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B Cell Activation
Micro 204. Molecular and Cellular Immunology2009
Lecturer: Jason Cyster
Antigen
Antibody 1
Antibody 2
Antibody 3
Lecture Outline
1. What factors promote B cell survival?
2. Why do some B cell responses require T cell help and others not?
3. What is ‘T cell help’?
4. What are plasma cells?
5. How do activated B cells undergo ‘affinity maturation’?
6. What is the mechanism for B cell memory?
7. How are antibody responses down-regulated?
CD22
IgM/IgD
β α45CD
syk
1SHP
lyn
promoted by-Ig crosslinking
2SH
2SH
2SH
2SH
2SH
3SH
1SHP
syk
lyn
Inactive Active
Early positive and negative events in BCR signaling
Constitutive BCR signaling is necessary for B cell survival
V-region
loxP loxP
IgM constant-region
Cre-recombinase
from Lam et al., Cell 90, 1073 (1997)
BAFF / Blys and B cell survival
• Member of TNF family• Made by stromal cells, macrophages, DCs• BAFF-receptor constitutively expressed on B
cells– BAFF also binds TACI and BCMA– APRIL is a second TACI, BCMA ligand
• Critical role in B cell homeostasis– BAFF knockout has ~100x less B cells– A/WySnJ mouse strain has mutation in
BAFFR and has ~10x reduced B cell numbers – BAFF over-expression increases total B cell
numbers and promotes autoantibody production
– BAFF serum levels elevated in lupus
TACIBCMA
BAFF
Stromal cell, MØ, DC
B cellBAFFR
NFB (alternative & classical pathways) -> Bcl2, Pim, other
-> Cell Survival
BAFF/Blys is necessary for B cell survival
from Schiemann et al., Science 293, 2111 (2001)
TACIBCMA
BAFF
B cell
BAFFR
MØ
(BAFFR mutant)
What is meant by ‘B cell activation’?
• Go -> G1 of cell cycle (increase in size)
• upregulate– MHC class II
– costimulatory molecules (B7-2)
– adhesion molecules (ICAM1)
– cytokine receptors (IL-2R)
• migrate to outer T zone– altered response to chemokines
• become receptive to T cell help– protected from fas
• enter mitosis if provided with submitogenic doses of other stimuli (LPS, CD40L, IL-4)
Early events in B cell activation by membrane associated antigen
Types of antigen• T-independent (TI) antigens
– induce division/differentiation by BCR signaling alone• bacterial polysaccharides, repeating surface molecules on viruses
• T-dependent (TD) antigens– activate via BCR but depend on additional signals from helper
T cells to cause division/differentiation• any antigen containing protein
• Most pathogens contain both T-I and T-D antigens• Only TD antigens can induce Germinal Center responses
• Polyclonal Mitogens– induce division/differentiation independently of BCR
• high dose LPS, polyclonal anti-IgM, anti-CD40• mostly observed in vitro
T cell
mitogenic BcR signal
'activation' signalbut not mitogenic
mitogenesis
differentiation
present
Ag
T-independent (TI) T-cell dependent (TD)
Types of B cell Antigens
Old Paradigm:
B cell plasma cellsB cell
T-Independent (type II) Responses
Multivalent Antigen
B cell plasma cells
DC/Mø
Emerging Model:
DC/Mø
B cell
(BAFF)
B cell plasma cells
T-Dependent Responses
DC
Antigen
T cell T cell
Dendritic Cell (DC)internalizes antigen (Ag), processes into peptides, presents peptides together with MHC molecules to T cells
B cell binds Ag via surface Ig, transmits BCR signals and presents peptides to T cells, receives T cell help (growth and differentiation factors)
Secretes Antibody (Ab)
B cells are antigen-presenting cells
• BCR cross-linking induces antigen internalization to endosomes
• antigen is proteolysed to peptides• peptides associate with MHC class II• MHC class II-peptide complexes traffic to
surface of B cell• B cells present antigen recognized by
their BCR ~105 x more efficiently than other antigens
B cell antigen presentation and the concept of linked help
protein
sugar
Polysaccharide SpecificB cell
T
Protein SpecificT cell
CD40LCytokinesBCR
MHC II
endosome
Haptens
• Small organic molecules of simple structure do not provoke antibodies by themselves. However, antibodies can be raised against them if the molecule is attached to a protein carrier.
