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B cell biology & development
 

B cell biology & development

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B cell biology & development

B cell biology & development

Presented by Jaichat Mekaroonkamol, MD.

November29, 2013

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    B cell biology & development B cell biology & development Presentation Transcript

    • B Cells biology and Development Jaichat Mekaroonkamol, MD.
    • The discovery of B cell immunity 1954 - Bruce Glick, Ohio State University Studies on the function of the bursa of Fabricius, a lymphoid organ in the cloacal region of the chicken Bursectomy – no apparent effect Bursectomised chickens were later used in experiments to raise antibodies to Salmonella antigens None of the bursectomised chickens made anti-Salmonella antibodies Bursa was later found to be the organ in which antibody producing cells developed – antibody producing cells were thereafter called B cells Mammals do not have a bursa of Fabricius
    • Origin of B cells and organ of B cell maturation Transfer marked 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
    • 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
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • 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
    • Bone marrow Abbas. Cellular and Molecular immunology. Seventh Edition
    • 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.
    • • 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
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • B cell receptor = surface immunoglobulin Rich. Clinical Immunology. Third Edition.
    • 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
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • The Mechanism of V(D)J Recombination Abbas. Cellular and Molecular immunology. Seventh Edition
    • • Combinatorial diversity • Junctional diversity Abbas. Cellular and Molecular immunology. Seventh Edition
    • Junctional diversity Abbas. Cellular and Molecular immunology. Seventh Edition
    • 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
    • • 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
    • Ligation of the pre-B cell receptor 1. Suppresses further H chain rearrangement 2. Triggers entry into cell cycle Large Pre-B Unconfirmed ligand of pre-B cell receptor 1. Ensures only one specificty of Ab expressed per cell Stromal cell 2. Expands only the pre-B cells with in frame VHDHJH joins ALLELIC EXCLUSION Expression of a gene on one chromosome prevents expression of the allele on the second chromosome
    • Evidence for allelic exclusion ALLOTYPE- polymorphism in the C region of Ig – one allotype inherited from each parent Allotypes can be identified by staining B cell surface Ig with antibodies B a (Refer back to Dreyer & Bennet hypothesis in Molecular Genetics of Immunoglobulins lecture topic) B AND b b B B Y Suppression of H chain rearrangement by pre-B cell receptor prevents expression of two specificities of antibody per cell Y b Y B Y a a/b b/b Y Y a/a a
    • Ligation of the pre-B cell receptor triggers entry into the cell cycle Proliferation Large Pre-B Large Large Pre-B Large Pre-B Large Pre-B Pre-B Small Large pre-B Proliferation stops Pre-receptor not displayed Many large pre-B cells with identical pre-B receptors IgM Intracellular VDJCH chain VL-JL rearranges Y Large pre-B Large Pre-B Large Large Pre-B Large Pre-B Large Pre-B Pre-B Immature B cell Light chain expressed IgM displayed on surface
    • Heavy and light chain rearrangement is potentially wasteful V D J C Germline V D D J C DH-JH joining 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
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • 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
    • B cell self tolerance: clonal deletion Small pre-B B B Immature B B YY Small pre-B cell assembles Ig Immature B cell recognises MULTIVALENT self Ag Clonal deletion by apoptosis
    • B cell self tolerance: anergy IgD normal IgM low Small pre-B B YY Immature B B IgM IgD B IgD IgD B Small pre-B cell assembles Ig Immature B cell recognises soluble self Ag No cross-linking Anergic B cell
    • Receptor editing A rearrangement encoding a self specific receptor can be replaced V V V B V D J C !!Receptor recognises self antigen!! Arrest development And reactivate RAG-1 and RAG-2 V B V V B D J Apoptosis or anergy C Edited receptor now recognises a different antigen and can be rechecked for specificity
    • 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
    • Coexpression of IgM and IgD. Abbas. Cellular and Molecular immunology. Seventh Edition
    • 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?
    • Rich. Clinical Immunology. Third Edition.
