B Cells and B Cell Development
The discovery of B cell immunity 1954 - Bruce Glick, Ohio State University Studies on the function of the bursa of Fabrici...
Origin of B cells and organ of B cell maturation After birth, development continues in the bone marrow B cell development ...
B cell development in the bone marrow Bone Marrow provides a MATURATION & DIFFERENTIATION MICROENVIRONMENT for B cell deve...
Bone Marrow E M M S
X X X Scheme of B Cell Development in the Bone Marrow Immature & mature B Central Sinus Progenitors Pre-B Stromal cells E ...
Bone marrow stromal cells nurture developing B cells Types of cytokines and cell-cell contacts needed at each stage of dif...
Bone marrow stromal cell Maturing B cells
B B Stromal cell
Stages of B cell development Each stage of development is defined by rearrangements of IgH chain genes, IgL chain genes, e...
Early pro-B Cytokines and cell-cell contacts at each stage of differentiation are different Kit Receptor Tyrosine kinase S...
Late pro-B Pre-B Cytokines and cell-cell contacts at each stage of differentiation are different Early pro-B Interleukin-7...
Stages of differentiation in the bone marrow are defined by Ig gene rearrangement B CELL STAGE IgH GENE CONFIGURATION Stem...
B cell receptor Transiently expressed when V H D H J H  C H   is productively rearranged VpreB/  5 - the surrogate light...
Ligation of the pre-B cell receptor 1. Ensures only one specificty of Ab expressed per cell 2. Triggers entry into cell cy...
Evidence for allelic exclusion Allotypes can be identified by staining B cell surface Ig with antibodies AND ALLOTYPE-  po...
Suppression of H chain gene rearrangement ensures only one specificity of Ab expressed per cell. Allelic exclusion prevent...
Allelic exclusion is needed for efficient clonal selection All daughter cells must express the same Ig specificity otherwi...
Allelic exclusion is needed to prevent holes in the repertoire  Exclusion of anti-brain B cells i.e. self tolerance Anti-b...
1. Suppresses further H chain rearrangement Ligation of the pre-B cell receptor 1. Ensures only one specificity of Ab expr...
Human IgG3 Heavy Chain nucleotide sequence ATGAAACANCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCACCTGCAGGAGTCGG...
Ligation of the pre-B cell receptor triggers entry into the cell cycle Many large pre-B cells with identical pre-B recepto...
V Germline D H -J H  joining V H -D H J H  joining V Germline V L -J L  joining Heavy and light chain rearrangement is pot...
B cells have several chances to successfully rearrange Ig genes Early Pro B Late Pro B Pre B V H -DJ H On first chromosome...
Small pre-B cell No antigen receptor at cell surface Unable to sense Ag environment !! May be self-reactive !! Immature B ...
B cell self tolerance: clonal deletion Immature  B cell recognises MULTIVALENT self Ag Clonal deletion by apoptosis B Y Y ...
Y B cell self tolerance: anergy Y Y Y Anergic B cell IgD normal IgM low Immature  B cell recognises soluble self Ag No cro...
Receptor editing A rearrangement encoding a self specific receptor can be replaced V V V V V V V V C D J Y B B !! Receptor...
Y Y Y Y Y Y Mature B cell exported to the periphery Y Y B cell self tolerance: export of self tolerant B cells Immature  B...
How can B cells express IgM and IgD simultaneously? N.B. Remember Molecular Genetics of Immunoglobulins lecture – No C   ...
Splicing of IgM and IgD RNA Two types of mRNA can be made simultaneously in the cell by differential usage of alternative ...
<ul><li>B cells develop in the foetal liver and adult bone marrow </li></ul><ul><li>Stages of B cell differentiation are d...
Although Fab fragments bind to membrane Ig (mIg), no signal is transduced through the B cell membrane Extensive cross link...
Ring staining Ig is evenly distributed around the cell surface Patching Ig is aggregated in uneven ‘clumps’ as a result of...
Transduction of signals by the B cell receptor Ig  Ig  Intracytoplasmic signalling domains Extracellular antigen recogni...
