Activation of B Cells.ppt
- 1. B cell development, activation and differentiation
- 2. The process is divided into three stages – Generation of mature immunocompetent B cells (maturation) – Activation of mature B cells – Differentiation into plasma and memory B cells
- 3. • Antigen-independent phase – This process is an orderly sequence of Ig-gene in the absence of antigen – These naive B cells are carried to the secondary lymphoid organs (spleen and lymph nodes) • Antigen-dependent phase – Upon activation cells proliferate (clonal expansion) and differentiate to generate plasma and memory B cells. – This involves affinity maturation (increase in the average affinity of antibodies) and class switching (change in isotype of the antibody produced by the B cell) Overview of B-cell development and differentiation B cell development and differentiation
- 4. • The generation of mature B cells starts in embryo and continues throughout life • Before birth, yolk sac, fetal liver and fetal bone marrow are major sites of B-cell maturation • After birth generation of mature B cells occurs in bone marrow B cell maturation
- 5. Progenitor B cells proliferate in bone marrow • Progenitor B cells (pro-B cells) proliferate within bone marrow, filling extravascular spaces between large sinusoids • Proliferation and differentiation of pro-B cells into precursor B cells (pre-B cells) requires microenvironment provided by bone marrow stromal cells • If pro-B cells are removed from bone marrow and cultured in vitro, they will not progress to mature B cell stages unless stromal cells are present • Stromal cells interact directly with pro-B and pre-B cells and secrete various cytokines (IL-7) which support developmental process Bone-marrow stromal cells are required for maturation of progenitor B cells into precursor B cells
- 6. Ig-gene rearrangment produces immature B Cells • First to occur in the pro-B cell stage is a heavy-chain DH-to-JH gene rearrangement followed by a VH-to-DHJH rearrangement • If first heavy-chain rearrangement is not productive, then VH-DH-JH rearrangement continues on the other chromosome • Upon completion of heavy-chain rearrangement, the cell is classified as a pre-B cell. • Development of a pre-B cell into an immature B cell requires a productive light- chain gene rearrangement • Because of allelic exclusion, only one light-chain isotype is expressed on the membrane of a B cell • Completion of a productive light-chain rearrangement commits the now immature B cell to a particular antigenic specificity determined by cell’s heavy-chain VDJ sequence and light-chain VJ sequence • Immature B cells express mIgM (membrane IgM) on cell surface
- 7. • Recombinase enzymes RAG-1 and RAG-2 are required for both heavy and light-chain gene rearrangements • Expressed during the pro-B and pre-B cell stages • Enzyme terminal deoxyribonucleotidyl transferase (TdT) which catalyzes insertion of N-nucleotides at the DH-JH and VH-DHJH coding joints is active during the pro-B cell stage • Because TdT expression is turned off during part of pre -B cell stage when light-chain rearrangement occurs, N- nucleotides are not usually found in VL-JL coding joints Overview of B-cell development and maturation
- 8. Thymus dependent and independent antigens have different requirements for response • Thymus-dependent (TD) antigens requires direct contact with TH cells • Thymus-independent (TI) antigens are divided into types 1 and 2 – TI-1 antigens (e.g., lipopolysaccharide) are polyclonal B-cell activators – TI-2 antigens (e.g., bacterial flagellin) activate B cells by extensively crosslinking mIg receptor Properties of thymus-dependent and thymus-independent antigens
- 9. Two types of signals drive B cells into and through cell cycle • Naive or resting B cells are in G0 stage of cell cycle • Activation drives the resting cell into cell cycle progressing through G1, S, G2 and M phase • The events could be grouped into – Competence signals which drive B cells from G0 into early G1 phase to receive next level of signals – Progression signals which drive cells from G1 into S phase and ultimately to cell division and differentiation (M phase) • Competence is achieved by signal 1 and signal 2 and are generated by different pathways with TI and TD antigens • These include signals generated when multivalent antigens bind and crosslink mIg An effective signal for B-cell activation involves two distinct signals induced by membrane events
- 10. Transduction of activating signals involves Ig-α/Ig-β heterodimers • Compartmentalization of function within receptor subunits • Activation by membrane- associated Src protein tyrosine kinases • Assembly of a large signaling complex with proteintyrosine- kinase activity • Recruitment of other signal- transduction pathways • Changes in gene expression Some of the many signal-transduction pathways activated by the BCR
- 11. B cell co-receptor complex can enhance B cell responses • B cell co-receptor is a complex of three proteins – CD19 (member of the immunoglobulin superfamily, has a long cytoplasmic tail and three extracellular domains) – CR2 (CD21) (a receptor of C3d, a breakdown product of the complement system) – TAPA-1 (CD81) • 104 molecules of mIgM had to be engaged by antigen for B cell activation to occur when co-receptor was not involved (only 102 molecules required when co-receptor was engaged) • Mice were immunized with either unmodified lysozyme or a hybrid of hen’s egg lysozyme to C3d (fusion protein responses were 1000 to 10,000 times greater than to lysozyme alone) B-cell coreceptor is a complex of three cell membrane molecules: TAPA-1 (CD81), CR2 (CD21), and CD19
- 12. In vivo sites for induction of humoral responses • In vivo activation and differentiation of B cells occurs in defined anatomic sites • Blood borne antigens are filtered by spleen, whereas antigens from tissue spaces are filtered by regional lymph nodes • As antigen percolates through cellular architecture of a node, it encounter antigen presenting cells • Once antigen mediated B cell activation takes place, small foci of proliferating B cells form at edges of T cell rich zone • Few activated B cells, together with a few TH cells migrate from foci to primary follicles which develop into secondary follicles • The secondary follicles provide a specialized microenvironment for interactions between B cells, activated TH cells and follicular dendritic cells • The follicular dendritic cells have Fc receptors and complement receptors which retain antigen-antibody complexes for long periods of time • Activated B cells may migrate towards the center of the secondary follicle and form a germinal center Schematic diagram of a peripheral lymph node showing anatomic sites at which various steps in B-cell ctivation, proliferation, and differentiation occur
- 13. Germinal centers and antigen induced B cell differentiation • Germinal centers arise within 7-10 days after initial exposure to a thymus dependent antigen • Proliferating B cells (centroblasts) appear in germinal centers as a well defined dark zone • Centroblasts give rise to centrocytes which are small, nondividing B cells expressing membrane Ig • Centrocytes move from dark zone into a region containing follicular dendritic cells called light zone • Germinal centers are important for affinity maturation, class switching and formation of plasma cells and memory B cells • Affinity maturation was first noticed by H. N. Eisen and G.W. Siskind when they immunized rabbits with the hapten carrier complex DNP- BGG • Average affinity of anti-DNP antibodies increased about 140-fold from 2 weeks to 8 weeks Overview of cellular events within germinal centers
- 14. B cells and germinal center Rapidly dividing and mutating B cells deep in the center Antigen-retaining FDCs Macrophage capturing dead B cells B cells being tested for antibody affinity Dark zone Light zone centrocytes centroblasts
- 15. Role of cytokines in B cell differentiation • Differentiation of B cells is dependent on cytokines • IL-2 is an inducer of proliferation for B cells • Involved in class switching, etc. The interactions of numerous cytokines with B cells generate signals required for proliferation and class switching during the differentiation of B cells into plasma cells