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Cytokines and autoimmunity

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Cytokines and autoimmunity

  1. 1. Autoimmunity: Lessons from Experimental Therapies and Special Cohorts November 3rd, 2006 Amit Bar-Or, MD, FRCPC Neuroimmunology Unit Montreal Neurological Institute Montreal, QC, Canada
  2. 2. <ul><li>Why study Special populations? </li></ul><ul><li>(i) Pediatric MS </li></ul><ul><li>Environmental contributors - Virology (EBV) </li></ul><ul><li>Immune dysregulation: T cell responses (self and environmental antigens) </li></ul><ul><li>Autoreactive antibodies </li></ul><ul><li>(ii) Experimental Therapies (ETP) </li></ul><ul><li>Antigen Specific </li></ul><ul><li>Global versus selective immune depletion </li></ul><ul><li>Immune modulation </li></ul>
  3. 3. Model of Multiple Sclerosis 2. Adhesion 3. Attraction Periphery BBB CNS 5. Reactivation 1. Activation Th 4. Invasion 6. Neural/glial responses B Cell B Cell
  4. 4. Immune cell infiltration and Dual MS Pathology Demyelination and Axonal compromise  TCR  T cell receptor) antibody Trapp et al. NEJM, 1998
  5. 5. T cells reactive to the CNS become activated in the periphery Inflammation and injury IFN  TNF  LT TH 1 TH 1 TH 1 Th 1 MBP-reactive IL-12 B7.1 APC (Monocytes) T Cell MBP-reactive Foreign/self Ag ? Molecular mimicry
  6. 6. Peripheral activation of Myelin-reactive T cells  Experimental Autoimmune Encephalomyelitis (EAE)
  7. 7. ‘ Altered Peptide Ligand’ (APL) therapy of MS <ul><li>APL induced ‘anti-inflammatory’ T cells and </li></ul><ul><li>was beneficial in EAE (Karin et al, J Exp Med, 1994). </li></ul>ENPVVHFFKNIVTPRTP MBP (85-101) XXPVVH X F X NIVTPRTP APL CGP 77116
  8. 8. Bielekova, et al Nat. Med , 2000 Kim et al, Clin Immunol , 2003 APL induced Antigen-specific T cells responses Week 4: Week 5: Relapse MBP APL 1 / (4.9x10 6 ) 0 1/840 1/620 Frequency Cross Reactivity 85% 78% (background < 1 %)
  9. 9. APL induced Antigen-specific T cells responses Bielekova, et al Nat. Med , 2000 Kim et al, Clin Immunol , 2003 Week 4: Week 5: Relapse MBP APL 1 / (4.9x10 6 ) 0 1/840 1/620 Frequency Cross Reactivity 85% 78% (background < 1 %)
  10. 10. Lessons MBP may be a real target in MS Induction of MBP-reactivity in periphery can result in human CNS inflammatory demyelination However, studies aimed at evaluating myelin-specific (T cell, antibody) responses in MS patients have been challenging
  11. 11. Genetic Predisposition Immune system Target organ (CNS) Birth Injury of the CNS New targets exposed (Epitope Spread) Issues of repair Compromise of the BBB Proliferation of T cells, B cells, Antibody formation Altered host Immune Response Environmental Exposure/s Viruses, etc ? 1st attack of demyelination (CIS) Diagnosis of MS
  12. 12. Wadsworth Pediatric MS Study Group Wadsworth Foundation
  13. 13. Epstein-Barr Virus Serological Results from Pediatric Multiple Sclerosis Patients and Controls Epstein-Barr virus (EBV) serologies multiple sclerosis (MS), Neurological controls, Autoimmune (AI) controls, and healthy controls. Children with MS were more likely to be positive for remote EBV infection and less likely to be EBV-negative than control patients. ** p < 0.001; * p < 0.05 EBV 0% 20% 40% 60% 80% 100% Acute Remote Negative Indeterminant MS (n=83) Neurol Control (n=29) AI Control (n=47) Healthy Control (n=26) ** ** * ** * * Tellier Lab