• Such small molecules are termed haptens (from the Greek haptein, to fasten).
• Some drugs such as Penicillin can act as haptens and induce antibody-mediated allergic reactions.
Making an antibody response to a hapten
proteinhapten
Hapten SpecificB cell
T
Haptenated-peptide Specific
T cell
CD40LCytokines
+
1. Hapten covalently attaches to self-protein2. Hapten specific B cell binds haptenated-protein
3. Complex is internalized and degraded to haptenated peptides
4. Haptenated peptides are presented to T cells5. B cell receives help and releases hapten specific
antibody
Innate features of pathogens act as B cell costimulators
• Pathogen multivalency– provides a level of BCR crosslinking optimal for activation
• Many pathogens activate TLRs – TLR signaling synergizes with BCR signal
• Some induce type I IFNs– IFNαβR signaling synergizes with BCR
• Many activate the complement cascade and become C3d coated– complement receptor (CR) crosslinking synergizes with BCR
signal
TLR signaling in B cells via adaptor Myd88 augments Ab response
Flagellin/alum i.p. d12
Pasare & Medzhitov, Nature (2005) 438,364
TLR
BCR
Myd88
Ag
WT
Flu, d5d10 of primary response
Type I IFN receptor signaling in B cells augments Ab response
Coro et al., JI (2006) 176, 4343Le Bon et al., JI (2006) 176, 2074
IFNR
BCRIFNαAg
• IFNαβ made by many cell types in response to viral infection• Many cell types, including B cells, express the IFNαβR
Antigen-C3d complexes cross-link BCR and CR2-CD19 complex - increase
sensitivity to antigen
syk
lyn
IgM
CR2/CD21
CD19
antigen C3d
PI3-KVav
Y
Y
HEL
HEL-C3d
Concentration (nM)
0 0.1 1 10 100 1000
Dempsey et al. (1996) Science 271, 348
Sensitivity of anti-HEL Ig-transgenic B cells to HEL and HEL-C3d
multiple downstream pathways
Key components of T cell help
• CD40L triggers CD40 -> synergizes with BCR signals to promote mitosis; cytokine (e.g. IL4) signals also contribute
• FasL triggers Fas -> BCR signaling protects from apoptosis
• T cell derived cytokines influence differentiation, isotype switching:– IL2, IL6 promote differentiation
– IL4 -> IgG1, IgE
– IFN -> IgG2a, IgG3
– TGFβ and IL5 -> IgA
• Many other molecules involved in T-B interactions – e.g. ICAM1/LFA1, ICOS/ICOSL, CD30/CD30L,
CD27L/CD27, OX40L/OX40, ...
Role of CD40 in B cell activation
• TCR triggering up-regulates CD40L on T cell
• CD40 signaling promotes B cell activation
• CD40L-deficiency = 'hyper-IgM syndrome' (no isotype switching, no Germinal Centers)
• CD40 signaling also important in DC, MØ function
CD4 T cell
CD40L (TNF family)
CD40 (TNF-R family)
TRAF2 TRAF3B cell
increased expression of cell cycle molecules,
survival molecules, cytokines,promotes isotype switching
antigen-binding B cell
MHC
B7.2
TCR
CD28
IL4
FAS
IL4R
CD40
FasL
CD40L
(death)MITOSIS
antigen-specific T cell
Plasma cell
GC cell
isotype switching,proliferation ?
