Heart transplantation becomes the only option for survival for patients with advanced heart failure who have exhausted other medical and surgical treatment options. 71,207 heart transplants have been recorded in the United Network of Organ Sharing (UNOS) database, each providing a medical ‘second chance’ for recipients otherwise facing death. Despite these remarkable successes cardiac transplantation is a temporary cure because rejection limits the lifespan of transplanted organs. Both rejection and tolerance require transendothelial migration of donorreactive T cells into allograft tissue. Whether or not T cell recognition of cognate antigen within the vasculature is prerequisite to transendothelial migration is unknown. Likewise little is known about the repertoire and specificity of T cell infiltrates within tolerant allografts. Our preliminary data in murine models of cardiac transplantation suggest that endothelial cell presentation of alloreactive major histocompatibility molecules (MHC) is an absolute requirement for transendothelial migration. Likewise, our preliminary data suggest that T cell infiltrates within cardiac isografts are completely self-restricted leading to the hypothesis that these are endogenous self-reactive regulatory cells. To better understand these questions fundamental to both rejection and tolerance we propose the following specific aims: 1) we will utilize a novel transplant model in which ovalbumin peptide presented in the context of the MHC molecule Kb is restricted to endothelial cells thereby allowing us to directly measure the ability of Kb restricted OT-1 transgenic T cells to extravasate in the presence and absence of cognate ligand on the endothelium; 2) we will utilize CDR3-based next generation sequencing to define the T cell receptor repertoire of isograft-infiltrating T cells; 3) we will perform in-vivo expansion of isograft infiltrates and test their regulatory capacity in adoptive immunotherapy models. These data will answer fundamental mechanistic questions about alloendothelium, generate rationale for manipulation of the endothelium in organ engineering, and better inform already-ongoing clinical trials of regulatory adoptive immunotherapy. Ultimately, these discoveries will translate into better therapy for the 3,897 patients currently awaiting cardiac transplantation.