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B CELL DEVELOPMENT IN THE BONE MARROW ORDERED B-CELL DEVELOPMENT immature B cell pre B cell ANTIGEN RECOGNIZING RECEPTOR H-chain + surrogate L-chain pro B cell H2L2 SIGNALING RECEPTOR NO ANTIGEN RECOGNIZING RECEPTOR B B Stromal cell Bone marrow stromal cells nurture developing B cells 1. Specific cell-cell contacts between stromal cells and developing B cells 2. Secretion of cytokines by stromal cells Cell-cell contact B Secreted Factors - CYTOKINES Stromal cell Types of cytokines and cell-cell contacts needed at each stage of differentiation are different Cytokines and cell-cell contacts at each stage of differentiation are different D-J rearranged VLA-4 Early pro-B Stem (Integrin) Receptor Tyrosine kinase Stem cell factor VCAM-1 (Ig superfamily) Cell adhesion molecules c-Kit Cell-bound growth factor Stromal cell Cytokines and cell-cell contacts at each stage of differentiation are different µ-chain made Interleukin-7 receptor V-D-J rearranged Interleukin-7 Growth factor Early pro-B Late pro-B VLA-4 (Integrin) VCAM-1 (Ig superfamily) Stromal cell Pre-B Pre- B cell receptor Heavy chain VHDHJH V-preB CHm l5 Iga & Igb signal transduction molecules Transiently expressed when VHDHJH CHm is productively rearranged VpreB/l5 - the surrogate light chain (SLC), is required for surface expression Ligand for the pre-B cell receptor is unknown Ligation of the pre-B cell receptor 1. Suppresses further H chain rearrangement 2. Triggers entry into cell cycle Large Pre-B Unknown 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 Ligation of the pre-B cell receptor triggers entry into the cell cycle 100X expansion V-J light chain rearranged Proliferation Large Pre-B Large Large Pre-B Large Pre-B Large Pre-B Pre-B V-J light chain expression is quite efficient with an 85% success rate From a single µ chain 85 receptor!!! 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 B cell receptor Heavy chain VHDHJH Light chain VLJLCL CHm Iga & Igb signal transduction molecules L chain is rearranged Stages of B cell development Stem Cell Early pro-B cell Late pro-B cell Large pre-B cell Peripheral Y Small pre-B cell Immature B cell Y Receptor H+L Mature B cell Receptor H+L Each stage of development is defined by IgH and IgL chain genes, expression of adhesion molecules and cytokine receptors RECEPTOR EXPRESSION DURING B-CELL DEVELOPMENT Allelic exclusion even though every B cell possesses a maternal and paternal locus of both genes, B cells express a single heavy and light chain. Does this „crippled” expression serve a purpose? Many of the genes (not all) are expressed co-dominantly, how could B cells manage to silence their other BCR-coding allels? ALLELIC EXCLUSION Evidence for allelic exclusion ALLOTYPE- a polymorphism in the Heavy chain C region of Ig Allotypes can be identified by staining B cell surface Ig with antibodies B a 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 Allelic exclusion is needed for efficient clonal selection Antibody S. typhi S. typhi 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 to prevent the emergence of new daughter specificities during proliferation after clonal selection Allelic exclusion prevents unwanted responses One Ag receptor per cell IF there were two Ag receptors per cell Y YY S. aureus Y Y Y Y Anti S. aureus Antibodies B S. aureus Anti Liver cell Abs Y Y Y Y Self antigen expressed by e.g. Liver cells YY B Anti S. aureus Antibodies Suppression of H chain gene rearrangement ensures only one specificty of Ab expressed per cell. Prevents induction of unwanted responses by pathogens Allelic exclusion is needed to prevent holes in the repertoire One specificity of Ag receptor per cell IF there were two specificities of Ag receptor per cell Anti-brain Ig Anti-brain Ig AND anti-S. Aureus Ig B B Exclusion of anti-brain B cells i.e. self tolerance B Deletion OR B BUT anti S.Aureus B cells will be excluded leaving a “hole in the repertoire” Anergy B S. aureus THE RESULT OF SOMATIC GENE REARRANGEMENTS 1. Combination of gene segments results in a huge number of various variable regions of the heavy and light chains expressed by different B-cells SOMATIC GENE REARRANGEMENT 2. Successful somatic rearrangement in one chromosome inhibits gene rearrangement in the other chromosome ALLELIC EXCLUSION 3. One B-cell produces only one type of heavy and one type of light chain COMMITMENT TO ONE TYPE OF ANTIGEN BINDING SITE 4. The B-cell pool consist of B-cells with differently rearranged immunoglobulin genes