Saturday October 12, 2019 from 08:00 to 08:30
Use of human pluripotent stem cells to produce human/pig hybrid thymus grafts to achieve immune tolerance to pig antigens with optimal immune function
Mohsen Khosravi Maharlooei1, Nichole Danzl1, Rafael Gras-Pena1, Haowei Li1, Markus Holzl1, Andrea Vecchione1, Aditya Misra1, Amanda Ruiz1, Rachel Madley1, Grace Nauman1, Guiling Zhao1, William Meng Suen Savage1, Kazuhiko Yamada1, Hans-Willem Snoeck1, Megan Sykes1.
1Columbia Center for Translational Immunology, Columbia University, New York, NY, United States
Introduction: Powerful immune responses to xenografts are difficult to control with conventional immunosuppression without excessive toxicity. Thymus transplantation is a promising approach to induce T-cell tolerance for xenotransplantation. Humanized mice generated with human hematopoietic stem cells (HSCs) and swine (SW) thymus grafts (SW/HU mice) are tolerant to human hematopoietic antigens. However, our data suggest that they 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). This problem and suboptimal positive selection of T-cells recognizing antigens presented by HLA might be overcome by creating a human/pig hybrid thymus. As the number of TECs in human adult thymi is limited due to thymic involution, we aim to generate TECs from patient-specific induced pluripotent stem cells (iPS) in order to generate hybrid thymus.
Methods: In initial studies, thymic stromal cells of human fetal (gestational age 20 weeks) and pediatric (4-month old) thymi were injected into freeze/thawed fetal SW thymic tissue and transplanted to NSG mice with human fetal liver CD34+ cells (SW-huTEC/HU mice). In order to develop a protocol for generation of pluripotent stem cell (PSC)-derived TECs, we began with a human embryonic stem cell (ES) line, RUES1. We developed an in vitro differentiation method to generate thymic epithelial progenitors (TEPs) by sequential induction of definitive endoderm, anterior foregut endoderm, and pharyngeal endoderm followed by specification of the thymus domain of the 3rd pharyngeal pouch. Using a similar protocol as for fetal and pediatric TECs, ES-derived TEPs were injected into fetal SW thymic tissue and transplanted to NSG mice with human fetal liver CD34+ cells (SW-huPSC-TEC/HU mice).
Results: HuTEC-injected SW thymi were functional and supported human thymopoiesis. Human mTECs and cTECs were admixed with pig TECs in hybrid thymi generated by both human fetal and pediatric stromal donors. Peripheral T-cells of SW-huTEC/HU mice were hyporesponsive to TEC donor-derived DCs. Cultured TEP cells formed colonies positive for FoxN1, the general TEC marker EpCAM, as well as specific cortical (K8) and medullary (K5, UEA-1) TEC markers. In thymic grafts of long-term SW-huPSC-TEC/HU mice, human PSC-derived TECs were detectable by co-staining of cytokeratin 8, 14 and HLA-DR in 6 of 7 recipients.
Conclusion: In conclusion, injection of fetal and pediatric human thymic stromal cells into pig thymus can generate a human/pig hybrid thymus. Human PSC-derived TEPs can be differentiated in vitro with our protocol and persist long-term in SW thymic grafts. We are currently testing tolerance to the PSC antigens in SW-huPSC-TEC/HU mice. Future studies will apply this TEP differentiation protocol to iPS cell lines.