Preparation of hybrid porcine thymus containing non‐human primate thymic epithelial cells in miniature swine

Abstract

Background We have achieved greater than a 6‐month survival of a life‐supporting kidney co‐transplanted with a vascularized thymic graft into non‐human primates (NHPs). Although we have achieved pig‐specific unresponsiveness in vitro, immunosuppression was not able to be fully weaned. Studies in mice and humanized mice suggest that a hybrid pig thymus (Hyb‐thy)‐containing host thymic epithelial cells (TECs) can optimize intra‐thymic selection, achieving xenograft tolerance with improved reconstitution of T‐cell function. Methods We have tested the feasibility of the preparation of a Hyb‐thy that contains NHP TECs in the donor thymic grafts. We first prepared the Hyb‐thy in the donor pigs 2‐3 weeks before xeno‐Tx. We performed six cases of Hyb‐thy preparation in six juvenile miniature swine. Two pigs received non‐manipulated cynomolgus monkey thymic cells that were isolated from an excised atrophic thymus via injection into their thymic lobes (Group 1). The remaining four received thymic cells that were isolated from non‐atrophic thymic glands (Groups 2 and 3). Pigs in Group 2 received unmanipulated thymic cells in one thymic lobe, as well as CD2‐positive cell‐depleted TEC‐enriched cells in the contralateral lobe. Pigs in Group 3 received TEC‐enriched cells alone. Results All thymus‐injected pigs received tacrolimus and rapamycin until endpoint (POD16). We detected cynomolgus monkey TEC networks in pig thymus from Groups 1 and 3, while pigs in Group 2 rejected the thymic cells. We demonstrated the preparation of Hyb‐thy in pigs using tacrolimus plus rapamycin therapy. Conclusions Our results suggest that the enrichment of TEC from the excised NHP thymus facilitated NHP TEC engraftment in pig thymus.

Reduced positive selection of a human TCR in a swine thymus using a humanized mouse model for xenotolerance induction

“…Adding a known TCR with defined specificity to this model allowed demonstration for the first time that cells expressing a human class I‐restricted TCR with therapeutic value are not selected efficiently in a swine thymus. To overcome this limitation in positive selection, we are developing approaches to generate hybrid thymi containing both human and swine TECs, building on approaches originally developed in the pig‐to‐mouse model that have begun to be explored in a non‐human primate model . The TCR transgenic humanized mouse described here provides an ideal in vivo model with which to assess the efficacy of this approach.…”

Xenotransplantation literature update, November/December 2019

“…However, to date no study has been able to achieve complete tolerance where all immunosuppression is stopped and they have long‐term functional survival, including the study by Rivard et al in 2018 . This has led Sekijima et al to develop a novel technique whereby they utilize the host non‐human primate (NHP) thymic cells to vascularize with the donor pig thymus (Hyb‐thy) and then finally transplant the Hyb‐thy with the pig organ to the NHP (Hyb‐VTL) . This study, the first in a series of two, aimed to investigate the feasibility of creating Hyb‐thy and determine the appropriate immunosuppression required for the preparation Hyb‐VTL.…”

Progress towards xenogenic tolerance

“…Therefore, the Sykes lab has studied the potential to create a recipient/swine hybrid thymus to provide self/donor-specific T-cell deletion and production of natural self/donor-reactive Tregs. 31,32,33 By injecting recipient TECs into porcine thymic grafts in a fetal pig-nude mice model, Fudaba 34 found that autoimmunity was suppressed, and regulatory cell function was enhanced. These studies were then extended into humanized and primate models.…”

“…31 This was first described in a humanized model by Maharlooe 31 using human thymic stromal cells injected into swine thymus, which was found to support human thymopoiesis in humanized mice. In the NHP model, Sekijima et al 32 introduced NHP TECs into hybrid thymic grafts from donor pigs and reported persistence of NHP TECs in miniature swine thymic lobes after 3 weeks. 32 Due to the lack of TECs in an adult thymus due to atrophy, induced pluripotent stem cells (PSC) for the generation of human TECs are being investigated to populate a hybrid thymus.…”

“…In the NHP model, Sekijima et al 32 introduced NHP TECs into hybrid thymic grafts from donor pigs and reported persistence of NHP TECs in miniature swine thymic lobes after 3 weeks. 32 Due to the lack of TECs in an adult thymus due to atrophy, induced pluripotent stem cells (PSC) for the generation of human TECs are being investigated to populate a hybrid thymus. 33 In vitro, it was possible to differentiate human PSC-derived thymic epithelial progenitors, which persisted in swine thymic grafts, and tolerance is currently being assessed for PSC specific antigens.…”