<ul><li>Phosphorylation is rapid, requires no protein synthesis or degradation to change the biochemical activity of a tar...
Regulation of Src kinases Phosphorylation of ‘Activating Tyrosine’ stimulates kinase activity Phosphorylation of ‘Inhibito...
Regulation of Src kinases by Csk and CD45 The balance between Csk and CD45 phosphatase activity sets the threshold for ini...
Phosphorylation of ITAMs by Src kinases 3. Src kinases bind to phosphorylated ITAMS and are activated to phosphorylate adj...
Syk protein Tyrosine kinases <ul><li>CD45 phosphatase allows activation of Src family kinases Blk, Fyn & Lyn  </li></ul><u...
CD45 The B cell co-receptor CD21 (C3d receptor) CD19 CD81 (TAPA-1) Ig  Ig  The B cell co-receptor
C3d opsonised bacterium <ul><li>mIg and CD21 are cross-linked by antigen that has activated complement </li></ul>P P <ul><...
BLNK Cell membrane-associated B cell Linker protein  - BLNK - contains many Tyrosine residues Activation of signals that a...
Transmission of signals from the cell surface to the nucleus <ul><li>B cell-specific parts of the signalling cascade are a...
<ul><li>MAP Kinase cascade </li></ul><ul><li>Small G-protein-activated MAP kinases found in all multicellular animals - ac...
B cell recognises non-self antigen in periphery  Ig-secreting plasma cell Differentiation in the periphery Mature peripher...
Plasma cells Surface  Surface  High rate  Growth  Somatic  Isotype Ig  MHC II  Ig secretion  hypermut’n  switch High Yes  ...
<ul><li>You should know: </li></ul><ul><li>Where B cells come from </li></ul><ul><li>What happens to B cells in the bone m...
Recirculating B cells normally pass through lymphoid organs B cells in blood Efferent lymph T cell area B cell area
Recirculating B cells are trapped by foreign antigens in lymphoid organs Antigen enters node in afferent lymphatic Y Y Y Y...
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...
Maturation of Follicular Dendritic cells Club-shaped tips of developing dendrites Filiform dendrites Bead formation on den...
Association of antigen with FDC in the form of an immune complex with C3b and antibodies attached Antigen enters the germi...
The veils of antigen-bearing dendritic cell surround the beads and the layer of immune complexes is thickened by transfer ...
Iccosomes bearing  different antigens Uptake of Iccosomes/Antigen by B cells Surface Ig captures antigen Cross-linking of ...
2. Binding and internalisation via  Ig induces expression of CD40 3. Antigen enters exogenous antigen processing pathway 4...
T cell help to B cells Signal 1 antigen & antigen receptor 1. T cell antigen receptor 2. Co-receptor (CD4) 3.CD40 Ligand Y...
T cell help - Signal 2  B cells are inherently prone to die by apoptosis Signal 1 & 2 upregulate Bcl-X L  in the B cell an...
T cell help - Signal 2 activates hypermutation  Signal 2, and thus T cells, regulate which B cells are clonally selected. ...
Control of Affinity & Affinity Maturation Only this cell, that has a high affinity for antigen can express CD40. Only this...
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  Cytokines IL-4 IL-5 IL-6 IFN-  TGF-  IgM IgG3 IgG1 IgG2b  IgG2a IgE IgA IL4 inh...
Regulation of specificity - Cognate recognition 1. T cells can only help the B cells that present  antigen to them 2. B ce...
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 Y Y Y Y...
Some interact with T cells and proliferate to form a primary focus Primary follicle formation HEV 1. Antigen loaded dendri...
T cell motility in the lymph node
Primary Follicles become secondary follicles when germinal centres develop Germinal Centre Microanatomy Dark zone Light zo...
Two B cell lineages B2 B cells IgM - no other isotypes B1 B cells ‘ Primitive’ B cells found in pleura and peritoneum CD5 ...
B-1 B Cells IgM uses a distinctive & restricted range of V regions Recognises repeating epitope Ag such as phospholipid ph...
