*Purpose: Thymus transplantation is a promising approach to induce T-cell tolerance for xenotransplantation. We have previously shown that humanized mice generated with human hematopoietic stem cells (HSCs) and swine (SW) thymus grafts (SW/HU mice) are tolerant to human hematopoietic antigens. Our recent data suggests that, unlike humanized mice generated with human HSCs and autologous thymus grafts (HU/HU mice), human T cells in SW/HU mice are not tolerant to human tissue-restricted antigens (TRAs), presumably due to the lack of expression of human TRAs by SW thymic epithelial cells (TECs). While there was no significant difference between HU/HU vs SW/HU Treg suppressive capacity in auto, allo and xeno responses, we found that human CD8 and Treg cells have lower survival rates in SW/HU compared to HU/HU mice. We hypothesized that these problems and suboptimal positive selection of T-cells recognizing antigens presented by HLA might be overcome by creating a pig thymus containing human TECs.

*Methods: In order to generate a “hybrid thymus”, stromal cells of human fetal thymi (gestational age 20 weeks) were injected into freeze/thawed fetal SW thymic tissue and transplanted to NSG mice with human fetal liver CD34+ cells.

*Results: As SW thymocytes might potentially reject human thymic stromal cells, we tested strategies to deplete pig thymocytes. Ex vivo treatment of SW thymic fragments with 2 deoxy glucose (2DG), which competitively inhibits the production of glucose-6-phosphate from glucose, for 12 hours most effectively depleted the thymocytes while preserving the capacity of SW thymic tissue to support thymopoiesis. HuTEC-injected SW thymi were functional and supported human thymopoiesis. Human mTECs and cTECs were admixed with pig TECs in long-term hybrid thymi. Peripheral T-cells of mice generated with fetal TEC-injected SW thymi were hyporesponsive to human TEC donor-derived DCs. However, dynamics of T cell development in mice with hybrid thymus were closer to those in SW/HU than HU/HU mice.

*Conclusions: In conclusion, injection of human thymic stromal cells into pig thymus can generate a human/pig hybrid thymus with an impact on tolerance.

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