B CELL DEVELOPMENT IN THE BONE MARROW

1. B CELL DEVELOPMENT IN THE BONE MARROW

2. a a ORDERED B-CELL DEVELOPMENT immature B cell pre B cell ANTIGEN RECOGNIZING RECEPTOR H2L2 pro B cell H-chain + surrogate L-chain SIGNALING RECEPTOR NO ANTIGEN RECOGNIZING RECEPTOR

3. B B Stromal cell

4. 1. Specific cell-cell contacts between stromal cells and developing B cells 2. Secretion of cytokines by stromal cells Secreted Factors - CYTOKINES B Cell-cell contact Stromal cell Bone marrow stromal cells nurture developing B cells Types of cytokines and cell-cell contacts needed at each stage of differentiation are different

5. c-Kit Receptor Tyrosine kinase Stem VLA-4 (Integrin) Stem cell factor Cell-bound growth factor VCAM-1 (Ig superfamily) Cell adhesion molecules Stromal cell Cytokines and cell-cell contacts at each stage of differentiation are different D-J rearranged Early pro-B

6. Interleukin-7 receptor Interleukin-7 Growth factor Early pro-B Stromal cell VLA-4 (Integrin) VCAM-1 (Ig superfamily) Cytokines and cell-cell contacts at each stage of differentiation are different µ-chain made V-D-J rearranged Late pro-B Pre-B

7. Heavy chain VHDHJH V-preB l5 CHm Iga & Igb signal transduction molecules Pre- B cell receptor 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

8. Large Pre-B Unknown ligand of pre-B cell receptor Stromal cell Ligation of the pre-B cell receptor 1.Suppresses further H chain rearrangement 2. Triggers entry into cell cycle 1. Ensures only one specificty of Ab expressed per cell 2. Expands only the pre-B cells with in frame VHDHJH joins ALLELIC EXCLUSION

9. Proliferation Large pre-B Y Immature B cell IgM Small pre-B Large pre-B Large Pre-B Large Pre-B Proliferation stops Pre-receptor not displayed Light chain expressed IgM displayed on surface Large Pre-B Large Pre-B Intracellular VDJCH chain VL-JL rearranges Large Pre-B Large Pre-B Large Pre-B Large Pre-B Large Pre-B Large Pre-B Ligation of the pre-B cell receptor triggers entry into the cell cycle 100X expansion Many large pre-B cells with identical pre-B receptors V-J lightchain rearranged V-J lightchainexpression is quiteefficientwith an 85% successrate From a single µ chain 85 receptor!!!

10. Heavy chain VHDHJH Light chain VLJLCL CHm Iga & Igb signal transduction molecules B cell receptor L chain is rearranged

11. RECEPTOR EXPRESSION DURING B-CELL DEVELOPMENT

12. 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

13. Y a B a/a Y b B b/b Y a B Y Y Y b a B B b a/b 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 AND Suppression of H chain rearrangement by pre-B cell receptor prevents expression of two specificities of antibody per cell

14. S. typhi S. typhi Allelic exclusion is needed for efficient clonal selection Antibody 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

15. Y Y Y Y Y B Self antigen expressed by e.g. Liver cells B Y Y Y Y S. aureus S. aureus Y Y Y Y Y Y Y Y Y Y Y Y Anti S. aureus Antibodies Anti S. aureus Antibodies Anti Liver cell Abs Y Y Y Y Y Y Y Y Allelic exclusion prevents unwanted responses One Ag receptor per cell IF there were two Ag receptors per cell Suppression of H chain gene rearrangement ensures only one specificty of Ab expressed per cell. Prevents induction of unwanted responses by pathogens

16. One specificity of Agreceptor per cell IF there were two specificitiesof Ag receptor per cell Y Y Y S. aureus Y B B Y B B B B Y OR Y Y B B Deletion Anergy Allelic exclusion is needed to prevent holes in the repertoire Anti-brain Ig Anti-brain Ig AND anti-S. Aureus Ig Exclusion of anti-brain B cells i.e. self tolerance anti S.Aureus B cells will be excluded leaving a “hole in the repertoire” BUT

