Affinity maturation in germinal center

1
Affinity maturation in germinal center
Germinal center
B cell foci
Proliferation Affinity maturation Development of
memory cells and long-lived plasma cells
B cell follicle
B cell
B cell foci
PNA germinal center
T cell
Day 12 after immunization
Medullary cord or Red pulp
Short-lived plasma cells Antibody secretion
PNA peanut agglutinin binds to germinal center B
cells
GC persists up to 3 weeks.
2
B cells undergo somatic hypermutation in germinal
center.
Naïve B cell
S?
S?1
S?2b
S?2a
S?
S?3
S?
?1
?2b
?3
?2a
?
?
?
?
IgH
B cell activation
5-GCTAACTTAGCTAGTTTTACGTC-3
5-GCTACCTTAACTAGTTTTATGTC-3
Mutations in V region
S?2a
S?
S?
Germinal center B cell

?2a
?2b
?
?
IgH
Mutation rate (1x103/bp per generation) is 106
fold higher than spontaneous mutation rate.
IgL loci also undergo somatic hypermutation.
Somatic hypermutation changes antigen binding
site.
Mutation in framework region may not
affect antigen binding
Mutation in CDRs change antigen binding affinity
3
AID induces somatic hypermutation.
DNA deamination model
V region
S?
S?1
S?2b
S?2a
S?3
S?
S?
?1
?2b
?3
?2a
?
?
?
?
5-GCTAACTTAGCTAGTTTTACGTC-3
Mutation hotspots WRCY (WA or T, RA or G, YC
or T)
3-CGATTGAATCGATCAAAATGCAG-5
AID
5-GCTAAUTTAGUTAGTTTTACGTC-3
3-CGATTGAATCGATCAAAATGCAG-5
Excision of U by uracil DNA glycosylase before
replication
Replication (top strand as template)
Xabasic site
5-GCTAAXTTAGXTAGTTTTACGTC-3
5-GCTAAUTTAGUTAGTTTTACGTC-3
3-CGATTGAATCGATCAAAATGCAG-5
3-CGATTAAATCAATCAAAATGCAG-5
Replication (top strand as template)
Replication (bottom strand as template)
Error-prone DNA polymerase
5-GCTAAXTTAGXTAGTTTTACGTC-3
NA,G,C,T
3-CGATTNAATCNATCAAAATGCAG-5
5-GCTAATTTAGTTAGTTTTACGTC-3
Replication (bottom strand as template)
3-CGATTAAATCAATCAAAATGCAG-5
5-GCTAANTTAGNTAGTTTTACGTC-3
Ncomplementary base against N
3-CGATTNAATCNATCAAAATGCAG-5
4
Germinal center is separated into dark zone and
light zone.
Light zone
Centrocytes
Stop prolifereation Surface expression of
Ig Selection for high affinity BCR
Secondary follicle (with germinal center)
Primary follicle
Apoptotic centrocyte
Follicular Dendritic cell (FDC)
TFH
Dark zone
Centroblasts
Rapid proliferation Somatic hypermutation Loss of
surface Ig expression No secretion of antibodies
Lymph node
Each germinal center is usually founded by 1-3 B
cells.
Mantle zone Resting B cells
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Selection for high affinity antigen binding site
Centrocytes with high affinity antigen binding
site interact with antigen on FDC. These cells
survive.
Light Zone
Centrocytes may move back into dark zone for
additional rounds of mutation and selection.
FDC
Fc?R
CR1 and CR2
Ag
Ag
Immune complex
C3d
Ag
Opsonized antigen
Complements facilitate affinity maturation
Centrocytes with low affinity antigen binding
site fail to interact with antigen on FDC. These
cells undergo apoptosis.
Dark zone
Somatic hypermutation generates antigen binding
sites with different affinity
6
Germinal center
Dark zone
Light zone
Mantle zone
Light zone
FDC
Immune complex
GC
Mantle zone
7
Primary antibody response against NP-KLH
Class switching to IgG1
Germinal center
Germinal center (PNA is germinal center B
cell marker
B cell foci
Short-lived plasma cells (Syndecan is plasma cell
marker)
plasma cell
8
No mutation in V region of D6-7 plasma cells
Few mutation in V region of D6-7 germinal
centerB cells
9
Mutation in V region of D12 germinal center B
cells
W33L mutation increases the affinity of antibody
toward NP.
(W33L)
10
Selection for mutations in CDRs
Unselected mutations are distributed in regions
within 1.5 kb downstream of the V reigon
promoter.
After selection for high affinity antibodies,
mutations are concentrated in CDRs.
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Long-lived plasma cells in bone
marrow Non-dividing
B cell memory
Long-term maintenance of high affinity
antibody in circulation
2nd lymphoid tissues
CXCL12
Blood circulation
CXCR4
CXCL12
Memory B cell
Plasmablast
Resting cells with high affinity BCR Do not
secrete antibodies. Re-activated by secondary
encounter with antigen Retain CCR7,
CXCR5 Circulate in blood and secondary lymphoid
tissues.
