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 mainline 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 depletion/reconstitution model and the costimulation blockade model. Our approach concentrates on the former. In this model, the recipient's immune system is partially ablated and then exposed to donor bone marrow. In our model polyclonal anti-thymocyte globulin (ATG) 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 5 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) identify and isolate the specific actions of polyclonal anti-lymphocyte serum that are responsible for the efficacy of this approach, (4) develop a nonhuman primate model to address specific pre-clinical questions and, (5) 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 Armed Forces Radiobiology Research Institute IACUC and supported by a MCRADA with Wyeth Ayerst has demonstrated that: (1) donor bone marrow that has been depleted of a variety of cell fractions (based upon phenotype) and purified sub fractions of donor marrow vary greatly with respect to tolerogenic efficacy, (2) the most effective fractions can be purified and amplified in culture without loss of efficacy, (3) substitution of profound recipient T cell depletion, prolonged costimulatory blockade or a combination of both in lieu of polyclonal rabbit anti-mouse ATG fails to produce chimerism and tolerance. Two manuscripts are currently in preparation regarding these rodent studies. Finally, to support these rodent studies, a protocol for production of polyclonal rabbit anti-mouse ATG was approved by the NIDDK IACUC. This capability will greatly facilitate our ability to manipulate ATG for mechanistic studies and eventually allow us to produce an effective rabbit anti-monkey ATG. 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 graft survival in excess of 80 days following cessation of immunosuppression. Unfortunately, the first primates treated with the regimen succumbed to opportunistic infections related to toxic levels of rapamycin achieved with a parenteral formulation and were euthanized with intact grafts. Following refinement of the regimen, these studies, while immature, have demonstrated that: (1) primates treated with dose levels of rapamycin, ATG and donor marrow translatable to humans tolerate the regimen well, (2) skin graft survival is prolonged, (3) no detectible donor chimerism can be detected following marrow infusion and, (4) no evidence of graft versus host disease is observed. The rodent and primate 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. While the IND was granted, the protocol was tabled by the IRB pending completion of additional safety studies in primates and completion of an ongoing protocol evaluating the use of ATG and rapamycin in combination. These conditions will be met and the protocol will be resubmitted for the November 2002 IRB meeting.
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