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The Generation of Retrogenic Mice

The Generation of Retrogenic Mice. MIG. Insertion: TCR alpha only or alpha_P2A_beta. Ψ. 5’LTR. SCID. pMIG. IRES. GFP. 3’LTR. Phoenix cells (293T cells transfected with pCL-Eco). CMV-env (Mo-MuLV) RSV-gag-pol (Mo-MuLV). SCID or C a -KO BM cells. pMIG. pMIG. pMIG.

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The Generation of Retrogenic Mice

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  1. The Generation of Retrogenic Mice MIG Insertion: TCR alpha only or alpha_P2A_beta Ψ 5’LTR SCID pMIG IRES GFP 3’LTR Phoenix cells (293T cells transfected with pCL-Eco) CMV-env (Mo-MuLV) RSV-gag-pol (Mo-MuLV) SCID or Ca-KO BM cells pMIG pMIG pMIG

  2. Anti-GAD TCR + or – B Lymphocytes TCR Retrogenic Induction High Levels GAD65Autoantibodies

  3. T Cell Islet Accumulation in Type 1 Diabetes is a Tightly Regulated, Cell-Autonomous Event Lennon et al Immunity 31, 643-653, 2009

  4. Unique autoreactive T cells recognize insulin peptides generated within the islets of Langerhans in autoimmune diabetes James F Mohan,…& Emil R Unanue Nature Immunology March 2010 InsulinomaType A Only islets + type B Rag-/- islets Type B CD11c islets Insulin granules

  5. Conserved T Cell Receptor Alpha Chain Induces Insulin Autoantibodies Kobayashi et al PNAS 105:10090-94 2008 Anti-B:9-23 TCR alpha Transgene No Transgene

  6. Nat Immunol. 2010 Mar;11(3):225-31. Epub 2010 Feb 7.Chromogranin A is an autoantigen in type 1 diabetes.Stadinski, …Kappler, Haskins Peptide WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-A(g7) in an atypical manner, occupying only the carboxy-terminal half of the I-A(g7) peptide-binding groove.

  7. T Cell Receptor Gene Segments Antigen Presenting Cell HLA Molecule Peptide V J V D J Chr. 14 Chr. 6

  8. T cell Vb Va T cell receptor class I MHC b2m b cell Antigen Recognition by CD8+ T Cells T. DiLorenzo

  9. V(D)J Rearrangement 1011-1016 different possible combinations. Kuby Immunology

  10. Peptide Presentation Model AA Peptide Side Chains Peptide I-Ag7

  11. GROOVE “TEETH” TEETH T Cell Receptor Analogy

  12. Child with IPEX syndrome Awaiting Bone Marrow Transplant 9 Months of Age

  13. IPEX: Immune Dysfunction, Polyendocrinopathy, Enteropathy, X-linked • Scurfin gene (Foxp3/JM2)- Controls Regulatory T Cells! • Approximately 80% of children with syndrome develop diabetes! • Bone marrow transplant can reverse

  14. “Typical” Insulitis of man differs from peri-insulitis NOD mouse: Bottazzo

  15. t t t A MODEL b-cells APC ISLET Ag T cells Pancreatic Lymph Node Mathis/Benoist

  16. MAN NOD MOUSE TRAV5D-04+TRAJ53 Restriction? ? Insulin B:9-23: SHLVEALYLVCGERG? IAg7 DR3/DR4 Homann 2006,JCI Natural T Regulatory Cells Antigen Specific T Reg IPEX Syndrome MAN: foxP3 mutant DM in days of birth! NOD anti-B9-23insulin TCR: foxP3 mutant DM

  17. ENVIRONMENTAL FACTORS Incidence type 1 DM Increasing 3%/year > 30 years! Protective Factor Decreasing -Hygiene Hypothesis – Bach NEJM 347:911, 2002 Triggering Factors -Congenital Rubella-Rubenstein Diabetes 31:1088, 1982 -Kilham Rat Virus (BB-DR rat)-Zipris J. Immunol 174:131, 2005 -Poly-IC Induction Interferon Alpha-Devendra Diabetes 54 2005 -Dietary Factors-Scott Ann Rev Nutr 26:175, 2006

