Prolonged Islet Allograft Survival in Diabetic NOD Mice by Targeting CD45RB and CD154 R. Damaris Molano, 1 Antonello Pileggi, 1 Thierry Berney, 1 Raffaella Poggioli, 1 Elsie Zahr, 1 Robert Oliver, 1 Camillo Ricordi, 1 David M. Rothstein, 2 Giacomo P. Basadonna, 2 and Luca Inverardi 1 Clinical islet transplantation is a successful procedure that can improve the quality of life in recipients with diabetes. A drawback of the procedure is the need for chronic administration of immunosuppressive drugs that, among other side effects, are potentially diabeto- genic. Definition of immunosuppressive protocols that utilize nondiabetogenic compounds could further im- prove islet transplantation outcome. We used the NOD mouse to assess the effect of targeting the T-lymphocyte surface receptors CD45RB and CD154 in preventing loss of allogeneic islet grafts as a result of recurrence of autoimmunity and allorejection. Administration of the two antibodies led to significantly prolonged allograft survival, with a percentage of grafts surviving long- term. The therapeutic efficacy of the treatment was paralleled by a shift in CD45RB isoform expression on T-lymphocytes, increased in vitro responsiveness to interleukin-7, and increased in vitro -interferon pro- duction after anti-CD3 antibody stimulation. Further- more, graft infiltration by CD8 T-cells was remarkably reduced. Recipient mice bearing functioning allografts were otherwise immunocompetent, as assessed in vivo and in vitro by numerous tests, including intragraft cytokine production, responsiveness to polyclonal stim- ulation and alloantigens, and analysis of cell subset phenotype. These data show that nondiabetogenic reg- imens of immunomodulation can lead to prolonged islet allograft survival in the challenging NOD mouse model. Diabetes 52:957–964, 2003 R ecent clinical trials demonstrated that islet transplantation can result in remarkable im- provement in the quality of life of patients with type 1 diabetes by maintaining tight glucose metabolic control in the absence of hypoglycemic epi- sodes (1– 4). However, the need for chronic immuno- suppression with its side effects still limits the use of this procedure to a small cohort of patients with brittle diabe- tes and severe hypoglycemic episodes, where the risks associated with transplantation and chronic immuno- suppression are justified. Furthermore, currently used immunosuppressive agents have intrinsic diabetogenicity, probably contributing to the observed need for more than one donor organ to achieve insulin independence (5). Immunomodulatory compounds that allow for pro- longed graft survival in the absence of diabetogenic effects could represent a valuable alternative for the treatment of transplant recipients. To this aim, blockade of signal 1 or 2 of T-cell activation by the use of biological modifiers such as monoclonal antibodies (mAb) and soluble recep- tor ligands has proved effective in preventing or delaying graft rejection as well as autoimmune diseases (6 –21), in the absence of -cell toxicity. Modulation of signal 1 by administration of anti-CD45RB mAb has shown efficacy in preventing kidney (6), pancreas (7), and islet allograft rejection in murine models (8 –11). CD45 is a family of protein phosphatases critically in- volved in T-cell receptor–mediated signal transduction (signal 1). Blockade of signal 2 by selectively targeting co-stimula- tory molecules has also yielded promising results in mod- ulating immune responses and has provided a precious tool to explore the immunological mechanisms underlying transplant rejection and autoimmunity (12). Treatment with anti-CD154 mAb induced long-term allograft accep- tance in several transplantation models (13–16) and effi- ciently prevented autoimmune diseases (17,18), including diabetes (19 –21). CD154 is a tumor necrosis factor recep- tor family member involved (via binding to CD40) in T-cell co-stimulation (signal 2) after antigen recognition. We have previously reported that simultaneous admin- istration of mAb targeting CD45RB and CD154 protected islet allografts in mice and allowed for the induction of tolerance in a large proportion of recipients in a nonauto- immune background (21). In addition, monotherapy with anti-CD154 mAb significantly prolonged survival of synge- neic and allogeneic islet transplants in spontaneously diabetic NOD mice (21). NOD mice spontaneously develop autoimmune diabe- tes, arguably representing the best available model for the study of allogeneic islet transplantation in type 1 diabetes. In NOD mice with already established autoimmune diabe- tes, few treatments lead to prolonged islet allograft sur- vival, and even fewer lead to indefinite acceptance of the graft (22,23). Therapeutic approaches that result in long- term islet graft survival and even immunological tolerance From the 1 Cell Transplant Center, Diabetes Research Institute, University of Miami School of Medicine, Miami, Florida; and the 2 Department of Transplant Surgery, Yale Medical School, New Haven, Connecticut. Address correspondence and reprint requests to Luca Inverardi, Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10th Avenue (R-134), Miami, FL 33136. E-mail: linverar@med.miami.edu. Received for publication 2 December 2002 and accepted in revised form 8 January 2003. R.D.M. and A.P. contributed equally to this work. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN-, -interferon; IL, interleukin; mAb, monoclonal antibodies; PMA, phorbol-myristate-acetate. 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