No Slide Title© Dr. Colin R.A. Hewitt
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
*
maturation
Transfer marked foetal
Bone Marrow provides a
B
B
B
B
*
Immature &
Bone marrow stromal cells nurture developing B cells
Types of cytokines and cell-cell contacts needed at each stage of differentiation are different
Secreted
B
Stromal cell
Cell-cell contact
*
Stages of B cell development
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
Peripheral
Early pro-B
Cytokines and cell-cell contacts at each stage of differentiation are different
Kit
Pre-B
Cytokines and cell-cell contacts at each stage of differentiation are different
Early pro-B
defined by Ig gene rearrangement
B CELL STAGE
Pre-B cell
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
Pre-
1. Ensures only one specificty of Ab expressed per cell
2. Triggers entry into cell cycle
ALLELIC EXCLUSION
Expression of a gene on one chromosome prevents expression of the allele on the second chromosome
1. Suppresses further H chain rearrangement
2. Expands only the pre-B cells with in frame VHDHJH joins
Large
Pre-B
*
Evidence for allelic exclusion
Allotypes can be identified by staining B cell surface Ig with antibodies
AND
ALLOTYPE- polymorphism in the C region of Ig – one allotype inherited from each parent
Suppression of H chain rearrangement by pre-B cell receptor prevents expression of two specificities of antibody per cell
(Refer back to Dreyer & Bennet hypothesis in Molecular Genetics of Immunoglobulins lecture topic)
a/a
b/b
a/b
Y
B
b
Y
B
a
B
b
Y
Y
Y
B
a
b
Y
B
a
ensures only one specificity of Ab expressed per cell.
Allelic exclusion prevents unwanted responses
One Ag receptor per cell
IF there were two Ag receptors per cell
Prevents induction of unwanted responses by pathogens
Y
Y
Y
Y
B
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
Antibody
Allelic exclusion is needed to prevent holes in the repertoire
Exclusion of anti-brain B cells i.e. self tolerance
Anti-brain Ig
Anti-brain Ig
anti S.aureus B cells will be excluded leaving a “hole in the repertoire”
BUT
Y
Y
B
B
B
B
Deletion
Anergy
OR
Y
Y
Y
B
B
1. Ensures only one specificity of Ab expressed per cell
2. Triggers entry into cell cycle
2. Expands only the pre-B cells with in frame VHDHJH joins
Large
Pre-B
*
ATGAAACANCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCACCTGCAGGAGTCGGGCCCAGGACTGGGGAAGCCTCCAGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCNNNGTGCCCAGCACCTGAACTCTTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCNNNNGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCGAGGAGATGACCAAGAACCAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
Large pre-B cells need in frame VHDHJH joins to mature
Translation in frame 2
Development
arrests
Development
arrests
*
Ligation of the pre-B cell receptor triggers entry into the cell cycle
Many large pre-B cells with identical pre-B receptors
Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B
Large
Pre-B
Proliferation
Y
Immature
With two “random” joins to generate a heavy chain
there is a 1:9 chance of a rearrangement of being in frame
With one “random” join to generate a light chain
there is a 1:3 chance of a rearrangement being of frame
There is, therefore, only a 1:27 chance of an in frame rearrangement
Out of frame rearrangements arrest further B cell maturation
V
D
J
C
D
J
V
C
D
J
D
J
V
C
V
J
C
V
C
J
Large
pre-B
Small
pre-B
rearrange Ig genes
Early Pro B
Late Pro B
Unable to sense Ag environment
!!May be self-reactive!!
Immature B cell
Can now be checked for self-reactivity
Acquisition of antigen specificity creates a need
to check for recognition of self antigens
Physical removal from the repertoire DELETION
Paralysis of function ANERGY
B
Y
Y
Y
Y
B
Immature
Y
Y
Y
A rearrangement encoding a self specific receptor can be replaced
V
V
V
V
V
V
V
D
J
V
C
Y
B
B
!!Receptor
recognises
rechecked for specificity
B cell self tolerance: export of self tolerant B cells
Immature
self Ag
IgM and IgD simultaneously?