additional cytokines,costimulatory signals
Cardinal features of B - T interaction
Altered signaling in anergic B cells
syk
lyn
Activated- acutely exposed to cross-linking antigen
β α
syk
lyn2SH
2SH
2SH
3SH
Anergic- chronically exposed
to antigen
negativefeedback
Reduced tyrosine phosphorylationElevated basal Ca - but reduced fluxConstitutive erk and NFATNo induction of NFkB, JNK
negative feedback
antigen-TOLERANT B cell
MHC TCR
CD28
FAS
IL4R
CD40
FasL
CD40L
DEATH(mitosis)
antigen-specific CD4+ T cell
antigen-ACTIVATED B cell
MHC
B7.2
TCR
CD28
IL4
FAS
IL4R
CD40
FasL
CD40L
(death)MITOSIS
antigen-specific CD4+ T cell
Anergic B cell
Anergic B cell
Oligovalent Self-Ag,Constitutively Exposed
Remains Anergic-> killed if presents to antigen specific T cell
Multivalent Foreign-Ag,Acutely Exposed
Becomes Activated-> can receive T cell help-> proliferate and secrete Ab
Anergic B cells can respond to 'stronger' antigens
membrane Ig
secretory Ig
B cell Plasma Cell
After appropriate activation the B cell differentiates into an antibody secreting cell, also known as a Plasma Cell
blimp1
XBP1
Production of membrane vs secreted Ig
membrane Ig
CH tmcy
CH tmcyAAAA
CH
AAAA
secretory Ig
splice, use poly A site 2
1 2
no splice, use poly A site 1
CH tmcy1 2
Plasma Cells are antibody secreting cells
Two types:
1. Plasma cells generated early in the primary response- short-lived (~ few days)
- typically low affinity
- form in T-independent and T-dependent responses
- home to red pulp of spleen, medullary cords of lymph nodes
- IgM but also IgG and other isotypes
2. Plasma cells generated later in the primary response and that predominate in secondary responses- arise predominantly from germinal centers (in primary) or from memory B
cells (in secondary)
- long-lived (possibly several months)
- often home to bone marrow, gut, lactating mammary gland
- predominantly isotype switched
naive B cell activated B cells
3 days 12 divisions
plasma cells
1 day differentiation
1 day104 Ab/cell/sec
antibodies
1 212 = 4,096 4,096 >1012
B cell antibody response - clonal replication enters into a higher order upon plasma cell differentation
bacteria - dividing every ~60 min5 days = 2120 divisions = 1.3x1036
BCR isotype switchingVDJ μ δ 3 1 2b 2a ε α
55 kb
( -4)IL T cell help
antigen
/ + IgM D naive B cell
1+ IgG memory
cell
1 IgG secreting
plasma cell
Ig Heavy chain class (isotype) switching
neutralization
Neutralization3wk t1/2
neutralization
Affinity Maturation
• Affinity maturation occurs in germinal centers and is the result of somatic hypermutation of Ig-genes in dividing B cells followed by selection of high affinity B cells by antigen displayed by FDCs
• The high affinity B cells emerging in germinal centers give rise to long-lived plasma cells and memory B cells
Antibody Affinity Maturation
Low affinity B High affinity B
mutation selection
Germinal Center
VH VL
Ag VH VL
VH VL
VH VL VH VL
AgVH VL
Germinal CentersFunction: to generate B cells that produce antibodies with increased
affinity for the inducing antigen
=> affinity maturation
Germinal Center Reaction:
Activated B cells give rise to Centroblasts
- localize in follicle, undergo rapid cell division and turn on machinery that causes somatic mutation in V-regions
Centroblasts give rise to Centrocytes
- migrate to the FDC-rich region of the Germinal Center
- survival is dependent on interaction with FDC-bound Ag and presentation of Ag to T cells
- centrocytes that successfully compete to bind antigen (e.g. by having higher affinity BCR) and to receive T cell help are selected and may differentiate into long-lived plasma cells or memory B cells
FDCFDC
FDC
Light Zone
Dark Zone
FDC
FDC
FDC
CBCB
CBCB
CBCB
CB
CB
T T
Tmemory B cell
plasma cell
CB
CB
MØ
T – T cellFDC – follicular dendritic cellMØ – macrophageCC - centrocyteCB - centroblast
CC
CCCC
CCCC
MØ
CB
Proliferation, somatic hypermutation
Selection, proliferation, differentiation
CB
CBCB CB
Germinal Center Organization
B
High Affinity
Low Affinity
CC
mantle zone
GC light zone (CD23++ FDCand centrocytes)
GC dark zone
T zone
(Ki67 cell cycle antigen+ centroblasts)
(CD23+ naive B cells)
Germinal Center
from Liu et al., Immunology Today 13, 17-21 (1992)
