This is a competitive renewal application. The goals of this project are to characterize specific costimulatory pathways invoked during allo and xeno immune responses to endothelium. Studies have defined common properties of the responses to human and porcine ECs but also have identified a major important difference regarding the expression of B7. There are substantial sequence differences in the cell surface antigens between these antigens and their contribution to signaling via ligand interactions may differ significantly. The response of human T cells is much stronger to porcine ECs than human allogeneic ECs. This is a consequence of direct recognition of MHC class I and class II antigens as well as the presence of pB7.2 on porcine ECs. The role of CD58 and CD59 in the CD2 pathway and pB7.2 in the CD28 pathway in xeno responses will be studied. The activation of human T cells will be assessed using complementary approaches. The contribution of distinct ligand interactions on transfected CHO cells individually or in combination will be examined. This will include normal and mutant forms of human CD58 and CD59 antigens as well as normal forms of these homologous porcine antigens. In addition, more complex antigen presenting cells will be studied after removing defined antigens. Several strategies will be employed to specifically inhibit the cell surface expression of SLA antigens and costimulatory molecules (pB7.2, CD59, CD58) on human and porcine ECs. Both primary as well as recall responses will be evaluated. The ability of human T cells to recognize SLA antigens will be studied by expressing these antigens on transfected cells. Recognition of SLA antigens in the absence of a costimulatory signal may result in induction of anergic human T cells. Transfections of NIH3T3 cells with SLA antigens plus additional costimulatory molecules will be utilized to define the requirements for breaking anergy. Comparisons between human and porcine systems will be made in order to define the critical pathways that could be targets for facilitating tissue transplants. Finally, the applicants will utilize an in vivo model in which the immune system of SCID mice has been reconstituted with human cells. The immunogenic properties of the transfected porcine ECs that have demonstrated altered recognition properties in culture will be evaluated in this model after formation of synthetic vascular networks in collagen gels.
