Les déficits immunitaires héréditaires et leurs thérapeutiques

Déficits immunitaires héréditaires (DIH)
•
Déficits immunitaires « classiques » :
> 200 déficits décrits, > 160 gènes décrits
Les déficits immunitaires héréditaires
et leurs thérapeutiques
1/5000 naissances
Classification :
• Déficits immunitaires combinés (T et B) (rares)
• Déficits immunitaires humoraux (B) (fréquents)
• Déficits de la phagocytose et de l’opsonisation
C. Picard
Centre d’étude des déficits immunitaires
• Déficits de l’immunité innée
• Déficits de l’homéostasie du système immunitaire
[email protected]
• Pathologies auto-inflammatoires
Déficit complet de l'immunité adaptative:
Déficit Immunitaire Combiné Sévère
NK cell
Déficit immunitaire combiné sévère (DICS)
Fréquence et transmission : 1/100 000, AR ou XR
Signes Cliniques = Infections
CD4+ T cell
Common lymphoid
progenitor
CD8+ T cell
B cell
Hematopoietic
stem cell
PMN
macrophage
Common myeloid
progenitor
-
opportunistes (Pneumocystose…)
-
fongiques (Candidose…)
-
virales (Parainfluenzae, Adv, CMV,VRS…)
-
bactériennes, BCGite
Signes Biologiques et radiologiques :
- Absence de thymus
- Numération Formule Sanguine: lymphopénie < 1500/mm3
dendritic cell
Déficit immunitaire combiné sévère :
Diagnostic
• Numération Formule Sanguine
lymphocytes +++
< 1500/mm3
- LYMPHOCYTES T = 0
Déficit immunitaire combiné sévère :
4 mécanismes
1. Mort des thymocytes/lymphocytes (T-B-NK-):
→ déficit en adénosine déaminase (ADA), déficit en PNP et AK2
2. Défaut de signalisation cytokinique (T-B+):
• Absence de thymus
• Phénotypage lymphocytaire
absence de lymphocyte T
• Hypogammaglobulinémie
(hypoIgG, IgA et IgM)
→ IL2RG, IL7RA, JAK3
3. Défaut de réarrangement de VDJ (T-B-NK+):
→ RAG1, RAG2, Artémis, DNA-Pks, Cernunnos, DNA-Ligase 4
4. Anomalies du TCR ou du pré-TCR (T-):
→ CD45, CD3E,CD3D,CD3G, CD3Z, ZAP70
Déficit en adénosine déaminase (AR)
DICS par défaut de signalisation cytokinique
Enzyme ubiquitaire
DNA
Cellules ADA-déficientes = accumulation
DICS T- B+ NK- XR (IL2RG):
• d-adénosine transformé par la deoxycytidine kinase (dCydK)
en:
• 2'-deoxyadénosine 5'-triphosphate (d-ATP).
d-Adénosine ↑
IL-4
IL-2
gc
IL-7
gc
IL-9
gc
IL-15
gc
IL-21
gc
gc
d-ATP ↑
d-CydK
ADA ↓
X
Ces 2 métabolites ont des effets sur le développement et la
fonction des lymphocytes (synthèse du DNA, blocage des
divisions cellulaires et de l’apoptose)
NK-lymphocyte development
T-lymphocyte development
→ lymphopénie T, B et NK
d-Inosine
DISC T-B+: IL7RA et JAK3 (AR)
DICS T-B-: anomalie de la recombinaison
NK
Jak3 deficiency
TcR: structure
CLP
NH2
T
CD4
IL-7
Va
s s
I I
s s
Vb
Ca
s s
I I
s s
Cb
Ja
Domaine
Variable
DbJb
Domaine
Constant
s s
HSC
T
CD8
COOH
Réarrangement
D
V
B
D
J
C
1) Recombinaison V(D)J
J
V
C
2) Epissage
DICS T- B+ NK- AR (JAK3):
DICS T- B+ NK+ AR (IL7RA):
gc
IL-7Ra
gc
IL-2R,-4R,-7R,
-9R,-15R,-21R
VDJ C
Recombinaison
- I-
- IIReconnaissance de
la cassure d ’ADN
Initiation de la recombinaison:
RAG-1 et RAG-2
RSS
Jak3
Configuration
réarrangée
- III Réparation (NHEJ)
Artémis, Ligase IV et Cernunnos
RSS
RAG-1 RAG-2
Déficit Immunitaire Combiné Sévère
Anomalies du TCR ou du préTCR
(CD45, CD3E,CD3D, CD3G, CD3Z, ZAP70) (AR)
ADA
deficiency
SCID T-B+
(γγc,Jak3)
NK cell
CID T+/-B+NK+
CD3D,E,G,Z
Common lymphoid
progenitor
CD4+ T cell
CD8+ T cell
SCID T-B+NK+
IL7RA
XX
X
B cell
SCID T-B-NK+
Rag1, Rag2, Artemis
Hematopoietic
stem cell
X
PMN
Common myeloid
progenitor
X
Reticular
Dysgenesia
(AK2)
Le Deist, médecine science 2007
macrophage
dendritic cell
Prise en charge thérapeutique
Survival in SCID patients after related HLA-mismatched HSCT
according to year at grafting
• Traitement anti-infectieux,antibioprophylaxie
european registry
• Immunoglobulines
• Traitement curatif:
– Greffe de cellules souches hématopoïétiques
• Geno-identique (fratrie)
• Phéno-identique (fichier)
• Haplo-identique (parents)
– Thérapie génique
(déficits en gammaC et ADA)
– Traitement substitutif (PEG-ADA)
Survival in SCID patients after related geno-id HSCT
Haplo identical HSCT for SCID as a function of years
of transplantation
1995-2005 (n=94)
Survival rate
(10 years) %
survival
< 1995 : 59%
<1995 (n=128)
> 1995 : 72%
P= 0.092
months
Buckley Annu Rev Immunol 2004
Kinetics of T cell developpment after HSCT
SCID with spontaneous reversion
= selective advantage of corrected T cells
Stephan V, et al. Atypical X-linked severe combined
immunodeficiency due to possible spontaneous reversion of the
genetic defect in T cells. N Engl J Med. 1996;335:1563-7.
Arredondo-Vega FX, et al. Adenosine deaminase deficiency with
mosaicism for a "second-site suppressor" of a splicing mutation:
decline in revertant T lymphocytes during enzyme replacement
therapy. Blood 2002 ;99:1005-13.
Hirschhorn R, e al. Spontaneous in vivo reversion to normal of an
inherited mutation in a patient with adenosine deaminase deficiency.
Nat Genet 1996; 13:290-5.
Gene therapy for SCID-X1/ The disease
SCID with spontaneous reversion
DICS T- B+ NK- XR (IL2RG):
= selective advantage of corrected T cells
IL-4
IL-2
Myeloid
lymphoid
progenitors
Hematopoietic
Stem cells
Peripheral
Lymphocytes
gc
IL-7
gc
gc
IL-9
IL-15
gc
IL-21
gc
gc
(2%)
Thymus
(10%)
(10%)
Mutated T cells
NK-lymphocyte development
T-lymphocyte development
Reverted T cells
*~ 60% of known SCID patients
*Lethal within one year of age
*Conventional therapy : HSCT
Gene therapy for
Primary Immuno-deficiencies
SCID-X1 n°1 gene therapy trial
Patients outcome
Phase I/II ; No HLA-id donor
Full correction of T-cell immunodeficiency in 8 out of 10 patients
MLV-based retroviral vector
10 treated IL2RG deficient patients (March 1999- April 2002)
Age : 1-11 months (mean 7 months)
Infections : all
CD34 (+) γc(+)/kg : 1-22 X 106 (median : 4.3x106/kg)
Transduction rate: 20-40%
P9: - received 1 X 10-6 CD34 (+) γc(+) / kg
- decline in immune function;
- HSCT 9/10 MUD 2 years post GT
- died from fungal pneumonia.