Cell morphology and migration dynamics
Affinity maturation is T-dependent
• T cells are enriched in the light zone
• Represent ~10% of GC cells
LZ
DZ
CD3ε
IgD
Most GC T Cells Are Not in Stable B-T Conjugates
Selection Model
• GC B cells that have improved affinity capture more antigen in a given amount of time, receiving a stronger BCR signal and presenting more MHC-peptide complexes to GC T cells, outcompeting surrounding B cells for T cell help
Summary of GC
Dynamics
Memory B cells
• Generated during the primary response– best characterized for T-dependent responses involving GC but some
evidence exists for memory to TI antigens
• Small, recirculating cells• Typically isotype switched (e.g. IgG+ or IgA+)• Typically have higher affinity for the inducing Ag• Longer lived than naïve B cells
– Persistence of memory B cells after an immune response ensures that we have increased numbers of B cells specific for the antigen and ready to respond on re-encounter
• May have intrinsic differences that promote greater clonal expansion and more rapid differentiation to plasma cells- differences in cytoplasmic domains of IgG vs IgM/D
- upregulation of TLRs
Down-regulation of B cell response by preformed IgGAntigen-IgG complexes cross-link BCR and FcRII - inhibit signaling
syk
lyn
IgM
FcRII
Y
calcium and lipid dependent signaling pathways
antigen
ITIM
SHIP
IgG
• The poly-Ig receptor is a special Fc receptor that selectively binds dimeric IgA• The process of transporting IgA across the cell is known as transcytosis• The IgA released into the gut lumen remains associated with part of the poly-Ig receptor (known as the secretory component) and this provides protection against proteolysis by gut proteases
Neonatal Immunity• Maternal IgG is transported by the neonatal Fc
receptor (FcRn)– across the placenta to the fetus– from colostrum across the gut epithelium
• Confers passive immunity in the newborn• The duration of protection is 3-4 months (or ~5 IgG
half-lives)– explains the high incidence of disease after this period by
bacteria such as Haemophilus influenzae
• Human milk contains IgA – provides some protection against gut pathogens
• Neonatal protection is only as good as the titer of IgG (and IgA) in the mother against the specific organism
IgG half-life• FcRn is also present in the adult and involved in protecting IgG
from degradation• Accounts for the long (3 week) half-life of IgG compared to other
Ig isotypes• Therapeutic agents that are fused to IgG Fc regions take
advantage of this property e.g. Enbrel (TNFR-Fc)
Features of primary and secondary antibody responses
Recommended ReadingPrimary papers:• Lakshmi Srinivasan…Klaus Rajewsky. (2009) PI3 Kinase Signals BCR-Dependent Mature B Cell
Survival. Cell 139, 573• Allen, C.D., Okada, T., Tang, H.L. and Cyster, J.G. (2007) Imaging germinal center selection
events during affinity maturation. Science 315: 528-31.• Schwickert T.A., … Nussenzweig, M.C. (2007) In vivo imaging of germinal centers reveals a
dynamic open structure. Nature 446: 83-7.• Horikawa, K.,…Goodnow, C.C. (2007) Enhancement and suppression of signaling by the
conserved tail of IgG memory-type B cell antigen receptors. 204: 759-69.• Gavin A.L., … Nemazee D. (2006) Adjuvant-enhanced antibody responses in the absence of toll-
like receptor signaling. Science 314: 1936-8.• Shaffer et al. (2004) XBP1, downstream of Blimp-1, expands the secretory apparatus and other
organelles, and increases protein synthesis in plasma cell differentiation. Immunity. 21:81-93
Reviews:
• Kurosaki T, Shinohara H, Baba Y.B Cell Signaling and Fate Decision. Annu Rev Immunol. 2009• Gupta, N. and Defranco, A.L. (2007) Lipid rafts and B cell signaling. Semin Cell Dev Biol 18, 616• Allen, C.D., Okada, T. and Cyster, J.G (2007) Germinal-center organization and cellular
dynamics.Immunity. 27:190-202• Shapiro-Shelef, M. and Calame, K. (2005) Regulation of plasma-cell development. Nat Rev
Immunol 5: 230-42.• McHeyzer-Williams L.J. and McHeyzer-Williams, M. (2005) Antigen-specific memory B cell
development. Annu Rev Immunol 23:487-513• Mackay, F., Schneider,P., Rennert,P. and Browning, J. (2003) BAFF and APRIL: A Tutorial on B
Cell Survival. Annu. Rev. Immunol.; 10.114 .