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Culture Documents
None of the
bursectomised
chickens made
anti-Salmonella
antibodies
Bursa was later found to be the organ in which antibody producing cells
developed antibody producing cells were thereafter called B cells
Mammals do not have a bursa of Fabricius
Mature marked
B cells
in periphery
No Mature
B cells
Defective bone marrow
Bone Marrow
S
M
M
E
E
n
d
o
o
s
t
e
u
m
Stromal cells
Pre-B
X
X
X
Macrophage
Immature &
mature B
Central
Sinus
Cell-cell contact
Secreted
Factors - CYTOKINES
Stromal cell
Maturing B cells
B
B
Stromal cell
Stem Cell
Immature B cell
Mature B cell
VLA-4
Early
pro-B
Stem
(Integrin)
Receptor
Tyrosine
kinase
Stem cell
factor
VCAM-1
(Ig superfamily)
Cell adhesion
molecules
Kit
Cell-bound
growth
factor
Stromal cell
Interleukin-7
Growth factor
Early
pro-B
Late
pro-B
Stromal cell
Pre-B
B CELL STAGE
Stem cell
Early pro-B
IgH GENE
CONFIGURATION
Germline
DH to JH
VHDHJH
Pre-B cell
receptor
expressed
VpreB
C H
5
Ig & Ig signal
transduction
molecules
Unconfirmed ligand
of pre-B cell receptor
1. Ensures only one specificty of
Ab expressed per cell
Stromal cell
ALLELIC EXCLUSION
Expression of a gene on one chromosome prevents expression of the
allele on the second chromosome
AND b
a/b
b/b
a/a
Y
Y
YB
Y
YB
Self antigen
expressed by
e.g. brain cells
YY
Anti
brain
Abs
Y Y
Y Y
Y
Anti
S. aureus
Antibodies
Y
Y
S. aureus
Y Y
Y Y
Y
YY
S. aureus
Anti
S. aureus
Antibodies
Antibody
S. typhi
S. typhi
Anti-brain Ig
B
Deletion
OR
Anergy
Anti-brain Ig
AND
anti-S. aureus Ig
S. aureus
Unconfirmed ligand
of pre-B cell receptor
1. Ensures only one specificity of
Ab expressed per cell
Stromal cell
Translation in frame 1
ATGAAACANCTGTGGTTCTTCCTTCTCCTGG
TGGCAGCTCCCAGATGGGTCCTGTCCCAGG
TGCACCTGCAGGAGTCGGGCCCAGGACTGG
GGAAGCCTCCAGAGCTCAAAACCCCACTTGG
TGACACAACTCACACATGCCCACGGTGCCCA
GAGCCCAAATCTTGTGACACACCTCCCCCGT
GCCCACGGTGCCCAGAGCCCAAATCTTGTG
ACACACCTCCCCCATGCCCACGGTGCCCAG
AGCCCAAATCTTGTGACACACCTCCCCCGTG
CCCNNNGTGCCCAGCACCTGAACTCTTGGG
AGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGATACCCTTATGATTTCCCGGACCC
CTGAGGTCACGTGCGTGGTGGTGGACGTGA
GCCACGAAGACCCNNNNGTCCAGTTCAAGT
GGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCTGCGGGAGGAGCAGTACA
ACAGCACGTTCCGTGTGGTCAGCGTCCTCA
CCGTCCTGCACCAGGACTGGCTGAACGGCA
AGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCC
AAAGCCAAAGGACAGCCCGAGGAGATGACC
AAGAACCAAGTCAGCCTGACCTGCCTGGTCA
AAGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAACACCACGCCTCCCATGCTGGACTCC
GACGGCTCCTTCTTCCTCTACAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACATCTTCTCATGCTCCGTGATGCATGAGGC
TCTGCACAACCGCTACACGCAGAAGAGCCTC
TCCCTGTCTCCGGGTAAATGA
MKXLWFFLLLVAAPRWVLSQV
HLQESGPGLGKPPELKTPLGD
TTHTCPRCPEPKSCDTPPPCP
RCPEPKSCDTPPPCPRCPEPK
SCDTPPPCXXCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDXXVQFKWYVDG
VEVHNAKTKLREEQYNSTFRV
VSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPEE
MTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYNTTPPMLD
SDGSFFLYSKLTVDKSRWQQG
NIFSCSVMHEALHNRYTQKSL
SLSPGK*
Translation in frame 2
(no protein)
Development
continues
Large
pre-B
Pre-B cell
receptor can
be activated
Development
arrests
*
Translation in frame 3
ETXVVLPSPGGSSQMGPVPGA
PAGVGPRTGEASRAQNPTW*
Development
arrests
Proliferation
Large
Pre-B
Large
Large
Pre-B
Large
Pre-B
Large
Pre-B
Pre-B
Small
Large
pre-B
Proliferation
stops
Pre-receptor
not displayed
Large
pre-B
Large
Pre-B
Large
Large
Pre-B
Large
Pre-B
Large
Pre-B
Pre-B
IgM
Intracellular VDJCH chain
VL-JL rearranges
Immature
B cell
Germline
D D J
DH-JH joining
VH-DHJH joining
D J
Large
pre-B
Germline
V V
VL-JL joining
Small
pre-B
NO
DH-JH
On second
chromosome
Late Pro B
YES
YES
VH-DJH
On first
chromosome
NO
VH-DJH
On second
chromosome
Pre B
YES
on first
YES
chromosome
Y
B
YES
on first
chromosome
IgM
on second
chromosome
NO
NO
NO
NO
IgM
YES
NO
YES
Immature B
on second
chromosome
NO
YES
Y
B
Immature B cell
DELETION
ANERGY
RECEPTOR EDITING
Small
pre-B
B
Immature
B
YY
Small pre-B cell
assembles Ig
Immature
B cell recognises
MULTIVALENT
self Ag
Clonal deletion by
apoptosis
Immature
B
IgD
IgD
Small
pre-B
YY
IgM
IgD
B
Small pre-B cell
assembles Ig
Immature
B cell recognises
soluble self Ag
No cross-linking
Anergic B cell
Receptor editing
A rearrangement encoding a self specific receptor can be replaced
V
D J
!!Receptor
recognises
self antigen!!
Arrest development
And reactivate
RAG-1 and RAG-2
B
V
D J
Apoptosis
or anergy
YY
Immature
B
IgD
IgM
Immature
B cell doesnt
recognise any
self Ag
YY
B
YY
YY
YY
Small
pre-B
IgM
IgD
Mature B cell
exported to the
periphery
C3
C1
C1
C2
C4
C
C
C2
S3
C3
C3
S1
C1
VDJ
C3
VDJ
C1
VDJ
C1
VDJ
C3
VDJ
C1
VDJ
C1
IgG3 produced.
Switch from IgM
IgA1 produced.
Switch from IgG3
IgA1 produced.
Switch from IgM
VD J
C1
C2
C3
C3
C1
C4
C1
C2
C3
pA2
pA1
DNA
Two types of mRNA can be made simultaneously in the cell by differential usage of
alternative polyadenylation sites and splicing of the RNA
VD J
C1
C2
C3
C4
AAA C1
V D J C
C2
C3
IgM mRNA
RNA cleaved and
polyadenylated at pA2
VD J
C1
C2
C3
C4
C1
C2
C3
pA1
V D J C
IgD mRNA
AAA
Summary
B cells develop in the foetal liver and adult bone marrow
Stages of B cell differentiation are defined by Ig gene
rearrangement
Pre-B cell receptor ligation is essential for B cell development
Allelic exclusion is essential to the clonal nature of immunity
B cells have several opportunities to rearrange their antigen
receptors
IgM and IgD can be expressed simultaneously due to differential
RNA splicing
So far, mostly about B cells in the bone marrow - what about mature
peripheral B cells?