  18. Natural T Regulatory Cells Antigen Specific T Reg T Cell Receptor

  19. Amino Acid Sequence of Mouse 1 and 2 and Human Insulin Leader 1: MAL LVHFLPLLALLALWEPKPTQA Leader 2: MALWMRFLPLLALLFLWESHPTQA Human : MALWMRLLPLLALLALWGPDPAAA 10 20 B Chain 1: FVKQHLCGPHLVEALYLVCGERGFFYTPKS B Chain 2: FVKQHLCGSHLVEALYLVCGERGFFYTPMS Human : FVNQHLCGSHLVEALYLVCGERGFFYTPKT 25 30 40 50 C-Peptide 1: RREVEDPQVEQLELGGSPG…..DLQTLALEVARQ C-Peptide 2: RREVEDPQVAQLELGGGPGAGDLQTLALEVAQQ Human : RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQ 55 60 70 80 A Chain 1: KR GIVDQCCTSICSLYQLENYCN A Chain 2: KR GIVDQCCTSICSLYQLENYCN Human : KR GIVEQCCTSICSLYQLENYCN 88 100 B:9-23

  20. Lack of progression to diabetes of NOD mice lacking both insulin native genes. 25 25 21 23 10 14 2 4 1 1 Ins1-, ins2-: n= Ins1+, ins2-: n= Life table update 5/19/05 Nakayama Nature 435:220,2005

  21. Insulin specific T cells Insultis Epitope and antigen spreading, expansion Diabetes Insulin is the primary antigen and precedes IGRP in hierarchy of autoantigens Krishnamurthy et al JCI:116:3258, 2006

  22. Is there a primary antigen or immune response to multiple antigens required for autoimmunity? T cells specific for multiple antigens T cells specific for one antigen Insulitis Insulitis OR Epitope and antigen spreading, expansion Expansion of T cells Diabetes Diabetes Krishnamurthy et al JCI:116:3258, 2006

  23. Natural peptides selected by diabetogenic DQ8 and murine I-Ag7 molecules show common sequence homology Suri et al JCI 115:2268, 2005Structure of Human insulin peptide DQ8, Lee et al Nature Immunology 6:501, 2001 Crystal DQ8;B:9-23: S H L V E A L Y L V C G E R G Wiley Nat Immunol P1 P4 P6 P9 Preferred AA in Bound Peptides I-Ag7 v,e,q I,L A,s D,E 12% 20% 30,11% 45% DQ8 E,d A,S A,V,s E,D 27,17% 19% 20% 60,25% % amino acid at position

  24. NOD Mouse anti-islet T Cell Clones/transgenics/retrogenics

  25. Hafler DRB1*0401 A1-15 Peakman DRB1*0401 C19-A3 Peakman A2 CD8 PPI:15-24 Gottlieb B:9-23 DQ8 Durinovic-Bello DRB1*0401 CD4 73-90

  26. Expanded T cells from pancreatic lymph nodes of type 1 diabetic subjects recognize an insulin epitopeKent et al, Nature 435:224, 2005 • Pancreatic LN: Single cell cloning - PHA • 2/3 Patients Clonal expansion duration diabetes 29 and 15 years • Vbeta29-1*03J2-3(50%)/Valpha8-3*02 J44*01(25%)Vbeta5-1*01 J2-3(52%)/Valpha39*01 J33*01(26%) • DRB1*0401 RestrictedInsulin A1-15 • Caveat: High concentration to stimulate 100uM BDC

  27. The insulin A-chain epitope recognized by human T cells is posttranslationally modifiedMannering et al, JEM 202:1191-1197, 2005 • CD4 T cells cloned with CFSE method from peripheral blood of one patient with diabetes and one child with insulin autoantibodies • Subset of the clones reacted with insulin A-chain 1-13 epitope • T Cells DR restricted and only reacted with peptide with vicinal disulfide bond between adjacent cysteines A6 and A7 • No reactivity with this peptide of clones from two normal controls with DR4 • Oral report by Sally Kent that their A1-15 peptide reactive T cells from pancreatic lymph node do not require vicinal disulfide cross-link