Comparison of B-1 and B-2 B cell properties Property B-1 cells B-2 cells N regions Few Extensive V region repertoire Restr...
T Independent Antigens (TI-2)  IgM Non-bone marrow derived B-1 cells are directly stimulated by antigens containing multiv...
LPS binding protein Bacterial Lipopolysaccharides, (TI-1 antigens), bind to host LPS binding protein in plasma LPS/LPSBP i...
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 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y T Independent Antigens (TI-1 e.g. LPS)...
No T Dependent Antigens TI-1 Antigens TI-2 Antigens Induce responses in babies Yes Yes Induce responses in athymics No Yes...
IgM Adult non-bone marrow derived B-1 cells are directly stimulated by antigens containing multivalent epitopes produce Ig...
Why are babies unresponsive to TI-2 antigens? As with adult B cells, immature B cells that bind multivalent self Ag underg...
Induces response in babies Yes Yes No Induces response in athymia No Yes Yes Primes T cells Yes No No Polyclonally activat...
Immune effector mechanisms against extracellular pathogens & toxins NEUTRALISATION NEUTRALISING ANTIBODIES Bacterium Toxin...
Effector mechanisms against extracellular pathogens OPSONISATION OPSONISATION Fc receptor binding Phagocytosis Bacteria in...
Effector mechanisms against extracellular pathogens COMPLEMENT Activation Lysis Bacteria in plasma Ab &  COMPLEMENT + Phag...
Summary <ul><li>B cell tolerance of self is by clonal deletion or anergy of self-reactive cells </li></ul><ul><li>Receptor...
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B Cell Development

  1. 1. B Cells and B Cell Development
  2. 2. 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
  3. 3. Origin of B cells and organ of B cell maturation After birth, development continues in the bone marrow B cell development starts in the foetal liver Transfer marked foetal liver cells No Mature B cells Normal bone marrow Defective bone marrow Mature marked B cells in periphery
  4. 4. B cell development in the bone marrow Bone Marrow provides a MATURATION & DIFFERENTIATION MICROENVIRONMENT for B cell development B Regulates construction of an antigen receptor 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 B
  5. 5. Bone Marrow E M M S
  6. 6. X X X Scheme of B Cell Development in the Bone Marrow Immature & mature B Central Sinus Progenitors Pre-B Stromal cells E n d o os t e u m Macrophage
  7. 7. Bone marrow stromal cells nurture developing B cells Types of cytokines and cell-cell contacts needed at each stage of differentiation are different Secreted Factors - CYTOKINES 2. Secretion of cytokines by stromal cells B Stromal cell 1. Specific cell-cell contacts between stromal cells and developing B cells Cell-cell contact
  8. 8. Bone marrow stromal cell Maturing B cells
  9. 9. B B Stromal cell
  10. 10. 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 Stem Cell Early pro-B cell Late pro-B cell Large pre-B cell Small pre-B cell Immature B cell Mature B cell
  11. 11. Early pro-B Cytokines and cell-cell contacts at each stage of differentiation are different Kit Receptor Tyrosine kinase Stem cell factor Cell-bound growth factor VLA-4 (Integrin) Stem Stromal cell Cell adhesion molecules VCAM-1 (Ig superfamily)
  12. 12. Late pro-B Pre-B Cytokines and cell-cell contacts at each stage of differentiation are different Early pro-B Interleukin-7 receptor Stromal cell Interleukin-7 Growth factor
  13. 13. Stages of differentiation in the bone marrow are defined by Ig gene rearrangement B CELL STAGE IgH GENE CONFIGURATION Stem cell Early pro-B Late pro-B Large pre-B Germline D H to J H V H to D H J H V H D H J H Ig light chain gene has not yet rearranged Pre-B cell receptor expressed
  14. 14. B cell receptor Transiently expressed when V H D H J H C H  is productively rearranged VpreB/  5 - 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- Ig  & Ig  signal transduction molecules C H  Heavy chain V H D H J H Light chain V L J L C L VpreB  5
  15. 15. Ligation of the pre-B cell receptor 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 V H D H J H joins Large Pre-B Stromal cell Unconfirmed ligand of pre-B cell receptor
