Download B cell development & function PPT

B Cells and B Cell Development
© Dr. Colin R.A. Hewitt
[email protected]
The discovery of B cell immunity
1954 - Bruce Glick, Ohio State University
Studies on the function of the bursa of Fabricius, a lymphoid organ in
the cloacal region of the chicken
Bursectomy – no apparent effect
Bursectomised chickens
were later used in
experiments to raise
antibodies to Salmonella
antigens
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
Origin of B cells and organ of B cell
maturation
Transfer marked foetal
liver cells
Normal bone marrow
Mature marked
B cells
in periphery
No Mature
B cells
Defective bone marrow
B cell development starts in the foetal liver
After birth, development continues in the bone marrow
B cell development in the bone marrow
B
Regulates construction of an antigen receptor
B
Ensures each cell has only one specificity
B
Checks and disposes of self-reactive B cells
B
Exports useful cells to the periphery
B
Provides a site for antibody production
Bone Marrow provides a
MATURATION & DIFFERENTIATION MICROENVIRONMENT
for B cell development
Bone Marrow
S
M
M
E
Scheme of B Cell Development in the Bone Marrow
Progenitors
E
n
d
o
o
s
t
e
u
m
Stromal cells
Pre-B
X
X
X
Macrophage
Immature &
mature B
Central
Sinus
Bone marrow stromal cells nurture
developing B cells
1. Specific cell-cell contacts between stromal cells and developing B cells
2. Secretion of cytokines by stromal cells
Cell-cell contact
B
Secreted
Factors - CYTOKINES
Stromal cell
Types of cytokines and cell-cell contacts needed at
each stage of differentiation are different
Maturing B cells
Bone marrow stromal cell
B
B
Stromal cell
Stages of B cell development
Stem Cell
Early pro-B cell
Late pro-B cell
Large pre-B cell
Peripheral
Small pre-B cell
Immature B cell
Mature B cell
Each stage of development is defined by rearrangements of IgH
chain genes, IgL chain genes, expression of surface Ig,
expression of adhesion molecules and cytokine receptors
Cytokines and cell-cell contacts at each
stage of differentiation are different
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
Cytokines and cell-cell contacts at each
stage of differentiation are different
Interleukin-7
receptor
Interleukin-7
Growth factor
Early
pro-B
Late
pro-B
Stromal cell
Pre-B
Stages of differentiation in the bone marrow are
defined by Ig gene rearrangement
B CELL STAGE
Stem cell
Early pro-B
IgH GENE
CONFIGURATION
Germline
DH to JH
Late pro-B Large pre-B
VH to DHJH
VHDHJH
Pre-B cell
receptor
expressed
Ig light chain gene has not yet rearranged
Pre- B cell receptor
Heavy chain
VHDHJH
Light chain
VLJLCL
VpreB
CHm
l5
Iga & Igb signal
transduction
molecules
Transiently expressed when VHDHJH CHm is productively rearranged
VpreB/l5 - the surrogate light chain, is required for surface expression
Ligand for the pre-B cell receptor may be galectin 1, heparan sulphate, other
pre-BCR or something as yet unknown
Ligation of the pre-B cell receptor
1. Suppresses further H chain rearrangement
2. Triggers entry into cell cycle
Large
Pre-B
Unconfirmed ligand
of pre-B cell receptor
1. Ensures only one specificty of
Ab expressed per cell
Stromal cell
2. Expands only the pre-B
cells with in frame VHDHJH joins
ALLELIC EXCLUSION
Expression of a gene on one chromosome prevents expression of the
allele on the second chromosome
Evidence for allelic exclusion
ALLOTYPE- polymorphism in the C region of Ig – one
allotype inherited from each parent
Allotypes can be identified by staining B cell surface Ig with antibodies
B
a
(Refer back to Dreyer & Bennet hypothesis in Molecular Genetics
of Immunoglobulins lecture topic)
B
AND b
b
B
B
Y
Suppression of H chain rearrangement by
pre-B cell receptor prevents expression of two
specificities of antibody per cell
Y
b
Y
B
Y
a
a/b
b/b
Y
Y
a/a
a
Allelic exclusion prevents unwanted responses
One Ag receptor per cell
IF there were two Ag receptors per cell
Y
YY
S. aureus
Y
Y
Y
Y
Anti
S. aureus
Antibodies
B
S. aureus
Anti
brain
Abs
Y
Y
Y
Y
Self antigen
expressed by
e.g. brain cells
YY
B
Anti
S. aureus
Antibodies
Suppression of H chain gene rearrangement
ensures only one specificity of Ab expressed per cell.
