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B cells: Beyond Antibodies November 1 st , 2006

Amit Bar-Or, MD, FRCPC Neuroimmunology Unit Montreal Neurological Institute Montreal, QC, Canada . B cells: Beyond Antibodies November 1 st , 2006. Outline. Antibody-independent roles of B cells antigen presentation (APC) innate immunity (environment/immune interface)

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B cells: Beyond Antibodies November 1 st , 2006

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  1. Amit Bar-Or, MD, FRCPC Neuroimmunology Unit Montreal Neurological Institute Montreal, QC, Canada B cells: Beyond AntibodiesNovember 1st, 2006

  2. Outline • Antibody-independent roles of B cells • antigen presentation (APC) • innate immunity (environment/immune interface) • immune regulation • lymphoneogeneis

  3. B Cell Development

  4. B Cell B cell roles in Neuroimmunological diseases

  5. Outline • Antibody-independent roles of B cells • antigen presentation (APC) • innate immunity (environment/immune interface) • immune regulation • lymphoneogeneis

  6. B cells as antigen-presenting cells (APC): T cell expansion and differentiation in vivo requires antigen presentation by B cells. Crawford et al. J Immunol 2006 Antigen-presenting B cells: role in induction of peripheral T cell tolerance Raimondi et al. J Immunol 2006.

  7. The Journal of Immunology, 2006, 176: 3498–3506.

  8. Background Though B cells are well documented as APC, their role in supporting and programming the T cell response in vivo is still unclear Studies using B cell-deficient (mMT) mice have given rise to contradictory results.

  9. Methods Mixed BM chimeric mice to define the contribution that B cells make as APC. Can generate TCR-Tg mice where B cell compartment is deficient in MHC class II, while other APC are largely normal. Other mice: CD40 deficient, LT deficient.

  10. Observations • T cell clonal expansion is significantly reduced • Differentiation of T cells into cytokine-secreting effector cells is impaired (in particular, Th2 cells) • (iii) Development of the memory T cell populations is also decreased. • Furthermore: Although MHC class II-mediated presentation by B cells was crucial for an optimal T cell response, neither a B cell-specific lack of CD40 (influencing costimulation) nor lymphotoxin (influencing lymphoid tissue architecture) had any effect on the T cell response.

  11. Reduced T cell proliferation in B cell deficient (mMT) mice

  12. Good reconstitution in mixed BM chimera (non-B cells mostly wild-type, all B cells MHC class II deficient)

  13. MHC class II on B cells in chimeric versus Wild type

  14. Preserved architecture in LN of chimerics and wild type(T cell: Green; B cell: red)

  15. Decreased ova-II T cell expansion in absence of MHC class II on B cells

  16. Deficient T cell expansion could be restored with addition of wild type B cells

  17. Impaired memory response to viral antigen in MHC-deficient B cell chimerics – assessed by T cell tetramer Primary response Recall response

  18. Impaired T cell cytokine production in chimeric mice

  19. T cell expansion in the chimerics is not dependent on CD40 signaling by B cells

  20. Similarly, T cell expansion in the chimerics is not dependent on LT signaling by B cells

  21. Conclusions In vivo, B cells provide extra and essential Ag presentation capacity over and above that provided by dendritic cells In this context, B cells optimize expansion and allow the generation of memory and effector T cells

  22. 1.Activation 4. Invasion 3. Attraction Th B Cell B Cell 2. Adhesion 5. Reactivation 6. Neural/glial responses Periphery BBB CNS Model of Human Autoimmune Disease (eg: MS)

  23. CD19 CD80 IgD CD27 Bar-Or et al, J Immunol, 2001 A human memory B cell subset expresses the CD80 (B7.1) costimulatory molecule

  24. 18% CD80 13% CD27 CD27+CD80+ Memory B Cell Subset Lower threshold of activation 3-10  more immunoglobulin Significantly stronger T cell responses Additional molecules (CD11b, CD72) important to B cell:T cell interaction Bar-Or A, et al, J Immunol. 2001;167:5669-5677. Alter A, et al. J Immunol. 2003;170:4497-4505. Duddy ME, et al. J Immunol. 2004;172:3422-3427. Bar-Or A. Adv Neurol. 2006;98:91-109.