17. THE RESULT OF SOMATIC GENE REARRANGEMENTS • 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

18. 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

19. Stem Cell Early pro-B cell Late pro-B cell Large pre-B cell Peripheral Small pre-B cell Immature B cell Mature B cell Stages of B cell development Y Receptor H+L Y Receptor H+L Each stage of development is defined by IgH and IgL chain genes, expression of adhesion molecules and cytokine receptors

20. SYNTHESIS OF IMMUNOGLOBULINS Secreted Ig Membrane Ig Golgi ER H and L chains are synthesized on separated ribosomes CHAPERONES Leader sequence Ribosome mRNA

21. DEVELOPMENT OF B-LYMPHOCYTES IN THE BONE MARROW Limphoid precursor B B B B c-kit/CD44 L rearrangement Surrogate L RAG-1/RAG-2 H rearrrangement  Selection clonal deletion • B cells recognizing self structures • Cell surface molecules • MHC proteins • Common molecules of haemopoetic cells • apoptosis, clonal deletion • Soluble molecules • House keeping genes • Metabolites • functional unresponsiveness • anergia H átrendeződés Other specificites PERIPHERAL LYMPHOID TISSUES

22. Self recognition Clonal deletion Self structure Negative selection of immature B-cells in the bone marrow Potential B-cell repertoire BONE MARROW RNA editing PERIPHERAL LYMPHOID ORGANS Available B-cell repertoire Foreign antigen independent About 30 billion mature naive B cells leave the bone marrow per day to circulate in blood

23. Immature B cells with specificity for multivalent self antigens are retained in the bone marrow.

24. Receptor Editing of Immature B cells with self-reactive BCR (Bone Marrow)

25. 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)

26. How can mature B-cells express surface IgM and IgD

27. Co-Expression of cell surface IgM and IgG On Mature B-cells is controlled by alternative RNA processing

28. RESULT OF SOMATIC GENE REARRANGEMENT AND ALLELIC EXCLUSION • Somatic rearrangement of Ig gene segments in a highly controlled manner • Single B-cells become committed to the synthesis of one unique H-chain and one unique L-chain variable domain, which determine their specificities • In one individual a large B-cell repertoire is generated consisting of B-cell clones with different H- and L-chain variable domains • This potential B-cell repertoire is able to recognize a wide array of various antigens • Immature B-cells express IgM and IgD surface Ig with the same variable domains

29. B – CELL ACTIVATION Where and how do all these things take place?

31. B cells in blood T cell area B cell area Efferens lymph B-cell recycling in the absence of antigen (lymph node)

32. B cells proliferate rapidly B cells leave blood & enter lymph node via high endothelial venules Antigen enters node in afferent lymphatic Y Y Y Y Y Y Y Y Y Y Y Y Y Y Germinal centre releases B cells that differentiate into plasma cells Y Y Y Y GERMINAL CENTRE Transient structure of Intense proliferation Recirculating B cells are trapped by foreign antigens in lymphoid organs

33. Germinal Center Reaction

34. „Dating” in the peripheral lymphoid organs

35. The structure of the germinal centre Somatic hypermutation LZ FDC DZ Somatic hypermutation LZ: light zone DZ: dark zone FDC: follicular dendritic cell

36. 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 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 B cells recognize Ag on the surface of FDC

37. T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID ORGANS IgM IgG IgA IgE

38. Ig domain + CHO a b ITAM ITAM Y Y Y Y SIGNALING UNITS OF THE B-CELL RECEPTOR Ig-a/CD79a Ig-b/CD79b ITAM: YxxLx7YxxI ITAM: Immunoreceptor Tyrosine-based Activation Motif

39. Main steps of B-cell signal transduction

40. THE CO-STIMULATORY ROLE OF CR2 (CD21) COMPLEMENT RECEPTOR IN B – LYMPHOCYTES C3d ANTIGÉN Antigenic determinant CD21/CR2 CD19 TAPA=CD81 Y Y B-CELL Enhanced B-cell activation

41. Mannose Tissue cells Bacterium Antigen B Cell THE NEURAMIC ACID RECEPTOR CD22 INHIBITS ACTIVATION THROUGH THE A B-CELL RECEPTOR Neuraminic acid CD22 Inhibited B cell activation

42. OPSONIZATION Binding of antibody increases phagocytosis FcR COMPLEMENT ACTIVATION Opsonization by C3b PLASMA CELL Complement C3b FcR FcR CR1 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 PHAGOCYTES ENGULFMENT, DEGRADATION