B cell biology & development

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B cell biology & development

Presented by Jaichat Mekaroonkamol, MD.

November29, 2013

Published in: Health & Medicine, Technology
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B cell biology & development

  1. 1. B Cells biology and Development Jaichat Mekaroonkamol, MD.
  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 Transfer marked fetal liver cells Normal bone marrow Mature marked B cells in periphery No Mature B cells Defective bone marrow B cell development starts in the fetal liver After birth, development continues in the bone marrow
  4. 4. Genesis • B cells - progeny of hematopoietic stem cells • Development – 7-8 wks gestation, pre-B cells appear in fetal liver – 22-23 wks gestation, pre-B cells appear in fetal bone marrow – After birth, pre-B cells limited to bone marrow
  5. 5. Abbas. Cellular and Molecular immunology. Seventh Edition
  6. 6. OVERVIEW OF LYMPHOCYTE DEVELOPMENT • The commitment of progenitor cells to the B cell or T cell lineage. • Proliferation • The sequential and ordered rearrangement of antigen receptor genes • Selection events • Differentiation of B and T cells into functionally and phenotypically distinct subpopulations Abbas. Cellular and Molecular immunology. Seventh Edition
  7. 7. Bone marrow Abbas. Cellular and Molecular immunology. Seventh Edition
  8. 8. 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 Rich. Clinical Immunology. Third Edition.
  9. 9. • In rodents: IL-7 drives proliferation of both T and B cell progenitors • In humans: IL-7 is required for the proliferation of T cell progenitors only • X-SCID: IL-7 receptor mutation – block in T cell and NK cell development – normal B cell development Abbas. Cellular and Molecular immunology. Seventh Edition
  10. 10. Abbas. Cellular and Molecular immunology. Seventh Edition
  11. 11. B cell receptor = surface immunoglobulin Rich. Clinical Immunology. Third Edition.
  12. 12. B cell Receptor Gene Rearrangement and Expression • Developing B cells use gene rearrangement to construct their antigen receptor • Immunoglobulin rearrangement is hierarchical – H chains first – L chains second, κ before λ • B cells must pass developmental checkpoints – First test immunoglobulin integrity – Then test binding specificity
  13. 13. Abbas. Cellular and Molecular immunology. Seventh Edition
  14. 14. Abbas. Cellular and Molecular immunology. Seventh Edition
  15. 15. The Mechanism of V(D)J Recombination Abbas. Cellular and Molecular immunology. Seventh Edition
  16. 16. • Combinatorial diversity • Junctional diversity Abbas. Cellular and Molecular immunology. Seventh Edition
  17. 17. Junctional diversity Abbas. Cellular and Molecular immunology. Seventh Edition
  18. 18. 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
  19. 19. • 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 Abbas. Cellular and Molecular immunology. Seventh Edition
  20. 20. 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
  21. 21. 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
  22. 22. 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
  23. 23. Heavy and light chain rearrangement is potentially wasteful V D J C Germline V D D J C DH-JH joining D J C VH-DHJH joining V J C Germline V V J C VL-JL joining V V Large pre-B Small pre-B
  24. 24. Abbas. Cellular and Molecular immunology. Seventh Edition
  25. 25. Acquisition of antigen specificity creates a need to check for recognition of self antigens B B Y Y Y Y Small pre-B cell Immature B cell No antigen receptor at cell surface Unable to sense Ag environment Cell surface Ig expressed Able to sense Ag environment 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
  26. 26. 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
  27. 27. 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
  28. 28. 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
  29. 29. Clinical Consequences • Failure to prevent the formation of antibodies with high avidity or high affinity to self-antigens can lead to autoimmunity – e.g. Myasthenia gravis, SLE
  30. 30. Coexpression of IgM and IgD. Abbas. Cellular and Molecular immunology. Seventh Edition
  31. 31. Summary • B cells develop in the fetal 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?
  32. 32. Rich. Clinical Immunology. Third Edition.
  33. 33. Late Stages of B cell Development • Exposure to antigen in the periphery – Activation – Class switching and somatic hypermutation – Selection for receptor specificity and affinity – Differentiation into plasma or memory B cells
  34. 34. Abbas. Cellular and Molecular immunology. Seventh Edition
  35. 35. Periphery 1 2 3 Effector phase Abbas. Cellular and Molecular immunology. Seventh Edition
  36. 36. B Lymphocyte antigen recognition 1. B cell receptor: Surface immunoglobulin (sIg)  Fab: antigen recognition 2. B cell epitope:  Conformational and linear epitope Abbas. Cellular and Molecular immunology. Seventh Edition
  37. 37. 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
  38. 38. Abbas. Cellular and Molecular immunology. Seventh Edition
  39. 39. JACI 2013
  40. 40. Signal transduction in B cells • Two "domains" in the BCR complex – Membrane Ig (mIg or the BCR) – Igα/Igβ (CD79) • BCR – binds antigen • Igα and Igβ transduce the signal – Cytoplasmic tails contain immunoreceptor tyrosine-based activation motif (ITAM) – Activate tyrosine kinases (Src family)
  41. 41. Ig-α (CD79a) Ig-β (CD79b) Specific cytoplasmic tail ITAM: immunoreceptor tyrosine-based activation motif Rich. Clinical Immunology. Third Edition.
