Project Abstract: Despite recent improvements in immunosuppression, graft/patient survival of small intestinal transplantation (ITx) remains suboptimal, limiting the broader application of this therapy. Induction of donor- specific tolerance to any organ is desirable to eliminate co-morbidities associated with immunosuppressive treatment. The induction of tolerance is particularly desirable for ITx due to: (1) the requirement for high levels of immunosuppression to prevent rejection of small bowel grafts; (2) complications associated with heavy immunosuppression; and (3) the young average age of the recipients. However, to our knowledge, tolerance of intestinal allografts has not been extensively studied in preclinical models. Even in rodent models, there are few reports demonstrating tolerance to small intestine, but one successful strategy has been through the induction of durable mixed allogeneic chimerism. The overall goal of this proposal is to develop a large animal preclinical model for tolerance induction following ITx and to develop a protocol appropriate for tolerance induction in parent to child (living donor LD) ITx. We recently reported that rejection rates appear to be higher in clinical recipients of isolated intestinal transplants (iITx) compared to multivisceral transplants (MVTx), which include donor liver, stomach and pancreas. Notably we have found, for the first time, that T cell mixed chimerism which develops without GVHD following iITx and even more commonly following MVTx, is associated with reduced rejection rates. We hypothesize that the presence of graft-vs-host-reactive (GVHR) clones in these MVTx recipients facilitates engraftment of donor progenitor cells contained within the grafts, and further hypothesize that transplantation of additional hematopoietic stem cells (HSCs) during this period of the GVHR will augment chimerism and tolerance induction even in iITx recipients. The early GVHR that migrates from the graft to the recipient’s peripheral immune system (lymphohematopoietic GVH response, or LGVHR) makes hematopoietic “space” for engraftment of these hematopoietic progenitors. In this proposal, we will utilize MHC inbred miniature swine, the only large animal model that allows reproducible transplantation with defined GVH and host-vs-graft (HvG) genetic barriers, to address the above hypotheses and develop a clinically relevant LD ITx tolerance induction model. We will first establish a porcine model of orthotopic iITx and MvTx that parallels our institution’s clinical protocol and determine the role of GVH and HvG alloreactivity in driving chimerism and clinical outcomes (Aim 1). We will then utilize the LGVHR and donor HSCs to achieve tolerance in long-term allograft acceptors in the models in Aim 1 (Aim 2). The studies in this proposal may have eventual clinical applicability that could solve the most problematic issues in ITx and vastly improve the outcomes of this therapeutic modality.