  16. 16. 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 Y B b Y Y B a b Y B a
  17. 17. Suppression of H chain gene rearrangement 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 Self antigen expressed by e.g. brain cells S. aureus Y Y Y Y Y B S. aureus Y Y Y Y Y Y Y Anti S. aureus Antibodies Y Y Y Y Y Y Anti brain Abs Y Y Y Y Y Y Y Anti S. aureus Antibodies
  18. 18. Allelic exclusion is needed for efficient clonal selection 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 S. typhi S. typhi
  19. 19. Allelic exclusion is needed to prevent holes in the repertoire Exclusion of anti-brain B cells i.e. self tolerance Anti-brain Ig AND anti- S. aureus Ig Anti-brain Ig anti S.aureus B cells will be excluded leaving a “hole in the repertoire” BUT Y Y B B One specificity of Ag receptor per cell S. aureus Y Y Y B B IF there were two specificities of Ag receptor per cell B B Deletion Anergy OR Y Y Y B B
  20. 20. 1. Suppresses further H chain rearrangement Ligation of the pre-B cell receptor 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 V H D H J H joins Large Pre-B Stromal cell Unconfirmed ligand of pre-B cell receptor
  21. 21. Human IgG3 Heavy Chain nucleotide sequence ATGAAACANCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCACCTGCAGGAGTCGGGCCCAGGACTGGGGAAGCCTCCAGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCNNNGTGCCCAGCACCTGAACTCTTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCNNNNGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCGAGGAGATGACCAAGAACCAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA Large pre-B cells need in frame V H D H J H joins to mature Translation in frame 2 (no protein) * Translation in frame 3 ETXVVLPSPGGSSQMGPVPGAPAGVGPRTGEASRAQNPTW* Translation in frame 1 MKXLWFFLLLVAAPRWVLSQVHLQESGPGLGKPPELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCXXCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDXXVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRYTQKSLSLSPGK* Large pre-B Development continues Pre-B cell receptor can be activated Development arrests Development arrests
  22. 22. 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 B cell Light chain expressed IgM displayed on surface IgM Large pre-B Large pre-B Intracellular VDJC H chain V L -J L rearranges Proliferation stops Pre-receptor not displayed Small pre-B
  23. 23. V Germline D H -J H joining V H -D H J H joining V Germline V L -J L joining Heavy and light chain rearrangement is potentially wasteful 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 V C D J V J C V C J Large pre-B Small pre-B
  24. 24. B cells have several chances to successfully rearrange Ig genes Early Pro B Late Pro B Pre B V H -DJ H On first chromosome V H -DJ H On second chromosome Immature B  on first chromosome  on second chromosome  on first chromosome  on second chromosome D H -J H On first chromosome D H -J H On second chromosome NO YES NO B NO NO NO NO YES YES NO YES YES YES YES Y IgM  B Y IgM  B YES NO
  25. 25. Small pre-B cell No antigen receptor at cell surface Unable to sense Ag environment !! May be self-reactive !! Immature B cell Cell surface Ig expressed Able 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 <ul><li>Physical removal from the repertoire DELETION </li></ul><ul><li>Paralysis of function ANERGY </li></ul><ul><li>Alteration of specificity RECEPTOR EDITING </li></ul>B Y Y Y Y B
  26. 26. B cell self tolerance: clonal deletion Immature B cell recognises MULTIVALENT self Ag Clonal deletion by apoptosis B Y Y B Immature B B Small pre-B Small pre-B cell assembles Ig
  27. 27. Y B cell self tolerance: anergy Y Y Y Anergic B cell IgD normal IgM low Immature B cell recognises soluble self Ag No cross-linking IgM IgD IgD IgD B B Y Y B Immature B B Small pre-B Small pre-B cell assembles Ig
  28. 28. Receptor editing A rearrangement encoding a self specific receptor can be replaced V V V V V V V V C D J Y B B !! Receptor recognises self antigen !! B Apoptosis or anergy Y B B Edited receptor now recognises a different antigen and can be rechecked for specificity C D J Arrest development And reactivate RAG-1 and RAG-2