Prevents induction of unwanted responses by pathogens
Allelic exclusion is needed for efficient clonal selection
Antibody
S. typhi
S. typhi
All daughter cells must express the same Ig specificity
otherwise the efficiency of the response would be compromised
Suppression of H chain gene rearrangement helps prevent the emergence of
new daughter specificities during proliferation after clonal selection
Allelic exclusion is needed to prevent holes in the repertoire
One specificity of Ag
receptor per cell
IF there were two specificities
of Ag receptor per cell
Anti-brain Ig
Anti-brain Ig
AND
anti-S. aureus Ig
B
B
Exclusion of anti-brain B cells
i.e. self tolerance
B
Deletion
OR
B
BUT anti S.aureus B cells
will be excluded
leaving a “hole in the
repertoire”
Anergy
B
S. aureus
Ligation of the pre-B cell receptor
1. Suppresses further H chain rearrangement
2. Triggers entry into cell cycle
Large
Pre-B
Unconfirmed ligand
of pre-B cell receptor
1. Ensures only one specificity of
Ab expressed per cell
Stromal cell
2. Expands only the pre-B
cells with in frame VHDHJH joins
Large pre-B cells need in frame VHDHJH joins to mature
Human IgG3 Heavy Chain
nucleotide sequence
Translation in frame 1
ATGAAACANCTGTGGTTCTTCCTTCTCCTGG
TGGCAGCTCCCAGATGGGTCCTGTCCCAGG
TGCACCTGCAGGAGTCGGGCCCAGGACTGG
GGAAGCCTCCAGAGCTCAAAACCCCACTTGG
TGACACAACTCACACATGCCCACGGTGCCCA
GAGCCCAAATCTTGTGACACACCTCCCCCGT
GCCCACGGTGCCCAGAGCCCAAATCTTGTG
ACACACCTCCCCCATGCCCACGGTGCCCAG
AGCCCAAATCTTGTGACACACCTCCCCCGTG
CCCNNNGTGCCCAGCACCTGAACTCTTGGG
AGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGATACCCTTATGATTTCCCGGACCC
CTGAGGTCACGTGCGTGGTGGTGGACGTGA
GCCACGAAGACCCNNNNGTCCAGTTCAAGT
GGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCTGCGGGAGGAGCAGTACA
ACAGCACGTTCCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAACGGCAA
GGAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGACAGCCCGAGGAGATGACCA
AGAACCAAGTCAGCCTGACCTGCCTGGTCAA
AGGCTTCTACCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAACACCACGCCTCCCATGCTGGACTCC
GACGGCTCCTTCTTCCTCTACAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACATCTTCTCATGCTCCGTGATGCATGAGGC
TCTGCACAACCGCTACACGCAGAAGAGCCTC
TCCCTGTCTCCGGGTAAATGA
MKXLWFFLLLVAAPRWVLSQV
HLQESGPGLGKPPELKTPLGD
TTHTCPRCPEPKSCDTPPPCP
RCPEPKSCDTPPPCPRCPEPK
SCDTPPPCXXCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDXXVQFKWYVDG
VEVHNAKTKLREEQYNSTFRV
VSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPEE
MTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYNTTPPMLD
SDGSFFLYSKLTVDKSRWQQG
NIFSCSVMHEALHNRYTQKSLS
LSPGK*
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
Ligation of the pre-B cell receptor triggers
entry into the cell cycle
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
Many large pre-B
cells with identical
pre-B receptors
IgM
Intracellular VDJCH chain
VL-JL rearranges
Y
Large
pre-B
Large
Pre-B
Large
Large
Pre-B
Large
Pre-B
Large
Pre-B
Pre-B
Immature
B cell
Light chain expressed
IgM displayed on surface
Heavy and light chain rearrangement is potentially wasteful
V
D
J
C
Germline
V
D D J
C
DH-JH joining
C
VH-DHJH joining
V
V
D J
Large
pre-B
With two “random” joins to generate a heavy chain
there is a 1:9 chance of a rearrangement of being in frame
V
J
C
Germline
V V
J
C
VL-JL joining
With one “random” join to generate a light chain
there is a 1:3 chance of a rearrangement being of frame
Small
pre-B
There is, therefore, only a 1:27 chance of an in frame rearrangement
Out of frame rearrangements arrest further B cell maturation
B cells have several chances to successfully
rearrange Ig genes
Early Pro B
DH-JH
On first
chromosome
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
k on first
YES
chromosome
Y
B
YES
l on first
chromosome
IgMl
k on second
chromosome
NO
NO
NO
NO
B
IgMk
YES
NO
YES
Immature B
l on second
chromosome
NO
YES
Y
B
Acquisition of antigen specificity creates a
need
to check for recognition of self antigens
Y
Small pre-B cell
B
Y
B
Y
Y
Immature B cell
No antigen receptor at cell surface
Cell surface Ig expressed
Unable to sense Ag environment
Able to sense Ag environment
!!May be self-reactive!!
