1. Type 1 insulin-dependent autoimmune diabetes. Ciriaco A. Piccirillo Canada Research Chair Department of Microbiology & Immunology McGill University Health Center Ciro.piccirillo@mcgill.ca

2. Diabetes Classification Type 1 –Immune Mediated –Insulin deficient, autoantibodies Type 2 –No Autoantibodies and treated without insulin Other Specific forms of Diabetes –Gestational Diabetes Monogenic:Single gene defect. APS-I: AIRE autosomal recessive IPEX: Scurfy Gene X-linked Monogenic: Single gene defect. APS-I: AIRE autosomal recessive IPEX: Scurfy Gene X-linked Polygenic:Summation of small effects of multiple genes creating diabetes susceptibility Polygenic: Summation of small effects of multiple genes creating diabetes susceptibility – e.g. NOD mouse

3. The Non-Obese Diabetic (NOD) mouse. Model of spontaneous Type 1 insulin-dependent diabetes (T1D).  -islet Insulin Normal Type 1 Autoimmune diabetes pLN pancreas Abnormal peripheral T cell tolerance: Self-reactive T cell Checkpoints 1. Peri-insulitis (Th2) 2.Insulitis (Th1) Cellularity heterogeneous Salivary glands Thyroid Nuclear antigens Testes Ovaries Multi-organ Autoimmunity l Cumulative incidence of T1D: 80% in females, 30% in males (at 30 weeks)

4. Activated TH1 CD4+ T Cell CD4+ Cell (TH2 ) CD4+ Cell (TH0 ) DR3, DR4,,DQ8/insulin peptide CD2 Macrophage/dendritic cell Fc R  IFN-  IL-12 CD40L CD40 , TCR IL-1, TNF, LT, NO, PGE-2 B Cell ?anti-insulin, GAD ab anti- Mog IL-4 Immunopathophysiology of Diabetes ?Antibody mediated injury Dendritic cell/ APC CD40L IL-4 CD40L CD8+ CTL FasL perforin  cell death  islet cells

5. LOSS OF FIRST PHASE INSULIN RESPONSE TIME Stages in Development of Type 1 Diabetes BETA CELL MASS DIABETES “PRE”- DIABETES GENETIC PREDISPOSITION INSULITIS BETA CELL INJURY NEWLY DIAGNOSED DIABETES MULTIPLE ANTIBODY POSITIVE GENETICALLY AT RISK (?Precipitating Event) Progressive loss insulin release Glucose normal Overt immunologic abnormalities Normal insulin release

6. Autoantibodies/Autoreactive B Cells Contribute to NOD Diabetes Immunoglobulin knockout prevention NOD DM Serreze et al, J. Immunol 1998, 161:3912-3918 I-Ag7 on B cells needed for NOD diabetes. Noorchashm et al, J. Immunol 1999, 163, 743-750 Anti-Insulin VH125 Heavy Chain Increases diabetes in NOD mice. Hulbert et al, J. Immunol, 2001, 167: 5535-5538 Transplacental autoantibodies accelerate NOD diabetes. Greeley et al, Nature Immunol. Progression to Diabetes increases with number of Autoantibodies (GAD, ICA512, Insulin)

7. T1D in NOD mice is T cell dependent NOD SCID NOD 0% 11% 70% *Diabetic T cell transfer into normal or immunodeficient NOD *T cell depletion studies *Combined contributions of CD4+ and CD8+ T cells: - Lessons from knockouts *CD4+ and CD8+ T cell clones can induce T1D alone.

8. Autoantigens: Lessons from diabetogenic T cells. Diverse T cell response - epitope spreading/cascade?

9. Checkpoints in T1D development Checkpoint 1 Insulitis (peri) -Starts at weaning: immunological changes related to food uptake and changes in the intestinal flora -Increased homing of T cells : expression of addressins MadCam and PNAd on pancreatic blood vessel epithelium -3-4 weeks of age, non-destructive -Th2 dominated Checkpoint 2 Beta cell loss & diabetes - T cells gain more aggressive effector mechanisms: Th1/Th2 balance, expression of Fas Ligand on CTLs, direct cytotoxicity. -Loss of protective mechanisms: -Protective cytokines, Regulatory T cells -Amplification : Epitope spreading -10-12 weeks of age, destructive -Th1 dominated

10. Diabetogenic MHC I-Ag7 The unusual H-2g7 MHC haplotype of NOD mice: Kd, I-Ag7,I-Enull, Db: Idd1 on chromosome 17. I-A  g7 and some HLA-DQB alleles: encode serine, alanine, or valine at position 57 and mediate T1D susceptibility Aspartic acid at position 57 is associated with resistance. Mutations to Aspartic acid reduce disease incidence but does not reduce insulitis. Homozygosity is required for disease: –Possible requirement for a threshold of MHC-peptide complexes for tolerance induction. –T1D incidence increases with HLA haplotype combinations (DR2/3) DQB1*0402 Asp57  Leu56   -chain  -chain

11. Defective Central Tolerance Diabetogenic MHC I-Ag7 1.I-Ag7 haplotype is poor peptide binder. 2.Failure to efficiently negatively select autoreactive T cells 3.Failure to positively select Treg cells.

12. Abnormal peripheral T cell Tolerance in NOD mice. Hyporesponsive T cell responses: –TCR induced proliferation and cytokine production (IL-2 / IL-4). –Deficient PKC/Ras/MAPK pathway –Weak MLR response Deficient frequency of NK-T cells: –IL-4 producing cells –Th1/Th2 balance Aberrant regulatory T cell network.

