Successful allotransplantation currently requires continuous administration of immunosuppressive medication to prevent immunological graft rejection. These drugs are expensive and their non-specific effects greatly increase the risk of infection and malignancy. Therapies with long lasting, antigen specific effects have thus been sought. Recently, it has been shown that T cells can be made specifically non-reactive by allowing for T cell receptor engagement while preventing signaling through co- stimulatory receptors including CD28 and possibly CD154. We have initiated extensive studies in non-human primates showing that monoclonal antibodies (Mabs) that block CD154 prevent and treat allograft rejection and that their effects appear, preliminary, to be long lasting and graft specific. Additional study has been initiated with agents that block CD28 activation by interrupting its association with its ligands CD80 and CD86. The role of this project will be to investigate Mabs that block the function of CD28 and CD154 in an applicable pre-clinical model and to provide information for the design of a clinical trial with these promising agents. We will utilize a well established rhesus monkey renal allograft model to study the efficacy and safety of induction with costimulation blocking agents alone and in combination with conventional agents. Extensive collaboration with project 2 will ensure that transplants will be performed between animals with defined donor and recipient MHC disparity. We will monitor the potential side effects of each agent, and optimize the duration and dosing of each induction regimen. We will test for allograft tolerance using secondary skin grafts from donor and third party animals complemented by assays for immune competence to environmental antigens. We will investigate the mechanism of costimulation blockade induced graft acceptance. We will assay the intragraft cytokine milieu by RT-PCR and with project 2 by immunohistochemistry. Particular attention will be directed to the intragraft transcription of cytokines, costimulation molecules, and adhesion molecules associated with ischemia re-perfusion injury. Peripheral donor-specific immune responses will be studied by mixed lymphocyte reaction, allo-antibody determination, limiting dilution analysis and cytotoxicity assays. We will also test whether graft acceptance established by costimulation blockade is dependent on the suppressed expression of Th1 cytokines IL-2 and IL-12. Using neutralizing antibodies against IL-12 and the administration of recombinant IL-2 and IL-12, the ability of these cytokines to reverse the effects of costimulation blockade will be assessed during induction and after prolonged graft survival. We will also apply mechanistic concepts derived from project 4 in vivo. In collaboration with project, we will test whether co-administered donor-specific hematopoietic stem cells at the time of transplantation promote graft acceptance, and adapt the methods for expansion of primitive marrow derived stem cells to the rhesus model. We will transition the most successful method for preventing graft rejection into clinical trials planned with project 2 and project 3 investigators.
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