Keratoplasty is the principal modality by which vision is restored in corneal blindness-the 2nd leading cause of blindness worldwide. Nevertheless, greater than 50% of corneal allografts placed in inflamed (or 'high-risk') graft beds fail despite maximal immune suppression. This incidence is completely overshadowed by the successful transplantation rates experienced in uncomplicated (or 'low-risk') graft beds, which boast a failure rate of only10% under cover of topical steroids. The epithelium of a corneal allograft has 'paradoxical'properties in alloimmunity: while it serves as a functional barrier and constitutive inflammation regulator/suppressor, it also carries a significant alloantigenic load. The latter property results in sensitization of the host against alloantigens of the graft, and importantly, these alloantigens are shared by the graft corneal endothelium-the critical target in rejection. It is therefore likely that composite grafts, consisting of allogeneic stoma-endothelium covered by an allodisparate 3rd-party donor epithelium, will sensitize the recipient to 3rd-party alloantigens-which are not shared by graft endothelium. I therefore hypothesize that this strategy will diminish allosensitization to graft endothelium and promote graft survival in a model of high-risk transplantation. Furthermore, this will provide epithelial barrier function along with the compliment of immunoregulatory factors to maximally promote allograft survival. Hence, the long-term objective of this study, which is to use such composite grafts to promote graft survival, is a goal relevant to the National Eye Institute mission and a highly feasible strategy in the clinical realm. In Specific Aim 1, I propose to examine the poorly understood roles of graft epithelium versus stroma- endothelium in high-risk hosts. This will be tested in vivo by identifying allosensitization in situ, as well as through the development of an in vitro assay to measure allosensitization, and subsequently verified in an in vivo setting. In Specific Aim 2, I propose to test whether such composite grafts promote corneal transplant survival. This will be tested by measuring T cell infiltration into the graft, assessing in vivo function of effector T cells via adoptive transfer assays, and finally the measurement of graft survival via biomicroscopy. LAY DESCRIPTION: Success rates for corneal transplantation are very poor in eyes with severe inflammation (so-called 'high-risk'hosts). These eyes are prone to graft failure by arousing certain proteins called 'alloantigens'on the transplant. In this proposal, I will test whether the reconstruction of transplants to exclude such alloantigens will promote transplant survival in mice. C