Heart/kidney co-transplantation in miniature swine provides the only consistent and reproducible model of nonablative cardiac allograft tolerance in large animals. It also mimics the beneficial effects of co-transplanting hearts and kidneys in humans. The mechanism by which a heart is protected from both acute and chronic rejection by the tolerant state induced by a kidney allograft is unknown. In our previous grant we characterized the role of the donor kidney and host thymus in tolerant heart/kidney recipients and began to investigate kidney-derived cell populations. Based on those studies, we now hypothesize that host thymus-derived regulatory T cells, generated by cells or antigens derived from the donor kidney, are responsible for the induction of tolerance to cardiac allografts in heart/kidney recipients. The corollary is that regulatory T cells are present in tolerant recipients bearing long-term cardiac allografts but are not present in chronically immunosuppressed recipients bearing long-term cardiac allografts. We will test this hypothesis by 1) determining the in vitro characteristics of regulatory T cells from tolerant heart/kidney recipients, 2) determining the role of regulatory T cells in the induction and maintenance phase of tolerance in heart/kidney recipients, 3) determining how a state of tolerance modifies the effects of intragraft cytokine excess in heart/kidney recipients, and 4) comparing the in vitro parameters of transplant immunity in animals bearing allogeneic heart transplants prolonged by tolerance induction vs. chronic immunosuppression. Understanding the mechanisms of T cell regulation may lead to novel tolerance strategies in human transplantation.