 immune
responses (viral, bacterial, antitumoral, autoimmune) [3, 9 –11].
Concerning primary immune responses, it is generally admitted
in many viral and bacterial systems that CD40 signaling appears
redundant (and CD4 T cells not required) during primary responses, as pathogen-derived products are recognized directly by
TLRs on the APCs, allowing CD40-independent (and CD4 T cell
help-independent) APC maturation [51, 52]. For those primary
responses that are CD4 T cell help-independent, it is, however,
well-demonstrated that CD4 T cells were required to allow memory differentiation [7, 8]. It is still unsettled through which mechanism CD4 T cells will help CD8 T cells in these infectious models. One may finally question whether if the infectious products
that bypass CD40 signaling to allow efficient primary response are
the same that would allow CD8 memory differentiation. As it be868 Journal of Leukocyte Biology
Volume 91, June 2012
comes obvious that distinct pathogens differently modulate
DC costimulatory capacity, they may also differently modulate
CD8 T cell responses [56]. In this regard, it is interesting to note
that CD8 T cells can receive cosignals from some but not all TLR
[56-58]. Notably, it has been shown that direct signaling through
TLR2 on CD8 T cells increased their functional properties [58].
Altogether, our results reveal a complex crosstalk among
APCs, CD4 T cells, and CD8 T cells. CD4 T cells provide
CD40-dependent and independent signals to allow complete
CD8 T cell effector and memory differentiation. Our data confirm the potential benefit of aCD40ab agonist therapy to improve memory differentiation. However, they suggest that future trials would have to take into account the whole complexity of CD4 T cell help: the cell subsets involved, the molecules
targeted, and their kinetics of expression to achieve maximal
efficacy, while reducing counteracting and toxic effects.
AUTHORSHIP
S.M. planned and did experiments, drew the figures, and contributed to the paper redaction. L.R. performed the initial experiments, designed the quantitative PCR experiments, and contributed to the paper redaction. L.B., C.P., and A.L. contributed to
the experiments. C.T. supervised the project, designed the experiments, and wrote the paper.
ACKNOWLEDGMENTS
This work was supported by the Agence Nationale de la Recherche. S.M. was supported by the Ministère de la recherche
and ARC. L.R. was supported by the Ministère de la recherche, ARC, and Fondation pour la Recherche Medicale. We
thank Drs. Christine Bourgeois, Eric Tartour, and Marianne
Mangeney for their helpful discussions and improvement of
the manuscript and all members of the animal facility.
DISCLOSURES
The authors declare no financial or commercial conflict of interest.
REFERENCES
1. Obar, J. J., Lefrançois, L. (2010) Memory CD81 T cell differentiation.
Ann. N. .Y Acad. Sci. 1183, 251–266.
2. Rocha, B., Tanchot, C. (2006) The Tower of Babel of CD81 T-cell memory: known facts, deserted roads, muddy waters, and possible dead ends.
Immunol. Rev. 211, 182–196.
3. Cox, M. A., Zajac, A. J. (2010) Shaping successful and unsuccessful CD8
T cell responses following infection. J. Biomed. Biotechnol. 2010, 159152.
4. Bourgeois, C., Veiga-Fernandes, H., Joret, A-M., Rocha, B., Tanchot, C. (2002)
CD8 lethargy in the absence of CD4 help. Eur. J. Immunol. 32, 2199–2207.
5. Bourgeois, C., Rocha, B., Tanchot, C. (2002) A role for CD40 expression
on CD81 T cells in the generation of CD81 T cell memory. Science 297,
2060 –2063.
6. Janssen, E. M., Lemmens, E. E., Wolfe, T., Christen, U., von Herrath, M. G.,
Schoenberger, S. P. (2003) CD41 T cells are required for secondary expansion and memory in CD81 T lymphocytes. Nature 421, 852– 856.
7. Shedlock, D. J., Shen, H. (2003) Requirement for CD4 T cell help in
generating functional CD8 T cell memory. Science 300, 337–339.
8. Sun, J. C., Bevan, M. J. (2003) Defective CD8 T cell memory following
acute infection without CD4 T cell help. Science 300, 339 –342.