  25. BLOOD CSF p<0.01 p<0.05 p<0.001 40 75 30 50 % B cells % B cells 20 25 10 0 0 RRMS SPMS PPMS OND RRMS SPMS PPMS OND One way ANOVA (p<0.0001) RRMS; n=11, SPMS; n=8, PPMS; n=11, OND; n=8 Increased percent of CD27+CD80+ B cells in the CSF of patients with Progressive forms of MS

  26. Outline • Antibody-independent roles of B cells • antigen presentation (APC) • innate immunity (environment/immune interface) • immune regulation • lymphoneogeneis

  27. Sorting human naïve B cells Naïve B cell proliferation (various stimuli) with or without CpG addition TLR9 stimulation (CpG) provides an essential signal for induction of human naive B cell proliferation

  28. TLR9 stimulation (CpG) provides an essential signal for induction of human naive B cell proliferation Sorting human naïve B cells Naïve B cell proliferation (various stimuli) with or without CpG addition

  29. Naive (TN) CD4+CD45RO–CCR7+ Central memory (TCM) CD4+CD45RO+CCR7+CXCR5– Follicular help (TFH) CD4+CD45RO+CCR7+CXCR5+ Effector memory (TEM) CD4+CD45RO+CCR7– Regulatory T cells (TREG) CD4+CD45RO+CD25+ Requirement for CpG is seen regardless of the source of T cell help

  30. Various TLR agonists provide ‘signal three’ either directly on naïve B cells, or indirectly through DC activation Anti-Ig + T Cell help + different TLR …

  31. TLR agonists provide ‘signal three’ either directly on naïve B cells, or indirectly through DC activation Anti-Ig + T Cell help + different TLR …

  32. 1 Anti-Ig, T help and CpG, induce strong RNA expression of: 1. AID (Ig Class switch) 2. xbp-1 (Plasma cell) 2 IgA G M TLR stimulation is required for induction of Ig isotype switch and differentiation to Ig-secreting (plasma) cells.

  33. 1 3 2 4 TLR stimulation is required for induction of Ig isotype switch and differentiation to Ig-secreting (plasma) cells. Anti-Ig, T help and CpG, induce strong RNA expression of: 1. AID (Ig Class switch) 2. xbp-1 (Plasma cell) IgA G M

  34. B cells and Toll-like receptors (TLRs): Toll-like receptors (TLRs) make it possible for B cells to ‘sense’ the environment (viruses, bacteria) and respond to external stimuli TLR signaling is important for normal B cell activation Ruprecht, Lanzavecchia, 2006; Medzhitov. Nature. Pasare, 2006. TLR signaling may cause B cells to become abnormally activated, and may lead to production of auto-antibodies Lau et al. J Exp Med. 2005; Martin, Elkon. J Exp Med, 2005; Fields et al. J Immunol 2006

  35. Model of Human Autoimmune Disease (eg: MS) 1.Activation 4. Invasion 3. Attraction Th B Cell B Cell 2. Adhesion 5. Reactivation 6. Neural/glial responses Periphery BBB CNS

  36. CD3 Comparing Migration of Monocytes, B cells and T cells across Human Brain Endothelial Cells (HBECs) Alter et al, J Immunol, 2003; Bar-Or et al Brain 2003

  37. Blocking VLA-4 inhibits B cell migration Counts Fluorescence Alter et al, J Immunol 2003

  38. Molecular Mechanisms of B cell Migration across Brain Endothelial Barriers Control IgG 30,000 20,000 aIL-8 aMCP-1 aICAM-1 aVLA-4 10,000 0 Blocking conditions Alter et al, J Immunol, 2003

  39. Distinct MMPs expressed by B cells and T cells MMP 11 MMP 17 p = 0.02 7.5 7.5 p = 0.03 7.5 5.0 5.0 5.0 2.5 2.5 2.5 0 0 0.0 B cells T cells B cells T cells MMP 11 B MMP 11 T TIMP-1 p =0 .02 MMP 28 30 2000 2000 p = 0.03 20 1000 1000 10 0 0 0 T cells B cells B cells T cells MMP 28 T MMP 28 T Bar-Or et al, Brain 2003

  40. B cell roles in Neuroimmunological diseases B Cell

  41. Outline • Antibody-independent roles of B cells • antigen presentation (APC) • innate immunity (environment/immune interface) • immune regulation • lymphoneogeneis

  42. CD40 alone Dual stimulation (BCR + CD40) b a CD40 alone 400 p = 0.0064 p < 0.0001 Dual stimulation (BCR + CD40) 600 500 300 400 IL-10 (pg/ml) 200 pg/ml 300 200 100 100 0 LT IL-10 0 0 100 200 300 400 500 600 LT (pg/ml) Reciprocal regulation of human B cell effector cytokines is context dependant Duddy et al J Immunol 2004; Duddy, Niino et al, in review

