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Abstract

In solid organ transplantation, the primary challenge is to overcome the alloimmune response that arises from immunological incompatibility between donor and recipient, leading to graft rejection. Current immunosuppressive therapies have significant long-term side effects and seldom achieve graft tolerance. Regulatory T cells (Tregs), critical for self-tolerance and suppressing excessive immune responses, have garnered considerable interest as an adoptive cell therapy in transplantation. One approach to enhance the potency of Treg therapy is to confer donor antigen-specificity by modifying Tregs to express a chimeric antigen receptor (CAR). Specifically, CAR-Tregs targeting MHC class I molecule HLA-A2 have shown prolonged graft survival in several preclinical models and are now entering clinical trials. However, the ability of CAR-Tregs to induce transplant tolerance has yet to be established. Recent experimental evidence has demonstrated that CAR-Tregs are susceptible to exhaustion caused by chronic stimulation, potentially limiting their efficacy. This thesis presents the development of new tools to investigate CAR-Treg exhaustion and to develop exhaustion-resistant CAR-Tregs. The first tool involved the generation of CARs that target MHC class II, expanding the potential targets for CAR-Tregs. A mouse CAR targeting the MHC class II molecule I-Eᵈ was created, and antigen-specific activation and proliferation of Tregs were demonstrated. In addition, hybridomas specific for HLA-DR4 were used to produce a human MHC class II CAR targeting HLA-DR4, and preliminary tests indicated antigen-specific effects in human T cells. These developments lay the groundwork for future research on optimal CAR-Treg targets that minimise Treg exhaustion. The second tool was an in vitro model to study exhaustion in the context of the HLA-A2-targeted CAR. This continuous antigen exposure model employed HLA-A2 expressing artificial antigen-presenting cells and, under optimised parameters, induced T cell dysfunction and an exhaustion-like phenotype in A2 CAR-T cells. This model provides a basis to investigate the transcriptional and epigenetic signature of Treg exhaustion due to chronic CAR stimulation. Together, this research developed tools that can facilitate the evidence-based design of CAR-Tregs, which can improve outcomes in solid organ transplantation.