  29. 29. Y Y Y Y Y Y Mature B cell exported to the periphery Y Y B cell self tolerance: export of self tolerant B cells Immature B cell doesn’t recognise any self Ag IgD and IgM normal IgM IgD IgD IgD IgD IgM IgM IgM Y Y B Immature B B Small pre-B Small pre-B cell assembles Ig B
  30. 30. How can B cells express IgM and IgD simultaneously? N.B. Remember Molecular Genetics of Immunoglobulins lecture – No C  switch region Consider similarities with mechanism allowing secreted and membrane Ig by the same cell C  2 C  C  4 C  2 C  1 C  1 C  3 C  C  C  C  C  3 VDJ S  3 C  C  C  3 VDJ C  1 S  1 C  1 C  3 VDJ C  1 C  3 VDJ IgG3 produced. Switch from IgM VDJ C  1 IgA1 produced. Switch from IgG3 VDJ C  1 IgA1 produced. Switch from IgM
  31. 31. 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 C  1 C  2 C  3 C  4 C  1 C  2 C  3 pA1 V D J C  1 C  2 C  3 C  4 C  1 C  2 C  3 V D J AAA RNA cleaved and polyadenylated at pA2 V D J C  IgM mRNA V D J C  IgD mRNA C  1 C  3 C  C  V D J C  1 C  2 C  3 C  4 C  1 C  2 C  3 DNA pA1 pA2 V D J RNA cleaved and polyadenylated at pA1 AAA
  32. 32. <ul><li>B cells develop in the foetal liver and adult bone marrow </li></ul><ul><li>Stages of B cell differentiation are defined by Ig gene rearrangement </li></ul><ul><li>Pre-B cell receptor ligation is essential for B cell development </li></ul><ul><li>Allelic exclusion is essential to the clonal nature of immunity </li></ul><ul><li>B cells have several opportunities to rearrange their antigen receptors </li></ul><ul><li>IgM and IgD can be expressed simultaneously due to differential RNA splicing </li></ul><ul><li>So far, mostly about B cells in the bone marrow - what about mature peripheral B cells? </li></ul>Summary
  33. 33. 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
  34. 34. Ring staining Ig is evenly distributed around the cell surface Patching 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
  35. 35. Transduction of signals by the B cell receptor Ig  Ig  Intracytoplasmic signalling domains Extracellular antigen recognition 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 - Y XX[L/V]X 6-9 Y XX[L/V]
  36. 36. <ul><li>Phosphorylation is rapid, requires no protein synthesis or degradation to change the biochemical activity of a target protein </li></ul><ul><li>It is reversible via the action of phosphatases that remove phosphate </li></ul><ul><li>Phosphorylation changes the properties of a protein by changing its conformation </li></ul><ul><li>Changes in conformation may activate or inhibit a biochemical activity or create a binding site for other proteins </li></ul>Phosphorylation by Src kinases Kinase domain Unique region SH3 domain SH2 domain Enzyme domain phosphorylates tyrosines (to give phosphotyrosine) Phosphotyrosine receptor domain Adaptor protein recruitment domain ITAM binding domain
  37. 37. Regulation of Src kinases Phosphorylation of ‘Activating Tyrosine’ stimulates kinase activity Phosphorylation of ‘Inhibitory Tyrosine’ inhibits kinase activity by blocking access to the Activating Tyrosine Residue Kinase domain Unique region SH3 domain SH2 domain Activating tyrosine residue Inhibitory tyrosine residue Kinase domain Unique region SH3 domain SH2 domain
  38. 38. 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
  39. 39. Phosphorylation of ITAMs by Src kinases 3. Src kinases bind to phosphorylated ITAMS and are activated to phosphorylate adjacent ITAMS P ITAM ITAM P P ITAM ITAM 2. Antigen clusters B cell receptors with CD45 phosphatases. Src kinases are phosphorylated 1. Csk inactived Src interacts with low affinity with the ITAMs of ‘resting’ receptors P ITAM P and are activated to phosphorylate ITAMS
  40. 40. Syk protein Tyrosine kinases <ul><li>CD45 phosphatase allows activation of Src family kinases Blk, Fyn & Lyn </li></ul><ul><li>Receptor cross-linking activates Src kinases that phosphorylate ITAMs in the Ig  and Ig  </li></ul>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 ITAM P P ITAM P P ITAM P P P P
  41. 41. CD45 The B cell co-receptor CD21 (C3d receptor) CD19 CD81 (TAPA-1) Ig  Ig  The B cell co-receptor
