Depletion of Donor Macrophages Permits Post-Transplant Tolerance Induction in a Pancreatic Islet Transplant Model

Abstract

Transplant tolerance induction permits survival of transplanted organs without the need for indefinite global immunosuppression (IS), thereby lowering medical and financial burdens of IS on transplant recipients1,2. Most clinical and preclinical models of tolerance induction, however, rely on pre-transplant modifications of the recipient. These models include donor chimerism induction and donor-specific transfusions3,4. As a result, these strategies are only applicable in living donor transplantation where donor cells are readily available days to weeks prior to transplantation. Here, we explore strategies for permitting tolerance induction entirely post-transplant, thereby expanding the applicability of tolerance induction regimens to additional clinically relevant settings. The first chapter of this thesis discusses transplant rejection and the current standard-of-care, including the limitations and drawbacks of traditional IS. While current IS strategies are highly successful in preventing acute transplant rejection, chronic rejection remains common. Additionally, IS produces negatives side effects including increased rates of opportunistic infections and malignancies. This chapter then goes on to discuss the clinical state of tolerance induction, a strategy which removes the need for pharmaceutical IS while still preventing organ rejection. In pre-clinical models of transplant tolerance induction, our lab has pioneered a strategy of recipient injections of donor splenocytes (SPs) treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) and has demonstrated its robust efficacy in several murine and non-human primate models of allogeneic transplantation. Donor ECDI-SPs are typically administered on days -7 and +1 relative to transplant at day 0, with the dose on day -7 being crucial for efficacy of the treatment. Consequently, the requirement of the day -7 dose also precludes its application in deceased donor transplantation. A need remains for a robust tolerance strategy that can be implemented entirely post-transplant, therefore applicable to deceased donor transplantation. This chapter then discusses pancreatic islet transplantation and its utility as a preclinical model. Islet transplantation is a less technical procedure than solid organ transplant, making it more feasible for high-throughput exploratory studies. Major mechanisms are preserved between islet transplant and solid organ transplant, making it a desirable preclinical model for early studies. The second chapter outlines the role of donor passenger immune cells in promoting transplant rejection. Our lab has demonstrated that donor tissue resident macrophages contribute to post-transplant immune infiltration of murine kidney allografts. The role of donor tissue resident macrophages is less well defined in pancreatic islet transplantation. Islets of Langerhans contain macrophages with a distinctly M1-like profile, expressing high levels of major histocompatibility class II (MHC II) and costimulatory molecules5,6. As such, these intra-islet macrophages are highly inflammatory7,8. Thus, we hypothesized that their depletion from the islet allograft prior to transplantation would reduce post-transplant inflammation and allow tolerance induction to be delayed until post-transplantation. This chapter then discusses the role of donor-derived chemokines in transplant rejection. Our lab has found that donor kidney resident macrophages release CCL8, contributing to graft infiltration and worse graft outcomes. This data supports a role for donor-derived chemokines in rejection, and raised interest in the role these chemokines may play in antagonizing rejection. In the third chapter, in a model of murine allogeneic islet transplantation, we show that depletion of intra-islet donor macrophages prior to transplantation abrogates the immediate influx of recipient innate immune cells to the islet allograft. When combined with post-transplant infusions of donor ECDI-SPs, this strategy results in donor-specific tolerance and indefinite islet allograft survival in the complete absence of immunosuppression. We demonstrate that this graft function is due to systemic donor-specific tolerance induction, as a same-donor secondary graft survives with no additional treatment, while a third party secondary graft is rejected as normal. In the fourth chapter, we demonstrate that intra-islet donor macrophages contribute to early graft infiltration. Flow cytometry was utilized to evaluate graft infiltration at multiple timepoints. At an early timepoint, donor macrophage depletion resulted in reduced innate graft infiltration. We also found that this change in early graft infiltration was in part due to islet macrophage release of chemokines CCL3, CCL4 and CCL5. In an ex vivo culture system, islets were analyzed using qPCR and supernatant was evaluated with multiplex assays. We found that donor macrophages contribute to chemokine release. In vivo, we utilized maraviroc, a small molecule CCR5 inhibitor, to demonstrate that perioperative blockade of CCR5 reduces early islet graft infiltration of recipient innate immune cells and permits tolerance induction by post-transplant donor ECDI-SP infusions, recapitulating the effect of donor macrophage depletion. These findings support that post-transplant tolerance induction by donor ECDI-SP infusions can be facilitated by donor macrophage depletion, likely via a reduction of chemokine-chemokine receptor interaction. In chapter five, we found that the observed islet allograft protection due to donor macrophage depletion was further characterized by a reduction of graft-infiltrating effector T cells and an increase of systemic FoxP3+ regulatory T cells (Tregs). We also demonstrate that donor macrophage depletion combined with post-transplant ECDI-SPs results in reduced donor-specific T cell activation using in vitro assays with recipient splenic T cells. The final chapter provides a review and discussion of our findings. This study demonstrates an effective strategy for post-transplant tolerization, thereby expanding the applicability of tolerance induction regimens to additional clinically relevant settings.