LB 0910

B lymphocytes
Subsets, Control of antibody
production and autoreactivity
Peripheral B cells
• Central selection during bone marrow
maturation
• Survival B cells incorporate in the
peripheral pool as small recirculating B
cells
• A fraction is selected in anatomically
distinct sites as sessile large cells with
partially activated phenotype -> MZB and
B1 cells
B lymphocyte subsets
• B1 lymphocytes
– CD5+
– Peritoneal cavity and MZB
– Autoreactive, low affinity
– IL-10 production upon TLR engagement
– Function: natural Ab ? First line of defense ?
• B2 lymphocytes
– Conventional peripheral B lymphocytes
– Develop in follicles
Peripheral B cell maturation
• Transitional B cells: HSA++, sIgM+, CD23(FcεRII), CD21/35low (C’R)
• Positive selection towards B2 cells (not clear?):
requirement for Igα chain
• Follicular B cells sIgM+, CD23+
• Tonic signaling by BCR required for survival
B1 lymphocytes
• Preferentially generated from fetal HSC as CD5+ B cells
• Partially autoreactive repertoire
• Important in the production of natural antibodies (IgM)
• Present in pleural and peritoneal cavities (Ab generation
for mucosal surfaces), IgA-producing cells
• Early responses to local infections
Natural antibodies
• Normal individuals produce large amounts
of autoantibodies
• Not synonym of pathology: proof requires
transfer of pathology by Ab
• Beneficial effect of IVIG
• Both IgM and IgG
• Multireactivity / connectivity
• Germline-encoded, limited clonal
expansion
Activation of autoreactive B
lymphocytes
• Naturally activated plasma cells: repertoire
is not random
• B cell transgenic mice: quasi normal AAb
repertoire due to escape of imposed B cell
reactivity
• AAb can be found in T –less and germfree mice
Marginal zone
• Border of white and red pulp
• Enriched in macrophages and marginal
zone B lymphocytes (MZB)
• In contact with blood stream
• Function: alarm, Ag uptake ?
Splenic architecture
MZB: CD19+, CD1dhi, CD23lo
Fol B: CD19+, CD1dlo, CD23hi
Marginal zone B cells (MZB)
• Essential component for response to blood-borne antigens
• 5% of splenic B cells, innate-like B cells
• Intermediate between innate and adaptive immunity: active within
hours versus weeks
• Mainly directed against non protein Ag (glycolipids, carbohydrate)
shared by microbes, product of complex metabolic pathways and
not susceptible to be mutated by the pathogens (costly)
• Inititiate T-independent responses
• Features of antigen-exposed B cells
• Lack of B1 and MZB in CD19 mutant mice, Notch2 required for their
development in spleen (?)
• Survival requires BAFF and APRIL (TNF superfamily) secreted by
DC
• Retention in MZ requires macrophages (scavenger receptor
MARCO+)
MZB repertoire
• Large clonal size, multireactivity, restricted variable gene usage and
limited junctional diversity
• Abundant germ-line heavy chain -> little or no affinity maturation
• Positive selection by self may help to produce autoreactive Ab
• In secreted IgM mutant mice, expansion of MZB pool: retroactive
control by soluble Ig or increased cellular debris -> argument in
favor of postive selection by self ?
• Role of microflora -> positive selection by commensal non self ?
• Escape from self-mediated peripheral negative selection (deletion,
editing or clonal silencing), due to low affinity ?
• Role of TLR / BCR synergism in activation
• Connectivity between Ig idiotypes ?
MZB activation
•
Low affinity and threshold
•
Circulating or cell-trapped Ag (PN, DC, M) is recognized by MZB
•
Upon BCR crosslinking (TLR?) development into red pulp plasma cells
•
High CD1d expression -> NKT activation through glycolipid recognition ?
•
Contribution to the production of natural antibodies (B1 ?) -> essential for
survival to endotoxic shock, microbe dissemination
•
Deleterious effect of splenectomy
•
Involved in the generation of autoreactive B cells in SLE ?
MZB and Ag uptake in spleen
(S1P antagonist)
IV Injection of TNP-Ficoll
C’-mediated uptake by MZB
MZB-mediated Ag transfer on FDC
IV Injection
of TNP-Ficoll
Impaired
migration
Impaired
Transport
On FDC
CD19 KO, no MZB; CXCR5 KO, no follicles
Bm chimera -> MZB come exclusively from CXCR5 wt or ko mouse
B2 lymphocytes
• Adaptive lymphocytes, mostly resting before Ag
exposure
• Require T help for full maturation
• Capable of isotype switch, affinity maturation
and memory cell development
• Colonize secondary lymphoid organs
Normal B cell differentiation
IgM memory
Antigen dependent
MZ
Somatic mutation
Class switch
Amplification of Ig diversity
Generation of B cell memory
T2
IgG,IgA
or IgE
Antigenic
challenge
FO
Plasma
cell
follicle
spleen
T1
Positive selection
Switched
memory
Transitional
Negative selection
Mature
T1
Antigen independent
New emigrants
Acquisition of Ig diversity
Pre-BCR
Bone marrow
Stem
cell
CLP
DH to JH
pro-B
VH to DJ H
Large
pre-B
BCR
Small
pre-B
immat.
