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Type 1 insulin-dependent autoimmune diabetes.

Type 1 insulin-dependent autoimmune diabetes. . Ciriaco A. Piccirillo Canada Research Chair Department of Microbiology & Immunology McGill University Health Center Ciro.piccirillo@mcgill.ca. Diabetes Classification. Type 1 Immune Mediated Insulin deficient, autoantibodies Type 2

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Type 1 insulin-dependent autoimmune diabetes.

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  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 • 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). Insulin b-islet Self-reactive T cell Normal Cellularity heterogeneous pLN Type 1 Autoimmune diabetes Checkpoints 1. Peri-insulitis (Th2) 2.Insulitis (Th1) Abnormal peripheral T cell tolerance: Salivary glands Thyroid Nuclear antigens Testes Ovaries • Cumulative incidence of T1D: 80% in females, 30% in males (at 30 weeks) pancreas Multi-organ Autoimmunity

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

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

  6. Autoantibodies/Autoreactive B Cells Contribute to NOD Diabetes • Immunoglobulin knockout prevention NOD DMSerreze 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 0% NOD SCID 11% 70% NOD *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. C l o n es /Li n e s CD4 / CD8 S o u rce A nt ig e n TC R T e tra m er T r an s ge ni c Co m m e n t A ut ho r BD C 2.5 CD4 NOD S p l een Unknown V 4, V 1 Ye s Ye s Ha s k i ns b a C hr o m o som e 6 BD C 6.9 CD4 NOD S p l een Unknown V 4 , V 1 3. 1 Ye s Ha s k i ns b a BD C 6-4 . 3 CD4 NOD I sl e t I nsu li n V 13 . 3- J 53 Weg m ann a a B : 9 - 23 V 13 . 3- J 53 BD C 12-2 . 4 CD4 NOD I sl e t I nsu li n Weg m ann a a B : 9 - 23 2H6 CD4 I nsu li n V 14 T GF be t a Z ekze r P er i pa n creat i c b L y mp h N o de B : 12 - 25 P ro te c ti ve 5A CD4 GAD Z ekze r I mmu n i zed S p l en o c y te s 524 - 543 4 . 1 CD4 Unknown Ye s Ye s S an t a m a ri a V 8, V 10 P hogr i n - 15 CD4 I mmu n i zed P hogr i n Ke l e m en b a L y mp h N o de P ep ti de 2 P hogr i n - 12 CD4 I mmu n i zed P hogr i n V 5,V 11 . 3 Ke l e m en b a L y mp h N o de P ep ti de 7 G9C8 CD8 NOD I sl e t I nsu li n Ye s Wong B : 15 - 23 8 . 3 CD8 NOD I sl e t NR P- A7 V 17 - J 42 Ye s Ye s S an t a m a ri a P erf o ri n a a i n d e pe n de n t mim o t ope F as m e d ia t ed V 8 - J 52 A / 4 CD8 NOD I sl e t Ye s S e r re z e a a G A D : 5 3 0 - 5 4 3 V 4 L in e s CD4 “D ri ve r ” Qu i nn b G A D : 5 2 4 - 5 3 8 CD4 P ro te c ti ve Qu i nn G A D : 5 4 6 - 5 5 4 V 1 2 b CD8 Qu i nn 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)  -chain Asp57 DQB1*0402 Leu56 -chain

  11. Defective Central ToleranceDiabetogenic MHC I-Ag7 I-Ag7 haplotype is poor peptide binder. Failure to efficiently negatively select autoreactive T cells 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 NODEvidence • 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 CD4+Foxp3+ nTreg Immunity Tolerance b-islet Ags Type 1 insulin dependent autoimmune diabetes

  15. Functional deficiency in CD4+CD25+ Treg cells in autoimmunity ? Self-reactive Teff cell CD4+CD25+ nTreg Autoimmune disease: Organ-specific - T1D, MS/EAE, Sjogren’s, Thyroiditis Systemic - SLE, APS, RA

  16. The Non-Obese Diabetic (NOD) mouse.Model of spontaneous type 1 insulin-dependent diabetes (T1D). Insulin b-islet Aberrant activation of effector T cells? Normal T1D Health T1D Health T1D Age Abnormal T cell tolerance to b-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. Are there functional deficiencies in CD4+ nTreg cells in NOD? Adoptive transfer NOD model of T1D. Diabetogenic TeffCD4+CD25- Protective nTreg CD4+CD25+ NOD NOD.TCR -/- BDC2.5 Islet-specific Vb4+CD4+ TCR Tg Wild- Type Diabetes?

  19. Functional CD4+CD25+ nTreg cells in NOD mice. BDC2.5 Teff Treg Diabetes incidence (%) Peripheral - + + + - + BDC2.5 Teff Treg Thymus Diabetes incidence (%) - + •  + + Days post-transfer

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

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

  22. Are there quantitative differences in the cellular frequency of CD4+ nTreg cells in NOD mice? NOD BDC2.5

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

  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. Established Primary 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. InheritedSusceptibility Loci: Both MHC and non-MHC genes are required. LOCUS CHROMOSOME CANDIDATE GENES IDDM1 6p21HLA-DQ\DR IDDM211p15INS VNTR IDDM3 15q26 D15s107 IDDM411q13MDU1, ZFM1, RT6,FADD/MORT1, LRP5 IDDM56q24-27 ESR,MnSOD IDDM6 18q12-q21D18s487, D18s64, JK (Kidd locus) IDDM72q31 D2s152, IL-1,NEUROD, GALNT3 IDDM8 6q25-27 D6s264, D6s446, D6s281 IDDM9 3q21-25 D3s1303 IDDM10 10p11-q11 D10s193, D10s208, D10s588 IDDM11 14q24.3-q31 D14s67 IDDM122q33CTLA-4, CD28 IDDM13 2q34 D2s137, D2s164, IGFBP2, IGFBP5 IDDM14 ?NCBI # 3413 IDDM15 6q21 D6s283, D6s434, D6s1580 IDDM16 ? NCBI # 3415 IDDM1710q25D10s1750-D10s1773

  30. The IDDM2 Locus IDDM2 Insulin Gene (INS) Predisposing Class I VNTR 26-63 repeats IDDM2 Insulin Gene (INS) Protective Class III VNTR 140-200 repeats 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. NOD.B6-chr3 .B6-Idd3 1 11 2 12 B6 B6 3 13 4 14 5 15 6 16 7 17 8 18 9 19 10 X 20% 1% Wicker LS et al. J Exp Med 1994 Lyons PA et al. Genome Res 2000 Low incidence of T1D in Idd3 recombinant congenic NOD mice NOD Chr 80%

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