  42. 42. C3d opsonised bacterium <ul><li>mIg and CD21 are cross-linked by antigen that has activated complement </li></ul>P P <ul><li>CD21 is phosphorylated and receptor-associated kinases phosphorylate CD19 </li></ul>Co-receptor phosphorylation <ul><li>Phosphorylated CD19 activates more Src family kinases </li></ul><ul><li>Ligation of the co-receptor increases B cell receptor signalling 1000 -10,000 fold </li></ul>Src family kinases then bind the phosphorylated CD19 Antigen recognition C3d binds to CD21, the complement receptor 2 (CR2) P P
  43. 43. BLNK Cell membrane-associated B cell Linker protein - BLNK - contains many Tyrosine residues Activation of signals that affect gene transcription Activated Syk phosphorylates BLNK P P P P BLNK binds Tec kinases Tec Tec Tec Tec Tec kinases activate phospholipase C-  (PLC-  ) PLC-  cleaves phosphotidylinositol bisphosphate (PIP 2 ) to yield diacylglycerol ( DAG ) and inositol trisphosphate ( IP 3 ) ITAM P P ITAM 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 MAP kinase cascade
  44. 44. Transmission of signals from the cell surface to the nucleus <ul><li>B cell-specific parts of the signalling cascade are associated with receptors unique to B cells - mIg, CD19 etc. </li></ul><ul><li>Subsequent signals that transmit signals to the nucleus are common to many different types of cell. </li></ul><ul><li>The ultimate goal is to activate the transcription of genes, the products of which mediate host defence, proliferation, differentiation etc. </li></ul><ul><li>Once the B cell-specific parts of the cascade are complete, signalling to </li></ul><ul><li>the nucleus continues via three common signalling pathways via: </li></ul><ul><li>The mitogen-activated protein kinase (MAP kinase) pathway </li></ul><ul><li>Increased in intracellular Ca2+ mediated by IP 3 </li></ul><ul><li>The activation of Protein Kinase C mediated by DAG </li></ul>
  45. 45. <ul><li>MAP Kinase cascade </li></ul><ul><li>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 . </li></ul><ul><li>Increases in intracellular calcium via IP 3 </li></ul><ul><li>IP 3 , 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 . </li></ul><ul><li>Activation of Protein Kinase C family members via DAG </li></ul><ul><li>DAG stays associated with the membrane and recruits protein kinase C family members. The PKC, serine/threonine protein kinases, ultimately activate the transcription factor NF  B </li></ul>The activated transcription factors AP-1, NFAT and NF  B induce B cell proliferation, differentiation and effector mechanisms Simplified scheme linking antigen recognition with transcription of B cell-specific genes
  46. 46. B cell recognises non-self antigen in periphery Ig-secreting plasma cell Differentiation in the periphery Mature peripheral B cell Y Y Y Y Y Y Y Y Y B Y Y Y Y Y Y Y B Y Y Y Y Y Y Y B
  47. 47. Plasma cells Surface Surface High rate Growth Somatic Isotype Ig MHC II Ig secretion hypermut’n switch High Yes No Yes Yes Yes Low No Yes No No No B B Mature B cell Plasma cell
  48. 48. <ul><li>You should know: </li></ul><ul><li>Where B cells come from </li></ul><ul><li>What happens to B cells in the bone marrow </li></ul><ul><li>How B cell differentiation is linked with Ig gene rearrangement </li></ul><ul><li>The B cell developmental ‘check points’ that ensure each cell produces a single specificity of antibody that does not react with self </li></ul><ul><li>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 </li></ul><ul><li>What do mature B cells do once activated by an antigen in the periphery? </li></ul>Summary
  49. 49. Recirculating B cells normally pass through lymphoid organs B cells in blood Efferent lymph T cell area B cell area
  50. 50. Recirculating B cells are trapped by foreign antigens in lymphoid organs Antigen enters node in afferent lymphatic Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y B cells leave blood & enter lymph node via high endothelial venules B cells proliferate rapidly GERMINAL CENTRE Transient structure of Intense proliferation Germinal centre releases B cells that differentiate into plasma cells
  51. 51. B cells (stained brown) in the Germinal Centre P = Paracortex, Mn = Mantle zone SC = Subcapsular zone
  52. 52. Follicular Dendritic cells (stained blue)in the Germinal Centre
  53. 53. 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
