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  • T-independent antigens bacterial polysaccharides low dose LPS repeating surface molecules on viruses TLR signaling can synergize with this type of signal T-dependent antigens usually require a protein component, but B cell may recognize a non-protein part of the complex (e.g. a surface sugar or lipid of a virus) most pathogens contain both T-independent and T-dependent antigens
  • Penicillin can induce both IgE and immediate type HS and IgG, leading to immune complex HS Why do some people respond to Penicillin if there are no costimulatory inputs? Perhaps there are I.e. as penicillin is given to people with bacterial infection Note that allergies of this type have a genetic basis and do tend to run in families
  • Pasare C, Medzhitov R. Control of B-cell responses by Toll-like receptors. Nature. 2005 Nov 17;438(7066):364-8. D12 after flagellin
  • Coro ES, Chang WL, Baumgarth N. Related Articles, Links Abstract Type I IFN receptor signals directly stimulate local B cells early following influenza virus infection. J Immunol. 2006 Apr 1;176(7):4343-51. D5 of flu infection Le Bon A, Thompson C, Kamphuis E, Durand V, Rossmann C, Kalinke U, Tough DF. Related Articles, Links Abstract Cutting edge: enhancement of antibody responses through direct stimulation of B and T cells by type I IFN. J Immunol. 2006 Feb 15;176(4):2074-8. Serum Ab at d10
  • Electron microscopy Images from Molecular Biology of the Cell Alberts et al., 3rd Edition., 1994
  • AID dependent
  • Certainly a lot of work has been done on fixed tissue sections and with isolated cells and this has led to a general model that proliferation and somatic hypermutation occur predominantly in the dark zone while selection occurs in the light zone. Strong experimental evidence has been obtained to indicate that there is a movement of cells from dark to light zone. In our own studies, work that was presented in workshop here 2 yrs ago by Chris Allen, we showed that CB positioning in the DZ is CXCR4 dependent whereas movement to the LZ involves CXCR5. But, we have known very little about the dynamics…
  • Superimposed tracks of 40 randomly selected cells.
  • Stably interacting cells moved at less than 10um/min. Only 32% of total GC T cells moved at less than 10um/min. Data suggest that majority of T cells in GCs were not engaged in stable interactions with B cells. Data suggest that each GC B cells encounters as many as 50 T cells per hour…Seems that T cell help may be limiting not only because there are fewer T cells than B cells but also because of additional mechanisms suppressing stable B-T interactions in GCs.
  • Secreted IgA functions by neutralizing pathogens or their toxins. Note: the J-chain polypeptide that holds the two IgA molecules together as a covalent dimer (and that functions to hold IgM together as a pentamer) is not the same as the joining (J) element that is part of the V-D-J region of the Ig heavy chain!
  • Colostrum is the protein-rich fluid secreted by the early postnatal mammary gland; human milk contains little IgG (but does contain IgA). After 6 months protection is essentially gone which explains the high incidence of disease caused by bacteria such as Haemophilus influenzae, especially H. flu meningitis. That is why the conjugate vaccine is so important. 2. Neonatal protection is only as good as the titer of IgG in the mother against the specific organism. So child could be infected if mother doesn't have IgG. Passive immunity can sometimes be harmful: if an Rh- mother mounts Ab response against Rh+ newborn due to red cell transfer during birth, those anti-Rh antibodies will be transferred into the blood supply of the next fetus and (if it is Rh+) cause lysis of the red cells
  • In the first week, before the GC response, IgM and some IgG are produced but mostly low affinity. By day 10 the GC response has gotten underway and you start to make high affinity antibody. Antibody secreting cells are the same thing as Plasma cells - they are present in secondary lymphoid tissues, bone marrow and in the mucosal epithelia. Memory B cells arising from GC are higher affinity. On secondary exposure you are able to make a more rapid response because you have more B cells present that are specific for the antigen (and they have other differences that make them fast responders). Plasma cells emerging from GC home preferentially to the bone marrow and live for many months or even years, contributing to long term antibody production.