Can now be checked for self-reactivity
1. Physical removal from the repertoire
2. Paralysis of function
3. Alteration of specificity
DELETION
ANERGY
RECEPTOR EDITING
B cell self tolerance: clonal deletion
Small
pre-B
B
B
Immature
B
B
YY
Small pre-B cell
assembles Ig
Immature
B cell recognises
MULTIVALENT
self Ag
Clonal deletion by
apoptosis
B cell self tolerance: anergy
IgD normal IgM low
Small
pre-B
B
YY
Immature
B
B
IgM
IgD
B
IgD
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
V
V
B
V
D J
C
!!Receptor
recognises
self antigen!!
Arrest development
And reactivate
RAG-1 and RAG-2
V
B
V
V
B
D J
Apoptosis
or anergy
C
Edited receptor now recognises
a different antigen and can be
rechecked for specificity
B cell self tolerance: export of self tolerant B cells
Small
pre-B
B
YY
Immature
B
B
IgD and IgM normal
IgD
IgM
IgM
IgD
B
IgD
IgM
IgM
IgD
Small pre-B cell
assembles Ig
Immature
B cell doesn’t
recognise any
self Ag
Mature B cell
exported to the
periphery
How can B cells express
IgM and IgD simultaneously?
Cm
Cd
Cg3
Cg1
Ca1
Cg2
Cg4
Cd
Ce
Cd
Ca2
Sg3
Cg3
Cg3
Cm
Sg1
Cm
Cg1
VDJ
Cg3
VDJ
Ca1
VDJ
Ca1
VDJ
Cg3
VDJ
Ca1
VDJ
Ca1
IgG3 produced.
Switch from IgM
IgA1 produced.
Switch from IgG3
IgA1 produced.
Switch from IgM
N.B. Remember Molecular Genetics of Immunoglobulins lecture – No Cd switch region
Consider similarities with mechanism allowing secreted and membrane Ig by the same cell
Splicing of IgM and IgD RNA
VD J
VD J
Cm1
Cm2
Cm
Cm3
Cd
Cg3
Cd1
Cm4
Cg1
Cd2
DNA
Cd3
pA2
pA1
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
Cm1
Cm2
Cm3
Cm4
AAA Cd1
V D J Cm
Cd2
Cd3
RNA cleaved and
polyadenylated at pA1
IgM mRNA
RNA cleaved and
polyadenylated at pA2
VD J
Cm1
Cm2
Cm3
Cm4
Cd1
Cd2
Cd3
pA1
V D J Cd
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?
What are the external signals that activate B cells?