  54. 54. Maturation of Follicular Dendritic cells Club-shaped tips of developing dendrites Filiform dendrites Bead formation on dendrites Bead formation on dendrites
  55. 55. 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 filiform dendrites of FDC develop beads coated with a thin layer of immune complexes
  56. 56. 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
  57. 57. Iccosomes bearing different antigens Uptake of Iccosomes/Antigen by B cells Surface Ig captures antigen Cross-linking of antigen receptor activates B cell Activated B cell expresses CD40 CD40 Y Y Y B Anti- B cell
  58. 58. 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 Fate of Antigens Internalised by B cells 1. Capture by antigen specific Ig maximises uptake of a single antigen B B
  59. 59. T cell help to B cells Signal 1 antigen & antigen receptor 1. T cell antigen receptor 2. Co-receptor (CD4) 3.CD40 Ligand Y Y Y B Th Th Signal 2 - T cell help
  60. 60. T cell help - Signal 2 B cells are inherently prone to die by apoptosis Signal 1 & 2 upregulate Bcl-X L in the B cell and Bcl-X L 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-  TGF-  Y Y Y B Th Signal 2 Signal 1 Cytokines
  61. 61. 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 CDR1 CDR2 CDR3 Day 6 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 Day 8 Day 12 Day 18 Deleterious mutation Beneficial mutation Neutral mutation Lower affinity - Not clonally selected Higher affinity - Clonally selected Identical affinity - No influence on clonal selection
  62. 62. 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 receptors - all specific for , but with different affinities due to somatic hypermutation of Ig genes in the germinal centre B B B B B
  63. 63. GC = Germinal Centre, TBM = Tingible Body Macrophages Germinal Centre Macrophages (stained brown) Clean Up Apoptotic Cells
  64. 64. Role of T cell cytokines in T cell help Cytokines IL-4 IL-5 IL-6 IFN-  TGF-  IgM IgG3 IgG1 IgG2b IgG2a IgE IgA IL4 inhibits inhibits induces inhibits induces IL-5 augments IFN-  inhibits induces inhibits induces inhibits TGF-  inhibits inhibits induces induces Proliferation & Differentiation Th Signal 2 Y Y Y B Signal 1 PC B B B B B B B B B B B B B B
  65. 65. 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
  66. 66. Synaptic tethering of a B cell (red) to a T cell (green)
  67. 67. T cell (in centre) surrounded by B cells with cytoskeleton stained green
  68. 68. Recirculating B cells are trapped by foreign antigens in lymphoid organs Antigen enters node in afferent lymphatic Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y B cells leave blood & enter lymph node via high endothelial venules B cells Rapidly proliferate in follicles GERMINAL CENTRE Transient structure of Intense proliferation Germinal centre releases B cells that differentiate into plasma cells
  69. 69. Some interact with T cells and proliferate to form a primary focus Primary follicle formation HEV 1. Antigen loaded dendritic cells migrate from subcapsular sinus to paracortical area of the lymph node DC B B B B B B T T T T 2. T cells migrate through HEV and are trapped by antigen on DC B 4. B cells migrate through HEV - most pass through the paracortex and primary follicle. T T T T 3. T cells proliferate B cells (90%) and T cells (10%) migrate to form a primary follicle B B B B B B T
  70. 70. T cell motility in the lymph node
  71. 71. Primary Follicles become secondary follicles when germinal centres develop Germinal Centre Microanatomy Dark zone Light zone B T Follicular dendritic cells select useful B cells 1. B cells (centroblasts) downregulate surface Ig, proliferate, somatically hypermutate their Ig genes. AFFINITY MATURATION 2. B cells (centrocytes) upregulate surface Ig, stop dividing and receive costimulatory signals from T cells and FDC 3. Apoptosis of self-reactive & unselected cells 4. Selected cells leave lymph node as memory cells or plasma cells
  72. 72. Two B cell lineages B2 B cells IgM - no other isotypes B1 B cells ‘ Primitive’ B cells found in pleura and peritoneum CD5 B B cell precursor B Mature B cell Plasma cell Y Y Y Y Y Y Y Y Y PC IgG B Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y B Distinct B cell precursor ? ?
