Local regulation and deletion of T cells to induce tolerance and establish long-term survival of high-risk corneal transplants

Abstract: Immunological rejection is the most common cause of corneal allo-transplant failure particularly when the recipient has a high risk vascularized corneal bed. Because corneal transplantation (CT) is the most common solid organ transplant performed in the United States (>50,000 /annum), induction of immune tolerance is a critical and unmet medical need according to the NEI (https://www.nei.nih.gov/sites/default/files/2021-12/NEI- StrategicPlan-VisionForTheFuture_508_edit.pdf). The studies proposed in this application, represent a translational basic science immunology project, which will directly apply our provocative pre-clinical experimental findings targeting TNFRSF25 in vivo to manipulate the Treg compartment to induce long-term tolerance in recipients of high-risk CT. Notably, Treg expansion has shown promise in graft vs host disease (GVHD)and type 1diabetes, however when IL-2 treatment is stopped the diseases return. New approaches are needed to induce long-term tolerance to CT and other allografts. The overall objective of this proposal is to develop combinatorial treatment strategies based on deletion and immune regulation. Here we will apply the concept of post-CT cyclophosphamide (Cy) treatment successfully used as singular prophylaxis for GVHD, for the first time to a vascularized solid organ, CT. Our new data identified an extended low dose Cy protocol administered post-CT (PTCy) that markedly prolonged transplant survival. Proposed studies will exploit the survival of Tregs after PTCy administration with novel TL1A and IL-2 based fusion proteins (FP) to target, expand and activate CD4+FoxP3+ T cells in vivo via TNFRSF25 and CD25. Importantly, studies here will develop an approach restricting Treg expansion to the ophthalmic compartment thereby minimizing potential global immune dysregulation. Experiments will first identify the optimal treatment protocol to prolong CT survival examining a unique IL2/CD25 FP currently in clinical trials. Additionally, we will test another recently developed, novel IL2-10/CD25 FP to test the hypothesis that together with Treg expansion, IL-10 mediated suppression of antigen presentation which underlies corneal allograft survival will be down-regulated. T cell receptor transgenic anti-allograft specific T cells dependent on either direct or indirect antigen presentation will be examined in vivo in transplanted mice to directly monitor their regulation (Aim 1). Experiments will investigate mechanistic pathways through in vivo readouts, TNFRSF25 knock-out and chimeric animals and cutting-edge spatial biology analyses via CODEX technology (Aim 2). Lastly, epigenetic regulation using a new bromodomain inhibitor to block local cytokine production will be tested to complement the treatment strategy (Aim 3). Overall, the research plan is to systemically build a tolerance platform by combining 3 highly complementary strategies for the establishment of allograft tolerance to maintain permanent CT. Importantly, because human mAbs and FPs specific for TNFRSF25, IL-2R and IL- 10R have recently been developed, the long-term goal of our pre-clinical research program is to translate our strategies into patients undergoing CT who experience high rates of immune mediated rejection.