PROJECT SUMMARY A major challenge in organ transplantation is the shortage of human organs. The Sykes lab has focused on investigating and developing new methods for inducing tolerance to prevent graft rejection. A method to address organ shortages is through the development and increased use of xenotransplantation, such as pig to human organ transplants. A major barrier of xenotransplantation is the necessity for long-term, high levels of immunosuppression due to increased polymorphisms compared to human to human organ transplantation. Although we have demonstrated the ability to achieve human T cell development in pig thymus grafts, we have also identified defects in positive and negative selection on a pig thymic epithelium of human T cells that must interact with human APCs in the periphery following transplantation. Our lab has been able to construct a hybrid porcine thymus that showed improved immunological functions. Thus, the objective of Project 3 in the parent proposal is to investigate the possibility of tolerance induction in humans through the use of mixed xenogeneic chimerism and two types of porcine thymic transplantation in HIS mice. Specific AIM #1 of Project 3 of the parent grant is to achieve optimal tolerance and immune function by combining mixed chimerism with either hybrid thymic transplantation or pig plus human thymic transplantation. Specific Aim #2 is to determine the mechanisms of T and B cell tolerance of this mixed chimerism and thymic transplantation in HIS mice. The proposed studies for this supplement relate to the investigation proposed for Specific Aim #2 of the parent grant, in which we will determine the mechanisms of T cell tolerance through mixed chimerism and thymic transplantation. This supplemental proposal aims to develop a method of clarifying the role of Treg mechanisms in xenotolerance. The supplement focuses on generating and understanding the human anti-pig Treg repertoire and the role of direct and indirect Treg responses in tolerance modeled by our two approaches in the parent grant. The proposed studies will establish a clear method for investigating the role of Tregs with direct and indirect specificity in induced human T cell tolerance to porcine xenografts These studies will also be of considerable relevance to studies in Project 2, in which expanded baboon Tregs are used to promote mixed chimerism from porcine donors.