    • 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
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • Periphery 1 2 3 Effector phase Abbas. Cellular and Molecular immunology. Seventh Edition
    • 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
    • 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
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • JACI 2013
    • 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)
    • Ig-α (CD79a) Ig-β (CD79b) Specific cytoplasmic tail ITAM: immunoreceptor tyrosine-based activation motif Rich. Clinical Immunology. Third Edition.
    • 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.
    • JACI 2013
    • 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
    • (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.
    • JACI 2013
    • 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)
    • Periphery 1 2 3 Effector phase Abbas. Cellular and Molecular immunology. Seventh Edition
    • Humoral immune response • T dependent responses • T independent responses Abbas. Cellular and Molecular immunology. Seventh Edition
    • B lymphocyte subsets Abbas. Cellular and Molecular immunology. Seventh Edition
    • 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-
    • 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
    • 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
    • Humoral immune response • T dependent responses • T independent responses Abbas. Cellular and Molecular immunology. Seventh Edition
    • Recirculating B cells normally pass through lymphoid organs T cell area B cells in blood B cell area Efferent lymph
    • Initial Activation and Migration of Helper T Cells and B Cells Abbas. Cellular and Molecular immunology. Seventh Edition
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • Periphery 1 2 3 Effector phase Abbas. Cellular and Molecular immunology. Seventh Edition
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • Affinity maturation
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • 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
    • Humoral immune response • T dependent responses • T independent responses Abbas. Cellular and Molecular immunology. Seventh Edition
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • T Independent Antigens (TI-1) LPS binding protein Bacterial Lipopolysaccharides, (TI-1 antigens), bind to host LPS binding protein in plasma LPS TLR 4 LPS/LPSBP is captured by CD14 on the B cell surface Toll - like receptor 4 (TLR4) interacts with the CD14/LPS/LPSBP complex CD14 B Cell Activation of B cell
    • T Independent Antigens (TI-1 e.g. LPS) LPS complexes with CD14, LPSBP & TLR4 B B B B B B Y Y Y Y Y Y Six different B cells will require 6 different antigens to activate them At high dose TI-1 antigens (like LPS) will POLYCLONALLY ACTIVATE all of the B cells irrespective fo their specificity. TI-1 antigens are called MITOGENS Y YY YY YY YY YY Y Y Y Y Y Y Y Y Y Y Y Y Y Y YY YY YY YY YY Y
    • T Independent Antigens (TI-2) TI-2 Antigen Immature B-2 Cell YY Mature B-1 Y Immature B cells that bind to multivalent self Ag undergo apoptosis B-2 cell repertoire is purged of cells recognising multivalent antigens during development in the bone marrow Y Y Y IgM Non-bone marrow derived B-1 cells are directly stimulated by antigens containing multivalent epitopes. No T cells are necessary Induces the expression of natural antibodies specific for TI-2 antigens
    • T 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
    • Why are babies unresponsive to TI-2 antigens? In adults: TI-2 Antigen Immature B-2 Cell YY Mature B-1 Y Adult immature B cells that bind to multivalent self Ag undergo apoptosis Y Y Y IgM Adult non-bone marrow derived B-1 cells are directly stimulated by antigens containing multivalent epitopes produce IgM WITHOUT T cell help.
    • Why are babies unresponsive to TI-2 antigens? In babies: All B cells, B-1 & B-2, are immature TI-2 Antigen Immature B-1 Cell Immature B cells that bind to multivalent self Ag undergo apoptosis YY As with adult B cells, immature B cells that bind multivalent self Ag undergo apoptosis Hence babies do not respond to TI-2 antigens. Babies are, therefore susceptible to pathogens with multivalent antigens such as those on pneumococcus
    • T Dependent & Independent Antigens 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
    • 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
    • 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.
    • Abbas. Cellular and Molecular immunology. Seventh Edition
    • 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
    • JACI 2013
    • THE END…..
    • 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)
    • Inhibition Rich. Clinical Immunology. Third Edition.