Tolerance of Adaptive and Innate Human Anti-Pig Immune Responses in Humanized Mice

The level of immunosuppression needed to maintain long-term xenograft survival in humans is expected to be high, resulting in toxicity, and may not fully prevent rejection. Induction of xenograft tolerance would overcome this limitation. We have developed two approaches to inducing human immune tolerance to the pig, namely mixed xenogeneic chimerism and porcine thymic transplantation, in a human immune system (HIS) mouse model. Each approach centrally tolerizes human T cells to the donor pig. Additionally, mixed xenogeneic chimerism tolerizes human NK cells and B cells producing anti-pig xenoantibodies, providing the basis for large animal studies in Projects 1 and 2. However, human T cells developing in porcine thymus grafts, while tolerant of the donor pig, demonstrate some homeostatic defects, lack tolerance to human tissue-restricted antigens (TRAs) and show reduced responses to antigens presented by HLA compared to T cells developing in a human thymus. In the project period, we have made progress in constructing a “hybrid” porcine thymus that contains human thymic epithelial cells (hTECs) derived from post-natal human thymus or human pluripotent stem cells (hPSCs) and demonstrated beneficial immunological effects of adding hTECs to the porcine thymus graft. In the renewal, we propose to build on these results to further improve the development of human immune systems tolerant to the pig, provoking further advances in the large animal models and ultimately in patients. In Aim 1, we will achieve optimal tolerance and immune function by combining mixed chimerism with either hybrid thymic transplantation or pig plus human thymic transplantation, modeling pig thymic transplantation without human recipient thymectomy. We will compare T cell reconstitution and tolerance to the pig and the human, including assessment of Treg mechanisms. We will utilize novel TCR transgenic tools to determine the impact of each method on thymic selection. We hypothesize that the ideal combination of thymus transplantation and mixed chimerism will generate a highly diverse, permanently and robustly tolerant TCR repertoire that recognizes both donor HLA- and SLA-restricted antigens. In Aim 2, we will determine the mechanisms of T and B cell tolerance via mixed chimerism and thymic transplantation. For T cell tolerance, we will generate and make use of directly and indirectly xenoreactive human anti-pig TCRs and human anti-pig Treg TCRs generated from human anti-pig responses. The impact of mixed chimerism and thymic transplantation on selection of these human anti-pig TCRs will be determined. We will also assess B cell tolerance to known porcine carbohydrate targets of human Nab-producing cells and use fluorochrome-labelled carbohydrate-protein conjugates, analysis of supernatants of stimulated B cells, and ELISPOT assays to determine the mechanisms of human B cell tolerance. Collectively, these studies will provide important proofs of principle and mechanistic insights into the human T and B cell tolerance achieved with mixed chimerism and xenogeneic thymic transplantation and will advance the approaches being tested in large animals in Projects 1 and 2 of this PPG, ultimately for application in humans.