  14. 14. T cell reactivities to self and environmental antigens in pediatric MS and Controls <ul><li>Children with MS harbor a circulating pool of peripherally primed myelin-reactive T cells </li></ul><ul><li>Children with other autoimmune diseases harbor circulating T cells reactive against disease-specific target tissues </li></ul><ul><li>Nutritional exposures early in life are associated with heightened reactivity of T cells against target organs </li></ul>
  15. 15. <ul><li>Methods </li></ul><ul><li>Primary stimulation assay using fresh PBMC </li></ul><ul><li>Adding no antigen or specific test antigens </li></ul><ul><li>Assessing T cell proliferation at day 7 </li></ul><ul><li>Blinded to source of PBMC (HSC Dosch lab) </li></ul>
  16. 16. T cell proliferative responses to tissue antigens in pediatric demyelinating disease and control cohorts Dosch Lab (Banwell, Dosch, Bar-Or, Submitted; Wadsworth Foundation) EX-2 MBP-1 MOG GFAP S-100 Tep69 69-349 GAD Gad555 PI 0 1 2 3 4 5 6 Pancreatic Myelin Glial EX-2 MBP-1 MOG GFAP S-100 Tep69 69-349 GAD Gad555 PI 0 1 2 3 4 5 6 Pancreatic Myelin Glial Diabetes Cohort EX-2 MBP-1 MOG GFAP S-100 Tep69 69-349 GAD Gad555 PI 0 1 2 3 4 5 6 Pancreatic Myelin Glial EX-2 MBP-1 MOG GFAP S-100 Tep69 69-349 GAD Gad555 PI 0 1 2 3 4 5 6 Pancreatic Myelin Glial MS Cohort CIS Cohort Healthy Cohort T cell proliferation (SI) CIS Cohort MS Cohort Diabetes Cohort Number 37 26 24 29 Average age (range) 14.3 (5.8-19.4) 9.7 (1-20) Healthy Cohort 9.8 (3.0-16.0) 10.6 (1.1-18.1)
  17. 17. CIS cohort BLG Casein BSA BSA-193 BSA-394 Abbos OVA 0 1 2 3 4 5 6 MS cohort BLG Casein BSA BSA-193 BSA-394 Abbos OVA 0 1 2 3 4 5 6 T cell responses to dietary antigens in inflammatory demyelination and controls Dosch Lab (Banwell, Dosch, Bar-Or, Submitted; Wadsworth Foundation) T1D cohort BLG Casein BSA BSA-193 BSA-394 Abbos OVA 0 1 2 3 4 5 6 Healthy Control cohort BLG Casein BSA BSA-193 BSA-394 Abbos OVA 0 1 2 3 4 5 6
  18. 18. Dosch Lab (Banwell, Dosch, Bar-Or, Submitted; Wadsworth Foundation) CIS Patients MS Patients 1.00 2.00 3.00 4.00 5.00 6.00 0 2 4 6 8 10 12 14 16 18 Age Activity BLG Casein BSA BSA-193 BSA-394 Abbos Tep69 69-349 GFAP S-100 IS4 GAD Gad555 PI b-IA2 EX-2 MBP-1 MOG Healthy Controls Neurological Controls T-cell reactivities to multiple antigens as a function of Age
  19. 19. B cell roles in Neuroimmunological diseases B Cell
  20. 20. Oligoclonal bands (OCB) in cerebrospinal fluid (CSF) B cells + Plasma Cells: ‘intra-clonal’ expansion Qin, 1998; Colombo, 2000; Baranzini, 2000; Smith-Jensen, 2000; Owens, 2000 MS lesion Pathology: Ig/complement Lucchinetti, Bruck, Lassmann, 1996, 2000 Anti-myelin Antibodies in phagocytes within lesions of MS and EAE Genain, 1999; Linnington; others Anti-myelin antibody extracted from MS brain pathology O’Connor 2005 Auto-antibodies in Multiple Sclerosis: History
  21. 21. Auto-antibodies in Neurological disease Role in peripheral diseases MG, neuropathies Treatment responsiveness Role in CNS inflammatory disease NMO-Ig and Neuromyelitis optica Multiple sclerosis?
