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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
Transfer marked foetalliver cells
Origin of B cells and organ of B cellmaturation
No MatureB cells
After birth, development continues in the bone marrow
Normal bone marrow
Defective bone marrow
Mature markedB cells
in periphery
B cell development starts in the foetal liver
B cell development in the bone marrow
B Regulates construction of an antigen receptor
Bone Marrow provides aMATURATION & DIFFERENTIATION MICROENVIRONMENT
for B cell development
Ensures each cell has only one specificityB
Checks and disposes of self-reactive B cellsB
Exports useful cells to the peripheryB
Provides a site for antibody productionB
Bone Marrow
E
M
M
S
Immature &mature B
CentralSinus
Progenitors Pre-B
Stromal cells
X
X
X
Endoosteum
Macrophage
Scheme of B Cell Development in the Bone Marrow
SecretedFactors - CYTOKINES
2. Secretion of cytokines by stromal cells
B
Bone marrow stromal cells nurture developing B cells
Types of cytokines and cell-cell contacts needed at each stage of differentiation are different
Stromal cell
1. Specific cell-cell contacts between stromal cells and developing B cells
Cell-cell contact
Bone marrow stromal cell
Maturing B cells
B
B
Stromal cell
Peripheral
Stages of B cell development
Stem Cell Early pro-B cell Late pro-B cell Large pre-B cell
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
Early pro-B
KitReceptor Tyrosinekinase
Stem cell factor
Cell-bound growthfactor
VLA-4(Integrin)
Stem
Cytokines and cell-cell contacts at each stage of differentiation are different
Stromal cell
Cell adhesionmolecules
VCAM-1(Ig superfamily)
Early pro-B
Interleukin-7receptor
Stromal cell
Late pro-B Pre-B
Interleukin-7Growth factor
Cytokines and cell-cell contacts at each stage of differentiation are different
Stages of differentiation in the bone marrow aredefined by Ig gene rearrangement
B CELL STAGE
IgH GENECONFIGURATION
Stem cell Early pro-B Late pro-B Large pre-B
Germline DH to JH VH to DHJH VHDHJH
Pre-B cellreceptor
expressed
Ig light chain gene has not yet rearranged
B cell receptor
Transiently expressed when VHDHJH CH is productively rearranged
VpreB/5 - the surrogate light chain, is required for surface expression
Ig & Ig signaltransductionmolecules
CH
Heavy chainVHDHJH
Light chainVLJLCL
VpreB
5
Ligand for the pre-B cell receptor may be galectin 1, heparan sulphate, other pre-BCR or something as yet unknown
Pre-
Ligation of the pre-B cell receptor
1. Ensures only one specificty ofAb expressed per cell
LargePre-B
Stromal cell
Unconfirmed ligand of pre-B cell receptor
2. Triggers entry into cell cycle
ALLELIC EXCLUSIONExpression 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-Bcells with in frame VHDHJH joins
Evidence for allelic exclusion
Allotypes can be identified by staining B cell surface Ig with antibodies
a/a b/b a/bYBb YBa YBb
Y
YB ab
YBa 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)
YY
Y Y
Suppression of H chain gene rearrangementensures only one specificity of Ab expressed per cell.
