b 细胞的活化. lecture outline 1. what is meant by ‘ b cell activation ’ ?1. what is meant...
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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!
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