Fab anti-mIg
mIg
(Fab)2 anti-mIg
Anti-(Fab)2
Although Fab fragments bind to
membrane Ig (mIg), no signal is
transduced through the B cell
membrane
Bridging (or ‘Cross-linking’) of
different mIg allows the B cell
receptor to transduce a weak signal
through the B cell membrane
Extensive cross linking of (Fab)2
bound to mIg using an anti-(Fab)2
antibody enhances the signal
through the B cell membrane
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
Transduction of signals by the B cell receptor
Extracellular antigen
recognition domains
Igb Iga
The cytoplasmic domains of the Iga and
Igb contain Immunoreceptor Tyrosine based Activation Motifs (ITAMS) - 2
tyrosine residues separated by 9-12
amino acids - YXX[L/V]X6-9YXX[L/V]
Intracytoplasmic
signalling domains
Phosphorylation by Src kinases
Kinase domain
Enzyme domain
phosphorylates tyrosines
(to give phosphotyrosine)
SH2 domain SH3 domain Unique region
Phosphotyrosine
receptor domain
Adaptor
protein
recruitment
domain
ITAM
binding
domain
• Phosphorylation changes the properties of a protein by changing its conformation
• Changes in conformation may activate or inhibit a biochemical activity or create a
binding site for other proteins
• Phosphorylation is rapid, requires no protein synthesis or degradation to change
the biochemical activity of a target protein
• It is reversible via the action of phosphatases that remove phosphate
Regulation of Src kinases
Kinase domain
Inhibitory tyrosine residue
SH2 domain SH3 domain Unique region
Activating tyrosine residue
Phosphorylation of ‘Activating Tyrosine’ stimulates kinase activity
Kinase domain
SH2 domain SH3 domain Unique region
Phosphorylation of ‘Inhibitory Tyrosine’ inhibits kinase activity
by blocking access to the Activating Tyrosine Residue
Regulation of Src kinases by Csk and CD45
C terminus
Kinase domain
SH2 domain
SH3 domain Unique region
Activated cells: a
phosphatase associated
with the Leukocyte
Common Antigen - CD45,
removes the C terminus
phosphate allowing the
activating tyrosine to be
phosphorylated
Resting cells: Src
kinase is inactivated
by a constitutively
expressed C terminal Src kinase (Csk)
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
Phosphorylation of ITAMs by Src kinases
ITAM
ITAM
ITAM
P
ITAM
P
ITAM
P
P
P
1. Csk inactived
Src interacts
with low affinity
with the ITAMs
of ‘resting’
receptors
2. Antigen clusters B cell receptors with
CD45 phosphatases.
Src kinases are phosphorylated
and are activated to phosphorylate
ITAMS
3. Src kinases bind to
phosphorylated ITAMS
and are activated to
phosphorylate adjacent
ITAMS
Syk protein Tyrosine kinases
• CD45 phosphatase allows activation of Src family kinases Blk, Fyn & Lyn
• Receptor cross-linking activates Src kinases that phosphorylate ITAMs in
the Iga and Igb
ITAM
P
P
Syk - 2 x SH2
domains spaced to
bind to two
phosphotyrosines on
an ITAM
ITAM
P
P
P
P
P
ITAM
P
One Syk binds to Iga, one to Igb - each Syk
transphosphorylates the other
The B cell co-receptor
CD21
(C3d receptor)
CD45
CD19
Igb Iga
CD81
(TAPA-1)
The B cell co-receptor
Co-receptor phosphorylation
C3d opsonised bacterium
C3d binds to
CD21, the
complement
receptor 2
(CR2)
Antigen
recognition
P P
P
P
Src family kinases
then bind the
phosphorylated
CD19
• 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
Activation of signals that affect gene transcription
BLNK
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
P
P
P
P
P
ITAM
P
Activated Syk phosphorylates BLNK
Activated Syk phosphorylates
Guanine-nucleotide exchange factors
(GEFS) that in turn activate small GTP
binding proteins Ras and Rac
Tec kinases activate
phospholipase C- g (PLC-g)
PLC-g cleaves
phosphotidylinositol
bisphosphate (PIP2) to yield
diacylglycerol (DAG) and
inositol trisphosphate (IP3)
Ras and Rac activate the MAP
kinase cascade
Transmission of signals from the cell
surface to the nucleus
• B cell-specific parts of the signalling cascade are associated with receptors
unique to B cells - mIg, CD19 etc.
• Subsequent signals that transmit signals to the nucleus are common to
many different types of cell.
• The ultimate goal is to activate the transcription of genes, the products of
which mediate host defence, proliferation, differentiation etc.
Once the B cell-specific parts of the cascade are complete, signalling to
the nucleus continues via three common signalling pathways via:
1.The mitogen-activated protein kinase (MAP kinase) pathway
2. Increased in intracellular Ca2+ mediated by IP3
3.The activation of Protein Kinase C mediated by DAG
Simplified scheme linking antigen recognition
with transcription of B cell-specific genes
• MAP Kinase cascade
Small G-protein-activated MAP kinases found in all multicellular animals activation of MAP kinases ultimately leads to phosphorylation of transcription
factors from the AP-1 family such as Fos and Jun.
• Increases in intracellular calcium via IP3
IP3, produced by PLC-g, binds to calcium channels in the ER and releases
intracellular stores of Ca++ into the cytosol. Increased intracellular [Ca++]
activate a phospatase, calcineurin, which in turn activates the transcription
factor NFAT.