Allelic exclusion prevents unwanted responses
BSelf antigenexpressed bye.g. brain cells
S. aureusY Y
YYYB
S. aureus
YY
Y
YY
Y
Y
AntiS. aureus
Antibodies
Y
Y Y
Y Y
YAntibrainAbs
One Ag receptor per cell IF there were two Ag receptors per cell
Y
Y Y
Y
YY Y
AntiS. aureus
Antibodies
Prevents induction of unwanted responses by pathogens
Allelic exclusion is needed for efficient clonal selection
All daughter cells must express the same Ig specificityotherwise the efficiency of the response would be compromised
Suppression of H chain gene rearrangement helps prevent the emergence ofnew daughter specificities during proliferation after clonal selection
S. typhi
Antibody
S. typhi
Allelic exclusion is needed to prevent holes in the repertoire
Exclusion of anti-brain B cells i.e. self tolerance
YYBB
One specificity of Agreceptor per cell
S. aureus
Anti-brain IgAND
anti-S. aureus IgYYYBB
IF there were two specificitiesof Ag receptor per cell
Anti-brain Ig
BB
Deletion Anergy
OR
anti S.aureus B cells will be excluded leaving a “hole in the repertoire”
BUT
YYYBB
1. Suppresses further H chain rearrangement
Ligation of the pre-B cell receptor
1. Ensures only one specificity ofAb expressed per cell
LargePre-B
Stromal cell
Unconfirmed ligand of pre-B cell receptor
2. Triggers entry into cell cycle
2. Expands only the pre-Bcells with in frame VHDHJH joins
Translation in frame 1
MKXLWFFLLLVAAPRWVLSQVHLQESGPGLGKPPELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCXXCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDXXVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRYTQKSLSLSPGK*
Human IgG3 Heavy Chain nucleotide sequence
ATGAAACANCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCACCTGCAGGAGTCGGGCCCAGGACTGGGGAAGCCTCCAGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCNNNGTGCCCAGCACCTGAACTCTTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCNNNNGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCGAGGAGATGACCAAGAACCAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTACACGCAGAAGAGCCTCTCC
CTGTCTCCGGGTAAATGA
Large pre-B cells need in frame VHDHJH joins to mature
Translation in frame 2(no protein)
*
Translation in frame 3
ETXVVLPSPGGSSQMGPVPGAPAGVGPRTGEASRAQNPTW*
Largepre-B
Development continues
Pre-B cell receptor can be activated
Developmentarrests
Developmentarrests
LargePre-B
LargePre-B
LargePre-B
LargePre-B Large
Pre-BLargePre-B Large
Pre-BLargePre-B Large
Pre-BLargePre-B
Proliferation
Y ImmatureB cell
Light chain expressedIgM displayed on surface
IgM
Ligation of the pre-B cell receptor triggers entry into the cell cycle
Largepre-B
Many large pre-B cells with identical pre-B receptors
Large pre-B
Intracellular VDJCH chainVL-JL rearranges
Proliferation stops
Pre-receptor not displayed
Small pre-B
V D J C
D JV CD J
V CD JV
Germline
DH-JH joining
VH-DHJH joining
V J C
V CJV
Germline
VL-JL joining
Heavy and light chain rearrangement is potentially wasteful
Largepre-B
Smallpre-B
With two “random” joins to generate a heavy chainthere is a 1:9 chance of a rearrangement of being in frame
With one “random” join to generate a light chainthere 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
B cells have several chances to successfullyrearrange Ig genes
Early Pro B Late Pro B Pre B
VH-DJH
On firstchromosome
VH-DJH
On secondchromosome
Immature B
NO
YES
NO
B
on firstchromosome
on secondchromosome
on firstchromosome
on secondchromosome
NO
NO
NO
NO
YES
YES
NO
YES
YES
YES
YES
YIgM
B
YIgM
B
DH-JH
On firstchromosome
DH-JH
On secondchromosome
YESNO
B Y
Y
Y
YB
Small pre-B cell
No antigen receptor at cell surfaceUnable to sense Ag environment
!!May be self-reactive!!
Immature B cell
Cell surface Ig expressedAble to sense Ag environment
Can now be checked for self-reactivity
Acquisition of antigen specificity creates a need
to check for recognition of self antigens
1. Physical removal from the repertoire DELETION2. Paralysis of function ANERGY3. Alteration of specificity RECEPTOR EDITING
B cell self tolerance: clonal deletion
Immature B cell recognisesMULTIVALENT
self Ag
B
Clonal deletion byapoptosis
YYBImmature
BB
Smallpre-B
Small pre-B cellassembles Ig
Y
B cell self tolerance: anergy
B
YY
YB
Anergic B cell
IgD normal IgM low
Immature B cell recognisessoluble self Ag
No cross-linking
YYB
ImmatureB
BSmallpre-B
Small pre-B cellassembles Ig
IgM
IgD
IgD
IgD
Receptor editingA rearrangement encoding a self specific receptor can be replaced
V CD JVV V
Y
BB!!Receptorrecognises
self antigen!!
