B 细胞的活化

Lecture Outline

• 1. What is meant by ‘B cell activation’? • 2. Why do some B cell responses require T cell he

lp and others not?• 3. What is ‘T cell help’?• 4. What are plasma cells?• 5. How do activated B cells undergo ‘affinity matu

ration’?• 6. What is the mechanism for B cell memory?

Antigen

Antibody 1

Antibody 2

Antibody 3

Antibody formats

VL

VH

CL

CH1

CH2

CH3

scFv

Fab

SS

Whole

Antibody

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)

Types of antigen• T-independent (TI) antigens - Type I

– induce division/differentiation independently of BCR (polyclonal mitogens)

• LPS, bacterial (CpG) DNA

• T-independent (TI) antigens - Type II– 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

T cell

mitogenic BcR signal

'activation' signalbut not mitogenic

mitogenesisdifferentiation

presentAg

T-independent (TI) T-cell dependent (TD)

Types of B cell Antigens

-> most pathogens contain both T-independent and T-dependent antigens

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

• many pathogens activate the complement cascade and become C3d coated– complement receptor (CR) crosslinking synergizes

with BCR signal

Antigen-C3d complexes cross-link BCR and CR2-CD19 complex - increase

sensitivity to antigen

syk

lyn

IgM

CR2/CD21

CD19TA

PA

1

antigen C3d

PI3-K Vav

Y

Y

HEL

HEL-C3d

[Ca

]2+

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

Current Paradigm :

B cell plasma cellsB cell

B cell plasma cells

DC

Emerging Model:

DC

B cell

T-Independent (type II) Responses

Multivalent Antigen

DCs support Ag-specific plasma cell differentation

PC-binding

Pla

sma

ce

ll m

ark

er

• 3 day co-cultures

-> DCs are a source of BAFF-> Costimulatory effect of DCs partly due to BAFF

Balazs et al., Immunity 17, 341 (2002)

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

T

Sugar SpecificB cell

Protein SpecificT cellAntigen internalization, proteolysis

-> presentation of peptides

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

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)

• Note: CD40 signaling also important in DC, Macrophage 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

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

– TGF and IL5 -> IgA

• Many other molecules involved in T-B interactions – e.g. ICAM1/LFA1, CD30/CD30L, CD27L/CD27,

OX40L/OX40, ...

Altered signaling in anergic B cells

syk

lyn

Activated - acutely exposed to cross-linking antigen

syk

lynSH2

SH2

SH2

SH3

Anergic - chronically exposed

to antigen

negative feedback

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

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Production of membrane vs secreted Ig

membrane Ig

CH tm cy

CH tmcyAAAA

CH

AAAA

secretory Ig

splice, use poly A site 2

1 2

no splice, use poly A site 1

CH tm cy1 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

Bblimp1

XBP1

Plasma cell differentiation

Plasma cell differentiation • Blimp1 represses PAX5 and genes associated with B cell identity,

induces increases in a subset of plasma cell genes (Syndecan, XBP1) and induces conversion of membrane to secretory Ig

• XBP1 is a component of the UPR (unfolded protein response) • Increased sIg production ‘stresses’ ER and promotes processing of

XBP1 mRNA into active transcript• XBP1 induces increases in

– Secretory pathway gene expression– Organelle biogenesis– Cell size and protein synthesis

Shaffer et al., Immunity. 21:81-93

B cell migration in the spleen during differentiation to plasma cells

1 day after antigen 5 days after antigen

F

T

RP

F

T

RP4 days

B cells Activated B cells B cells Plasma cells

SpleenLymph Node

J

venoussinsus

B cellT cell Plasmablast Plasma cell

blood

BLC

BLCBLC

+Ag

ELCSLC

SLC

SLC

ELC

SDF1

SDF1

SDF1

SDF1

SDF1T

B

RP

MZ

SDF1

SDF1

SDF1

SDF1

Bone Marrow

B

T

M

BLC

SLC

ELCELC

SLCSLC

ELC

SDF1

SDF1

SDF1SDF1

bloodlymph

Small Intestine

Colon

blood

IgA Plasmablast IgA Plasma cell

SDF1

TEC

K

TECK

MEC

MECblood

BLCBLC

BLC

+Ag

SLC

SDF1

Plasma Cell Migration Summary

SDF1 for splenic red-pulp, LN medullary cords, Bone Marrow TECK/CCR9 for small intestine (IgA) MEC/CCR10 for large intestine, stomach, salivary glands (IgA)

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

Ig Heavy chain class (isotype) switching

VDJ

55 kb

(cytokines, CD40L)T cell help

antigen

IgM+ naive B cell

IgG+ memory cell

IgGsecreting plasma cell

Ig Heavy chain class (isotype) switching

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

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 that receive T cell help are selected and may differentiate into long-lived plasma cells or memory B cells

Germinal Center

Dark Zone- activated B cells (centroblasts, CB) undergo somatic mutation of Ig V genes

FDC

FDC

FDC

CB

CC

CB

CB CBCB

CB CBCB

T

T T

TCC

CC

CC

CCCC

memory B cell

plasma cell

CBCB

CBMØ

BT

Light Zone- B cells (centrocytes, CC) compete to bind Ag and receive T cell help- selects for cells with higher affinity BCR

AID dependent mutator complex

DNA replicationerror

ATG ... GGC TAT GTT CAC CGT ...