• Activation of Protein Kinase C family members via DAG
DAG stays associated with the membrane and recruits protein kinase C
family members. The PKC, serine/threonine protein kinases, ultimately
activate the transcription factor NFkB
The activated transcription factors AP-1, NFAT and NFkB induce B cell
proliferation, differentiation and effector mechanisms
Differentiation in the periphery
B
B
YY
YY
Mature peripheral
B cell
B cell recognises
non-self antigen
in periphery
Y
Y
Y
Y
YY
B
Ig-secreting plasma cell
Plasma cells
Surface Surface High rate Growth Somatic Isotype
Ig
MHC II Ig secretion
hypermut’n switch
B
High
Yes
No
Yes
Yes
Yes
Low
No
Yes
No
No
No
Mature B cell
B
Plasma cell
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
What do mature B cells do once activated by an
antigen in the periphery?
Recirculating B cells normally pass through
lymphoid organs
T cell area
B cells in
blood
B cell
area
Efferent
lymph
Recirculating B cells are trapped by foreign
antigens in lymphoid organs
Antigen enters
node in afferent
lymphatic
YY
Y
B cells
proliferate
rapidly
B cells leave blood &
enter lymph node via
high endothelial venules
Y
YY
Y
Y
Y
GERMINAL CENTRE
Transient structure of
Intense proliferation
YY
Y
Germinal centre
releases B cells
that differentiate
into plasma cells
B cells (stained brown) in the Germinal Centre
P = Paracortex, Mn = Mantle zone SC = Subcapsular zone
Follicular Dendritic cells (stained blue)in the Germinal Centre
Retention of Antigens on Follicular Dendritic Cells
Radiolabelled antigen localises on the surface of Follicular Dendritic cells
and persists there, without internalisation, for very long periods
Maturation of Follicular Dendritic cells
Club-shaped tips of developing dendrites
Filiform dendrites
Bead formation on dendrites
Bead formation on dendrites
Association of antigen with FDC
Antigen enters the germinal centre
in the form of an immune complex with
C3b and antibodies attached
The Immune complexes bind to
Fc and complement receptors
on the FDC dendrites
Ig Fc receptor
Complement receptor 3
FDC surface
The filiform dendrites of FDC develop
beads coated with a thin layer of immune
complexes
Iccosome formation and release
DC veils
The veils of antigen-bearing dendritic
cell surround the beads and the layer of
immune complexes is thickened by
transfer from the dendritic cell.
These beads are then released and are
then called ICCOSOMES
Iccosomes (black coated particles)
bind to and are taken up by B cell
surface immunoglobulin
Uptake of Iccosomes/Antigen by B cells
Anti-
Y
Iccosomes bearing
different antigens
Surface Ig captures antigen
Cross-linking of antigen receptor activates B cell
Activated B cell expresses CD40
B cell
B
CD40
Fate of Antigens Internalised by B cells
B
1. Capture by antigen
specific Ig maximises
uptake of a single antigen
2. Binding and internalisation via
Ig induces expression
of CD40
3. Antigen enters exogenous antigen
processing pathway
4. Peptide fragments of antigen are loaded
onto MHC molecules intracellularly.
MHC/peptide complexes are
expressed at the cell surface
B
T cell help to B cells
Signal 2 - T cell help
B
Y
Signal 1
antigen & antigen
receptor
Th
Th
1. T cell antigen receptor
2. Co-receptor (CD4)
3.CD40 Ligand
T cell help - Signal 2
Signal 2
Cytokines
Th
B
IL-4
IL-5
IL-6
IFN-g
TGF-b
Y
Cytokines
Signal 1
B cells are inherently prone to die by apoptosis
Signal 1 & 2 upregulate Bcl-XL in the B cell and
Bcl-XL prevents apoptosis
Signal 1 & 2 thus allow the B cell to survive
T cells regulate the survival of B cells and thus
control the clonal selection of B cells
T cell help - Signal 2 activates hypermutation
Signal 2
Receipt of signal 2
by the B cell also
activates
hypermutation in the
CDR - encoding
parts of the Ig genes
Th
B
Signal 1
Y
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
Signal 2, and thus T cells,
regulate which B cells are
clonally selected.
Low affinity Ig takes up and
presents Ag to T cells
inefficiently.
Inefficient presentation to T
cells does not induce CD40.
Deleterious mutation
Beneficial mutation
Neutral mutation
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
With no signal 2 delivered
by CD40, low affinity B cells
die.
