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Abstract

Regulatory T cell (Treg) therapy is a promising approach to treat type 1 diabetes (T1D). Current methods of Treg therapy rely on non-specific Tregs which have limited potency and potential off-target immunosuppressive effects. As a result, there is a need for optimized islet antigen-specific Treg therapies for T1D. To generate islet-specific human Tregs, I engineered chimeric antigen receptors (CARs) that recognize diabetes-relevant peptides presented on cell surface proteins known as Major Histocompatibility Complex (MHC) molecules. CAR functionality was assessed by T cell and Treg reactivity to relevant or irrelevant peptide-MHC complexes expressed on a human leukemia cell line. I identified two promising candidate CARs, referred to as 5F5 and G3H8, that responded specifically to their respective peptide-MHC targets. When transduced in Tregs, both CARs improved Treg suppressive ability compared to untransduced controls. The 5F5 CAR, however, had low peptide sensitivity and consequent limited reactivity to primary islets. Studies are ongoing to test different CAR formats to increase sensitivity. CAR-Treg therapy can also benefit T1D patients who have received an islet transplant. Our lab has recently shown that infusion of Tregs transduced with an HLA-A2 targeting CAR (A2-CAR Tregs) can prevent autoreactive T cell induced diabetes in mice that have received an HLA-A2+ islet transplant in the anterior chamber of their eye. To better understand the spatial dynamics of A2-CAR Tregs in this model, I optimized an immunofluorescence microscopy protocol to detect immune cells at the site of transplantation and in the native pancreas. Both A2-CAR Tregs and diabetogenic T cells were present in the transplanted and endogenous islets. Interestingly, I observed an increased frequency of Tregs in the graft of A2-CAR Treg treated-mice even after A2-CAR Treg depletion, suggesting that the diabetogenic T cells may have been tolerized. These data showed that immunofluorescence microscopy can be a useful tool to image the immune environment in an induced diabetes, islet transplantation mouse model. Overall, my work provides a foundation from which T1D relevant CARs can be further optimized and characterized to unlock their full potential for use in Tregs as a cell therapy.