  42. 42. 1. Phosphorylation by Src family kinases: Lyn, Fyn, Blk 2. Syk binding to ITAM 3. Transcription factor activation 2 1 Rich. Clinical Immunology. Third Edition.
  43. 43. JACI 2013
  44. 44. Modulation of B cell signals Activation: • CD19, CD21, & TAPA-1 Complex – CD19, a member of Ig superfamily – CD21 (CR2), a receptor for C3d – CD81 (TAPA-1), a transmembrane protein • ITAM Motifs
  45. 45. (C3d receptor: CR2) (TAPA-1) The B cell co-receptor • 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 coreceptor increases B cell receptor signalling 1,000 10,000 fold Rich. Clinical Immunology. Third Edition.
  46. 46. JACI 2013
  47. 47. Clinical Consequences • Abnormal antibody responses in the presence of B cells (CVID, IgAD) o Alteration of BCR signals  CD19 deficiency (AR) o Defective B cell survival signals  TACI, BAFFR alterations or deficiency (AR, AD)
  48. 48. Periphery 1 2 3 Effector phase Abbas. Cellular and Molecular immunology. Seventh Edition
  49. 49. Humoral immune response • T dependent responses • T independent responses Abbas. Cellular and Molecular immunology. Seventh Edition
  50. 50. B lymphocyte subsets Abbas. Cellular and Molecular immunology. Seventh Edition
  51. 51. Three Types of Mature B cells B-1 cells • Self-renewing and preferentially produced in the fetus • Spontaneously produce polyreactive Igs • Low affinity • Response to CHO and Lipid antigen • Natural antibody in mucosa tissue • 5% of total B cell • In mouse: CD5+, Mac-1+ • *In human: CD20+ CD27+ CD43+ CD70-
  52. 52. Three Types of Mature B cells Marginal Zone B cells • Found in the marginal zone of the spleen – May also be found in the blood • Respond quickly to antigens, e.g. polysaccharides
  53. 53. Three Types of Mature B cells B-2 cells (‘Conventional’ B cells) • Preferentially produced after birth • Replaced in bone marrow • Typically respond to protein Ag, requiring T cell help
  54. 54. Humoral immune response • T dependent responses • T independent responses Abbas. Cellular and Molecular immunology. Seventh Edition
  55. 55. Recirculating B cells normally pass through lymphoid organs T cell area B cells in blood B cell area Efferent lymph
  56. 56. Initial Activation and Migration of Helper T Cells and B Cells Abbas. Cellular and Molecular immunology. Seventh Edition
  57. 57. Abbas. Cellular and Molecular immunology. Seventh Edition
  58. 58. Abbas. Cellular and Molecular immunology. Seventh Edition
  59. 59. Periphery 1 2 3 Effector phase Abbas. Cellular and Molecular immunology. Seventh Edition
  60. 60. Abbas. Cellular and Molecular immunology. Seventh Edition
  61. 61. Affinity maturation
  62. 62. Abbas. Cellular and Molecular immunology. Seventh Edition
  63. 63. Plasma cells Surface Surface High rate Growth Somatic Isotype Ig MHC II Ig secretion hypermut’n switch B High Yes No Yes Yes Low No Yes No No Yes Mature B cell B Plasma cell No
  64. 64. Humoral immune response • T dependent responses • T independent responses Abbas. Cellular and Molecular immunology. Seventh Edition
  65. 65. Abbas. Cellular and Molecular immunology. Seventh Edition
  66. 66. 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
  67. 67. 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 Y Y Y Y Y Y Y Y Y Y Y Y Y YY YY YY YY YY Y
  68. 68. 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
  69. 69. T Dependent & Independent Antigens T Dependent Antigens Induce responses in babies Induce responses in athymics Prime T cells Polyclonally activate B cells Require repeating epitopes TI-1 Antigens TI-2 Antigens Yes No Yes No No Yes Yes No Yes No No Yes No No Yes
  70. 70. 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.
  71. 71. 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
  72. 72. T Dependent & Independent Antigens T Dependent Antigens Induces response in babies Induces response in athymia Primes T cells Polyclonally activates B cells Requires repeating epitopes 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
  73. 73. Key Concepts Peripheral B cell Development • T-independent activation of naive B cells causes them to develop into either short-lived plasma cells or long-lived B1 cells. • T-dependent activation of B cells leads to germinal center formation, permitting somatic hypermutation and class switch recombination
  74. 74. Key Concepts Peripheral B cell Development • T-dependent activation of naive B cells results in differentiation into either memory B cells or short-lived plasma cells. • T-dependent activation of memory B cells either results in expansion of memory B cells or differentiation into long-lived plasma cells.
  75. 75. Abbas. Cellular and Molecular immunology. Seventh Edition
  76. 76. Summay Antibody insufficiency can best be interpreted by taking into account where B cell development has failed: 1. Inability to produce immunoglobulin 2. Inability to produce a B cell receptor complex 3. Inability to signal properly through the BCR 4. Failure to receive T cell signals 5. Failure to modify immunoglobulin genes 6. Failure to receive or act on survival signals
  77. 77. JACI 2013
  78. 78. THE END…..
  79. 79. Clinical Consequences • B cell development can be monitored by examining the pattern of expression of lymphoidspecific surface proteins – Antibodies against these surface proteins can be used to eliminate B cells • e.g. Rituximab (anti-CD20)
  80. 80. Inhibition Rich. Clinical Immunology. Third Edition.

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