  22. 22. Antel and Bar-Or, NEJM , 2003
  23. 23. O’Connor et al , JNI , 2003 Anti-MBP (rabbit) Anti-GAD T1D Scatchard analysis of serum autoantibodies bound/free 0 . 0 4 0 . 0 9 0 . 1 4 0 . 1 9 0 . 2 4 1 8 0 0 3 8 0 0 5 8 0 0 bound [cpm] MS serum NHD serum Anti-MBP ADEM
  24. 24. 2.2 0.8 1.4 1.5 F:M 11.4 (5.8-17.6) 12.1 (2.2-18.5) 12.4 (2.0-20.1) 14.1 (2.2-19.6) Average age at enrollment (range) 48 59 80 137 Number Autoimmune Controls Neurological Controls Healthy Controls MS Cohort
  25. 25. Lopez- Amaya et al
  26. 26. Cohort MS Autoimmune Neurological Healthy % Positive 22.3 28.6 23.1 19.2 Frequency of Seropositivity for IgG/M/A anti-MBP antibodies Lopez- Amaya et al
  27. 27. <ul><li>Suggests that development of humoral (antibody) responses to myelin antigen can be normal </li></ul><ul><li>Anti-MBP antibody: no major role in MS initiation </li></ul>In the MS children with anti-MBP ‘+’: significant higher proportion of ‘ADEM-like presentation’ Acute Disseminated EncephaloMyelitis Typically MONOPHASIC, seen post-immunization Multi-focal, often quite dramatic inflammatory demyelinating attack, but is not a chronic disease
  28. 28. Suggests that: IF you have first attack of MS, AND IF you have circulating anti-myelin antibodies, you are more likely to get ‘ADEM-like’ episode. Consistent with EAE animal model observations: Anti-myelin antibody alone, cannot ‘transfer’ disease However, with anti-myelin antibodies, less T cells are needed to transfer EAE and disease is more severe
  29. 29. <ul><li>Why study Special populations? </li></ul><ul><li>(i) Pediatric MS </li></ul><ul><li>Environmental contributors - Virology (EBV) </li></ul><ul><li>Immune dysregulation: T cell responses (self and environmental antigens) </li></ul><ul><li>Autoreactive antibodies </li></ul><ul><li>(ii) Experimental Therapies (ETP) </li></ul><ul><li>Antigen Specific </li></ul><ul><li>Global versus selective immune depletion </li></ul><ul><li>Immune modulation </li></ul>
  30. 30. Immunomodulation with MBP encoding DNA plasmid (BHT-3009) Amit Bar-Or*, Farzaneh Jalili*, Masaaki Niino*, Caroline Anita Bodner*, Jack Antel*, Fu-Dong Shi^, Mary Price^, Susan Rhodes^, Timothy Vollmer^, Jill Gianettoni # , Frank Valone # , Lawrence Steinman # , Hideki Garren # ENS, 2006 Promoter Ori pA hMBP Kan r BHT-3009 3485 bp IM at weeks 1, 3, 5, 9 Administration of BHT-3009 0.5 mg and 1.5 mg dose cohorts complete Current status 10 Patients per dose cohort 80 mg daily Dose of atorvastatin 0.5 mg, 1.5 mg, or 3 mg Dose of BHT-3009 <ul><li>Placebo </li></ul><ul><li>BHT-3009 </li></ul><ul><li>BHT-3009 + atorvastatin </li></ul>Arms 30 Total patients RR-MS or SP-MS Patients I/II Phase
  31. 31. Antigen-specific T cell responses proliferation and cytokine production using CFSE and Intracellular Cytokine Staining No Antigen Tetanus CFSE IFN 
  32. 32. Immunomodulation with MBP encoding DNA plasmid (BHT-3009) ECTRIMS, 2005 Baseline: IFN  Week 9 of dose: CFSE Patient 002 (CD4+) TT W-MBP Patient 003 (CD4+) TT MBP 83-99 Patient 013 (CD8+) TT MBP 83-99 7.0% 13.3% 10.1% 5.4%
  33. 33. Immune ablation and AHST Reconstitution 2. Adhesion 3. Attraction Periphery BBB CNS 5. Reactivation 1. Activation Th 4. Invasion 6. Neural/glial responses B Cell B Cell
  34. 34. Periphery BBB CNS 5. Reactivation 6. Neural/glial responses Immune ablation and AHST Reconstitution B Cell
  35. 35. Periphery BBB CNS 5. Reactivation 6. Neural/glial responses Immune ablation and AHST Reconstitution
  36. 36. Periphery BBB CNS 5. Reactivation 6. Neural/glial responses Immune ablation and AHST Reconstitution Th New immune system B Cell
  37. 37. Young, early diagnosis, highly active patients Treatment had dramatic effects: No new relapses; no new inflammatory brain lesions However, brain continues to shrink (atrophy) …
  38. 38. BMT in MS Important window into disease pathophysiology and human immune reconstitution The continued brain atrophy over 3 years is probably beyond ‘destined’ tissue loss May reflect under-recognized CNS toxicity of chemotherapy regimen May emphasize ongoing process within CNS (relatively independent of periphery)
