ENDOTHELIAL MODIFICATIONS THAT REDUCE T CELL ACTIVATION

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DESCRIPTION (Adapted from the Applicant's abstract): Cultured human and pig endothelial cells (ECs) can present MHC molecules directly to human T cells and can provide sufficient costimulatory signals such that activation of resting T cells results. In the current funding period, the investigators have shown that human peripheral blood adherent cells can activate resting naive and memory T cells, that human ECs can activate resting memory but not naive T cells, and that non-immune cell types (e.g. fibroblasts) are unable to activate resting T cells and can only restimulate activated T blasts. These differences arise from differences in costimulator molecules expressed by these various cell types. The investigators hypothesize that human ECs will function in vivo to initiate recall responses by presenting antigen to circulating memory T cells but fail to initiate primary immune reactions involving naive T cells. They further hypothesize that alterations in the costimulator molecules expressed by ECs will alter T cell activation in vitro and alter the outcome of immune reactions in vivo. In the renewal period the investigators propose to test these hypotheses in five specific aims. (1) The costimulator molecules on cultured human ECs will be altered by reducing LFA-3 (CD58), expressing transfected B7.1 (CD80) or B7.2 (CD86), blocking CD40, or pretreating with cytokines and the consequences for T cell subset activation will be assessed using in vitro assays (cytokine transcription and secretion, proliferation, differentiation and development of anergy); (2) Pig ECs, which express B7.2, will be compared to human ECs in the same assays and pig B7.2 will be analyzed when expressed in human ECs; (3) The role of costimulators (LFA-3, B7.1, B7.2, CD40) on ECs will be studied in a human peripheral blood lymphocyte-severe combined immunodeficient (huPBL-SCID) mouse model of vascularized human skin allograft rejection, developed by the investigators; (4) The huPBL-SCID mouse model will be extended to examine human T cell-pig EC interactions by engraftment of vascularized pig skin grafts; (5) New huPBL-SCID mouse models will be developed to examine human T cell interactions with human ECs that have been genetically modified to alter the expression of costimulator molecules. These experiments will (1) provide a test of the hypotheses; (2) test the concept that genetic alterations of ECs can alter the course of T cell-mediated injury in allografts, xenografts and other settings; and (3) develop new and useful in vivo models for evaluating the efficacy of immunosuppressive drugs, anti-human antibodies, and other therapeutic interventions on human immune responses.