Département Hospitalo-Universitaire AUToimmune and HORmonal diseases

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1 Uth DHU Département Hospitalo-Universitaire AUToimmune and HORmonal diseases rs DIABETES ADRENAL DISEASES VASCULITIDES L immunomodulation du diabète. Quelles applications? quel avenir?

2 incidence [105/year] insulitis TYPE 1 DIABETES MELLITUS an autoimmune disease islet cell autoantibodies b cell selectivity anti-gad anti-ia2 anti-insulin anti-znt8 increasing incidence ( 3-4%/year) & geographical incidence variation (> 65/10 5 /year in Finland) Gianani R 2010, Diabetologia genes numerous numerous s Bottazzo & Doniach 1974, Lancet environment Gepts W 1965, Diabetes absence of a unique environmental factor

3 age Controls mortality rate /10 3 person-yr standardized mortality rate Skrivarhaug et al. Diabetologia 2006 [Norway] T1D MORTALITY Allegheny County childhood-onset (< 18) T1D registry diagnosed increased mortality compared to the general population n = 1075 acute renal cardio vascular Intermediate disease compared to high risk autoimmune diseases infections cancer Secrest et al. Diabetes 2010

4 ß cell mass TYPE 1 DIABETES: A CHRONIC DISEASE patient duration of diabetes heterogeneity diabetes recurrence Insulitis 1 22 years 60 days CD8+ T cells years 44 days CD8+ T cells years 92 days CD8+ T cells + ICA Bingley et al. Diabetologia 2006 diabetes hyperglycemia

5 TYPE I DIABETES NKT IL-4, IFN-g NK IFN-g autoantibodies CD1d APC NKG2D/MIC Cl II T CD4 TNF-a, IFN-g Cl II Cl I T reg b cell IL-10, TGF-b GRZ/perforin Pancreatic islet

6 TYPE I DIABETES immunosuppression APC Cl II insulin replacement therapy T CD4 b cell mass restoration graft regeneration TNF-a, IFN-g Cl II Cl I b cell tolerance induction GRZ/perforin Pancreatic islet

7 TYPE I DIABETES anti-tnfa [Etanercept] IL1 antagonists [Anakinra] autoantibodies anti-cd20 [Rituximab] APC Cl II T CD4 TNF-a, IFN-g Cl II Cl I cyclosporin A azathioprine + corticoids anti-thymoglobulin ± corticoids anti-cd3 GRZ/perforin intensive Insulin therapy b cell nicotinamide Pancreatic islet

8 TREATMENT OF DIABETES WITH ANTI-CD ± 1.86 nmol/l per month ± 1.30 nmol/l per month Herold KC et al. 2002, N Engl J Med

9 TREATMENT OF DIABETES WITH ANTI-CD3 months months months n = 40 (placebo) n = 40 (anti-cd3) Keymeulen B et al. 2005, N Engl J Med

10 TREATMENT OF DIABETES WITH ANTI-CD3 Keymeulen B et al. N Engl J Med 200

11 TYPE I DIABETES intrinsic T cell tolerance anergy changes in gene expression profiles peptides APC Cl II T CD4 TNF-a, IFN-g Cl II low-dose IL-2 Rapamycin/IL-2 T reg Cl I insulin IL-10, TGF-b extrinsic T cell tolerance GRZ/perforin GAD IA2,ZnT8 b cell DiaPep277 Pancreatic islet

12 TREATMENT OF PREDIABETES WITH INSULIN first and second degree relatives 3152 autoantibody positive 5 year risk > 50% in 372/2103 randomization to observation (n = 70) versus s.c. insulin twice daily 0.25 units/kg (n = 69) mean follow up 3.7 years s.c./i.v. & oral insulin (DPT1, 2002, N Engl J Med) Nasal insulin (Näntö-Salonen, 2008, Lancet)

13 TREATMENT OF PREDIABETES WITH ORAL INSULIN Diabetes Prevention Trial-Type first and second degree relatives 3483 autoantibody positive 5 year risk 26-50% in 388/2103 randomization to IAA 80 nu/ml 6.2%/year 10.4%/year 8.2%/year 6.4%/year oral insulin 7.7 mg/24h (n = 186, mean IAA 382 ± 555 nu/ml) versus placebo (n = 186, mean IAA 346 ± 336 nu/ml) Skyler J et al Diabetes Care 2005

14 GAD VACCINATION IN RECENT-ONSET T1D [Intention to treat analysis] n = 35 n = 34 sponse to GAD up to 15 months IL-5/-10/-13/-17/IFNg/TNFa IL-6/ mg GAD-alum days 1 & 30 within 6 months following onset GAD-induced expression of oxp3 & TNFa Ludvigsson et al, N Engl J Med, 2008

15 TrialNet intervention studies: within 100 days from diagnosis Anti-IL1β [Canakinumab] CTLA-4 Ig [Abatacept] in Recent Onset Diabetes Glutamic acid decarboxylase (GAD) in New Onset T1DM Metabolic Control in New Onset Diabetes Rituximab Study (Anti-CD20) Mycophenolate mofitil / dacliumab (MMF/DZB) study TrialNet prevention studies Oral insulin for prevention of Type 1 diabetes Study Anti-CD3 [Teplizumab] for prevention in relatives CTLA4-Ig [Abatacept] for prevention in relatives The Nutritional Intervention to Prevent Type 1 diabetes Study accessed 1st March, 2014.