13. T cell immunoregulation in the NOD Evidence Delay between insulitis onset and diabetes Prediabetic T cells prevent adoptive transfer of disease into NOD.scid mice. Thymectomy Cyclophosphamide Treg cells are numerous and heterogeneous

14. Balance of effector and regulatory mechanisms determines peripheral tolerance Type 1 insulin dependent autoimmune diabetes  -islet Ags nT reg CD4 + Foxp3 +

15. Functional deficiency in CD4 + CD25 + Treg cells in autoimmunity ? nT reg Self-reactive T eff cell Autoimmune disease: Organ-specific - T1D, MS/EAE, Sjogren’s, Thyroiditis Systemic - SLE, APS, RA CD4 + CD25 +

16.  -islet Insulin Normal T1D Age T1D Aberrant activation of effector T cells? Health T1D Health The Non-Obese Diabetic (NOD) mouse. Model of spontaneous type 1 insulin-dependent diabetes (T1D). Abnormal T cell tolerance to  -islet antigens

17. CD4+CD25+ nTreg cells in T1D. Functional deficiency in CD4+CD25+ Treg cells: – NOD mice succumb to T1D more rapidly in their absence – Delayed administration blocks disease. – NOD mice deficient for B7.1/2, CD40, and CD28 molecules have a more aggressive disease course. CD4+CD25+ Treg cells are absent Faulty “signals” in NOD mice? Development, activation requirement, survival or function?

18. Adoptive transfer NOD model of T1D. Diabetogenic T eff CD4 + CD25 - Protective nT reg CD4 + CD25 + Diabetes? NOD.TCR  -/- Wild- Type BDC2.5 Islet-specific V  4 + CD4 + TCR Tg NOD Are there functional deficiencies in CD4 + nTreg cells in NOD?

19. BDC2.5 T eff T reg + - ++ -+ Thymus Peripheral Days post-transfer    Diabetes incidence (%) BDC2.5 T eff T reg + - ++ Functional CD4 + CD25 + nTreg cells in NOD mice.

20. Age-dependent loss in nTreg cells? Roland Tisch JEM 2005

21. Immunomonitoring of nTreg cells in health and disease. nTregX,Y,Z nTreg Peripheral CD4 + T cells expressing CD25 Activated nTreg Activated effectors Anergized effectors Induced Treg Health Pre-clinical or symptomatic disease Immune activation NormalPeri-insulitisInsulitis/T1D Neonatal Adult

22. NODBDC2.5 Are there quantitative differences in the cellular frequency of CD4 + nTreg cells in NOD mice?

23. Pancreatic LN Non- draining LN CFSE BDC2.5 + Treg Pancreatic LN Non-draining LN CD4 + CD25 + nTreg cells do not affect the activation or proliferation of diabetogenic T cells. CD69

24. Resistance to T1D correlates with an increased infiltrate of CD4 + Foxp3 + nTreg cells in pancreatic environments. - nTreg function + nTreg cells - nTreg cells

25. Use of nTreg cells for the cure of T1D. Primary Established Tarbell et al. JEM 2004

26. Genetic determinants of nTreg cell development in NOD mice?

27. Foxp3 dependent development of nTreg cells.

28. Deficiency of Foxp3+ nTreg cells promotes T1D. Primary cause or consequence?

29. Inherited Susceptibility Loci: Both MHC and non-MHC genes are required. LOCUS CHROMOSOME CANDIDATE GENES IDDM16p21HLA-DQ\DR IDDM211p15INS VNTR IDDM315q26D15s107 IDDM411q13MDU1, ZFM1, RT6, FADD/MORT1, LRP5 IDDM56q24-27ESR, MnSOD IDDM618q12-q21D18s487, D18s64, JK (Kidd locus) IDDM72q31D2s152, IL-1, NEUROD, GALNT3 IDDM86q25-27D6s264, D6s446, D6s281 IDDM93q21-25D3s1303 IDDM1010p11-q11D10s193, D10s208, D10s588 IDDM1114q24.3-q31D14s67 IDDM122q33CTLA-4, CD28 IDDM132q34D2s137, D2s164, IGFBP2, IGFBP5 IDDM14?NCBI # 3413 IDDM156q21D6s283, D6s434, D6s1580 IDDM16?NCBI # 3415 IDDM1710q25D10s1750-D10s1773

30. Insulin Gene (INS) Class I VNTR 26-63 repeats Predisposing IDDM2 Insulin Gene (INS) Class III VNTR 140-200 repeats IDDM2 Protective The IDDM2 Locus VNTR = Variable Number of Tandem Repeats VNTR stimulates INS steady-state transcription in ß-cells VNTR length inversely correlates with INS mRNA levels in ß-cells in vivo Class III VNTR alleles = LOWER (~30%) INS transcription than predisposing class I VNTR alleles Class III VNTR alleles = Higher thymic INS transcription than predisposing alleles

31. Low incidence of T1D in Idd3 recombinant congenic NOD mice Chr 2 3 4 5 6 7 8 9 101010101 12121212 13131313 14141414 15151515 16161616 17171717 18181818 19191919 X11 20% Wicker LS et al. J Exp Med 1994 Lyons PA et al. Genome Res 2000 B6.B6- Idd3 NOD 1% B6 NOD.B6-chr3 80%