Lower affinity - Not clonally selected
Higher affinity - Clonally selected
Identical affinity - No influence on clonal selection
Only B cells with high affinity
Ig survive - This is affinity
maturation
Control of Affinity & Affinity Maturation
Five B cell antigen
receptors all specific
for
, but with
different affinities
due to somatic
hypermutation
of Ig genes in
the germinal centre
B
B
B
B
B
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
Germinal Centre Macrophages (stained brown)
Clean Up Apoptotic Cells
GC = Germinal Centre, TBM = Tingible Body Macrophages
Role of T cell cytokines in T cell help
Signal 2
Cytokines
IL-4
IL-5
IL-6
IFN-g
TGF-b
Th
B
Y
Signal 1
B
B B
B
B
B
B
PC
B
B
B
B
B
B
B
Proliferation & Differentiation
IgM
IgG3
IgG1
IgG2b IgG2a IgE
IgA
IL4
inhibits inhibits induces
inhibits induces
IL-5
augments
IFN-g inhibits induces inhibits
induces inhibits
TGF-b inhibits inhibits
induces
induces
Regulation of specificity - Cognate recognition
1. T cells can only help the B cells that present
antigen to them
2. B cells are best at presenting antigens that they
take up most efficiently
3. B cells are most efficient at taking up antigens that
their B cell antigen receptors bind to
4. T and B cells help each other to amplify immunity
specific for the same antigen
i.e. Regulates the Characteristics of Adaptive Immunity
Sharply focuses specificity - Pathogen specificity
Improves specificity & affinity - Better on 2nd exposure
Is specific antigen dependent - Learnt by experience
Seeds memory in T and B cell pools
Synaptic tethering of a B cell (red)
to a T cell (green)
T cell (in centre) surrounded by B cells with
cytoskeleton stained green
Recirculating B cells are trapped by foreign
antigens in lymphoid organs
Antigen enters
node in afferent
lymphatic
YY
Y
B cells
Rapidly
proliferate
in follicles
B cells leave blood &
enter lymph node via
high endothelial venules
Y
YY
Y
Y
Y
GERMINAL CENTRE
Transient structure of
Intense proliferation
YY
Y
Germinal centre
releases B cells
that differentiate
into plasma cells
B cells (90%) and T cells
(10%) migrate to form
a primary follicle
B
B
B
T
B
B
B
1. Antigen loaded dendritic cells
migrate from subcapsular
sinus to paracortical area
of the lymph node
Primary follicle
formation
3. T cells
proliferate
T
DC
B B
B T T
B T T T
B B
4. B cells migrate through
HEV - most pass through the B
paracortex and primary follicle.
Some interact with T cells and
proliferate to form a primary focus
T
T
HEV
2. T cells migrate
through HEV and
are trapped by
antigen on DC
T cell motility in the lymph node
Germinal Centre Microanatomy
2. B cells (centrocytes) upregulate
surface Ig, stop dividing and
receive costimulatory signals
from T cells and FDC
Light zone
4. Selected cells leave lymph
node as memory
cells or plasma cells
Primary Follicles become
secondary
follicles
B
Dark zone
when germinal centres develop
T
Follicular dendritic
cells select useful
B cells
3. Apoptosis of
self-reactive &
unselected cells
1. B cells (centroblasts) downregulate
surface Ig, proliferate, somatically
hypermutate their Ig genes.
AFFINITY MATURATION
Two B cell lineages
B cell precursor
Mature B cell
B
B
Plasma cell
PC
B2 B cells
CD5
?
B
Distinct B cell
precursor
B
Y
Y
Y
Y
Y
YY
Y
Y
Y
?