P3: - Failure after cell trapping in enlarged spleen;
- successful BMT = A.W
Absence of Conditioning Regimen
But 4 out of 8 patients developed acute T cell leukemia
F.U : 8- 11 years
Early Kinetic of T cell reconstitution
Kinetic of T cell reconstitution
CD34+gc+/kg
7000
P8
CD3+ lymphocytes/µl
5000
P2
4000
3000
P1
2000
1000
P6
P1 : 3x106
P2 : 5x106
P8 : 22x106
P6 :
1.2x106
0
0
1
2
3
4
5
6
7
8
9
7000
10
11
P7
6000
12
13
P4 : 18x106
P5 : 20x106
P10 :11x106
P4
P5
5000
4000
3000
P10
2000
P7 : 4x106
1000
0
0
1
2
3
4
5
6
7
8
9
10
11
12
9000
P5
8000
CD3+ lymphocytes/µl
6000
P4
7000
6000
5000
P7
P10
4000
P8
3000
P2
2000
P6
1000
P1
0
0
20
40
60
80
100
120
Months
13
Months
Thymic output
BV2
P2
BV6b
7,4 %
BV22
4,6 %
1,1 %
7,1 %
Kinetics of Igs levels
BV21
BV11
4,5 %
PBMC (41)
IgG
1600
10 aa
10 aa
11,7 %
10 aa
10 aa
10 aa
5,3 %
2,2 %
4,4 %
1200
7%
P7
PBMC (64)
10 aa
10,5 %
10 aa
10 aa
10 aa
0
10 aa
0
12,1%
15 %
2,6 %
7,8 %
20
40
10 aa
10 aa
TRECs/ 105 PBMC
P7
100
P10
P4
10
P8
100
120
IgM
P1
400
400
300
300
200
P2
P1
P6
P2
200
P2
100
P7
0
P5
1
IgA
10 aa
10000
1000
80
500
500
10 aa
60
9,8 %
PBMC (97)
10 aa
P2
P4
400
PBMC (81)
10 aa
P1
10 aa
9,1 %
1,9 %
4%
6,6 %
10 aa
10 aa
P8
P5
800
10 aa
0
20
P5
40
60
100
P8
80
100
P7
P5
0
120
0
20
40
P8
60
P1
80
100
120
0,1
0
20
40
60
80
100
120
Months
Patient 4 Clonality study
Longitudinal analysis
of vector integration sites
6 13 17 24
31 34
-C
37
Insertional mutagenesis /LTR driven oncogene activation
Sequential Immunoscope Study of
T Vδ
δ1 Population
M
T g/d
control
M23
P4 (M+30), P5 (M+34) , P10 (M+33) and P7 (M+68)
MFG c
1
M6
*
* *
*
*
MFG c
2
P4
P5
3‘LTR
MFG c
3
4
P10
5
LMO2, chr11
M27
*
198 pbs
M13
M31
BMI1
1
4
MFG c
25.240 pb
M17
M34
Monoclonal γδ T cell clone (immunoscope + TCR sequence)
Blast like cell but mature T cell phenotype
Integration site increased at least from m13
RV induced leukemia = Primary T-ALL ?
Oncogenic rearrangements after the first insertional hit = those
observed in primary T-ALL
All the patients have aditional genetic alterations:
Gain-of function NOTCH1 mutation in P5 and P10
49.499 pb
P10
SPAG6, chr10p12.31
CCND2
MFG c
8
P7
2.440 pb
Intergenic, chr12p13.32
Gene therapy for SCID-X1 : UK trial
Another SCID-X1 gene therapy trial in UK (A. Thrasher)
10 patients median FU 6 Y
Similar MLV-derived RV vector
Good Immune reconstitution
1/10 T ALL at M+24 : LMO2
6
SCID-X1
Gene therapy vs haploidentical HSCT
Gene therapy for SCID-X1
Conclusion
*RV-mediated γc gene transfer into CD34+ cells leads to
o No GVHD
a sustained correction of the T cell immunodeficiency;
o Short term T cell reconstitution=lower risque of infections
10 years follow-up!
*Leukemia occurrence related to RV Insertional mutagenesis. LTR
driven activation of oncogenes (5 /20)
o Stable NK cell reconstitution: often poor
o Restoration of Humoral response after GT (frequent) but
dysfunctional recipient B cell immunity after HSCT
*4 out of 5 patients are in CR after chemotherapy + regeneration of
a polyclonal T cell repertoire
ADA Deficiency
SCID-ADA gene therapy trials
Trials in Italy, UK, USA and Japan :
Adenosine
ADA
*Fatal AR form of
SCID (T- B- NK-)
*Incidence: 15-20%
*Accumulation of toxic levels
of purines metabolites
Adenosine-deaminase
inosine
PNP
30 infants treated with Retroviral vectors :
hypoxanthine
adenosine
deoxyadenosine
*sustained gene marking in most patients
*pre-conditioning allow engraftment of transduced progenitor cells
d-ATP
*HSCT with HLA-id sibling= Tt of choice
ribonucleotide reductase Inhibition
DNA synthesis inhibition
* PEG-ADA withdrawal ensure a selective advantage for infused cells
*PEG-ADA corrects
the metabolic alterations, improves the clinical
condition but often fails to sustain correction of
the ID (neutralizing Abs)
SCID-ADA gene therapy trial
(Aiuti et al 2009)
Kinetic of T cell reconstitution
Phase I/II ,
10 patients ( July 2000- Sept 2006)
ADA patients * who lacked HLA-id sibling
* failure of 6 months PEG-ADA therapy