9. Bourgeois, C., Tanchot, C. (2003) Mini-review CD4 T cells are required
for CD8 T cell memory generation. Eur. J. Immunol. 33, 3225–3231.
10. Bevan, M. J. (2004) Helping the CD8(1) T-cell response. Nat. Rev. Immunol. 4, 595– 602.
www.jleukbio.org
Meunier et al. CD40 signaling on CD8 responses
11. Castellino, F., Germain, R. N. (2006) Cooperation between CD41
and CD81 T cells: when, where, and how. Annu. Rev. Immunol. 24, 519 –
540.
12. Grewal, I. S., Flavell, R. A. (1998) CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16, 111–35.
13. Ridge, J. P., Di Rosa, F., Matzinger, P. (1998) A conditioned dendritic
cell can be a temporal bridge between a CD41 T-helper and a T-killer
cell. Nature 393, 474 – 478.
14. Bennett, S. R., Carbone, F. R., Karamalis, F., Flavell, R. A., Miller, J. F.,
Heath, W. R. (1998) Help for cytotoxic-T-cell responses is mediated by
CD40 signalling. Nature 393, 478 – 480.
15. Schoenberger, S. P., Toes, R. E., van der Voort, E. I., Offringa, R., Melief,
C. J. (1998) T-cell help for cytotoxic T lymphocytes is mediated by
CD40 –CD40L interactions. Nature 393, 480 – 483.
16. Lefrançois, L., Altman, J. D., Williams, K., Olson, S. (2000) Soluble antigen and CD40 triggering are sufficient to induce primary and memory
cytotoxic T cells. J. Immunol. 164, 725–732.
17. Diehl, L., den Boer, A. T., Schoenberger, S. P., van der Voort, E. I., Schumacher, T. N., Melief, C. J., Offringa, R., Toes, R. E. (1999) CD40 activation in
vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance
and augments anti-tumor vaccine efficacy. Nat. Med. 5, 774–779.
18. Otahal, P., Knowles, B. B., Tevethia, S. S., Schell, T. D. (2007) Anti-CD40
conditioning enhances the T(CD8) response to a highly tolerogenic
epitope and subsequent immunotherapy of simian virus 40 T antigeninduced pancreatic tumors. J. Immunol. 179, 6686 – 6695.
19. Sarawar, S. R., Lee, B. J., Reiter, S. K., Schoenberger, S. P. (2001) Stimulation via CD40 can substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus. Proc. Natl. Acad. Sci. USA 98, 6325– 6329.
20. Kedl, R. M., Jordan, M., Potter, T., Kappler, J., Marrack, P., Dow, S.
(2001) CD40 stimulation accelerates deletion of tumor-specific CD8(1)
T cells in the absence of tumor-antigen vaccination. Proc. Natl. Acad. Sci.
USA 98, 10811–10816.
21. Murugaiyan, G., Martin, S., Saha, B. (2007) CD40-induced countercurrent
conduits for tumor escape or elimination? Trends Immunol. 28, 467– 473.
22. Bergmann, S., Pandolfi, P. P. (2006) Giving blood: a new role for CD40
in tumorigenesis. J. Exp. Med. 203, 2409 –2412.
23. Reinders, M. E. J., Sho, M., Robertson, S. W., Geehan, C. S., Briscoe,
D. M. (2003) Proangiogenic function of CD40 ligand–CD40 interactions.
J. Immunol. 171, 1534 –1541.
24. Chiodoni, C., Iezzi, M., Guiducci, C., Sangaletti, S., Alessandrini, I., Ratti,
C., Tiboni, F., Musiani, P., Granger, D. N., Colombo, M. P. (2006) Triggering CD40 on endothelial cells contributes to tumor growth. J. Exp.
Med. 203, 2441–2450.
25. Bartholdy, C., Kauffmann, S. Ø., Christensen, J. P., Thomsen, A. R.
(2007) Agonistic anti-CD40 antibody profoundly suppresses the immune
response to infection with lymphocytic choriomeningitis virus. J. Immunol.
178, 1662–1670.
26. Hernández, J., Aung, S., Marquardt, K., Sherman, L. A. (2002) Uncoupling of proliferative potential and gain of effector function by CD8(1)
T cells responding to self-antigens. J. Exp. Med. 196, 323–333.