B Apoptosisor anergy
YBB
Edited receptor now recognisesa different antigen and can be
rechecked for specificity
CD JVV VV
Arrest developmentAnd reactivate
RAG-1 and RAG-2
YY
Y
YYY
Mature B cellexported to the
periphery
Y
Y
B cell self tolerance: export of self tolerant B cells
IgD and IgM normal
IgMIgD
IgD
IgD
IgD
IgMIgM
IgM
Immature B cell doesn’t recognise any
self Ag
YYB
ImmatureB
BSmallpre-B
Small pre-B cellassembles Ig
B
How can B cells expressIgM and IgD simultaneously?
C2CC4C2C1C1C3CC
C
C
C3VDJ
S3
C
C
C3
VDJ
C1
S1
C1
C3
VDJ C1
C3VDJ
IgG3 produced.Switch from IgM
VDJ C1
IgA1 produced.Switch from IgG3
VDJ C1
IgA1 produced.Switch from IgM
N.B. Remember Molecular Genetics of Immunoglobulins lecture – No C switch regionConsider similarities with mechanism allowing secreted and membrane Ig by the same cell
Splicing of IgM and IgD RNA
C1 C2 C3 C4 C1 C2 C3
pA1V D J
C1 C2 C3 C4 C1 C2 C3V D J
AAA
RNA cleaved and polyadenylated at pA2
V D J C IgM mRNA
V D J C IgD mRNA
C1C3CCV D J
C1 C2 C3 C4 C1 C2 C3 DNApA1 pA2
V D J
Two types of mRNA can be made simultaneously in the cell by differential usage of alternative polyadenylation sites and splicing of the RNA
RNA cleaved and polyadenylated at pA1
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
Although Fab fragments bind to membrane Ig (mIg), no signal is transduced through the B cell membrane
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
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?
Ring stainingIg is evenly distributedaround the cell surface
PatchingIg is aggregated inuneven ‘clumps’ asa result of mildcross-linking of Ig
CappingIg 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
IgIg
Intracytoplasmicsignalling domains
Extracellular antigenrecognition domains
The cytoplasmic domains of the Ig and Ig contain Immunoreceptor Tyrosine -based Activation Motifs (ITAMS) - 2 tyrosine residues separated by 9-12 amino acids - YXX[L/V]X6-9YXX[L/V]
Kinase domain Unique regionSH3 domainSH2 domain
Enzyme domainphosphorylates tyrosines(to give phosphotyrosine)
Phosphotyrosinereceptor domain
Adaptor protein
recruitment domain
ITAM binding domain
• 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
Regulation of Src kinases
Kinase domain Unique regionSH3 domainSH2 domain
Activating tyrosine residueInhibitory tyrosine residue
Phosphorylation of ‘Activating Tyrosine’ stimulates kinase activity
Kinase domain Unique regionSH3 domainSH2 domain
Phosphorylation of ‘Inhibitory Tyrosine’ inhibits kinase activityby blocking access to the Activating Tyrosine Residue
Regulation of Src kinases by Csk and CD45
Kinase domain Unique regionSH3 domainSH2 domain
Kinase domain
Resting cells: Src kinase is inactivated by a constitutively expressed C -terminal Src kinase - (Csk)
Kinase domain Unique regionSH3 domainSH2 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
The balance between Csk and CD45 phosphatase activity sets the threshold for initiating receptor signalling
3. Src kinases bind to phosphorylated ITAMS and are activated to phosphorylate adjacent ITAMS
P
ITAM
ITAM P PITAM
ITAM
2. Antigen clusters B cell receptors with CD45 phosphatases.Src kinases are phosphorylated
Phosphorylation of ITAMs by Src kinases
1. Csk inactived Src interacts with low affinity with the ITAMs of ‘resting’ receptors
P
ITAM P
and are activated to phosphorylate ITAMS
One Syk binds to Ig, one to Ig - each Syk transphosphorylates the other
Syk - 2 x SH2 domains spaced to bind to two phosphotyrosines on an ITAM
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 Ig and Ig
ITAMP
PITAM
P
PITAM
P
P
P P
CD21(C3d receptor)
CD19
CD81(TAPA-1)