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    1. 1. B Cell Activation Micro 204. Molecular and Cellular Immunology 2009 Lecturer: Jason Cyster
    2. 2. Antigen Antibody 1 Antibody 2 Antibody 3
    3. 3. Lecture Outline <ul><li>1. What factors promote B cell survival? </li></ul><ul><li>2. Why do some B cell responses require T cell help and others not? </li></ul><ul><li>3. What is ‘T cell help’? </li></ul><ul><li>4. What are plasma cells? </li></ul><ul><li>5. How do activated B cells undergo ‘affinity maturation’? </li></ul><ul><li>6. What is the mechanism for B cell memory? </li></ul><ul><li>7. How are antibody responses down-regulated? </li></ul>
    4. 5. Early positive and negative events in BCR signaling
    5. 6. Constitutive BCR signaling is necessary for B cell survival V-region loxP loxP IgM constant-region from Lam et al., Cell 90, 1073 (1997) Cre-recombinase
    6. 7. BAFF / Blys and B cell survival <ul><li>Member of TNF family </li></ul><ul><li>Made by stromal cells, macrophages, DCs </li></ul><ul><li>BAFF-receptor constitutively expressed on B cells </li></ul><ul><ul><li>BAFF also binds TACI and BCMA </li></ul></ul><ul><ul><li>APRIL is a second TACI, BCMA ligand </li></ul></ul><ul><li>Critical role in B cell homeostasis </li></ul><ul><ul><li>BAFF knockout has ~100x less B cells </li></ul></ul><ul><ul><li>A/WySnJ mouse strain has mutation in BAFFR and has ~10x reduced B cell numbers </li></ul></ul><ul><ul><li>BAFF over-expression increases total B cell numbers and promotes autoantibody production </li></ul></ul><ul><ul><li>BAFF serum levels elevated in lupus </li></ul></ul>TACI BCMA BAFF Stromal cell, MØ, DC B cell BAFFR NF  B (alternative & classical pathways) -> Bcl2, Pim, other -> Cell Survival
    7. 8. BAFF/Blys is necessary for B cell survival from Schiemann et al., Science 293, 2111 (2001) TACI BCMA BAFF B cell BAFFR MØ (BAFFR mutant)
    8. 9. What is meant by ‘B cell activation’? <ul><li>Go -> G1 of cell cycle (increase in size) </li></ul><ul><li>upregulate </li></ul><ul><ul><li>MHC class II </li></ul></ul><ul><ul><li>costimulatory molecules (B7-2) </li></ul></ul><ul><ul><li>adhesion molecules (ICAM1) </li></ul></ul><ul><ul><li>cytokine receptors (IL-2R) </li></ul></ul><ul><li>migrate to outer T zone </li></ul><ul><ul><li>altered response to chemokines </li></ul></ul><ul><li>become receptive to T cell help </li></ul><ul><ul><li>protected from fas </li></ul></ul><ul><li>enter mitosis if provided with submitogenic doses of other stimuli (LPS, CD40L, IL-4) </li></ul>
    9. 10. Early events in B cell activation by membrane associated antigen
    10. 11. Types of antigen <ul><li>T-independent (TI) antigens </li></ul><ul><ul><li>induce division/differentiation by BCR signaling alone </li></ul></ul><ul><ul><ul><li>bacterial polysaccharides, repeating surface molecules on viruses </li></ul></ul></ul><ul><li>T-dependent (TD) antigens </li></ul><ul><ul><li>activate via BCR but depend on additional signals from helper T cells to cause division/differentiation </li></ul></ul><ul><ul><ul><li>any antigen containing protein </li></ul></ul></ul><ul><li>Most pathogens contain both T-I and T-D antigens </li></ul><ul><li>Only TD antigens can induce Germinal Center responses </li></ul><ul><li>Polyclonal Mitogens </li></ul><ul><ul><li>induce division/differentiation independently of BCR </li></ul></ul><ul><ul><ul><li>high dose LPS, polyclonal anti-IgM, anti-CD40 </li></ul></ul></ul><ul><ul><ul><li>mostly observed in vitro </li></ul></ul></ul>
    11. 12. 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
    12. 13. Old Paradigm: B cell plasma cells B cell T-Independent (type II) Responses Multivalent Antigen B cell plasma cells DC/Mø Emerging Model: DC/Mø B cell (BAFF)