27. Ichikawa, H. T., Williams, L. P., Segal, B. M. (2002) Activation of APCs
through CD40 or Toll-like receptor 9 overcomes tolerance and precipitates autoimmune disease. J. Immunol. 169, 2781–2787.
28. Roth, E., Schwartzkopff, J., Pircher, H. (2002) CD40 ligation in the presence of self-reactive CD8 T cells leads to severe immunopathology. J. Immunol. 168, 5124 –5129.
29. Rapetti, L., Meunier, S., Pontoux, C., Tanchot, C. (2008) CD4 help regulates expression of crucial genes involved in CD8 T cell memory and sensitivity to regulatory elements. J. Immunol. 181, 299 –308.
30. Elgueta, R., Benson, M. J., de Vries, V. C., Wasiuk, A., Guo, Y., Noelle,
R. J. (2009) Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol. Rev. 229, 152–172.
31. Lantz, O., Grandjean, I., Matzinger, P., Di Santo, J. P. (2000) g Chain
required for naïve CD41 T cell survival but not for antigen proliferation.
Nat. Immunol. 1, 54 –58.
32. Tanchot, C., Lemonnier, F. A., Pérarnau, B., Freitas, A. A., Rocha, B.
(1997) Differential requirements for survival and proliferation of CD8
naïve or memory T cells. Science 276, 2057–2062.
33. Kieper, W. C., Burghardt, J. T., Surh, C. D. (2004) A role for TCR affinity
in regulating naive T cell homeostasis. J. Immunol. 172, 40 – 44.
34. Sallusto, F., Lenig, D., Förster, R., Lipp, M., Lanzavecchia, A. (1999) Two
subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708 –712.
35. Unsoeld, H., Krautwald, S., Voehringer, D., Kunzendorf, U., Pircher, H.
(2002) Cutting edge: CCR71 and CCR72 memory T cells do not differ
in immediate effector cell function. J. Immunol. 169, 638 – 641.
www.jleukbio.org
36. Quezada, S. A., Jarvinen, L. Z., Lind, E. F., Noelle, R. J. (2004) CD40/
CD154 interactions at the interface of tolerance and immunity. Annu.
Rev. Immunol. 22, 307–328.
37. Bereznaya, N. M., Chekhun, V. F. (2007) Expression of CD40 and CD40L
on tumor cells: the role of their interaction and new approach to immunotherapy. Exp. Oncol. 29, 2–12.
38. Vonderheide, R. H. (2007) Prospect of targeting the CD40 pathway for
cancer therapy. Clin. Cancer Res. 13, 1083–1088.
39. Lu, Z., Yuan, L., Zhou, X., Sotomayor, E., Levitsky, H. I., Pardoll, D. M.
(2000) CD40-independent pathways of T cell help for priming of
CD8(1) cytotoxic T lymphocytes. J. Exp. Med. 191, 541–550.
40. Bullock, T. N. J., Yagita, H. (2005) Induction of CD70 on dendritic cells
through CD40 or TLR stimulation contributes to the development of CD81
T cell responses in the absence of CD41 T cells. J. Immunol. 174, 710 –717.
41. Lee, S-J., Myers, L., Muralimohan, G., Dai, J., Qiao, Y., Li, Z., Mittler,
R. S., Vella, A. T. (2004) 4-1BB and OX40 dual costimulation synergistically stimulate primary specific CD8 T cells for robust effector function.
J. Immunol. 173, 3002–3012.
42. Hendriks, J., Xiao, Y., Rossen, J. W. A., van der Sluijs, K. F., Sugamura, K., Ishii,
N., Borst, J. (2005) During viral infection of the respiratory tract, CD27, 4-1BB,
and OX40 collectively determine formation of CD81 memory T cells and their
capacity for secondary expansion. J. Immunol. 175, 1665–1676.
43. Dawicki, W., Bertram, E. M., Sharpe, A. H., Watts, T. H. (2004) 4-1BB
and OX40 act independently to facilitate robust CD8 and CD4 recall responses. J. Immunol. 173, 5944 –5951.