  39. 39. Uccelli, Corcione, Pistoia, Serafini, Aloisi
  40. 40. Reproduced with permission from Kutzelnigg A et al. Brain. 2005;128:2705-2712. Subpial cortical demyelination in progressive MS Kutzelnigg, A. et al. Brain 128:2705-2712; 2005
  41. 41. B cell Activating Factor (BAFF) produced by astrocytes and up-regulated in MS lesions Krumbholz M et al; J Exp Med . 17;201(2):195-200, 2005
  42. 42. Abnormal B cell cytokine production in patients with Multiple Sclerosis IL-10 p = 0.008 p = 0.037 0 200 400 CD40 alone Dual stimulation Cytokine Secretion (pg/ml) Normals Multiple Sclerosis cpm (x 10 -3 ) Proliferation CD40 alone Dual stimulation 0 5 10 15 ns ns LT 0 200 400 CD40 alone Dual stimulation Cytokine Secretion (pg/ml) ns ns TNF  0 100 200 CD40 alone Dual stimulation Cytokine Secretion (pg/ml) ns ns
  43. 43. Mitoxantrone therapy reciprocally regulates B cell cytokine production Untreated MS Mitoxantrone treated MS Cytokine Secretion (pg/ml) IL-10 p = 0.007 p = 0.004 0 300 600 CD40 alone Dual stimulation TNF  0 100 200 CD40 alone Dual stimulation ns p = 0.006 Cytokine Secretion (pg/ml) LT 0 150 300 CD40 alone Dual stimulation ns p = 0.008 Cytokine Secretion (pg/ml) cpm (x 10 -3 ) Proliferation CD40 alone Dual stimulation 0 5 10 15 ns ns
  44. 44. Mitoxantrone treatment results in decreased proportion of circulating CD27+ B cells CD27 MFI 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 Untreated Mitoxantrone Treated 64% 36% 89% 11% CD19 MFI a Untreated Mitoxantrone Treated 0 10 20 30 40 50 % CD27+ B cells p = 0.004 b
  45. 45. CD20 CD19 Pre D8 M10 Rituximab (anti-CD20) depletion of circulating B cells
  46. 46. Stuve O, et al. Clinical stabilization and effective B-lymphocyte depletion in the cerebrospinal fluid and peripheral blood of a patient with fulminant relapsing-remitting multiple sclerosis . Arch Neurol. 2005;62(10):1620-3. Monson NL, et al. Effect of rituximab on the peripheral blood and cerebrospinal fluid B cells in patients with primary progressive multiple sclerosis . Arch Neurol. 2005;62(2):258-64. Cross AH, et al. Rituximab reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis patients. J Neuroimmunol. 2006 Aug 10. (Cree BA, et al. An open label study of the effects of rituximab in neuromyelitis optica. Neurology. 2005;64(7):1270-2). Rituximab in MS
  47. 47. 0 200 400 LT IL-10 M12 BL M12 BL Cytokine Secretion (pg/ml) BL M3 M6 M9 M12 Tx 0 50 100 150 Absolute B cell Number (per  l) AHSCT (BMT) Effects of selective and non-selective B cell depletion on effector cytokine network CD27 - B cells CD27 + B cells M12 BL M12 BL 0 200 400 LT IL-10 Cytokine Secretion (pg/ml) 0 50 100 150 BL M3 M6 M9 M12 Tx Absolute B cell Number (per  l) Rituximab
  48. 48. B Cells as Active Immune Regulators via Effector Cytokines - role in immune regulation <ul><li>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 </li></ul><ul><li>Naive B cells (normally harboring autoreactive repertoire), stimulated in “bystander” context (CD40 only), secrete IL-10 that could acquiesce otherwise undesired response </li></ul>
  49. 49. 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 Many thanks to: CIHR, MSSC, ITN/NIH, Wadsworth, CIHR/IHRT, MSSC Scientific Research Foundation Virology /HERV Raymond Tellier - HSC Chris Power - Edmonton T cell Assays Hans-Michael Dosch - HSC Roy Chung 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 Pediatric MS/CIS Brenda Banwell Julia Kennedy Lauren Krupp Doug Arnold Dessa Sadovnick DNA Vaccine Hideki Garren - BHT Nogo Project Alyson Fournier - MNI Tanja Kuhlmann - Gottingen Migration/MMP Wee Yong - Calgary Robert Nuttall - UEA Dylan Edwards - UEA Fabrizio Giuliani - Edmonton Jack Antel - MNI APC Projects Heinz Wiendl - Wurzburg Scott Zamvil - UCSF Sergio Baranzini - UCSF Jack Antel - MNI Canadian BMT Study Group Mark Freedman - Ottawa Harry Atkins - Ottawa Rafick Sekaly - CHUM Remi Cheynier - Inst. Pasteur Clare Baecher Alan - Harvard

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