(Fab)2 anti-mIg
Anti-(Fab)2
Ring staining
Patching
Ig is evenly distributed
around the cell surface
Ig is aggregated in
uneven clumps as
a result of mild
cross-linking of Ig
Capping
Ig is collected at a pole of the cell
in a cap as a result of extensive
cross-linking of Ig
Ig Ig
Kinase domain
Enzyme domain
phosphorylates tyrosines
(to give phosphotyrosine)
Phosphotyrosine
receptor domain
Adaptor
protein
recruitment
domain
ITAM
binding
domain
Kinase domain
Kinase domain
SH2 domain
Activated cells: a
phosphatase associated
with the Leukocyte Common
Antigen - CD45, removes
the C terminus phosphate
allowing the activating
tyrosine to be
phosphorylated
Kinase domain
Kinase domain
SH2 domain
SH3 domain
Unique region
The balance between Csk and CD45 phosphatase activity sets the threshold for
initiating receptor signalling
ITAM
ITAM
ITAM
P
ITAM
ITAM
P
1. Csk inactived
Src interacts
with low affinity
with the ITAMs
of resting
receptors
ITAM
P
P
Syk - 2 x SH2
domains spaced to
bind to two
phosphotyrosines on
an ITAM
ITAM
ITAM
CD45
CD19
Ig Ig
CD81
(TAPA-1)
Co-receptor phosphorylation
C3d opsonised bacterium
C3d binds to
CD21, the
complement
receptor 2
(CR2)
Antigen
recognition
P P
mIg and CD21 are cross-linked by antigen that has activated complement
CD21 is phosphorylated and receptor-associated kinases phosphorylate CD19
Phosphorylated CD19 activates more Src family kinases
Ligation of the co-receptor increases B cell receptor signalling 1000 -10,000 fold
P P P P
Tec Tec Tec Tec
Cell membrane-associated B
cell Linker protein - BLNK contains many Tyrosine
residues
BLNK binds Tec kinases
ITAM
ITAM
PLC- cleaves
phosphotidylinositol
bisphosphate (PIP2) to yield
diacylglycerol (DAG) and
inositol trisphosphate (IP3)
Ras and Rac activate the MAP
kinase cascade
The activated transcription factors AP-1, NFAT and NFB induce B cell
proliferation, differentiation and effector mechanisms
YY
YY
Mature peripheral
B cell
YY
YY
B cell recognises
non-self antigen
in periphery
Y
Y
Y
YY
YY
YY
YY
YY
Plasma cells
Surface Surface High rate Growth Somatic Isotype
Ig
MHC II Ig secretion
hypermutn switch
High
Yes
No
Yes
Yes
Low
No
No
Yes
No
No
Mature B cell
B
Plasma cell
Yes
Summary
You should know:
Where B cells come from
What happens to B cells in the bone marrow
How B cell differentiation is linked with Ig gene rearrangement
The B cell developmental check points that ensure each cell
produces a single specificity of antibody that does not react with
self
How B cells transmit information from the shape and charge of an
antigen through the cell membrane to allow the expression of
genes in the nucleus
B cells in
blood
B cell
area
Efferent
lymph
Antigen enters
node in afferent
lymphatic
YY
Y
B cells
proliferate
rapidly
Y
Y
YY
YY
Y
YYY
Y
Y
GERMINAL CENTRE
Transient structure of
Intense proliferation
YY
Y
Germinal centre
releases B cells
that differentiate
into plasma cells
Filiform dendrites
Ig Fc receptor
Complement receptor 3
FDC surface
The filiform dendrites of FDC develop
beads coated with a thin layer of immune
complexes
DC veils
Anti-
Y
Y
Y
Iccosomes bearing
different antigens
B cell
B
CD40
1. Capture by antigen
specific Ig maximises
uptake of a single antigen
YYY
Signal 1
antigen & antigen
receptor
Th
Th
1. T cell antigen receptor
2. Co-receptor (CD4)
3.CD40 Ligand
Signal 2
Cytokines
Th
IL-4
IL-5
IL-6
IFN-
TGF-
Cytokines
YYY
Signal 1
Receipt of signal 2
by the B cell also
activates
hypermutation in the
CDR - encoding
parts of the Ig genes
Th
Signal 1
Day 6
Day 8
Day 12
Day 18
Clone 1
Clone 2
Clone 3
Clone 4
Clone 5
Clone 6
Clone 7
Clone 8
Clone 9
Clone 10
Deleterious mutation
Beneficial mutation
Neutral mutation
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
Only this cell, that has a high affinity for antigen can express CD40.