    13. 14. B cell plasma cells T-Dependent Responses DC Antigen T cell 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. 15. B cells are antigen-presenting cells <ul><li>BCR cross-linking induces antigen internalization to endosomes </li></ul><ul><li>antigen is proteolysed to peptides </li></ul><ul><li>peptides associate with MHC class II </li></ul><ul><li>MHC class II-peptide complexes traffic to surface of B cell </li></ul><ul><li>B cells present antigen recognized by their BCR ~10 5 x more efficiently than other antigens </li></ul>
    15. 16. B cell antigen presentation and the concept of linked help protein sugar Polysaccharide Specific B cell BCR MHC II T Protein Specific T cell CD40L Cytokines endosome
    16. 17. Haptens <ul><li>Small organic molecules of simple structure do not provoke antibodies by themselves. However, antibodies can be raised against them if the molecule is attached to a protein carrier. </li></ul><ul><li>Such small molecules are termed haptens (from the Greek haptein , to fasten). </li></ul><ul><li>Some drugs such as Penicillin can act as haptens and induce antibody-mediated allergic reactions. </li></ul>
    17. 18. Making an antibody response to a hapten protein hapten Hapten Specific B cell T Haptenated-peptide Specific T cell CD40L Cytokines + <ul><li>Hapten covalently attaches to self-protein </li></ul><ul><li>Hapten specific B cell binds haptenated-protein </li></ul><ul><li>Complex is internalized and degraded to haptenated peptides </li></ul><ul><li>Haptenated peptides are presented to T cells </li></ul><ul><li>B cell receives help and releases hapten specific antibody </li></ul>
    18. 19. Innate features of pathogens act as B cell costimulators <ul><li>Pathogen multivalency </li></ul><ul><ul><li>provides a level of BCR crosslinking optimal for activation </li></ul></ul><ul><li>Many pathogens activate TLRs </li></ul><ul><ul><li>TLR signaling synergizes with BCR signal </li></ul></ul><ul><li>Some induce type I IFNs </li></ul><ul><ul><li>IFN  R signaling synergizes with BCR </li></ul></ul><ul><li>Many activate the complement cascade and become C3d coated </li></ul><ul><ul><li>complement receptor (CR) crosslinking synergizes with BCR signal </li></ul></ul>
    19. 20. TLR signaling in B cells via adaptor Myd88 augments Ab response Flagellin/alum i.p. d12 Pasare & Medzhitov, Nature (2005) 438,364 TLR BCR Myd88 Ag
    20. 21. Type I IFN receptor signaling in B cells augments Ab response WT Flu, d5 d10 of primary response Coro et al., JI (2006) 176, 4343 Le Bon et al., JI (2006) 176, 2074 IFNR BCR IFN  Ag <ul><li>IFN  made by many cell types in response to viral infection </li></ul><ul><li>Many cell types, including B cells, express the IFN  R </li></ul>
    21. 22. Antigen-C3d complexes cross-link BCR and CR2-CD19 complex - increase sensitivity to antigen
    22. 23. Key components of T cell help <ul><li>CD40L triggers CD40 -> synergizes with BCR signals to promote mitosis; cytokine (e.g. IL4) signals also contribute </li></ul><ul><li>FasL triggers Fas -> BCR signaling protects from apoptosis </li></ul><ul><li>T cell derived cytokines influence differentiation, isotype switching: </li></ul><ul><ul><li>IL2, IL6 promote differentiation </li></ul></ul><ul><ul><li>IL4 -> IgG1, IgE </li></ul></ul><ul><ul><li>IFN  -> IgG2a, IgG3 </li></ul></ul><ul><ul><li>TGF  and IL5 -> IgA </li></ul></ul><ul><li>Many other molecules involved in T-B interactions </li></ul><ul><ul><li>e.g. ICAM1/LFA1, ICOS/ICOSL, CD30/CD30L, CD27L/CD27, OX40L/OX40, ... </li></ul></ul>
    23. 24. Role of CD40 in B cell activation <ul><li>TCR triggering up-regulates CD40L on T cell </li></ul><ul><li>CD40 signaling promotes B cell activation </li></ul><ul><li>CD40L-deficiency = 'hyper-IgM syndrome' (no isotype switching, no Germinal Centers) </li></ul><ul><li>CD40 signaling also important in DC, MØ function </li></ul>increased expression of cell cycle molecules, survival molecules, cytokines, promotes isotype switching
    24. 25. Cardinal features of B - T interaction
    25. 26. 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
    26. 28. Anergic B cells can respond to 'stronger' antigens
