Animal model of immune dysregulation

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  • Lists the loci linked to diabetes, will mention green loci

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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 [email_address]
  • 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).  -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
    • 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? 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 BD C 6.9 CD4 NOD S p l een Unknown V  4 , V  1 3. 1 Ye s C hr o m o som e 6 Ha s k i ns BD C 6-4 . 3 CD4 NOD I sl e t I nsu li n B : 9 - 23 V  13 . 3- J  53 Weg m ann BD C 12-2 . 4 CD4 NOD I sl e t I nsu li n B : 9 - 23 V  13 . 3- J  53 Weg m ann 2H6 CD4 P er i pa n creat i c L y mp h N o de I nsu li n B : 12 - 25 V  14 T GF be t a P ro te c ti ve Z ekze r 5A CD4 I mmu n i zed S p l en o c y te s GAD 524 - 543 Z ekze r 4 . 1 CD4 Unknown Ye s Ye s S an t a m a ri a P hogr i n - 15 CD4 I mmu n i zed L y mp h N o de P hogr i n P ep ti de 2 V  8, V  10 Ke l e m en P hogr i n - 12 CD4 I mmu n i zed L y mp h N o de P hogr i n P ep ti de 7 V  5,V  11 . 3 Ke l e m en G9C8 CD8 NOD I sl e t I nsu li n B : 15 - 23 Ye s Wong 8 . 3 CD8 NOD I sl e t NR P- A7 mim o t ope V  17 - J  42 Ye s Ye s P erf o ri n i n d e pe n de n t F as m e d ia t ed S an t a m a ri a A / 4 CD8 NOD I sl e t V  8 - J  52 Ye s S e r re z e L in e s CD4 CD4 CD8 G A D : 5 3 0 - 5 4 3 G A D : 5 2 4 - 5 3 8 G A D : 5 4 6 - 5 5 4 V  4 V  1 2 “ D ri ve r ” P ro te c ti ve Qu i nn Qu i nn Qu i nn
  • 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
    • 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 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. Immunity Tolerance 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 (%) 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. nTreg X,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 Normal Peri-insulitis Insulitis/T1D Neonatal Adult
  • 22. NOD BDC2.5 Are there quantitative differences in the cellular frequency of CD4 + nTreg cells in NOD mice?
  • 23. Pancreatic LN Non-draining LN CFSE BDC2.5 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 IDDM1 6p21 HLA-DQDR IDDM2 11p15 INS VNTR IDDM3 15q26 D15s107 IDDM4 11q13 MDU1 , ZFM1 , RT6 , FADD/MORT1 , LRP5 IDDM5 6q24-27 ESR , MnSOD IDDM6 18q12-q21 D18s487 , D18s64, JK (Kidd locus) IDDM7 2q31 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 IDDM12 2q33 CTLA -4, CD28 IDDM13 2q34 D2s137, D2s164, IGFBP2, IGFBP5 IDDM14 ? NCBI # 3413 IDDM15 6q21 D6s283, D6s434, D6s1580 IDDM16 ? NCBI # 3415 IDDM17 10q25 D10s1750-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 NOD 80% 2 3 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 X 11 20% Wicker LS et al. J Exp Med 1994 Lyons PA et al. Genome Res 2000 B6 .B6- Idd3 1% B6 NOD.B6-chr3