IgG
B1 B cells
‘Primitive’ B cells found in
pleura and peritoneum
IgM - no other isotypes
B-1 B Cells
IgM uses a distinctive & restricted range of V regions
CD5
B
Y
Y
Few non-template encoded (N) regions in the IgM
Y
Y
IgM
Recognises repeating epitope Ag such as
phospholipid phosphotidyl choline & polysaccharides
NATURAL ANTIBODY
NOT part of adaptive immune response:
No memory induced
Not more efficient on 2nd challenge
Present from birth
Can make Ig without T cell help
Comparison of B-1 and B-2 B cell properties
Property
N regions
V region repertoire
Location
Renewal
Spontaneous Ig production
Isotypes
Carbohydrate
specificity
Carbohydrate specificity
Proteinspecificity
specificity
Protein
Need T cell
cellhelp
help
Somatic hypermutation of Ig
Memory development
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
T Independent Antigens (TI-2)
TI-2 Antigen
Immature
B-2 Cell
YY
Mature
B-1
Y
Immature B cells that bind to
multivalent self Ag undergo
apoptosis
B-2 cell repertoire is purged
of cells recognising
multivalent antigens during
development in the bone
marrow
Y
Y
Y
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
T Independent Antigens (TI-1)
LPS binding
protein
Bacterial Lipopolysaccharides, (TI-1
antigens), bind to host LPS binding
protein in plasma
LPS
TLR 4
LPS/LPSBP is captured by CD14
on the B cell surface
Toll - like receptor 4 (TLR4) interacts
with the CD14/LPS/LPSBP complex
CD14
B Cell
Activation of B cell
T Independent Antigens (TI-1 e.g. LPS)
LPS complexes with CD14, LPSBP & TLR4
B
B
B
B
B
B
Y Y Y Y Y Y
Six different B cells will require 6 different antigens to activate them
At high dose TI-1 antigens (like LPS) will POLYCLONALLY ACTIVATE all of
the B cells irrespective fo their specificity.
TI-1 antigens are called MITOGENS
Y YY YY YY YY YY Y
YY YY YY YY YY YY
YY YY YY YY YY YY
T Dependent & Independent Antigens
Induce responses in babies
Induce responses in athymics
Prime T cells
Polyclonally activate B cells
Require repeating epitopes
T
Dependent
Antigens
TI-1
Antigens
TI-2
Antigens
Yes
No
Yes
No
No
Yes
Yes
No
Yes
No
No
Yes
No
No
Yes
Why are babies unresponsive to TI-2 antigens?
In adults:
TI-2 Antigen
Immature
B-2 Cell
YY
Mature
B-1
Y
Adult immature B cells that
bind to multivalent self Ag
undergo apoptosis
Y
Y
Y
IgM
Adult non-bone marrow derived B-1
cells are directly stimulated by
antigens containing multivalent
epitopes produce IgM WITHOUT T
cell help.
Why are babies unresponsive to TI-2 antigens?
In babies:
All B cells, B-1 & B-2, are immature
TI-2 Antigen
Immature
B-1 Cell
Immature B cells that bind to
multivalent self Ag undergo
apoptosis
YY
As with adult B cells, immature B cells that bind multivalent self Ag
undergo apoptosis
Hence babies do not respond to TI-2 antigens.
Babies are, therefore susceptible to pathogens with multivalent
antigens such as those on pneumococcus
T Dependent & Independent Antigens
Induces response in babies
Induces response in athymia
Primes T cells
Polyclonally activates B cells
Requires repeating epitopes
T
Dependent
Antigens
TI-1
Antigens
TI-2
Antigens
Yes
No
Yes
No
No
Yes
Yes
No
Yes
No
No
Yes
No
No
Yes
TD: Activate B-1 and B-2 B cells
TI-1: Activate B-1 and B-2 B cells
TI-2: Activate only B-1 B cells
Examples
TD: Diptheria toxin, influenza heamagglutinin, Mycobacterium tuberculosis
TI-1: Bacterial lipopolysaccharides, Brucella abortis
TI-2: Pneumococcal polysaccharides, Salmonella polymerised flagellin
Immune effector mechanisms against
extracellular pathogens & toxins
NEUTRALISATION
Toxin
Bacterium
`
Adhesion to
host cells blocked
Toxin release
blocked
Prevents
invasion
Prevents
toxicity
NEUTRALISING ANTIBODIES
`
Y
Effector mechanisms against
extracellular pathogens
OPSONISATION
Bacteria in extracellular space
+
Ab
OPSONISATION
Fc receptor
binding
Phagocytosis
Effector mechanisms against
extracellular pathogens
COMPLEMENT Activation
Bacteria in plasma
Lysis
+
Ab &
COMPLEMENT
Opsonisation
Complement &
Fc receptor
binding
Phagocytosis
Summary
• B cell tolerance of self is by clonal deletion or anergy of self-reactive cells
• Receptor editing increases the efficiency of B cell development
• Follicular dendritic cells acquire antigen and transfer it to B cells
• T cell help to B cells is via CD40L and cytokines
• CD40 expression indirectly leads to Ig affinity maturation
• Germinal centre microanatomy & function
• There are two lineages of B cells - B1 and B2 B cells
• The dependency of B cells upon T cells varies