B
VL to J L
Mature
Trans.
B
mature
B
Checkpoints during B cell development in the bone marrow
pre-BCR
mediated selection
B cell
engagement
BCR mediated
emigration
Pre-BCR
CLP
Pro-B
DH to JH
pre-BI
VH to DJ H
Large
pre-BII
BCR
Small
pre-BII
immat.
B
mature
B
VL to J L
Tissue specificity, kinetics of Ig gene rearrangements, allelic exclusion, repertoire selection
(5x106 pro-B ---> 2x107 immature B cells/day ---> 5-10% will reach the periphery)
Steps of B cell differentiation in the bone marrow
Human
Mouse
stem cell
early
pro-B
pro-B
CLP
pro-B
A
pre-B
pre-B I
B
large
pre-B II
C
small
pre-B II
C’
immature B
mature B
immature B
mature B
D
E
F
non-productiv e
VHDJ H
ΨH Ψ L
non-productiv e VLJ L
µ-SLC
TdT
TdT
cµ +
SLC+
D
JH
VH
DJ H
cµ +
SLC+/-
IgM
cµ +
VL
JL
IgM
IgD
Stem cells
B cell differentiation
in BM
Pro-B
Stromal cells
Pre-BI
Pre-BII
Immature B
-a centrally directed sequence of
differentiation
-B cell precursors in contact with
stromal reticular cells
-B lymphocytes delivered in the
blood after an intravascular
maturation
Jacobsen and Osmond, 1990, EJI, 11, 2395
Morphological construction of the bone marrow
Hematopoiesis occurs in the extravascular spaces between sinues. The medullery vascular
sinues are lined with endothelial cells and surronded by adventitial recicular cells.
Nagasawa, Nat. Rev. 2006
The pre-BCR checkpoint
Pre-BCR
Stem
cell
CLP
DH to JH
pro-B
VH to DJ H
Large
pre-B
BCR
Small
pre-B
VL to J L
immat.
B
mature
Trans.
B
The pre-BCR and the SLC
Igm
SLC
VH
EL
VpB
VpreB
SLC
l5
Jλ
EL
Iga/Igb
l 5/l -like
Y Y
Y Y
Cell
surface
VpreB
gene
l5
gene
L
L EL
V preB
EL Jλ
EL
Cl region
Pre-BCR functions
Igm
VH
VpB
SLC
l5
Iga/Igb
Y Y
Y Y
Cell surface
Differentiation
Large --> small pre-B
Fr C --> Fr D
Proliferation
VH repertoire
selection
Allelic exclusion
Evidence for a pre-BCR stromal cell ligand
Galectin-1, a S type lectin
pre-B
pre-B + GAL1
pre-B cell
GAL1
m
Stromal
cell
DIC
VpreB
without stroma
GAL1
DIC
VpreB
The pre-BCR and GAL1 colocalize and are polarized at the contact zone between ptre-B
and stromal cells. The relocalization process depends on the presence of stromal cells
immature B
GAL1
m
µ
DIC
GAL1 faintly accumulates at the contact zone
between the 2 cells, but the BCR is never relocalized
DIC
Possible role of Galectin-1+ cells for pre-B cell functions
Stromal cell
Human and mouse
pre-B cell
pre-BCR
GAL-1
GAL-2
integrins
ADAM-15
fibronectin
GAL-1 produced by stromal cells binds to integrins and pre-BCRs. GAL1acts as a supramolecular organizer of a 3D lattice that clusters together the
relocalizing pre-B cell integrins and the pre-BCRs
Stromal cell
Synapse formation
Pre-BCR clustering
and signaling
pre-B cell
pre-BCR
GAL-1
GAL-2
integrins
ADAM-15
fibronectin
Formation of the pre-B/stromal cell synapse results
in pre-BCR clustering and in the initiation of pre-BCR signaling
Rossi et al., J Immunol., 2006, 177, 796
Espeli et al., Seminar Immunol., 2006, 18, 56
Functional role of the pre-B/stromal cell synapse?
Immature B
pre-B II
SLC-/-
without stroma
Clustering of different pre-BCRs could be necessary to reach the activation threshold
necessary for pre-BII cell differentiation and proliferation. Ligand engagement could also
enhance the pre-BCR tonic signaling, thus increasing the efficiency of these programs.
S. Mancini poster 19
Movements of HSC and developing B cells in BM
Pre-BCR selection
Cell proliferation
Bone marrow
?