Age: median: 1.7y (0.6-5.6 y)
Non-myeloablative CR : busulfan 4 mg/kg
CD34+ mean dose :
8.2 x106 /kg (0.9+2.2 –13.6)
28.6 % transduced CFU
MLV derived vector
Aiuti et al. NEJM 2009
F.U median : 4 y (1.8- 8 y)
Safety
Large-Scale Integration sites analysis in
SCID trials
No abnormal expansion or clonal outgrowth
SCID-X1 Paris trial (Deichman et al, 2007)
SCID-X1 London trial (Schwarzwaelder et al, 2007)
ADA Milan trial (Aiuti et al, 2007)
P3: prolonged neutropenia and thrombocytopenia
P6: EBV reactivation,
* Bias for integration: Transcription factors; cell-cycle control and
signal transduction genes and cancer-associated genes
P7: AI hepatitis
P8: prolonged neutropenia and AI thrombocytopenia
( PEG-ADA restrarted 0.4y after GT)
PEG-ADA restarted
* Vector integration is clustered in Common Insertion Sites (CIS) :
LMO2, CCND2
in P8 0.4y after GT
in P2 4.5y after GT
They strongly confer a selective advantage to the cell
= Clonal dominance ………… Malignant transformation
Perspectives
Déficits phagocytaires
*SCID-X1 n°2 gene therapy trial (Paris, London, Cincinnatti)
with Retroviral Self-inactivating (SIN) Vector
Absence of HLA-id sibling + Severe infections
NK cell
CD4+ T cell
Common lymphoid
progenitor
CD8+ T cell
SIN : lacking viral enhancers/promoters in their 3' long terminal
repeat (LTR)
B cell
Hematopoietic
stem cell
PMN
Common myeloid
progenitor
macrophage
dendritic cell
Fonctions des phagocytes
Déficits héréditaires de la phagocytose
Fusion de la
membrane
fusion des
lysosomes
• Infections bactériennes et/ou fongiques
o Défaut quantitatif
Opsonisation
Formation
du phagosome
Activation
des mécanismes
Bactériolytiques
Génération de H2O2
+ radicaux libres
NADPH oxydase
Neutropénie auto-immunes
Neutropénies congénitales
phagolysosome
o Défaut qualitatif
Déficit d’adhésion leucocytaire (déficit CD18)
Migration
Exocytose des
débris bactériens
Granulomatose septique chronique (CGD)
Neutropénies congénitales
Granulomatose septique chronique (CGD)
• Déficit phagocytaire
maladies métaboliques : glycogénose 1b
maladie de Shwachman :
• Fréquence : 1/200 000
•AR, SDBS,
•Ins. pancréas exocrine, anomalies osseuses
• XR (gp91phox),
syndrome de Kostmann :
• AR (p47phox, p22phox, p67phox)
•AR, HAX1
• Porteur mutation récurrente p47phox:
1/2000
neutropénie cyclique :
•AD, ELA2
• Anomalie de l’explosion oxydative
(NADPH)
Granulomatose septique chronique (CGD)
• Pneumopathies
79%
• Abcès
68%
• Adénites suppuratives
53%
• Ostéomyélites
25%
• Septicémies
18%
Biopsie pulmonaire
Histologie : granulome
• Cellulites
5%
• Méningites
4%
• Otites
15%
Aspergillus fumigatus
• Germes : Aspergillus, S.aureus, entérobactéries
(Winkelstein et al. Medicine 2000)
Nitroblue tetrazolium reduction (NBT)
Explorations :
Prise en charge thérapeutique
Controle
Patient
vectrice
• Antibiothérapie et traitement anti-fongiques
• Bactrim et Sporanox prophylactique
• +/- :
HSCT
Thérapie génique
Clonal dominance in X-CGD trial
Clonal dominance in CGD trial
(Ott et al 2006)
(Ott et al 2006)
Neutrophil disorder; defect of NADPH oxidase subunit (gp91phox)
HSCT if HLA-matched donor
91 RIS in MDS1/Evi1 :
P1=42, P2=49
Gene therapy trial in Frankfurt :
*2 adult patients included
*MLV-derived retroviral vector (SFFV promotor)
*Transgene-positive in PBMC : 30%
*Sustained superoxide production in neutrophils/ clearance of
infections
Conclusion GT
*Efficacy of gene therapy related to transgene-based selective
advantage : SCID gc or SCID ADA
*Insertional mutagenesis related efficacy of gene therapy in absence
of intrinsic selective advantage in the required lineage : CGD .....
*Requirement to use safer vectors
SIN-RV or SIN-LV vectors
Insertion with a strong preference in some genes =
selective advantage to the myeloid cells and then clonal expansion.
Transgene silencing (LTR promotor’CpG methylation)
but maintenance of LTR enhancer transactivation potential Myelodysplasia
(monosomy 7) : P1 died from sepsis (M+27); P2 MUD HSCT