Postdoctoral research fellow The University of British Columbia Vancouver, British Columbia, Canada
Disclosure(s):
Dominic A. Boardman, PhD: No financial relationships to disclose
Abstract Text: 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. 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. When stimulated with HLA-A2+PDL1+ K562 cells, PD1-deficient CAR Tregs expressed higher levels of activation markers than PD1-sufficient CAR Tregs, demonstrating a greater sensitivity to antigen stimulation. However, these cells maintained their characteristic expression of FOXP3 and Helios following transfer into HLA-A2-transgenic NSG mice, suggesting PD1-ablation did not compromise Treg stability. Upon co-culture with mature HLA-A2+ dendritic cells, PD1-deficient CAR Tregs suppressed the expression of MHC class II and co-stimulatory molecules more effectively than PD1-sufficient CAR Tregs, demonstrating PD1-ablation enhanced the suppressive potency of CAR Tregs. Future work will test the therapeutic efficacy of PD1-deficient CAR Tregs in a humanised mouse model.