INDEPENDENT OF ANTIGEN OCCURS DURING B-CELL DEVELOPMENT IN THE BONE MARROW Allelic exclusion helps diagnose and monitor lymphoma: Due to clonal expansion of a single cell that contains a unique rearrangement the amount of cancer cells in blood or in bone marrow can be determined Can be used to monitor residual tumor cells upon treatment SYNTHESIS OF IMMUNOGLOBULINS Secreted Ig Membrane Ig Golgi ER H and L chains are synthesized on separated ribosomes CHAPERONES Leader sequence Ribosome mRNA DEVELOPMENT OF B-LYMPHOCYTES IN THE BONE MARROW Limphoid precursor B cells recognizing self structures Cell surface molecules MHC proteins Common molecules of haemopoetic cells c-kit/CD44 RAG-1/RAG-2 H H rearrrangement átrendeződés m apoptosis, clonal deletion Soluble molecules House keeping genes Metabolites Surrogate L L rearrangement B functional unresponsiveness anergia Other specificites Selection clonal deletion B B B PERIPHERAL LYMPHOID TISSUES Negative selection of immature B-cells in the bone marrow BONE MARROW 1 2 3 4 5 n Potential B-cell repertoire Self recognition Clonal deletion Self structure PERIPHERAL LYMPHOID ORGANS RNA editing Foreign antigen independent Available B-cell repertoire About 30 billion mature naive B cells leave the bone marrow per day to circulate in blood Immature B cells with specificity for multivalent self antigens are retained in the bone marrow. Receptor Editing of Immature B cells with self-reactive BCR (Bone Marrow) Immature B cells specific for monovalent self antigens develop a state of anergy. Anergic B cells have a half life of 4-5 days (10% that of regular B cells) How can mature B-cells express surface IgM and IgD Co-Expression of cell surface IgM and IgG On Mature B-cells is controlled by alternative RNA processing RESULT OF SOMATIC GENE REARRANGEMENT AND ALLELIC EXCLUSION 1. Somatic rearrangement of Ig gene segments in a highly controlled manner 2. Single B-cells become committed to the synthesis of one unique H-chain and one unique L-chain variable domain, which determine their specificities 3. In one individual a large B-cell repertoire is generated consisting of B-cell clones with different H- and L-chain variable domains 4. This potential B-cell repertoire is able to recognize a wide array of various antigens 5. Immature B-cells express IgM and IgD surface Ig with the same variable domains B – CELL ACTIVATION Where and how do all these things take place? B-cell recycling in the absence of antigen (lymph node) T cell area B cells in blood B cell area Efferens lymph Recirculating B cells are trapped by foreign antigens in lymphoid organs Antigen enters node in afferent lymphatic YY Y B cells proliferate rapidly B cells leave blood & enter lymph node via high endothelial venules Y YY Y Y Y GERMINAL CENTRE Transient structure of Intense proliferation YY Y Germinal centre releases B cells that differentiate into plasma cells Germinal Center Reaction „Dating” in the peripheral lymphoid organs The structure of the germinal centre Somatic hypermutation LZ FDC DZ Somatic hypermutation LZ: light zone DZ: dark zone FDC: follicular dendritic cell Antigen is bound on the surface of follicular dendritic cells (FDC) FDC FDC-s bind immune complexes (Ag-Ab ) Ag detectable for 12 months following immunization A single cell binds various antigens B cells recognize Ag on the surface of FDC Fig. 9.15. On the surface of FDC-s immune complexes form the so-called iccosomes,that can be released and taken up later by the surrounding germinal center B cells T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID ORGANS IgM IgG IgA IgE SIGNALING UNITS OF THE B-CELL RECEPTOR Ig-a/CD79a Ig-b/CD79b a b Y ITAM Y Ig domain + CHO Y Y ITAM ITAM: YxxL x7 YxxI ITAM: Immunoreceptor Tyrosine-based Activation Motif Main steps of B-cell signal transduction THE CO-STIMULATORY ROLE OF CR2 (CD21) COMPLEMENT RECEPTOR IN B – LYMPHOCYTES C3d ANTIGÉN Antigenic determinant CD21/CR2 CD19 B-CELL TAPA=CD81 Y Y Enhanced B-cell activation THE NEURAMIC ACID RECEPTOR CD22 INHIBITS ACTIVATION THROUGH THE A B-CELL RECEPTOR Mannose Tissue cells Bacterium Neuraminic acid Antigen B Cell CD22 Inhibited B cell activation EFFECTOR FUNCTIONS OF ANTIBODIES INHIBITION Binding of bacteria to epithelial cells Binding of viruses to receptor Binding of bacterial toxins to target cells NEUTRALIZATION Small proportion of antibodies OPSONIZATION COMPLEMENT ACTIVATION Binding of antibody increases phagocytosis PLASMA CELL FcR Opsonization by C3b Complement C3b FcR FcR CR1 PHAGOCYTES ENGULFMENT, DEGRADATION