B 细胞的活化

1. B 细胞的活化

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

4. Antigen Antibody 1 Antibody 3 Antibody 2

5. Antibody formats VH scFv VL CH1 CL CH2 Fab Whole Antibody CH3 SS

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. Types of B cell Antigens T-independent (TI) T-cell dependent (TD) T cell present Ag mitogenic 'activation' signal mitogenesis BcR signal but not mitogenic differentiation -> 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. T-Independent (type II) Responses Emerging Model: DC DC plasma cells B cell B cell Current Paradigm : Multivalent Antigen B cell B cell plasma cells

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

13. T-Dependent Responses Antigen DC T cell T cell B cell plasma 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) Dendritic Cell (DC) internalizes antigen (Ag), processes into peptides, presents peptides together with MHC molecules to T cells

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 ~105 x more efficiently than other antigens

15. B cell antigen presentation and the concept of linked help protein sugar T Protein Specific T cell Sugar Specific B 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 • IFNg -> IgG2a, IgG3 • TGFb 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 negative feedback Reduced tyrosine phosphorylation Elevated basal Ca - but reduced flux Constitutive erk and NFAT No induction of NFkB, JNK

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

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

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. Plasma cell differentiation blimp1 B 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 F T T 4 days RP RP B cellsActivated B cells B cellsPlasma 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. bacteria - dividing every ~60 min 5 days = 2120 divisions = 1.3x1036 B cell antibody response - clonal replication enters into a higher order upon plasma cell differentation 3 days 12 divisions 1 day differentiation 1 day 104 Ab/cell/sec naive B cell activated B cells plasma cells antibodies 4,096 >1012 1 212 = 4,096

29. Ig Heavy chain class (isotype) switching g m e a VDJ 55 kb T cell help (cytokines, CD40L) IgG+ antigen memory cell IgM+ naive B 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 Light Zone - B cells (centrocytes, CC) compete to bind Ag and receive T cell help - selects for cells with higher affinity BCR FDC T T CC T CC FDC FDC memory B cell T CC CC CC CC plasma cell MØ CB CB CB Dark Zone - activated B cells (centroblasts, CB) undergo somatic mutation of Ig V genes CB CB CB CB CB CB CB CB B T

34. DNA replication error 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 V CH1 Met ... Gly Tyr Val His Arg ... ...Gly, Pro... ...GGC, CCT... ATG ... GGC TAT GTT CAC CGT ... A AID dependent mutator complex AID = Activation Induced Deaminase

35. Met ... Gly Tyr Val His Arg ... Asp ...Gly, Pro... ...GGC, CCT... ATG ... GGC TAT GTT CAC CGT ... A Somatic mutation of Ig V region in GC B cell V CH1 -> 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. Affinity maturation improves the ‘fit’ of the antibody for the inducing antigen CDR CDR 1 2 1 3 2 3 Ag Ag before after - increasing the binding affinity Mutations are targeted to antigen binding region of antibody Ag VL VH CH1 CL CH2 CDR = complementarity determining region, also known as the hypervariable region (part of V domain that binds the antigen) CH3

37. Affinity maturation and antibody responses

38. Germinal Center mantle zone (CD23+ naive B cells) GC light zone (CD23++ FDC and centrocytes) GC dark zone (Ki67 cell cycle antigen+ centroblasts) T zone 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 mature B cell helper T cell bacteria Centroblast -CLASS SWITCH RECOMBINATION -SOMATIC HYPERMUTATION DARK ZONE LIGHT ZONE FDC centrocyte Memory B cell apoptosis anergic 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 FcgRII - inhibit signaling

50. Features of primary and secondary antibody responses