ENDOTHELIAL MODIFICATIONS THAT REDUCE T CELL ACTIVATION

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The investigators have previously shown that cultured human endothelial cells (EC) can activate allogeneic CD4+ and CD8+ T cells to secrete cytokines, display effector molecules (e.g., CD40L, FasL) and receptors (e.g., CD25, the IL-2Ralpha chain), and proliferate in response to IL-2. The response of T cells to cultured EC depends upon EC display of MHC molecules, of costimulators and of adhesion molecules. These observations have led to a two part hypothesis. First, the investigators propose that human EC in vascularized allografts can initiate rejection reactions. Second, that modifications to reduce expression or function of graft EC costimulators and/or adhesion molecules will reduce graft rejection. In the current application, they propose to extend their studies of the role of specific costimulator molecules (LFA-3 and CD40) and adhesion molecules (ICAM-1 and ICAM-2) on these and other early T cell responses (e.g., receptor ITAM, ZAP 70 and linker molecule phosphorylation, transcription factor activation, gene expression, and proliferation in primary and secondary cultures) in vitro. They also will examine the role of costimulators and adhesion molecules and of selected other proteins (e.g., gap junction proteins) in the formation of immune synapses between alloreactive T cells and EC. They will use established and develop new limiting dilution approaches to evaluate the extent of recognition of allogeneic EC by the direct and indirect (i.e., dendritic cell-dependent) pathways, evaluate the effects of immunoregulatory cytokines (IL-10 and IL-11) on these responses, and evaluate novel methods of acquiring and displaying alloantigens (e.g., membrane transfer or DNA incorporation from phagocytosed nuclei). Finally, they will extend their study of human EC-mediated activation of T cells to in vivo settings by examining the roles played by EC costimulators and adhesion molecules in huPBL-SCID mice chimeric animals engrafted with vascularized skin or with synthetic vessels formed with cultured human EC. The results of these studies may lead to new therapeutic strategies for allograft preservation and may also apply to other clinical settings where T cells interact with vascular endothelium, e.g., in atherosclerosis and receptors and acute coronary syndromes.