IL7
IL7
Fibroblast
Cell fate decision
CXCL12
sinusoidal
endothelium
GAL1
Pre-B
IL7
?
Pro-B
CXCL12
HSC
Immat. B
HSC
Secondary
lymphoid
organs
Pre-pro-B
HSC
CXCL12
IL7
osteoblast
HSC move from the bone surface or endothelial cells toward CXCL12+ cells. Differentiating HSC
move along the processes of CXCL12+ cells. Pre-pro-B cells in contact with CXCL12+ bodies then
migrate and reach IL-7 + cells. Pre-BCR+ cells interact with GAL1+ /IL-7+ cells.
The BCR checkpoint
Pre-BCR
Stem
cell
CLP
DH to JH
pro-B
VH to DJ H
Large
pre-B
BCR
Small
pre-B
immat.
B
mature
Trans.
B
VL to J L
Autoreactive
Deletion
Anergy
Receptor editing
VH replacement
Non autoreactive
Periphery
Central B cell tolerance checkpoint
Bone marrow
Periphery
BCR dependent
Antigen independent
Pro-B
Pre-B
Immature B
Igµ
sIgM
SLC
Igα/Igβ
B cell
commitment
stem
cell
sIgM
Pro-B
Large
pre-B
small
pre-B
Self-reactive
Early
Immature B
Immature
B
76%
43%
55%
7%
new
Emigrant B
(nuclear and cytoplasmic Ag)
Poly-reactive
(ssDNA, dsDNA, insulin, LPS)
Central B cell tolerance
checkpoint
Wardemann et al., Science 2003
Self antigens in the BM can lead to deletion
or inactivation of immature B cells
Deletion or
Receptor Editing
anti-MHC
anti-Hel x mHel
Anergy
anti-Hel x sHel
Ignorance
Normal
maturation
Gene defects leading to agammaglobulinemia
B cell homeostasis
• B cell fitness required for survival: normal B cell
development in xid (Btk deficient ) or CD19mice but impaired development in mixed bm
chimeras
• Mixed monoclonal Ig / polyclonal bm ->
advantage to polyclonal bm -> competition for
poorly defined ligand ?
• Prerequisite for constant renewal of B cell
repertoire and homeostatic expansion
• Double aTNP/aHEL Ig Tg B cells show
enhanced survival in the presence of low levels
of soluble HEL (low BCR engagement)
B cell activators
• T-independent antigens
– Type 1: components of bacteria (LPS, …) ->
polyclonal expansion
– Type 2: polymeric, polysaccharidic antigens (TNPFicoll)
• T-dependent antigens
–
–
–
–
Clonal expansion
Isotype switch
Affinity maturation
Immune memory
Contribution of innate receptors
• LPS: TLR4 agonist, polyclonal B cell
mitogen
• B cells express most TLR
• Some (TLR7, 9) are intracellular; specific
for nucleic acids (self ?) they may not have
direct access to self Ag and may require
additional BCR specificity and delivery to
endosomes ?
TLR role in T-dependent Ab production
• Myd88 deficient mice have impaired Tdependent Ab responses
• TLR in B cells may be required for the
production of some isotypes (TLR9 -> IgG2a ?)
or the control of cytokine production (IL-6
required for IgM)
• Be1 and Be2 subsets (B effectors) 6> distinct
cytokines -> Th1 development requires Myd88
expressing B cells
T and B cells homing into LN
B zone
B zone
CCR7
CXCR4
CXCL13
B CXCR5
HEV
T CCR7
CXCR4
CCR7 -> CCL21/19
CXCR4 -> CXCL12
CXCR5 -> CXCL13
T zone
CCL21/19
Ag trafic in lymph node
Ag encounter by B cells
• Occurs in secondary lymphoid organs (around
HEV with DC or in subcapsular sinus with MΦ)
• Soluble Ag diffuse via the (FRC) conduit system
• Larger Ag are actively transported by myeloid
cells
• FDC store large amounts of Ag via FcγRIIB and
CD21 (C’ coated Ag)
B cells in antigen presentation
• Bm chimeric mice with MHC II-deficient B
cells show delayed T cell priming -> early
impact on T cell activation ?
• Fast accumulation of Ag / MHC peptides
by B cells
Do B cells contribute to T cell
priming ?
• Intuitively no, since there are very few Agspecific B cells before immunization
• However, mice with MHC II-deficient B
cells have unpaired T cell priming ?
• Unconventional capture ? MHC tickling ?