44. Bansal-Pakala, P., Halteman, B. S., Cheng, M. H-Y., Croft, M. (2004) Costimulation of CD8 T cell responses by OX40. J. Immunol. 172, 4821– 4825.
45. Soroosh, P., Ine, S., Sugamura, K., Ishii, N. (2006) OX40 –OX40 ligand
interaction through T cell-T cell contact contributes to CD4 T cell longevity. J. Immunol. 176, 5975–5987.
46. Soong, L., Xu, J. C., Grewal, I. S., Kima, P., Sun, J., Longley Jr., B. J.,
Ruddle, N. H., McMahon-Pratt, D., Flavell, R. A. (1996) Disruption of
CD40 –CD40 ligand interactions results in an enhanced susceptibility to
Leishmania amazonensis infection. Immunity 4, 263-273.
47. Borrow, P., Tishon, A., Lee, S., Xu, J., Grewal, I. S., Oldstone, M. B., Flavell,
R. A. (1996) CD40L-deficient mice show deficits in antiviral immunity and have
an impaired memory CD81 CTL response. J. Exp. Med. 183, 2129–2142.
48. Thomsen, A. R., Nansen, A., Christensen, J. P., Andreasen, S. O., Marker, O.
(1998) CD40 ligand is pivotal to efficient control of virus replication in mice
infected with lymphocytic choriomeningitis virus. J. Immunol. 161, 4583–4590.
49. Huster, K. M., Busch, V., Schiemann, M., Linkemann, K., Kerksiek, K. M.,
Wagner, H., Busch, D. H. (2004) Selective expression of IL-7 receptor on
memory T cells identifies early CD40L-dependent generation of distinct
CD81 memory T cell subsets. Proc. Natl. Acad. Sci. USA 101, 5610 –5615.
50. Marzo, A. L., Vezys, V., Klonowski, K. D., Lee, S-J., Muralimohan, G.,
Moore, M., Tough, D. F., Lefrançois, L. (2004) Fully functional memory
CD8 T cells in the absence of CD4 T cells. J. Immunol. 173, 969 –975.
51. Montfort, M. J., Bouwer, H. G. A., Wagner, C. R., Hinrichs, D. J. (2004) The
development of functional CD8 T cell memory after Listeria monocytogenes
infection is not dependent on CD40. J. Immunol. 173, 4084 – 4090.
52. Sun, J. C., Bevan, M. J. (2004) Cutting edge: long-lived CD8 memory
and protective immunity in the absence of CD40 expression on CD8 T
cells. J. Immunol. 172, 3385–3389.
53. Lee, B. O., Hartson, L., Randall, T. D. (2003) CD40-deficient, influenzaspecific CD8 memory T cells develop and function normally in a CD40sufficient environment. J. Exp. Med. 198, 1759 –1764.
54. Johnson, S., Zhan, Y., Sutherland, R. M., Mount, A. M., Bedoui, S., Brady,
J. L., Carrington, E. M., Brown, L. E., Belz, G. T., Heath, W. R., Lew,
A. M. (2009) Selected Toll-like receptor ligands and viruses promote
helper-independent cytotoxic T cell priming by upregulating CD40L on
dendritic cells. Immunity 30, 218 –227.
55. Martin, S., Pahari, S., Sudan, R., Saha, B. (2010) CD40 signaling in
CD81CD401 T cells turns on contra-T regulatory cell functions. J. Immunol. 184, 5510 –5518.
56. Manicassamy, S., Pulendran, B. (2009) Modulation of adaptive immunity
with Toll-like receptors. Semin. Immunol. 21, 185–193.
57. Arens, R., Schoenberger, S. P. (2010) Plasticity in programming of effector and memory CD8 T-cell formation. Immunol. Rev. 235, 190 –205.
58. Mercier, B. C., Cottalorda, A., Coupet, C-A., Marvel, J., Bonnefoy-Bérard, N.
(2009) TLR2 engagement on CD8 T cells enables generation of functional
memory cells in response to a suboptimal TCR signal. J. Immunol. 182, 1860–
1867.
KEY WORDS:
immune responses z effector functions z CD4 z CD40 –CD40L pathway z aCD40ab administration
Volume 91, June 2012
Journal of Leukocyte Biology 869