Introduction: Pig thymus transplantation can tolerize human T cells to xenografts in human immune system (HIS) mice. However, studies in immunocompetent mice have shown that recipient thymectomy is essential for the success of this approach. Avoidance of recipient thymectomy would enhance the appeal of this approach in humans. Additionally, human T cells developing in pig thymus lack tolerance to human tissue-restricted antigens (TRAs) and show reduced responses to antigens presented by HLA and suboptimal homeostasis in the periphery.  Pig/human mixed chimerism (MC) can induce central deletional T cell tolerance as well as NK cell and B cell tolerance. We hypothesized that combining MC with pig thymus transplantation in HIS mice in the presence of a human thymus might overcome all of these limitations. Human thymus could delete human TRA-reactive T cells and generate TRA-specific regulatory T (Treg) cells while enhancing HLA-restricted responses and homeostasis. The pig thymus would generate pig-specific Tregs and SLA-restricted T cells to protect the xenografts from infections. MC could assure central deletion in both thymi and allow T cells selected by both human HLA and pig SLA molecules to receive HLA and SLA-restricted antigenic stimulation and homeostatic signals in periphery. In addition, this approach would permit tolerance induction without thymectomy in human recipients.
Method: Thymectomized NSG, pig cytokine-transgenic (PCT) NSG or PCT-NOD/SCID mice were sublethally irradiated followed by injection of pig bone marrow cells and implantation of fetal pig thymus and human fetal thymus tissue under the capsule of each kidney. Human fetal CD34+ cells hematopoietic stem cells (HSCs) were injected intravenously. Control groups received dual pig/human thymus plus human HSCs, human thymus plus mixed chimerism, pig thymus plus human HSCs and human thymus plus human HSCs. Human and pig chimerism were monitored. 17-20 weeks post-transplantation, tolerance of peripheral human T cells to donor pig and human antigens was assessed by mixed lymphocyte reaction. 
Results: Human T cells from chimeric recipients of human thymus alone and of dual pig and human thymus were specifically hyporesponsive to both donor human and pig antigens, indicating tolerance. Depletion of Tregs led to increased anti-donor responses in animals with pig thymus alone without MC, but tolerance was largely deletional in animals with dual thymus and MC. Human and pig antigen presenting cells were detected in pig and human thymi of mixed chimeric mice with dual thymus, consistent with ongoing clonal deletion in both thymi.
Conclusion: Transplantation of dual pig and human thymus combined with MC induction leads to tolerance of human T cells to autologous and pig antigens. These data suggest that combined MC and porcine thymic transplantation could achieve tolerance in humans without thymectomy, supporting the clinical application of this approach for xenograft tolerance induction.