    27. 29. 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 blimp1 XBP1
    28. 30. Production of membrane vs secreted Ig
    29. 31. Plasma Cells are antibody secreting cells <ul><li>Two types: </li></ul><ul><li>1. Plasma cells generated early in the primary response </li></ul><ul><ul><li>- short-lived (~ few days) </li></ul></ul><ul><ul><li>- typically low affinity </li></ul></ul><ul><ul><li>- form in T-independent and T-dependent responses </li></ul></ul><ul><ul><li>- home to red pulp of spleen, medullary cords of lymph nodes </li></ul></ul><ul><ul><li>- IgM but also IgG and other isotypes </li></ul></ul><ul><li>2 . Plasma cells generated later in the primary response and that predominate in secondary responses </li></ul><ul><ul><li>- arise predominantly from germinal centers (in primary) or from memory B cells (in secondary) </li></ul></ul><ul><ul><li>- long-lived (possibly several months) </li></ul></ul><ul><ul><li>- often home to bone marrow, gut, lactating mammary gland </li></ul></ul><ul><ul><li>- predominantly isotype switched </li></ul></ul>
    30. 32. naive B cell activated B cells 3 days 12 divisions plasma cells 1 day differentiation 1 day 10 4 Ab/cell/sec antibodies 1 2 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
    31. 33. BCR isotype switching
    32. 34. Ig Heavy chain class (isotype) switching neutralization Neutralization 3wk t1/2 neutralization
    33. 35. Affinity Maturation <ul><li>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 </li></ul><ul><li>The high affinity B cells emerging in germinal centers give rise to long-lived plasma cells and memory B cells </li></ul>
    34. 36. Antibody Affinity Maturation Low affinity B High affinity B mutation selection Germinal Center V H V L Ag V H V L V H V L V H V L V H V L Ag V H V L
    35. 37. Germinal Centers <ul><li>Function: to generate B cells that produce antibodies with increased affinity for the inducing antigen </li></ul><ul><li>=> affinity maturation </li></ul><ul><li>Germinal Center Reaction: </li></ul><ul><li>Activated B cells give rise to Centroblasts </li></ul><ul><li>- localize in follicle, undergo rapid cell division and turn on machinery that causes somatic mutation in V-regions </li></ul><ul><li>Centroblasts give rise to Centrocytes </li></ul><ul><li>- migrate to the FDC-rich region of the Germinal Center </li></ul><ul><li>- survival is dependent on interaction with FDC-bound Ag and presentation of Ag to T cells </li></ul><ul><li>- centrocytes that successfully compete to bind antigen (e.g. by having higher affinity BCR) and to receive T cell help are selected and may differentiate into long-lived plasma cells or memory B cells </li></ul>
    36. 38. FDC FDC FDC Light Zone Dark Zone FDC FDC FDC CB CB CB CB CB CB CB CB T T T memory B cell plasma cell CB CB MØ T – T cell FDC – fol licular dendritic cell MØ – macrophage CC - centrocyte CB - centroblast CC CC CC CC CC MØ CB Proliferation, somatic hypermutation Selection, proliferation, differentiation CB CB CB CB Germinal Center Organization B High Affinity Low Affinity CC
    37. 39. 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)
    38. 40. Cell morphology and migration dynamics
    39. 41. Affinity maturation is T-dependent <ul><li>T cells are enriched in the light zone </li></ul><ul><li>Represent ~10% of GC cells </li></ul>LZ DZ CD3  IgD
    40. 42. Most GC T Cells Are Not in Stable B-T Conjugates
    41. 43. Selection Model <ul><li>GC B cells that have improved affinity capture more antigen in a given amount of time, receiving a stronger BCR signal and presenting more MHC-peptide complexes to GC T cells, outcompeting surrounding B cells for T cell help </li></ul>
    42. 45. Summary of GC Dynamics