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
Ca2
Ce
Cg4
Cg2
Ca1
Cg1
Cg3
Cd
Cm
Cm
Cd
Cg3
VDJ
Sg3
Cm
Cd
Cg3
VDJ
Cg1
Sg1
Ca1
Cg3
VDJ
Ca1
Cg3
VDJ
Splicing of IgM and IgD RNA
Two types of mRNA can be made simultaneously in the cell by differential usage of alternative polyadenylation sites and splicing of the RNA
V
D
J
Cm1
Cm2
Cm3
Cm4
Cd1
Cd2
Cd3
pA1
V
D
J
Cm1
Cm2
Cm3
Cm4
Cd1
Cd2
Cd3
AAA
V
D
J
Cm
AAA
*
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?
Summary
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Although Fab fragments bind to membrane Ig (mIg), no signal is transduced 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
What are the external signals that activate B cells?
mIg
Fab anti-mIg
(Fab)2 anti-mIg
Bridging (or ‘Cross-linking’) of different mIg allows the B cell receptor to transduce a weak signal through the B cell membrane
Anti-(Fab)2
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
Iga
Igb
Intracytoplasmic
signalling domains
Extracellular antigen
recognition domains
*
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
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 by Src kinases
by blocking access to the Activating Tyrosine Residue
Kinase domain
Unique region
SH3 domain
SH2 domain
Regulation of Src kinases by Csk and CD45
The balance between Csk and CD45 phosphatase activity sets the threshold for initiating receptor signalling
Kinase domain
Unique region
SH3 domain
SH2 domain
Kinase domain
Resting cells: Src kinase is inactivated by a constitutively expressed C -terminal Src kinase - (Csk)
Kinase domain
Unique region
SH3 domain
SH2 domain
C terminus
Activated cells: a phosphatase associated with the Leukocyte Common Antigen - CD45, removes the C terminus phosphate allowing the activating tyrosine to be phosphorylated
Phosphorylation of ITAMs by Src kinases
3. Src kinases bind to phosphorylated ITAMS and are activated to phosphorylate adjacent ITAMS
ITAM
P
ITAM
P
ITAM
P
ITAM
Src kinases are phosphorylated
ITAM
P
1. Csk inactived Src interacts with low affinity with the ITAMs of ‘resting’ receptors
P
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
One Syk binds to Iga, one to Igb - each Syk transphosphorylates the other
Syk - 2 x SH2 domains spaced to bind to two phosphotyrosines on an ITAM
ITAM
P
P
ITAM
P
P
ITAM
P
P
P
P
CD45
C3d opsonised bacterium
mIg and CD21 are cross-linked by antigen that has activated complement
P
P
Co-receptor phosphorylation
Phosphorylated CD19 activates more Src family kinases
Ligation of the co-receptor increases B cell receptor signalling 1000 -10,000 fold
Src family kinases then bind the phosphorylated CD19
Antigen
recognition
P
P
BLNK
Cell membrane-associated B cell Linker protein - BLNK - contains many Tyrosine residues
Activation of signals that affect gene transcription
Activated Syk phosphorylates BLNK
ITAM
P
P
ITAM
P
P
P
P
Guanine-nucleotide exchange factors (GEFS) that in turn activate small GTP binding proteins Ras and Rac
Ras and Rac activate the MAP
kinase cascade
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:
The mitogen-activated protein kinase (MAP kinase) pathway
Increased in intracellular Ca2+ mediated by IP3
The activation of Protein Kinase C mediated by DAG
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
Simplified scheme linking antigen recognition with transcription of B cell-specific genes
B cell recognises
Ig MHC II Ig secretion hypermut’n switch
High Yes No Yes Yes Yes
Low No Yes No No No
B
B
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?
