Development of a Pre-Implantation Regulatory T Cell-Permissive Immunosuppression Protocol in a Rat Model of Ex Vivo Lung Perfusion Followed by Lung Transplantation

Abstract

CD4+CD25highFoxp3+ regulatory T cells (Treg) can establish immunologic tolerance in animal models. We have developed a method to deliver recipient-derived Tregs to donor lungs during ex-vivo lung perfusion (EVLP). Our goal is to maximize the effectiveness of this therapy by developing a Treg-permissive post transplant immunosuppression protocol. We hypothesized that combination therapy with interleukin-2 (IL-2)/anti-IL-2 antibody complexes (IL-2c) and tacrolimus (Tac) would facilitate the expansion of endogenous Tregs and attenuate acute rejection, thus creating an environment permissive to Treg therapy. We performed rat left lung transplantation (LTx) experiments using Fischer 344 (F344, RT-1lv1) donors and Wistar Kyoto (WKy, RT-1l) recipients with or without antecedent 3h EVLP. Animals received: no treatment (NT); IL-2c (a single low [30ug] or high [60ug] dose) alone; Tac (0.1mg/kg/day, 7 doses) alone; or both IL-2c and Tac, intraperitoneally starting on the day of transplantation. Allografts that underwent EVLP had lower acute rejection grades in IL-2c/Tac-treated rats than in NT and Tac-treated rats (A Grade p = 0.046; B Grade p = 0.064; Fig1). In contrast, there were no differences among these groups in allografts that had not undergone EVLP. In the grafts, the frequency of CD4+CD25highFoxp3+ cells increased in both low and high dose IL-2c/Tac groups compared with Tac group, regardless of EVLP (p = 0.012, p = 0.107; Fig 2). Although the frequency of CD4+CD25+Foxp3- effector T cells also increased with high dose IL-2c in the non-EVLP group, this was not seen with EVLP (p = 0.804) (Fig 2). Despite the presence of Tac, IL-2c increased Tregs in the graft after transplantation and attenuated acute rejection in EVLP-treated lungs. This work lays the groundwork for pre-implantation allograft-directed regulatory T cell therapy.