Th26 - CRISPR-Mediated Deletion of PD1 to Enhance the Efficacy of Human CAR Tregs

Regulatory T cells (Tregs) are being tested clinically as therapeutic tools for treating transplant rejection and autoimmunity. Pre-clinical studies have shown that the efficacy of Tregs can be enhanced by using chimeric antigen receptors (CARs) to confer specificity for disease-relevant antigens, such as allogeneic HLA molecules in a transplant setting. However, recent data has suggested that PD1 signalling limits the activation potential of Tregs and may contribute to Treg dysfunction. This is a particularly important consideration in the context of type I diabetes and islet transplantation as β-cells are known to express PDL1, a ligand of PD1. We hypothesised that CRISPR-mediated removal of PDCD1 (encoding PD1) would improve the sensitivity of CAR Tregs to antigen stimulation, thereby enhancing their therapeutic efficacy. Human CAR Tregs were generated using a previously characterised HLA-A2-specific CAR, PD1 was removed by CRISPR and successfully edited cells were identified by incorporation of a homology directed repair DNA template that encoded a truncated CD19 (ΔCD19) reporter. A high degree of gene editing was achieved with >50% of the CAR⁺ Tregs expressing ΔCD19. Upon stimulation with HLA-A2⁺ cells, PD1-deficient CAR Tregs upregulated activation markers (e.g. 4-1BB) to a greater degree than PD1-sufficient CAR Tregs. Thus, PD1-deficient CAR Tregs are more sensitive to antigen stimulation and may be more therapeutically efficacious upon adoptive transfer. Future work will explore the functional consequences of removing PD1 in CAR Tregs and test the therapeutic efficacy of these cells in a humanised mouse islet transplant model.