    43. 46. Memory B cells <ul><li>Generated during the primary response </li></ul><ul><ul><li>best characterized for T-dependent responses involving GC but some evidence exists for memory to TI antigens </li></ul></ul><ul><li>Small, recirculating cells </li></ul><ul><li>Typically isotype switched (e.g. IgG + or IgA + ) </li></ul><ul><li>Typically have higher affinity for the inducing Ag </li></ul><ul><li>Longer lived than naïve B cells </li></ul><ul><ul><li>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 </li></ul></ul><ul><li>May have intrinsic differences that promote greater clonal expansion and more rapid differentiation to plasma cells </li></ul><ul><ul><li>differences in cytoplasmic domains of IgG vs IgM/D </li></ul></ul><ul><ul><li>upregulation of TLRs </li></ul></ul>
    44. 47. Down-regulation of B cell response by preformed IgG Antigen-IgG complexes cross-link BCR and Fc  RII - inhibit signaling
    45. 48. <ul><li>The poly-Ig receptor is a special Fc receptor that selectively binds dimeric IgA </li></ul><ul><li>The process of transporting IgA across the cell is known as transcytosis </li></ul><ul><li>The IgA released into the gut lumen remains associated with part of the poly-Ig receptor (known as the secretory component) and this provides protection against proteolysis by gut proteases </li></ul>
    46. 49. Neonatal Immunity <ul><li>Maternal IgG is transported by the neonatal Fc receptor (FcRn) </li></ul><ul><ul><li>across the placenta to the fetus </li></ul></ul><ul><ul><li>from colostrum across the gut epithelium </li></ul></ul><ul><li>Confers passive immunity in the newborn </li></ul><ul><li>The duration of protection is 3-4 months (or ~5 IgG half-lives) </li></ul><ul><ul><li>explains the high incidence of disease after this period by bacteria such as Haemophilus influenzae </li></ul></ul><ul><li>Human milk contains IgA </li></ul><ul><ul><li>provides some protection against gut pathogens </li></ul></ul><ul><li>Neonatal protection is only as good as the titer of IgG (and IgA) in the mother against the specific organism </li></ul>
    47. 50. IgG half-life <ul><li>FcRn is also present in the adult and involved in protecting IgG from degradation </li></ul><ul><li>Accounts for the long (3 week) half-life of IgG compared to other Ig isotypes </li></ul><ul><li>Therapeutic agents that are fused to IgG Fc regions take advantage of this property e.g. Enbrel (TNFR-Fc) </li></ul>
    48. 51. Features of primary and secondary antibody responses
    49. 52. Recommended Reading <ul><li>Primary papers: </li></ul><ul><li>Lakshmi Srinivasan…Klaus Rajewsky. (2009) PI3 Kinase Signals BCR-Dependent Mature B Cell Survival. Cell 139, 573 </li></ul><ul><li>Allen, C.D., Okada, T., Tang, H.L. and Cyster, J.G. (2007) Imaging germinal center selection events during affinity maturation. Science 315: 528-31. </li></ul><ul><li>Schwickert T.A., … Nussenzweig, M.C. (2007) In vivo imaging of germinal centers reveals a dynamic open structure. Nature 446: 83-7. </li></ul><ul><li>Horikawa, K.,…Goodnow, C.C. (2007) Enhancement and suppression of signaling by the conserved tail of IgG memory-type B cell antigen receptors. 204: 759-69. </li></ul><ul><li>Gavin A.L., … Nemazee D. (2006) Adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling. Science 314: 1936-8. </li></ul><ul><li>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 </li></ul><ul><li>Reviews: </li></ul><ul><li>Kurosaki T, Shinohara H, Baba Y. B Cell Signaling and Fate Decision. Annu Rev Immunol . 2009 </li></ul><ul><li>Gupta, N. and Defranco, A.L. (2007) Lipid rafts and B cell signaling. Semin Cell Dev Biol 18, 616 </li></ul><ul><li>Allen, C.D., Okada, T. and Cyster, J.G (2007) Germinal-center organization and cellular dynamics. Immunity . 27:190-202 </li></ul><ul><li>Shapiro-Shelef, M. and Calame, K. (2005) Regulation of plasma-cell development. Nat Rev Immunol 5: 230-42. </li></ul><ul><li>McHeyzer-Williams L.J. and McHeyzer-Williams, M. (2005) Antigen-specific memory B cell development. Annu Rev Immunol 23:487-513 </li></ul><ul><li>Mackay, F., Schneider,P., Rennert,P. and Browning, J. (2003) BAFF and APRIL: A Tutorial on B Cell Survival. Annu. Rev. Immunol .; 10.114 </li></ul><ul><li>. </li></ul>