Prolonged Diabetes Reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates Hering et al, Nature Medicine 12: , 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

Five-Year Follow-Up After Clinical Islet Transplantation Ryan et al, Diabetes 54: , Months of Follow-up Percent derived from life Table

Issues in Islet Graft Transplantation Allograft immunity Xenograft immunity Autoimmunity

Transplant Immunity Autoimmunity Islets

Number of transplants Islet Pancreas Heart Kidney World Experience in Organ / Tissue Transplantation Transplantation >500,000 >50,000>13,000<500

Autografts: islets obtained from the recipient to prevent pancreatectomy-induced diabetes 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

Islet Transplantation Registry Autografts (n=50, 50%) Allografts (n=15, 40%) Allografts + IDDM (n=200, 8%) Months post-transplantation % Insulin-Independence

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

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

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

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

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

a.m.p.m. Post-transplant Pre-transplant Time of day Blood glucose (mg/dl) Shapiro et al. N Engl J Med 2000; 343:

Islet Transplantation: The NIH Experience Diabetes Care 2003: 26: Major Procedure Complications Partial portal vein thrombosis Intra-abdominal hemorrhage Immunosupression Complications Kidney Toxicity Sirolimun-induced Pneumonitis Three Patients Discontinued Immunotherapy ½ Patients insulin-independent at one year Decreased Hypoglycemia and less severe Hypoglycemia

Percent 4 Year Survival with Pancreas Transplant Ventrom et al JAMA 2003; 290:

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

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

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

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

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

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

Anti-LFA-1 Therapy Facilitates Long-Term Islet Allograft Acceptance ( C57Bl/6 --> BALB/c ) % Grafts Functioning Days Post Transplantation Anti-LFA-1 (n = 20) Control Ig (n = 10)

Failure of Anti-LFA-1 to Prevent Disease Recurrence (NOD --> NOD) % Grafts Functioning Days Post Transplantation Untreated (n = 10) Anti-LFA-1 (n = 8) Young SZ-NOD (n = 3)

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

C C C APC T (C) T T CoS (1) (1) (2) No Response C C C  cell The Stimulator Cell Model

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

TOLERANCE