IgIg
CD45
The B cell co-receptor
The B cell co-receptor
Src family kinases then bind the phosphorylated CD19
Antigen recognition
C3d opsonised bacterium
• mIg and CD21 are cross-linked by antigen that has activated complement
C3d binds to CD21, the complement receptor 2 (CR2)
P P
• CD21 is phosphorylated and receptor-associated kinases phosphorylate CD19
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
BLNKCell membrane-associated B cell Linker protein - BLNK - contains many Tyrosine residues
Activated Syk phosphorylates BLNK
P P P P
BLNK binds Tec kinases
Tec Tec Tec Tec
Tec kinases activatephospholipase C- (PLC-)
PLC- cleaves phosphotidylinositol bisphosphate (PIP2) to yield diacylglycerol (DAG) and inositol trisphosphate (IP3)
ITAMP
PITAM
P
P
P P
Activated Syk phosphorylates Guanine-nucleotide exchange factors (GEFS) that in turn activate small GTP binding proteins Ras and Rac
Ras and Rac activate the MAPkinase cascade
Activation of signals that affect gene transcription
Transmission of signals from the cellsurface 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 tothe nucleus continues via three common signalling pathways via:
1.The mitogen-activated protein kinase (MAP kinase) pathway2. Increased in intracellular Ca2+ mediated by IP3
3.The activation of Protein Kinase C mediated by DAG
• MAP Kinase cascadeSmall 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-, 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 DAGDAG stays associated with the membrane and recruits protein kinase C family members. The PKC, serine/threonine protein kinases, ultimately activate the transcription factor NFB
The activated transcription factors AP-1, NFAT and NFB induce B cell proliferation, differentiation and effector mechanisms
Simplified scheme linking antigen recognition with transcription of B cell-specific genes
B cell recognisesnon-self antigen
in periphery
Ig-secreting plasma cell
Differentiation in the periphery
YY Y YY YYY Y
B
Y Y
Y
Y
YYY
BY Y
YY
YYYB
Mature peripheralB cell
Plasma cells
Surface Surface High rate Growth Somatic Isotype Ig MHC II Ig secretion hypermut’n switch
B
BMature B cell
Plasma cell
High Yes No Yes Yes Yes
Low No Yes No No No
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?
Summary
Recirculating B cells normally pass throughlymphoid organs
B cells inblood
Efferentlymph
T cell area
B cell area
Antigen entersnode in afferent
lymphatic
Y
Y
Y
Y
Y
YY
Y
Y
Y
Y
Y
Y
Y
YY
Y
YB cells leave blood & enter lymph node via
high endothelial venulesB cellsproliferate
rapidly
GERMINAL CENTRETransient structure ofIntense proliferation
Germinal centrereleases B cellsthat differentiateinto plasma cells
Recirculating B cells are trapped by foreign antigens in lymphoid organs
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 cellsand 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
in the form of an immune complex with C3b and antibodies attached
Antigen enters the germinal centre
Complement receptor 3Ig Fc receptor
FDC surface
The filiform dendrites of FDC develop beads coated with a thin layer of immune complexes
The Immune complexes bind to Fc and complement receptors on the FDC dendrites
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
DC veils
Iccosomes (black coated particles) bind to and are taken up by B cell
surface immunoglobulin
Y YY
Iccosomes bearing different antigens
B
Uptake of Iccosomes/Antigen by B cells
Anti- B cell
Surface Ig captures antigen
Cross-linking of antigen receptor activates B cell
Activated B cell expresses CD40
CD40
B
B
2. Binding and internalisation via Ig induces expressionof CD40