Only this cell can receive signal 2
Only this cell is rescued from apoptosis i.e. clonally selected
The cells with lower affinity receptors die of apoptosis by neglect
Cytokines
IL-4
IL-5
IL-6
IFN-
TGF-
Th
YYY
Signal 1
B
B B
B
B
B
B
B
B
B
PC
B
B
B
IgG3
IgG1
IgA
IL4
inhibits inhibits induces
inhibits induces
IL-5
augments
IFN- inhibits induces inhibits
induces inhibits
TGF- inhibits inhibits
induces
induces
Antigen enters
node in afferent
lymphatic
YY
Y
B cells
Rapidly
proliferate
in follicles
Y
Y
YY
YY
Y
YYY
Y
Y
GERMINAL CENTRE
Transient structure of
Intense proliferation
YY
Y
Germinal centre
releases B cells
that differentiate
into plasma cells
B
T
B
B
B
Primary follicle
formation
3. T cells
proliferate
B B
B T T
B T T T
B B
DC
T
T
HEV
2. T cells migrate
through HEV and
are trapped by
antigen on DC
Light zone
Follicular dendritic
cells select useful
B cells
3. Apoptosis of
self-reactive &
unselected cells
Mature B cell
Plasma cell
PC
B2 B cells
Y
Y
Y
YY
YYYY
?
CD5
Y
Y
Y
Y
Y
Y
Y
Y
Distinct B cell
precursor
IgG
B1 B cells
Primitive B cells found in
pleura and peritoneum
B-1 B Cells
IgM uses a distinctive & restricted range of V regions
CD5
Y
Y
Y
Y
Y
Y
Y
Y
IgM
B-1 cells
Few
Restricted
Peritoneum/pleura
Self renewal in situ
High
IgM
Yes
Yes
Rarely
Rarely
No
No
No
No
B-2 cells
Extensive
Diverse
Everywhere
Bone marrow
Low
IgM/G/A/D/E
Rarely
Yes
Yes
Yes
Yes
High
Yes
Specificity & requirement for T cell help suggests strikingly different types
of antigens are seen by B-1 and B-2 B cells
Y
Mature
B-1
Y
Y
Y
Y
Y
Y
Y
Y
YY
B-2 cell repertoire is purged
of cells recognising
multivalent antigens during
development in the bone
marrow
Y
Y
Y
Immature
B-2 Cell
TI-2 Antigen
IgM
Non-bone marrow derived B-1 cells
are directly stimulated by antigens
containing multivalent epitopes.
No T cells are necessary
Induces the expression of natural
antibodies specific for TI-2 antigens
CD14
B Cell
Activation of B cell
Y Y Y Y Y Y
Y YY YY YY YY YY Y
YY YY YY YY YY YY
YY YY YY YY YY YY
T
Dependent
Antigens
TI-1
Antigens
TI-2
Antigens
Yes
No
Yes
No
No
Yes
Yes
No
Yes
No
No
Yes
No
No
Yes
TI-2 Antigen
Y
Mature
B-1
Y
Y
Y
Y
Y
Y
Y
Y
YY
Adult immature B cells that
bind to multivalent self Ag
undergo apoptosis
Y
Y
Y
Immature
B-2 Cell
IgM
Adult non-bone marrow derived B-1
cells are directly stimulated by
antigens containing multivalent
epitopes produce IgM WITHOUT T
cell help.
YY
T
Dependent
Antigens
TI-1
Antigens
TI-2
Antigens
Yes
No
Yes
No
No
Yes
Yes
No
Yes
No
No
Yes
No
No
Yes
Examples
TD: Diptheria toxin, influenza heamagglutinin, Mycobacterium tuberculosis
TI-1: Bacterial lipopolysaccharides, Brucella abortis
TI-2: Pneumococcal polysaccharides, Salmonella polymerised flagellin
Bacterium
Toxin
Toxin release
blocked
Prevents
invasion
Prevents
toxicity
Adhesion to
host cells blocked
NEUTRALISING ANTIBODIES
+
Ab
OPSONISATION
Fc receptor
binding
Phagocytosis
Lysis
+
Ab &
COMPLEMENT
Opsonisation
Complement &
Fc receptor
binding
Phagocytosis
Summary