V CH1

A ...GGC, CCT...

Met ... Gly Tyr Val His Arg ... ...Gly, Pro...

AID = Activation Induced Deaminase

Somatic mutation of Ig V region in GC B cell-> mutations are actively induced in the V-regions of the

antibody heavy and light chain genes

ATG ... GGC TAT GTT CAC CGT ...

Met ... Gly Tyr Val His Arg ...

A

Asp

...GGC, CCT...

...Gly, Pro...

V CH1

Somatic mutation of Ig V region in GC B cell

-> now encodes antibody molecule with slightly altered antigen binding site

-> sometimes, by chance, this site will have an improved ability to bind the inducing antigen (i.e. a higher affinity)

VH VL

CLCH1

CDR1 2 3

CH2

CH3

CDR1 2 3

Ag

Mutations are targeted to antigen binding region of antibody

CDR = complementarity determining region, also known as the hypervariable region (part of V domain that binds the antigen)

Ag

before

Affinity maturation improves the ‘fit’ of the antibody for the inducing antigen

after

Ag

- increasing the binding affinity

Affinity maturation and antibody responses

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)

Germinal Center

from Hutloff et al., Nature 397, 263-6 (1999)

B cell differentiation in the germinal centerB cell differentiation in the germinal center

centrocyte

FDC

mature B cell

helperT cell

bacteria

LIGHTZONE

-CLASS SWITCH RECOMBINATION

-SOMATIC HYPERMUTATION

DARK ZONE

Centroblast

apoptosis

anergic

MemoryB cell

Plasma B cell

Memory B cells

• Generated in germinal centers– therefore we only have humoral memory to T-

dependent 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

Antigen-IgG complexes cross-link BCR and FcRII - inhibit signaling

syk

lyn

IgM

FcRII

Y

calcium and lipid dependent signaling pathways

antigen

I T I M

SHIP

IgG

Features of primary and secondary antibody responses

Tumors of B Cells

Lymphoid progenitor

Tumor Equivalent normal cell Location

Chronic lymphocytic leukemia CD5 B-1 B cell Blood

Acute lymphoblastic leukemiaBone marrow

BloodPre-B cell leukemia Pre-B cell

Follicular center cell lymphomaBurkitt’s lymphoma

Mature B cellPeriphery

Waldenstrom’s macroglobulinemia B cellIgM secreting

Multiple myeloma Plasma cell secretingvarious Ig isotypes

Bone marrow

Related information:

Effects on Cells of Immune System

LymphocyteMacrophageMacrophage

Bcell T cell NK LAK

Proliferation,Differentiation,

Ig secretion and selection

Proliferation,Differentiation,

Cytokineproduction

Activation of cells of immune system Cytokine production

IL1 IL2 IL4

IL1 IL2 IL4 IL5 IL6 IFNγ

cytokines

Related information:

Recommended ReadingPrimary papers:• Jun, J.E. et al. (2003) Identifying the MAGUK protein Carma-1 as a

central regulator of humoral immune responses and atopy by genome-wide mouse mutagenesis. Immunity 18:751-62

• Lesley, R. et al. (2004) Reduced competitiveness of autoantigen-engaged B cells due to increased dependence on BAFF. Immunity. 2004 20:441-53

• 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. (2002) Regulation of B cell fates by BCR signaling

components. Curr Opin Immunol. 14(3):341-7.• Mackay, F., Schneider,P., Rennert,P. and Browning, J. (2002) BAFF and

APRIL: A Tutorial on B Cell Survival. Annu. Rev. Immunol.; 10.114 • Kurosaki, T. (1999) Genetic analysis of B cell antigen receptor signaling.

Annu. Rev. Immunol. 17: 555-592 • Healy, J.I., and C.C. Goodnow. 1998. Positive versus negative signaling

by lymphocyte antigen receptors. Annu Rev Immunol 16:645-670.• Ravetch, J.V. and Bolland, S. (2001) IgG Fc Receptors. Annu. Rev.

Immunol. 19, 275• MacLennan, I.C.M. 1994. Germinal Centers. Annu. Rev. Immunol.

12:117-139

• Thank you!