Solid organ transplantation is currently standard therapy for those individuals suffering from kidney, liver, pancreas, heart and lung failure. Unfortunately, the compelling benefits afforded to patients by transplantation are tempered by the requirement for indefinite immunosuppression that can be associated with an increased susceptibility to certain infections, malignancy and renal failure secondary to the nephrotoxicity of current mainstream immunosuppressive agents. This project aims to develop a regimen that conditions the transplant recipient with a transient period of immunosuppression that produces a permanent state of donor specific immunologic unresponsiveness, otherwise known as tolerance. Only two approaches for tolerance induction have demonstrated significant success in the primate model. These include the mixed chimerism model and the costimulation blockade model. Our approach concentrates on the former. In this model, the recipient is exposed to a conditioning regimen consisting of lymphocyte depletion and immunosuppression at the time of transplantation. Following conditioning, donor bone marrow is infused. In our model polyclonal anti-thymocyte globulin (ATG), busulfan and sirolimus are used to prepare the recipient for donor bone marrow infusion. This approach has been demonstrated in various models to induce the development of recipient regulatory cells that inhibit rejection and induce the deletion of recipient allospecific T cells. This project has 4 parts: (1) identify the cell populations in donor bone marrow responsible for the efficacy of this approach, (2) develop methods of culturing, storing, amplifying and enriching bone marrow for the most efficacious cell populations or fractions, (3) develop a nonhuman primate model to address specific pre-clinical questions and, (4) initiate human protocols utilizing this approach. Over the past year, the following progress can be reported. Work conducted under a rodent protocol approved by the NIDDK IACUC and supported by a MCRADA with Wyeth Ayerst has demonstrated that: (1) Addition of a nonmyeloablative dose of busulfan to the regimen results in a dramatic reduction in the amount of donor bone marrow required to produce chimerism and tolerance, (2) Mice rendered stable mixed chimeras through this approach possess cells of donor origin that are capable of specifically inhibiting anti-donor activity of recipient strain lymphocytes, (3) The presence of these regulatory cells is transient, (4) The regulatory effect is contact dependent and (5) The regulatory effect can be abrogated through blockade of CD25. A primate protocol dedicated to this project was approved by the NIDDK IACUC and is supported by a three way MCRADA with Wyeth Ayerst and Sangstat. This protocol outlines experiments designed to test the safety of this approach as well as translational issues such as dosage and timing of administration of the components of the regimen in order to facilitate the application of this protocol in humans. Application of this protocol in a stringent primate skin allograft model has demonstrated that: (1) Skin graft survival is prolonged but not indefinite following cessation of immunosuppression, (2) No consistent donor chimerism can be detected following infusion of between 1 x 10-7 and 1.5 x 10-9 donor bone marrow cells/kg, (3) No evidence of graft versus host disease is observed and (4) Early development of anti-donor alloantibody can be prevented through the addition of rituximab to the conditioning regimen. Application of this approach to the less stringent kidney allograft model has resulted in long term graft survival (>100 days) following cessation of immunosuppression. The animal based laboratory work is designed to support the conduct of tolerance induction protocols in the clinical center. Toward that end, an application for an IND from the FDA was submitted and a protocol for application of this model to humans was submitted to the NIDDK IRB in November 2001. The IND was granted in August 2002 and the human protocol was eventually approved by the IRB in August 2003. Four patients have been accrued to date. The first patient experienced a reversible rejection episode following withdrawal of immunosuppression. No peripheral blood or bone marrow chimerism has been detected at any time point post bone marrow infusion. The second patient remains on monotherapy immunosuppression. Withdrawal of immunosuppression was halted due to the development of focal segmental glomerulosclerosis. This patient demonstrated <1% donor chimerism in the peripheral blood one week following bone marrow infusion. No chimerism was detected two weeks later. Patients 3 and 4 demonstrated aggressive but ultimately reversible rejection episodes shortly following bone marrow infusion and are currently on two and three drug maintenance immunosuppression respectively. Further patient accrual is currently on hold pending approval of modifications to the conditioning regimen.
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