Summary
lymphoid organs
*
Recirculating B cells are trapped by foreign antigens in lymphoid organs
Antigen enters
*
*
Radiolabelled antigen localises on the surface of Follicular Dendritic cells
and persists there, without internalisation, for very long periods
*
Filiform dendrites
Association of antigen with FDC
in the form of an immune complex with C3b and antibodies attached
Antigen enters the germinal centre
The Immune complexes bind to Fc and complement receptors on the FDC dendrites
Complement receptor 3
Ig Fc receptor
FDC surface
*
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
Iccosome formation and release
surface immunoglobulin
Surface Ig captures antigen
Activated B cell expresses CD40
CD40
Y
Y
Y
B
Ig induces expression
processing pathway
onto MHC molecules intracellularly.
Fate of Antigens Internalised by B cells
1. Capture by antigen
B
B
Signal 1
antigen & antigen
2. Co-receptor (CD4)
*
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
Cytokines
IL-4
IL-5
IL-6
IFN-g
TGF-b
Y
Y
Y
B
Th
T cell help - Signal 2 activates hypermutation
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.
With no signal 2 delivered by CD40, low affinity B cells die.
Only B cells with high affinity Ig survive - This is affinity maturation
Y
Y
Y
B
Th
Signal 2
Signal 1
Receipt of signal 2 by the B cell also activates hypermutation in the CDR - encoding parts of the Ig genes
Clone 1
Clone 2
Clone 3
Clone 4
Clone 5
Clone 6
Clone 7
Clone 8
Clone 9
Clone 10
Higher affinity - Clonally selected
Control of Affinity & Affinity Maturation
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
Five B cell antigen
Germinal Centre Macrophages (stained brown)
Clean Up Apoptotic Cells
Cytokines
IL-4
IL-5
IL-6
IFN-g
TGF-b
IL4 inhibits inhibits induces inhibits induces
IL-5 augments
TGF-b inhibits inhibits induces induces
Proliferation & Differentiation
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
*
to a T cell (green)
*
cytoskeleton stained green
*
Recirculating B cells are trapped by foreign antigens in lymphoid organs
Antigen enters
Primary follicle
migrate from subcapsular
paracortex and primary follicle.
(10%) migrate to form
*
surface Ig, proliferate, somatically hypermutate their Ig genes.
AFFINITY MATURATION
receive costimulatory signals
3. Apoptosis of
node as memory
CD5
B
NOT part of adaptive immune response:
No memory induced
Present from birth
NATURAL ANTIBODY
CD5
B
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
IgM
Property B-1 cells B-2 cells
N regions Few Extensive
Location Peritoneum/pleura Everywhere
Spontaneous Ig production High Low
Isotypes IgM IgM/G/A/D/E
Memory development No Yes
Specificity & requirement for T cell help suggests strikingly different types
of antigens are seen by B-1 and B-2 B cells
Yes Rarely
Rarely Yes
No Yes
Carbohydrate specificity
Protein specificity
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
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
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Mature
B-1
Y
Y
Immature
Bacterial Lipopolysaccharides, (TI-1 antigens), bind to host LPS binding protein in plasma
LPS/LPSBP is captured by CD14
on the B cell surface
TLR 4
with the CD14/LPS/LPSBP complex
Activation of B cell
T Independent Antigens (TI-1)
T Independent Antigens (TI-1 e.g. LPS)
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
LPS complexes with CD14, LPSBP & TLR4
B
B
B
B
B
B
Induce responses in athymics No Yes Yes
Prime T cells Yes No No
Polyclonally activate B cells No Yes No
Require repeating epitopes No No Yes
T Dependent & Independent Antigens
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 adults:
Adult immature B cells that bind to multivalent self Ag undergo apoptosis
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Mature
B-1
Y
Y
Immature
Why are babies unresponsive to TI-2 antigens?
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
In babies:
Y
Y
Immature
B-1 Cell
TI-2 Antigen
*
Primes T cells Yes No No
Polyclonally activates B cells No Yes No
Requires repeating epitopes No No Yes
T Dependent & Independent Antigens
TI-1: Bacterial lipopolysaccharides, Brucella abortis
TI-2: Pneumococcal polysaccharides, Salmonella polymerised flagellin
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
T
Dependent
Antigens
TI-1
Antigens
TI-2
Antigens
NEUTRALISATION
OPSONISATION
OPSONISATION
COMPLEMENT Activation
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
*