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 .