16 TYPE 1 DIABETES T CELLS TO DIABETES PREVENTION APC T CD4 TNF-a, IFN-g b cell T CD8

17 HUMAN INSULIN 1 MALWMRLLPL 11 LALLALWGPD 21 PAAA FVNQHL 31 CGSHLVEALY 41 LVCGERGFFY 51 TPK PEPTIDE SIGNAL CHAINE B TRREAED 61 LQVGQVELGG 71 GPGAGSLQPL 81 ALEGSLQKRG 91 IVEQCCTSIC 101 SLYQLENYCN PEPTIDE C CHAINE A MALLVHFLPLLALLALWEPKPTQAFVKQHLCGPHLVEALYLVCGERGFFYTPK SRREVEDPQVEQLELGGSPGDLQTLALEVARQKRGIVDQCCTSICSLYQLENYCN B9-23 MALWMRFLPLLALLFLWESHPTQAFVKQHLCGSHLVEALYLCGERGFFYTPM SRREVEDPQVAQLELGGGPGAGDLQTLALEVAQQKRGIVDQCCTSICSLYQLENYCN

18 CLASS I ALLELE-SPECIFIC PEPTIDES peptide sequence HLA class I HLVEALYLV A ALYLVCGER A3 A LYLVCGERGF A24 LVCGERGFFY A LVCGERGFFY A3 A11 VCGERGFFYT A1 VCGERGFFYT A2 VCGERGFFYT B8 VCGERGFFYT B18 GERGFFYT A1 GERGFFYT B ERGFFYTPK A FYTPKTRRE B TPKTRREAEDL B8 peptide sequence class I allele 2-11 A2 ALWMRLLPLL A24 ALWMRLLPLL B RLLPLLALL A2 RLLPLLALL A RLLPLLALLAL A LALWGPDPAA A2 LALWGPDPAA A ALWGPDPAAA A2 ALWGPDPAAA A26 Toma et al, PNAS 2005; Diabetes 2009

19 CD8+ T CELL RESPONSE TO A2-RESTRICTED PREPROINSULIN PEPTIDES [ELISPOTassay] peptide binding % CD8+ IFN response 10-4 M 10-6 M long standing recent onset total 34-42* 58% 37% 2/4 4/10 6/ * 36% 65% 7/13 1/4 8/ * 96% 71% 5/15 2/4 7/ * 44% 36% 7/13 0/4 44% 7/ * 96% 94% 7/13 1/4 8/17 * Val 42, L 14, L 16, A 23 & A 24 were identified as C-terminal residues generated by proteasome digestion in vitro. ** 6-14 or 6-16 immunization anti-6-14 or cross-reactives responses anti-6-16 to 6-14 and 6-16 T cell clones Toma et al, PNAS2005; Toma et al. Diabetes 2009

20 PE-A: TTM PE-A PE-A: TTM PE-A PE-A: TTM PE-A TETRAMER RECOGNITION OF BLOOD CD8 + T CELLS TCD8 + b cells class I peptide streptavidin biotin A2.1-restricted peptides class I preproinsulin peptide A A A A A A A A controls 10 4 PDHase MATA Nef Dt1A5 TTM A2 PPIh_Dt1A5 TTM A fcsÉCD3+ CD8+ Dt1A5 TTM A2 PPIh_Dt1A5 TTM A fcsÉCD3+ CD _D124D 6-14.fcsÉCD3+ CD TCD PerCP-A: CD8 PerCP-A PerCP-A: CD8 PerCP-A PerCP-A: CD8 PerCP-A Luce et al. Diabetes 2011 Sandrine Luce

21 TETRAMER RECOGNITION OF BLOOD CD8 + T CELLS A2.1-restricted peptides single cell PCR central memory T cells effector memory T cells Luce et al. Diabetes 2011

22 AUC mg.h/dl mg NEW PRECLINICAL T1D MODELS beta cell mass x A2/DQ8 Tg Human insulin OUF YES Insulin A2.1 DQ8 genes insulin/a2.1/dq8 mice Luce et al. unpublished Intraperitoneal glucose tolerance test s.c./i.v. & oral insulin (DPT1, 2002, N Engl J Med) Nasal insulin (Näntö-Salonen, 2008, Lancet) YES C ol YES C ol

23 DIABETES IN RIP-B7 TRANSGENIC YES MICE % total islets diabetes (%) TCR CD11c CD8 CD28 TCR T cell CD19 CD4 D4 MHC B7.2 dapi non anti-glucagon diabetic anti-cd3 diabetic CD11b RIP-B7 YES Age (weeks) Luce et al. unpublished

24 DIABETES IN RIP-B7 TRANSGENIC YES MICE rdu proliferation assay CD8 + IFNγ Elispot assay CD8 + TMr + expansion assay Luce et al. unpublished

25 FROM RESEARCH TO CLINIC Uth DHU rs FROM PALLIATIVE TREATMENT TO PREVENTION OF TYPE 1 DIABETES diagnosis immunotherapy prediabetes damaged islet of Langerhans diabetes diagnostics T NKT T reg APC NK T CD8 B T CD4 Class II-restricted epitopes modified by inclusion of a thiol-disulfide oxidoreductase motif within flanking residues peptide autoantigen HLA Immunotherapy EU FP7 project Carlier et al. PloSOne 2012 YES mice Luce S et al. Diabetes 2011

26 Uth DHU Département Hospitalo-Universitaire AUToimmune and HORmonal diseases rs DIABETES ADRENAL DISEASES VASCULITIDES Andrea Toma Sandrine Luce Etienne Larger Roberto Mallone François Lemonnier Agnès Lehuen Sylvianne Muller, Jean Paul Briand (CNRS, Strasbourg) Decio Eizirik (Brussels) Charbel Masaad (Paris) Laurent Drouot, Olivier Boyer (Rouen)