CXCR4
CCR7, CXCR5
Centrocytes with high affinity BCR for antigen
Germinal center
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ELISPOT assay
Determine the frequency of cells secreting a
cytokine or antibody.
Before activation
Frequency of T cells secreting IFN-g after
activation.
13
B cell memory after LCMV infection in mice
Persistent, high affinity antibody production
Immediate protection
Rapid response
High frequency of antigen-specific memory B and
T cells
ELISA
Memory B cells have undergone affinity maturation
and class switching
Efficient binding to antigen Diverse effector
functions
Assay for memory B cell
Spleen or bone marrow cells from immunized mice
ELISPOT
Stimulate with antigen
MBC also detected in lymph node and blood
Memory B cells proliferate and differentiate into
plasma cells
ELISPOT
Count the number of ASC by ELISPOT after 6 days
14
Predominance of class switching to IgA in Peyers
patches
Antibody levels (?g/ml)
IgM
IgG
IgA
8
166
4
Intestinal fluid
240-827
1
Colonic fluid
Trace amount
Distribution of plasma cells producing different
antibody classes
1 IgD
1 IgD
6 IgG
13 IgM
30 IgA
52 IgG
17 IgM
80 IgA
2.5 x 1010 plasma cells
Systemic
gt 6 x 1010 plasma cells
Mucosal
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TGF-? induces class switching to IgA.
Defective IgA production in response to
immunization in TGF-?R deficient mice
Lack of IgA B cells in Peyers patches of
TGF-?R deficient mice
WT
Deficient
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IgA plasma cells and memory B cells from Peyers
patches home to mucosal tissues.
Brown MAdCAM-1 on HEV of mucosal tissues
Yellow mucosal plasma cells in lamina propria
Induction site Peyers patch
IgA
?4?7
CCR9
GC
IgA
Effector sites
(TECK) CCL25
MAdCAM1
Plasma cells
Memory B cells
Intestine lamina propria
intestine
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B1 cell and Marginal zone B cell
B1 cells constitute 30-50 (1 x 106 cells) of B
cells in peritoneal and pleural cavities.
B1 cells represent 2 (1 x 106 cells) of B cells
in spleen, but rare in lymph nodes.
B-2/follicular B cells
Marginal zone B cells
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B1 cells are generated during fetal and neonatal
stage.
Fetal liver
Neonatal B cell development in spleen
Adult (2-4 weeks after birth) B cell development
in bone marrow
B-1a cell
Fetal/neonatal
B-1 precursor
Transitional B-1 cell
B1 cells are self-renewing in adult.
B-1b cell, marginal zone B cell
Adult
B-2 precursor
Transitional B-2 cell
B-2 cell
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Natural antibodies provide early protection
against infection.
Natural antibodies (IgM) are polyreactive against
many pathogen surface carbohydrates.
IgM against pathogens are detected in unimmunized
mice.
Serum from unimmunized mice protect from
infection.
OD405
2x dilution
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Thymus independent (TI) response
TI-I response
High concentration of LPS (B cell mitogen)
TLR4
TLR4
TLR4
TLR4
TLR4
BCR
BCR
BCR
BCR
BCR
Polyclonal activation of B cells
Class switching to IgG3, IgG2b No affinity
maturation or memory.
Low concentration of LPS
LPS
bacteria
TLR4
Antibodies specific for antigen
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TI-2 response
Repetitive bacterial surface antigens
(carbohydrates, lipids) crosslink BCR.
Antibodies against TI-2 antigens
IgM, IgG3 B1 cells produce IgA in intestine.
No affinity maturation
No memory
B1 cell, Marginal zone B cells
MZ B cells and B1 cells mediate early TI response
against infection.
MZ B cell
Cell number
Follicular B cells are activated by 48-72 hr.
Forward size scatter correlates with cell
size (activated B cells become larger)
Syndecan (plasma cell marker)
MZ B cells are activated faster than follicular B
cells in response to iv injection of LPS (TI-I)
22
B1 cells and MZ B cells mediate early TI-2
response against blood-borne bacteria (S.
pneumoniae).
B1 cells (T15) and MZ B cells (M167) develop into
plasma cells 3 days after iv immunization of S.
pneumoniae.
T15 and M167 represent BCRs specific for
phosphoryl choline on bacteria surface.
Both B1 and MZ B cells respond to blood
immunization (iv).
Peritoneal immunization (ip) preferentially
induces B1 cell.
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Questions
Which cell types contribute to B cell memory?
Where are these cells generated? Where do these
cells distribute?
How are somatic hypermutations generated? How do
somatic hypermutations improve antibody affinity?
What is the difference between ELISA and ELISPOT
assay?
What are the differences between mucosal and
systemic antibody production?
What are B1, B2 and MZ B cells?
Which cell type produce natural antibodies?
What are TI-1 and TI-2 responses? What are the
functions of TI responses?
Relevant part in textbook
p125-127, p292-297, p278-279.