3. Antigen enters exogenous antigenprocessing pathway
4. Peptide fragments of antigen are loadedonto MHC molecules intracellularly.MHC/peptide complexes areexpressed at the cell surface
Fate of Antigens Internalised by B cells
1. Capture by antigenspecific Ig maximisesuptake of a single antigen
YYY
T cell help to B cells
B
Signal 1antigen & antigen
receptor
Th
1. T cell antigen receptor
2. Co-receptor (CD4)
3.CD40 Ligand
Th
Signal 2 - T cell help
T cell help - Signal 2
YYY
B Th
Signal 2
Signal 1
B cells are inherently prone to die by apoptosisSignal 1 & 2 upregulate Bcl-XL in the B cell andBcl-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
CytokinesIL-4IL-5IL-6
IFN-TGF-
T cell help - Signal 2 activates hypermutation
YYYB 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
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
Clone 1Clone 2Clone 3Clone 4Clone 5Clone 6Clone 7Clone 8Clone 9Clone 10
CD
R1
CD
R2
CD
R3
Day 6
CD
R1
CD
R2
CD
R3
CD
R1
CD
R2
CD
R3
CD
R1
CD
R2
CD
R3
Day 8 Day 12 Day 18
Deleterious mutationBeneficial mutationNeutral mutation
Lower affinity - Not clonally selectedHigher affinity - Clonally selectedIdentical affinity - No influence on clonal selection
Control of Affinity & Affinity Maturation
Five B cell antigenreceptors - all specificfor , but withdifferent affinitiesdue to somatichypermutationof 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
GC = Germinal Centre, TBM = Tingible Body Macrophages
Germinal Centre Macrophages (stained brown)Clean Up Apoptotic Cells
Role of T cell cytokines in T cell help
Th
Signal 2
YYYB
Signal 1
CytokinesIL-4IL-5IL-6
IFN-TGF-
IgM IgG3 IgG1 IgG2b IgG2a IgE IgA
IL4 inhibits inhibits induces inhibits inducesIL-5 augmentsIFN-inhibits induces inhibits induces inhibitsTGF- inhibits inhibits induces induces
PC
BBB B
BB
B
BB
BB
B
BB
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 specificityImproves specificity & affinity - Better on 2nd exposureIs 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 withcytoskeleton stained green
Antigen entersnode in afferent
lymphatic
Y
Y
Y
Y
Y
YY
Y
Y
Y
Y
Y
Y
Y
YY
Y
YB cells leave blood & enter lymph node via
high endothelial venulesB cellsRapidly
proliferatein follicles
GERMINAL CENTRETransient structure ofIntense proliferation
Germinal centrereleases B cellsthat differentiateinto plasma cells
Recirculating B cells are trapped by foreign antigens in lymphoid organs
HEV
1. Antigen loaded dendritic cellsmigrate from subcapsular sinus to paracortical area
of the lymph node
DC
BB
B
B
B
B
T
T
T
T
2. T cells migratethrough HEV and are trapped by antigen on DC
B
4. B cells migrate throughHEV - most pass through theparacortex and primary follicle.
T
TTT
3. T cellsproliferate
Some interact with T cells andproliferate to form a primary focus
B cells (90%) and T cells (10%) migrate to form
a primary follicle
BB
B
B
B
BT
Primary follicleformation
T cell motility in the lymph node
Primary Follicles becomesecondary follicles
when germinal centres developDark zoneLight zone B
T
Follicular dendriticcells select useful
B cells
Germinal Centre Microanatomy
1. B cells (centroblasts) downregulatesurface Ig, proliferate, somatically
hypermutate their Ig genes.AFFINITY MATURATION
2. B cells (centrocytes) upregulatesurface Ig, stop dividing andreceive costimulatory signals
from T cells and FDC
3. Apoptosis ofself-reactive &
unselected cells
4. Selected cells leave lymphnode as memory
cells or plasma cells
CD5
Two B cell lineages
B
B cell precursor
B
Mature B cell
B2 B cells
Plasma cell
YY Y YY Y
YY Y
PC
IgG
B
YYYY
YYYYY
Y
YYYY
YYYYY
Y
IgM - no other isotypes
B
Distinct B cellprecursor
?
