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

2. Anti-GAD TCR + or – B Lymphocytes TCR Retrogenic Induction High Levels GAD65 Autoantibodies

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 Rag-/- isletsOnly islets + type B CD11c islets Insulinoma Type A Insulin granules Type B

5. Conserved T Cell Receptor Alpha Chain Induces Insulin Autoantibodies Kobayashi et al PNAS 105:10090-94 2008 Anti-B:9-23 TCR alpha TransgeneNo 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 Chr. 14Chr. 6 VJVJ VDJVDJ Peptide Antigen Presenting Cell

8. Antigen Recognition by CD8 + T Cells T cell receptor VV VV T cell class I MHC  2m  cell T. DiLorenzo

9. 10 11 -10 16 different possible combinations. Kuby Immunology

10. I-A g7 Peptide Peptide Presentation Model AA Peptide Side Chains

11. TEETH GROOVE “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

17.  -cells Pancreatic Lymph Node T cells ISLET A MODEL Ag APC Mathis/Benoist

18. TRAV5D-04+TRAJ53 Restriction? Insulin B:9-23: SHLVEALYLVCGERG? Natural T Regulatory CellsAntigen Specific T Reg IPEX Syndrome MAN: foxP3 mutant DM in days of birth! NOD anti-B9-23insulin TCR: foxP3 mutant DM ? MANNOD MOUSE IA g7 DR3/DR4 Homann 2006,JCI

19. 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

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

21. Leader 1: MAL LVHFLPLLALLALWEPKPTQA Leader 2: MALWMRFLPLLALLFLWESHPTQA Human : MALWMRLLPLLALLALWGPDPAAA 10 20 B Chain 1: FVKQHLCG PHLVEALYLVCGERG FFYTPKS B Chain 2: FVKQHLCG SHLVEALYLVCGERG FFYTPMS Human : FVNQHLCG SHLVEALYLVCGERG FFYTPKT 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 Amino Acid Sequence of Mouse 1 and 2 and Human Insulin B:9-23

22. Lack of progression to diabetes of NOD mice lacking both insulin native genes. Ins1-, ins2-: n= Ins1+, ins2-: n= 25 10 14 21 23 1111 2424 Life table update 5/19/05 Nakayama Nature 435:220,2005

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

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

25. Natural peptides selected by diabetogenic DQ8 and murine I-A g7 molecules show common sequence homology Suri et al JCI 115:2268, 2005 Structure 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 P1P4P9 I-A g7 v,e,q I,L A,s D,E 12% 20% 30,11% 45% P6 DQ8 E,d A,S A,V,s E,D 27,17% 19% 20% 60,25% Preferred AA in Bound Peptides Wiley Nat Immunol % amino acid at position

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

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

28. Expanded T cells from pancreatic lymph nodes of type 1 diabetic subjects recognize an insulin epitope Kent 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 Restricted Insulin A1-15 Caveat: High concentration to stimulate 100uM BDC

29. The insulin A-chain epitope recognized by human T cells is posttranslationally modified Mannering 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 A 1-15 peptide reactive T cells from pancreatic lymph node do not require vicinal disulfide cross-link

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

31. 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-2 b mice! ELISPOT Responses to Insulin B:9-23 though mice do not have I-A g7. MYSTERIES: Need for Ins1-/-; Lack protection H-2 b ; Presentation B:9-23 by H- 2 b.

32. 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 New Responses PROINSULIN PREPROINSULIN

33. “Santamria” NRP(IGRP206-214): Class I (K d )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-K d Tetramer Produced NRP=KYNKANWFL; NRP-V7: KYNKANVFL Higher Avidity Peptide/Tetramer V7 30% Intra-islet post 9 weeks >.5% in Blood Predicts Diabetes NOD Mice

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

35. 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

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

37. GAD, glutamic acid decarboxylase; IAPP, islet amyloid polypeptide AntigenPositionMHCWhy examined GAD65114-123A2MHC binding IAPP5-13A2MHC binding InsulinB10-18A2Proteasome cleavage B14-22A3, A11Proteasome cleavage B15-23A24MHC binding B15-24A24Proteasome cleavage B17-26A1, A3, A11Proteasome cleavage B18-27A1, A2, B8, B18Proteasome cleavage B20-27A1, B8Proteasome cleavage B21-29A3Proteasome cleavage B25-C1B8Proteasome cleavage B27-C5B8Proteasome cleavage Antigens for CD8 + T Cells in Type 1 Diabetes Patients DiLorenzo

38. AntigenPositionMHCT cellsHow identified DMK138-146H-2D b AI4; islet Positional scanning libraries IGRP206-214H-2K d 8.3; islet MHC purification and mass spectrometry 21-29H-2K d IsletMHC binding 225-233H-2D b IsletMHC binding 241-249H-2D b IsletMHC binding 324-332H-2K d IsletMHC binding Insulin 1/2B15-23H-2K d G9C8; isletExpression cloning DMK, dystrophia myotonica kinase GAD, glutamic acid decarboxylase IGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein Antigens for Islet-infiltrating CD8 + T Cells in NOD Mice DiLorenzo

39. 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

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