  73. 73. 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 induced Not more efficient on 2nd challenge Present from birth Few non-template encoded (N) regions in the IgM NATURAL ANTIBODY Can make Ig without T cell help CD5 B Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y IgM
  74. 74. Comparison of B-1 and B-2 B cell properties Property B-1 cells B-2 cells N regions Few Extensive V region repertoire Restricted Diverse Location Peritoneum/pleura Everywhere Renewal Self renewal in situ Bone marrow Spontaneous Ig production High Low Isotypes IgM IgM/G/A/D/E Carbohydrate specificity Yes Rarely Protein specificity Rarely Yes Need T cell help No Yes Somatic hypermutation of Ig No High 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 Need T cell help
  75. 75. T Independent Antigens (TI-2) 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 B-2 Cell TI-2 Antigen
  76. 76. LPS binding protein 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 Toll - like receptor 4 (TLR4) interacts with the CD14/LPS/LPSBP complex Activation of B cell T Independent Antigens (TI-1) LPS CD14 B Cell
  77. 77. 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 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y 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
  78. 78. No T Dependent Antigens TI-1 Antigens TI-2 Antigens Induce responses in babies Yes Yes 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
  79. 79. 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 B-2 Cell TI-2 Antigen
  80. 80. 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: All B cells, B-1 & B-2, are immature Y Y Immature B-1 Cell TI-2 Antigen Immature B cells that bind to multivalent self Ag undergo apoptosis
  81. 81. Induces response in babies Yes Yes No Induces response in athymia No Yes Yes Primes T cells Yes No No Polyclonally activates B cells No Yes No Requires repeating epitopes No No Yes T Dependent & Independent Antigens Examples TD: Diptheria toxin, influenza heamagglutinin, Mycobacterium tuberculosis 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
  82. 82. Immune effector mechanisms against extracellular pathogens & toxins NEUTRALISATION NEUTRALISING ANTIBODIES Bacterium Toxin Y ` ` Y ` ` Y ` ` Toxin release blocked Prevents toxicity Adhesion to host cells blocked Prevents invasion Y ` `
  83. 83. Effector mechanisms against extracellular pathogens OPSONISATION OPSONISATION Fc receptor binding Phagocytosis Bacteria in extracellular space Ab +
  84. 84. Effector mechanisms against extracellular pathogens COMPLEMENT Activation Lysis Bacteria in plasma Ab & COMPLEMENT + Phagocytosis binding Complement & Fc receptor Opsonisation
  85. 85. Summary <ul><li>B cell tolerance of self is by clonal deletion or anergy of self-reactive cells </li></ul><ul><li>Receptor editing increases the efficiency of B cell development </li></ul><ul><li>Follicular dendritic cells acquire antigen and transfer it to B cells </li></ul><ul><li>T cell help to B cells is via CD40L and cytokines </li></ul><ul><li>CD40 expression indirectly leads to Ig affinity maturation </li></ul><ul><li>Germinal centre microanatomy & function </li></ul><ul><li>There are two lineages of B cells - B1 and B2 B cells </li></ul><ul><li>The dependency of B cells upon T cells varies </li></ul>
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