  43. CD27 - Naive B cells CD27 + Memory B cells Proliferation IL-10 p = 0.004 ns 200 20 ns ns 100 cpm (x 10-3) Cytokine Secretion (pg/ml) 10 0 0 CD40 alone Dual stimulation CD40 alone Dual stimulation TNFa LT ns p = 0.002 ns p = 0.002 300 200 Cytokine Secretion (pg/ml) 150 Cytokine Secretion (pg/ml) 100 0 0 CD40 alone Dual stimulation CD40 alone Dual stimulation Normal memory and naïve B cells express distinct patterns of cytokine production

  44. B Cells as Active Immune Regulators via Effector Cytokines - role in immune regulation • Memory B cells, stimulated by their Ag (BCR) and subsequent T cell help (CD40), secrete TNF and LT, thereby actively contributing to efficiency of adaptive memory immune responses • Naive B cells (normally harboring autoreactive repertoire), stimulated in “bystander” context (CD40 only), secrete IL-10 that could acquiesce otherwise undesired response

  45. 15 Proliferation IL-10 ns ns ns ns ns ns p = 0.008 p = 0.037 400 10 Normals Multiple Sclerosis 5 cpm (x 10-3) 200 Cytokine Secretion (pg/ml) 0 CD40 alone Dual stimulation 0 CD40 alone Dual stimulation 400 TNFa LT 200 200 Cytokine Secretion (pg/ml) 100 Cytokine Secretion (pg/ml) 0 CD40 alone Dual stimulation 0 CD40 alone Dual stimulation Abnormal B cell cytokine production in patients with Multiple Sclerosis

  46. Outline • Antibody-independent roles of B cells • antigen presentation (APC) • innate immunity (environment/immune interface) • immune regulation • lymphoneogeneis

  47. Many thanks to: Migration/MMP Wee Yong - Calgary Robert Nuttall - UEA Dylan Edwards - UEA Fabrizio Giuliani - Edmonton Jack Antel - MNI Pediatric MS/CIS Brenda Banwell Julia Kennedy Lauren Krupp Doug Arnold Dessa Sadovnick Bar-Or Lab Farzaneh Jalili Christine Ghorayeb Sarah Ekdawi Stefan Sawoszczuk Melissa Wright Claudia Calder Madeline Pool Tarik Touil Donald Gagne Thierry Vincent Isabel Rambaldi Experimental Therapeutics Gregory Cosentino Boli Fan Caroline Bodner Sudy Alatab Alumni Martin Duddy - Belfast Masaaki Niino -Sapporo Andrea Alter - McGill Ho Jin Kim - Seoul anti-CNS Antibodies Clara Lopez-Amaya - HSC Mario Moscarello U of T Kevin O’Connor – Harvard Bill Robinson - Stanford David Hafler - Harvard Larry Steinman - Stanford Canadian BMT Study Group Mark Freedman - Ottawa Harry Atkins - Ottawa Rafick Sekaly - CHUM Remi Cheynier - Inst. Pasteur Clare Baecher Alan - Harvard Nogo Project Alyson Fournier - MNI Tanja Kuhlmann - Gottingen T cell Assays Hans-Michael Dosch - HSC Roy Chung APC Projects Heinz Wiendl - Wurzburg Scott Zamvil - UCSF Sergio Baranzini - UCSF Jack Antel - MNI DNA Vaccine Hideki Garren - BHT Virology/HERV Raymond Tellier - HSC Chris Power - Edmonton CIHR, MSSC, ITN/NIH, Wadsworth, CIHR/IHRT, MSSC Scientific Research Foundation

  48. Many thanks to: Pediatric MS/CIS Brenda Banwell Doug Arnold Dessa Sadovnick Julia Kennedy Maria Tantses Lauren Krupp Jack Antel Pierre Duquette Alexnadre Prat Nathalie Arbour Claudia Calder Tarik Touil Christine Ghorayeb Donald Gagne Farzaneh Jalili Madelaine Pool Alumni Masaaki Niino – Sapporo Martin Duddy - Belfast Ho Jin Kim - Seoul Canadian BMT Study Group Mark Freedman - Ottawa Harry Atkins - Ottawa David Haegert - McGill Rafick Sekaly - CHUM Remi Cheynier - Inst. Pasteur Clare Baecher Alan Virology/HERV Raymond Tellier - HSC Chris Power - Edmonton T cell Assays/BHT Hans-Michael Dosch - HSC Clare Baecher-Allan Roy Chung Larry Steinman Hideki Garren ETP Projects Greg Cosentino Sudy Alatab Caroline Bodner Boli Fan Alyson Fournier Tim Kennedy Phil Barker anti-CNS Antibodies Clara Lopez-Amaya - HSC Mario Moscarello U of T Kevin O’Connor - Harvard Bill Robinson - Stanford Larry Steinman - Stanford Support: CIHR/IHRT, MSSC, ITN/NIH, Wadsworth MSSC Scientific Research Foundation

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