  28. Ins2 deficiency augments spontaneous HLA-A*0201-restricted T cell responses to Insulin Jarchum and DiLorenzo J Immunol 2010 vol 184 Humanized HLA-A2 Mice lacking Insulin 2! Ins B:10-18 CD8 Elispot

  29. Thymus-specific deletion of insulin induces autoimmune diabetes.Fan et al EMBO Journal 28:2812-2824 2009 • Ins1-/- mice (only) with cre mediated thymic mTEC deletion of ins2 develop autoimmune diabetes by age 3 weeks. • Diabetes develops in H-2b mice! • ELISPOT Responses to Insulin B:9-23 though mice do not have I-Ag7. • MYSTERIES: Need for Ins1-/-; Lack protection H-2b; Presentation B:9-23 by H-2b.

  30. The frequency and immunodominance of Islet-specific CD8+ T-cell responses change after Type 1 Diabetes Diagnosis and Treatment Martinuzzi et al Diabetes 57:1312-1320, 2008 7-16 months post onset most ELSIPOT (HLA-A2) responses decrease PREPROINSULIN PROINSULIN New Responses

  31. “Santamria” NRP(IGRP206-214): Class I (Kd)Recognized Peptide • CD8 T cell from NOD islets (e.g. clone 8.3) • Conserved Valpha/ nDn/ J alpha (Va17,Ja42)Vbeta not conserved but contributes • Accelerated diabetes TCR Transgenic • Mimotope Defined-Kd Tetramer ProducedNRP=KYNKANWFL; NRP-V7: KYNKANVFL • Higher Avidity Peptide/Tetramer V7 • 30% Intra-islet post 9 weeks>.5% in Blood Predicts Diabetes NOD Mice

  32. % NRP-V7 tetramer+ CD8+ cells % NRP-V7 tetramer+ CD8+ cells Age (weeks) Age (weeks) Tan et al., JCI 1/2003 Female NOD Mice Peripheral Blood Kd NRP-V7 Peptide (KYNKANVFL) Tetramer Analysis Avidin Kd Kd Kd Diabetes No Diabetes

  33. CTLs are targeted to kill β cells in patientswith type 1 diabetes through recognition ofa glucose-regulated preproinsulin epitope Ania Skowera,…. and Mark Peakman JCI 2008:118 3268-3271. Interferon gamma HLA-A2 ELISPOT peripheral blood Patients+ (SI>3) DM vs Controls

  34. CD8+ CTL CTL Screening of Peptide Fractions by 51Cr Release Cytotoxicity Assay Method Used to Discover IGRP APC T. DiLorenzo

  35. Antigens for CD8+ T Cells in Type 1 Diabetes Patients DiLorenzo GAD, glutamic acid decarboxylase; IAPP, islet amyloid polypeptide

  36. Antigens for Islet-infiltrating CD8+ T Cells in NOD Mice DMK, dystrophia myotonica kinase GAD, glutamic acid decarboxylase IGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein DiLorenzo

  37. Toma et al. Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients PNAS 102:10581, 2005 • Selected 8-11mer peptides of proinsulin • PBMCs of 29/32 (90%) recent+long term diabetics responded IFNgamma in ELISPOT assay versus minimal response of normals • Significant Peptides often overlapped insulin B chain peptide B:9-23(=Proins 33-47); Proins 34-42(B10-18); Proins 41-50(B17-26);Proins 42-51;Prins44-51; but also B chain-C-peptide (49-57) • Multiple class I alleles, including A1 and B8

  38. T cell epitopes on the diabetes autoantigen Phogrin (IA-2beta) are conserved among different species C terminus TM PTP N terminus Peptide 2: Amino acids 643-658 Peptide 7: Amino acids 762-777 GADPSADATEAYQEL (rat) GADPSADATEAYQEL (mouse) GGDPGADATAAYQEL (human) KNRSLAVLTYDHSRI (rat) KNRSLAVLTYDHSRI (mouse) KNRSLAVLTYDHSRV (human)

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