?B1 B cells‘Primitive’ B cells found in pleura and peritoneum
CD5
B
YYYY
Y
YYYY
Y
YYYY
Y
YYYY
Y
IgM
B-1 B Cells
IgM uses a distinctive & restricted range of V regions
Recognises repeating epitope Ag such as phospholipid phosphotidyl choline & polysaccharides
NOT part of adaptive immune response:No memory inducedNot more efficient on 2nd challengePresent from birth
Few non-template encoded (N) regions in the IgM
NATURAL ANTIBODY
Can make Ig without T cell help
Comparison of B-1 and B-2 B cell properties
Property B-1 cells B-2 cells
N regions Few ExtensiveV region repertoire Restricted DiverseLocation Peritoneum/pleura EverywhereRenewal Self renewal in situ Bone marrowSpontaneous Ig production High LowIsotypes IgM IgM/G/A/D/ECarbohydrate specificity Yes RarelyProtein specificity Rarely YesNeed T cell help No YesSomatic hypermutation of Ig No HighMemory development No Yes
Yes RarelyRarely YesNo Yes
Carbohydrate specificityProtein specificityNeed T cell help
Specificity & requirement for T cell help suggests strikingly different typesof antigens are seen by B-1 and B-2 B cells
T Independent Antigens (TI-2)
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
YYYY
Y
YYYY
Y
YYYY
Y
YYYY
Y
IgM
YYY
YY
MatureB-1
YY
ImmatureB-2 Cell
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
TI-2 Antigen
LPS
LPS bindingprotein
CD14
B Cell
Bacterial Lipopolysaccharides, (TI-1 antigens), bind to host LPS binding protein in plasma
LPS/LPSBP is captured by CD14on the B cell surface
TLR 4
Toll - like receptor 4 (TLR4) interactswith the CD14/LPS/LPSBP complex
Activation of B cell
T Independent Antigens (TI-1)
Y Y Y Y Y YB B B B B B
Y Y Y Y Y YY Y Y Y Y YY Y Y Y Y YY Y Y Y Y YY Y Y Y Y YY Y Y Y Y YT 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
TDependentAntigens
TI-1Antigens
TI-2Antigens
Induce responses in babies Yes YesInduce responses in athymics No Yes YesPrime T cells Yes No NoPolyclonally activate B cells No Yes NoRequire repeating epitopes No No Yes
T Dependent & Independent Antigens
No
Adult immature B cells that bind to multivalent self Ag
undergo apoptosis
YYYY
Y
YYYY
Y
YYYY
Y
YYYY
Y
IgM
YYY
YY
MatureB-1
YY
ImmatureB-2 Cell
Adult non-bone marrow derived B-1 cells are directly stimulated by antigens containing multivalent
epitopes produce IgM WITHOUT T cell help.
TI-2 Antigen
Why are babies unresponsive to TI-2 antigens?
In adults:
YYImmatureB-1 Cell
TI-2 Antigen
Why are babies unresponsive to TI-2 antigens?
Immature B cells that bind to multivalent self Ag undergo
apoptosis
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:All B cells, B-1 & B-2, are immature
TDependentAntigens
TI-1Antigens
TI-2Antigens
Induces response in babies Yes Yes NoInduces response in athymia No Yes YesPrimes T cells Yes No NoPolyclonally activates B cells No Yes NoRequires repeating epitopes No No Yes
T Dependent & Independent Antigens
ExamplesTD: Diptheria toxin, influenza heamagglutinin, Mycobacterium tuberculosisTI-1: Bacterial lipopolysaccharides, Brucella abortisTI-2: Pneumococcal polysaccharides, Salmonella polymerised flagellin
TD: Activate B-1 and B-2 B cellsTI-1: Activate B-1 and B-2 B cellsTI-2: Activate only B-1 B cells
Y ``
Y`
`
Immune effector mechanisms against extracellular pathogens & toxins
NEUTRALISATION
Y` `
Toxin releaseblocked
Preventstoxicity
NEUTRALISING ANTIBODIES
Adhesion tohost cells blocked
Preventsinvasion
Bacterium
Y ``
Toxin
Fc receptorbinding
Effector mechanisms against extracellular pathogens
OPSONISATION
OPSONISATION Phagocytosis
Bacteria in extracellular space
Ab
+
Effector mechanisms against extracellular pathogens
COMPLEMENT ActivationBacteria in plasma
Ab & COMPLEMENT
+
Phagocytosis
binding
Complement &Fc receptor
Lysis
Opsonisation
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
