1. B 细胞的活化

2. Lecture Outline 1. What is meant by ‘ B cell activation ’ ?1. What is meant by ‘ B cell activation ’ ? 2. Why do some B cell responses require T cell help and others not?2. Why do some B cell responses require T cell help and others not? 3. What is ‘ T cell help ’ ?3. What is ‘ T cell help ’ ? 4. What are plasma cells? 5. How do activated B cells undergo ‘ affinity maturation ’ ?5. How do activated B cells undergo ‘ affinity maturation ’ ? 6. What is the mechanism for B cell memory?

4. Antigen Antibody 1 Antibody 2 Antibody 3

5. Antibody formats VLVL VHVH CLCL C H1 C H2 C H3 scFv F ab SS Whole Antibody

6. What is meant by ‘B cell activation’? Go -> G1 of cell cycle (increase in size) upregulate – MHC class II – costimulatory molecules (B7-2) – adhesion molecules (ICAM1) – cytokine receptors (IL-2R) migrate to outer T zone – altered response to chemokines become receptive to T cell help – protected from fas enter mitosis if provided with submitogenic doses of other stimuli (LPS, CD40L, IL-4)

7. Types of antigen T-independent (TI) antigens - Type I –induce division/differentiation independently of BCR (polyclonal mitogens) LPS, bacterial (CpG) DNA T-independent (TI) antigens - Type II –induce division/differentiation by BCR signaling alone bacterial polysaccharides, repeating surface molecules on viruses T-dependent (TD) antigens –activate via BCR but depend on additional signals from helper T cells to cause division/differentiation any antigen containing protein Most pathogens contain both T-I and T-D antigens Only TD antigens can induce Germinal Center responses

8. T cell mitogenic BcR signal 'activation' signal but not mitogenic mitogenesis differentiation present Ag T-independent (TI) T-cell dependent (TD) Types of B cell Antigens -> most pathogens contain both T-independent and T-dependent antigens

9. Innate features of pathogens act as B cell costimulators pathogen multivalency –provides a level of BCR crosslinking optimal for activation many pathogens activate TLRs –TLR signaling synergizes with BCR signal many pathogens activate the complement cascade and become C3d coated –complement receptor (CR) crosslinking synergizes with BCR signal

10. Antigen-C3d complexes cross-link BCR and CR2- CD19 complex - increase sensitivity to antigen

11. Current Paradigm : B cellplasma cellsB cell plasma cells DC Emerging Model: DC B cell T-Independent (type II) Responses Multivalent Antigen

12. DCs support Ag-specific plasma cell differentation PC-binding Plasma cell marker 3 day co-cultures -> DCs are a source of BAFF -> Costimulatory effect of DCs partly due to BAFF Balazs et al., Immunity 17, 341 (2002)

13. B cellplasma cells T-Dependent Responses DC Antigen T cell Dendritic Cell (DC) internalizes antigen (Ag), processes into peptides, presents peptides together with MHC molecules to T cells B cell binds Ag via surface Ig, transmits BCR signals and presents peptides to T cells, receives T cell help (growth and differentiation factors) Secretes Antibody (Ab)

14. B cells are antigen-presenting cells BCR cross-linking induces antigen internalization to endosomes antigen is proteolysed to peptides peptides associate with MHC class II MHC class II-peptide complexes traffic to surface of B cell\ B cells present antigen recognized by their BCR ~10 5 x more efficiently than other antigens

15. B cell antigen presentation and the concept of linked help protein sugar T Sugar Specific B cell Protein Specific T cell Antigen internalization, proteolysis -> presentation of peptides

16. Cardinal features of B - T interaction

17. Role of CD40 in B cell activation TCR triggering up- regulates CD40L on T cell CD40 signaling promotes B cell activation CD40L-deficiency = 'hyper- IgM syndrome' (no isotype switching, no Germinal Centers) Note: CD40 signaling also important in DC, Macrophage function increased expression of cell cycle molecules, survival molecules, cytokines, promotes isotype switching

18. Key components of T cell help CD40L triggers CD40 -> synergizes with BCR signals to promote mitosis; cytokine (e.g. IL4) signals also contribute FasL triggers Fas -> BCR signaling protects from apoptosis T cell derived cytokines influence differentiation, isotype switching: – IL2, IL6 promote differentiation – IL4 -> IgG1, IgE – IFN  -> IgG2a, IgG3 – TGF  and IL5 -> IgA Many other molecules involved in T-B interactions – e.g. ICAM1/LFA1, CD30/CD30L, CD27L/CD27, OX40L/OX40,...

