Five-Year Follow-Up After Clinical Islet Transplantation Ryan et al, Diabetes 54:2060-2069, 2005.

1. Prolonged Diabetes Reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates Hering et al, Nature Medicine 12:301-303, 2006 • Reversal Diabetes > 100 days Porcine islet transplants into streptozotocin diabetic cynomolgus macaques • Required Rx with toxic regimen including anti-CD154, leflunomide, basiliximab, FTY720 and everolimus • No Gal-specific antibody mediated hyperacute rejection

2. Five-Year Follow-Up After Clinical Islet TransplantationRyan et al, Diabetes 54:2060-2069, 2005. Percent derived from life Table Months of Follow-up

3. Issues in Islet Graft Transplantation • Allograft immunity • Xenograft immunity • Autoimmunity

4. Transplant Immunity Autoimmunity Islets

5. >13 ,000 World Experience in Organ / Tissue Transplantation Kidney >500,000 Heart >50,000 Pancreas Islet <500 0 200000 100000 300000 400000 500000 600000 Number of transplants

6. Autografts:islets obtained from the recipient to prevent pancreatectomy-induced diabetes Allografts: islets obtained from an unrelated donor to prevent pancreatectomy-induced diabetes Allografts + IDDM: islets obtained from an unrelated donor after or at the same time as kidney transplantation Clinical Experience in Islet Transplantation

7. Islet Transplantation Registry 1990 - 1997 100 90 80 Autografts (n=50, 50%) 70 60 % Insulin-Independence 50 40 Allografts (n=15, 40%) 30 20 Allografts + IDDM (n=200, 8%) 10 0 10 1 7 8 2 3 5 9 11 12 0 4 6 Months post-transplantation

8. Possible Reasons for Islet Graft Failure Insufficient islet mass Poor quality of islets Failure to engraft Toxicity of anti- rejection drugs Islets Insulin resistance Disease recurrence Allograft rejection

9. `Edmonton’ Protocol University of Alberta, Edmonton, Alberta J. Shapiro, M.D. R. Rajotte, Ph.D. • Islet-alone transplantation trial • Type 1 diabetic patients received two transplants of large numbers of high quality islets. • Standard immunosuppressive therapy was • replaced with a steroid-free protocol: Daclizumab (DZB) (anti-IL2 receptor antibody) Daily doses of sirolimus and low-dose tacrolimus

10. Eligibility Criteria • 18-55 years of age, have had IDDM for >5 years • Hypoglycemia unawareness requiring medical assistance • Uncontrolled blood sugars despite intensive insulin therapy (“ brittle diabetes”). • Evidence of early diabetic nephropathy or other secondary complications

11. Exclusion Criteria • cardiac disease or psychiatric illness • active alcohol or substance abuse • previous transplant • a history of malignancy or abnormal liver function • an active infection (HIV, Hepatitis B or C, TB)

12. `Edmonton Protocol’ Results • 13 consecutive cases of insulin-independence with a duration of (longest > 2.5 years) • normalization of HbA1C values (mean of 5.7% at 3 and 6 months post-transplantation) • no episodes of hypoglycemia • no episodes of acute rejection and minimal toxicity from anti-rejection drug therapy

13. Pre-transplant 600 500 400 Blood glucose (mg/dl) 300 200 100 0 Shapiro et al. N Engl J Med 2000; 343:230-238 600 500 Post-transplant 400 Blood glucose (mg/dl) 300 200 100 0 2 4 6 12 2 4 8 10 12 6 8 10 a.m. p.m. Time of day

14. Islet Transplantation: The NIH ExperienceDiabetes Care 2003: 26:3288-95 • Major Procedure ComplicationsPartial portal vein thrombosisIntra-abdominal hemorrhage • Immunosupression ComplicationsKidney ToxicitySirolimun-induced Pneumonitis • Three Patients Discontinued Immunotherapy ½ Patients insulin-independent at one year Decreased Hypoglycemia and less severe Hypoglycemia

15. Percent 4 Year Survival with Pancreas Transplant Ventrom et al JAMA 2003; 290: 2817-2823

16. Future Directions • Reduce requirement to single pancreas / recipient • Interventions to reduce peri-transplant inflammation • Progress towards ‘tolerizing’ strategies

17. Alternative sources of tissue • insulin-producing cell lines • stem cells • xenografts (other species)

18. Experimental Islet Transplantation C57Bl/6 mouse (H-2b) Remove pancreas Streptozotocin-induced diabetic BALB/c mouse (H-2d) Isolate islets Transplant islets under kidney capsule

19. Key Components to Islet Allograft Cellular Rejection • Donor-derived APCs • Donor MHC class I expression • Host CD8 T cells • Variable requirement for CD4 T cell help

20. Key Components to Islet Xenograft Cellular Rejection • Host-derived APCs • Host MHC class II expression • Host CD4 T cells

21. Conclusion / Hypothesis • Allograft Rejection --> Predominant CD8- dependent ‘direct’ recognition • Xenograft Rejection and Autoimmune pathogenesis --> Predominant CD4- dependent ‘indirect’ recognition

22. Anti-LFA-1 Therapy Facilitates Long-Term Islet Allograft Acceptance(C57Bl/6 --> BALB/c) 100 80 60 % Grafts Functioning Anti-LFA-1 (n = 20) 40 Control Ig (n = 10) 20 0 0 20 40 60 80 100 Days PostTransplantation

23. Failure of Anti-LFA-1 to Prevent Disease Recurrence(NOD --> NOD) 100 80 Untreated (n = 10) 60 % Grafts Functioning Anti-LFA-1 (n = 8) 40 Young SZ-NOD (n = 3) 20 0 0 20 40 60 80 100 Days Post Transplantation

24. Anti-CD4 but not anti-CD8 therapy prevents acute disease recurrence in NOD mice 1 0 0 8 0 Untreated (n=8) Anti-CD8 (116-13.1; n=10) 6 0 % Grafts Functioning Anti-CD4 (GK1.5; n=9) 4 0 0 2 0 0 8 0 2 0 4 0 6 0 1 0 0 Days Post-transplantation

25. C (1) T C APC T (C) (C) (2) C CoS C (1) No b cell T C Response (C) C The Stimulator Cell Model

26. DIRECT A (1) DONOR T T A APC (A) (A) (2) A Shed Graft Antigens INDIRECT (X) x B (1) T HOST T x B (B.X) (B.X) APC (2) B x

27. CD4 T Cell B Cell Delayed-Type CD8 T Cell Help Help Hypersensitivity GRAFT

29. TOLERANCE