BCR/Ig mediated antigen presentation
LB
X 100-1000 less efficient
T cell activation potential
LB
Ag-specific B cell = Dendritic cell
BCR-mediated activation
• BCR: mIgH + L with IGαβ heterodimer
• BCR engagement -> Tyr Phosph of ITAM
by Lyn -> Syk activation
• Signalosome assembly (Vav, Btk, PI3K,
PLC-γ2, Blnk
Immune synapse on B cells
• BCR microclusters (10-100 BCRs/IgM, IgD) in phosphatase-free
areas (CD45, CD148) -> Ca signalling
• Exclusion of other negative regulators (CD22, FcγRIIB)
• Wide variations in BCR affinities (>>TCR) for membrane bound Ag:
threshold Ka=10 µM in the absence of cosignal
• Propagation by BCR spreading requires CD19 involvement (in a
complex with CD21 = CR2, CD81, leu13) -> role of C’
• Impaired affinity maturation and GC formation in CD19 or CD21
mutant mice
Immune synapse in B cells
Consequences
• Gene expression
• Cytoskeletal rearrangement -> Immune
synapse
• BCR internalization -> Ag uptake and
processing
Role of complement
• T-independent B cell activation
– Type 1: C’ independent -> multivalent BCR
ligation
– Type 2: repetitive Ag, often C’ dependent ->
cosignal
• T-dependent B cell activation
T cell dependent activation of B cells
MHC II
SIGNAL 1
TCR
TCR
CD4
B7
LB
CD28
CD28
B7
CR
C’
C’
SIGNAL 2
TCR
CD4
LB
CD40L
SWITCH
CD40
SIGNAL 2
HELP
T-B: from encounter to help
T/B migration patterns
- CXCR5 expressed by mature B -> access to follicles
- Ag capture by B cells -> upregulation of CCR7
- Mobilization towards boundary with T zone: T-B encounter
Parameters influencing follicular T cells entry in GC
- Ag mediated T cell activation -> CXCR5 expression (days)
- CD40-activated DC -> Ox40L - Ox40 on T cells
Lack of Ox40, no follicular migration
GC formation requires T help
- Localization in the light zone (CXCL13 production)
- CD40/40L involvement -> prolif, switch, memory B
- Other costimuli: ICOS -> Ab production (IgG1 ?)
- Cytokines: IL-4 (proliferation), IL-10/5 (Ab production), IL-21/6 (plasma cells),
control of switch (IFNg, IL-4, TGFb, …)
Germinal center dynamics
B cell selection in GC
Antibody repertoire
• Stems from a unique and conserved
combinatorial system present only in jawed
vertebrates (IgV-like domains found in sponges
lack J segments and thus CDR3 loops)
• VH3-53 : 57% identity between shark and
human -> encompasses 450 Millions years of
evolution
• Basic features: epitope promiscuity and
polyreactivity, illustrated by the natural antibody
repertoire
Antibody crossreactivity
• A single paratope binds several epitopes
• Explainable by shared ligand chemistry
and molecular mimicry
• Crossreactivity may explain some cases of
autoimmunity and allergy
• But how can one be promiscuous and
specific ?
Antibody multispecificity
• 1940: Pauling’s hypothesis : « specific binding
sites were selected out of an ensemble of
preexisting Ab conformations »
• Different paratopes bind different epitopes
• Mechanisms may include
– Induced-fit conformational changes upon ligand
binding (documented by crystallographic data
comparing bound and free mAb)
– Equilibrium between different isomers
Schematic energy landscapes
in protein-protein interactions
The SPE7 anti-DNP mAb
• Monoclonal IgE
• Binds DNP with high affinity (Kd 20 nM)
• No crossreactivity with DNP analogs (Kd >
100 µM)
• Binds unrelated compounds with variable
affinities (Kd µM to nM range)
Isomers of an anti-DNP mAb
Unbound
Bound
Electrostatic potential is visualized
Blue for +; red for -
Pre-steady state kinetics of complex
formation reveal two preexisting isomers
Equilibrium between preexisting isomers
(intermediate phase)
Ab2 -> Ab1 relaxation time
Ab2 binds the hapten (fast phase)
Induced-fit
Isomerisation
Affinity x 500
(slow phase)
Highest affinity due to the number
and nature of hydrogen bonds
Deep binding site
Specific AND promiscuous
• A kinetic proof-reading mechanism compensates the
lack of discrimination of Ab2 by an induced-fit
optimisation of antigen binding : this mechanism
determines both the crossreactivity and the high
affinity of this mAb for its ligand
• Conformational diversity : the preexistence of
multiple isomers confers further multispecificity to
the mAb (Peptide library scanning reveals a third
antibody conformer)
Affinity maturation : a model
system to study protein evolution ?
• A feature of adaptive humoral immunity
• Requires T-B cell cooperation in functional
lymphoid organs
• Conformational diversity and functional
promiscuity can be considered to be traits of the
ability of a protein to evolve
• Can one define general rules ?
– Variations around interaction hot spots : Change from a pliable germline
Ab-combining site to epitope-template structural rigidity
– Highest affinity correlates with a minimal distortion from unbound state,
thus minimizing entropic penalty