19. Altered signaling in anergic B cells Reduced tyrosine phosphorylation Elevated basal Ca - but reduced flux Constitutive erk and NFAT No induction of NFkB, JNK negative feedback

21. Anergic B cells can respond to 'stronger' antigens

22. membrane Ig secretory Ig B cell Plasma Cell After appropriate activation the B cell differentiates into an antibody secreting cell, also known as a Plasma Cell

23. Production of membrane vs secreted Ig

24. Plasma Cells are antibody secreting cells Two types: 1. Plasma cells generated early in the primary response - short-lived (~ few days) - typically low affinity - form in T-independent and T-dependent responses - home to red pulp of spleen, medullary cords of lymph nodes - IgM but also IgG and other isotypes 2. Plasma cells generated later in the primary response and that predominate in secondary responses - arise predominantly from germinal centers (in primary) or from memory B cells (in secondary) - long-lived (possibly several months) - often home to bone marrow, gut, lactating mammary gland - predominantly isotype switched

25. B blimp1 XBP1 Plasma cell differentiation Blimp1 represses PAX5 and genes associated with B cell identity, induces increases in a subset of plasma cell genes (Syndecan, XBP1) and induces conversion of membrane to secretory Ig XBP1 is a component of the UPR (unfolded protein response) Increased sIg production ‘stresses’ ER and promotes processing of XBP1 mRNA into active transcript XBP1 induces increases in –Secretory pathway gene expression –Organelle biogenesis –Cell size and protein synthesis Shaffer et al., Immunity. 21:81-93

26. B cell migration in the spleen during differentiation to plasma cells 1 day after antigen 5 days after antigen F T RP F T 4 days B cells Activated B cellsB cells Plasma cells

27. Plasma Cell Migration Summary  SDF1 for splenic red-pulp, LN medullary cords, Bone Marrow  TECK/CCR9 for small intestine (IgA)  MEC/CCR10 for large intestine, stomach, salivary glands (IgA)

28. naive B cell activated B cells 3 days 12 divisions plasma cells 1 day differentiation 1 day 10 4 Ab/cell/sec antibodies 12 12 = 4,096 4,096>10 12 B cell antibody response - clonal replication enters into a higher order upon plasma cell differentation bacteria - dividing every ~60 min 5 days = 2 120 divisions = 1.3x10 36

29. Ig Heavy chain class (isotype) switching VDJ    55 kb (cytokines, CD40L) T cell help antigen IgM+ naive B cell IgG+ memory cell IgG secreting plasma cell

30. Ig Heavy chain class (isotype) switching

31. Affinity Maturation Affinity maturation occurs in germinal centers and is the result of somatic hypermutation of Ig-genes in dividing B cells followed by selection of high affinity B cells by antigen displayed by FDCs The high affinity B cells emerging in germinal centers give rise to long-lived plasma cells and memory B cells

32. Germinal Centers Function: to generate B cells that produce antibodies with increased affinity for the inducing antigen => affinity maturation Germinal Center Reaction: Activated B cells give rise to Centroblasts - localize in follicle, undergo rapid cell division and turn on machinery that causes somatic mutation in V-regions Centroblasts give rise to Centrocytes - migrate to the FDC-rich region of the Germinal Center - survival is dependent on interaction with FDC-bound Ag and presentation of Ag to T cells - centrocytes that successfully compete to bind antigen (e.g. by having higher affinity BCR) and that receive T cell help are selected and may differentiate into long-lived plasma cells or memory B cells

33. Germinal Center Dark Zone - activated B cells (centroblasts, CB) undergo somatic mutation of Ig V genes FDC CB CC CB T T T T CC memory B cell plasma cell CB MØMØ B T Light Zone - B cells (centrocytes, CC) compete to bind Ag and receive T cell help - selects for cells with higher affinity BCR

34. AID dependent mutator complex DNA replication error ATG... GGC TAT GTT CAC CGT... V C H1 A...GGC, CCT... Met... Gly Tyr Val His Arg......Gly, Pro... AID = Activation Induced Deaminase Somatic mutation of Ig V region in GC B cell -> mutations are actively induced in the V-regions of the antibody heavy and light chain genes

35. ATG... GGC TAT GTT CAC CGT... Met... Gly Tyr Val His Arg... A Asp...GGC, CCT......Gly, Pro... V C H1 Somatic mutation of Ig V region in GC B cell -> now encodes antibody molecule with slightly altered antigen binding site -> sometimes, by chance, this site will have an improved ability to bind the inducing antigen (i.e. a higher affinity)

36. VHVH VLVL CLCL C H1 CDR 1 23 C H2 C H3 CDR 12 3 Ag Mutations are targeted to antigen binding region of antibody CDR = complementarity determining region, also known as the hypervariable region (part of V domain that binds the antigen) Ag before Affinity maturation improves the ‘fit’ of the antibody for the inducing antigen after Ag - increasing the binding affinity

37. Affinity maturation and antibody responses

38. mantle zone GC light zone (CD23 ++ FDC and centrocytes) GC dark zone T zone (Ki67 cell cycle antigen + centroblasts) (CD23 + naive B cells) Germinal Center from Liu et al., Immunology Today 13, 17-21 (1992)

39. Germinal Center from Hutloff et al., Nature 397, 263-6 (1999)

41. B cell differentiation in the germinal center centrocyte FDC mature B cell helper T cell bacteria LIGHT ZONE -CLASS SWITCH RECOMBINATION -SOMATIC HYPERMUTATION DARK ZONE Centroblast apoptosis anergic Memory B cell Plasma B cell

48. Memory B cells Generated in germinal centers –therefore we only have humoral memory to T-dependent antigens Small, recirculating cells Typically isotype switched (e.g. IgG + or IgA + ) Typically have higher affinity for the inducing Ag Longer lived than naïve B cells –Persistence of memory B cells after an immune response ensures that we have increased numbers of B cells specific for the antigen and ready to respond on re-encounter

49. Antigen-IgG complexes cross-link BCR and Fc  RII - inhibit signaling

50. Features of primary and secondary antibody responses

51. Tumors of B Cells Lymphoid progenitor Tumor Equivalent normal cell Location Chronic lymphocytic leukemia CD5 B-1 B cellBlood Acute lymphoblastic leukemia Bone marrow Blood Pre-B cell leukemia Pre-B cell Follicular center cell lymphoma Burkitt’s lymphoma Mature B cell Periphery Waldenstrom’s macroglobulinemia B cell IgM secreting Multiple myelomaPlasma cell secreting various Ig isotypes Bone marrow Related information:

52. Effects on Cells of Immune System Lymphocyte Macrophage B cell T cell NKLAK Proliferation, Differentiation, Ig secretion and selection Proliferation, Differentiation, Cytokine production Activation of cells of immune system Cytokine production IL1 IL2 IL4 IL1 IL2 IL4 IL5 IL6 IFNγ cytokines Related information:

53. Recommended Reading Primary papers: Jun, J.E. et al. (2003) Identifying the MAGUK protein Carma-1 as a central regulator of humoral immune responses and atopy by genome-wide mouse mutagenesis. Immunity 18:751-62 Lesley, R. et al. (2004) Reduced competitiveness of autoantigen-engaged B cells due to increased dependence on BAFF. Immunity. 2004 20:441-53 Shaffer et al. (2004) XBP1, downstream of Blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation. Immunity. 21:81-93 Reviews: Kurosaki, T. (2002) Regulation of B cell fates by BCR signaling components. Curr Opin Immunol. 14(3):341-7. Mackay, F., Schneider,P., Rennert,P. and Browning, J. (2002) BAFF and APRIL: A Tutorial on B Cell Survival. Annu. Rev. Immunol.; 10.114 Kurosaki, T. (1999) Genetic analysis of B cell antigen receptor signaling. Annu. Rev. Immunol. 17: 555-592 Healy, J.I., and C.C. Goodnow. 1998. Positive versus negative signaling by lymphocyte antigen receptors. Annu Rev Immunol 16:645-670. Ravetch, J.V. and Bolland, S. (2001) IgG Fc Receptors. Annu. Rev. Immunol. 19, 275 MacLennan, I.C.M. 1994. Germinal Centers. Annu. Rev. Immunol. 12:117-139

54. Thank you!