🎗CAR-Tregs synergize with anti-thymocyte globulin and rapamycin to delay skin allograft rejection

Introduction: Solid-organ transplantation is the only curative therapy for end-stage organ failure. Nevertheless, recipients must undergo intensive immunosuppressive treatment, which increases their susceptibility to secondary infections and malignancies. As an alternative, regulatory T cell (Treg) therapy is being tested as a new way to induce allograft tolerance. We previously showed that Tregs engineered to express an HLA-A2-specific chimeric antigen receptor (A2-CAR) delayed HLA-A2+ skin graft rejection. However, their persistence and suppressive effect was transient. We hypothesized that adjunct immunosuppression may synergize with A2-CAR Tregs. To reflect the clinical context in which A2-CAR Tregs are being tested, we aimed to investigate the ability of a combination therapy including anti-thymocyte globulin (ATG, rabbit anti-mouse), rapamycin, and A2-CAR-Tregs to delay rejection in a model of F1 (BALB/c x B6.HLA-A2) skin to C57BL/6J mice (B6 mice) recipient transplant model.
Methods: Wild type B6 mice were transplanted with circular skin patch from F1 skin (using BALB/c mice which additionally expressed the 2W-OVA transgene, BALB/c 2W-OVA x B6.HLA-A2) or syngeneic B6 mice. ATG recipients received two intraperitoneal injections (500ug/dose) on day -1 pre and day 3 post-transplant. Rapamycin (1mg/kg) was administered for 10 consecutive days from day 14. Mice were infused with 1M A2-CAR-Tregs into the tail vein on day 20 post-transplant, a time point at which we determined the T cell depleting effects of ATG were minimal. Skin grafts were monitored at least three times per week, and weekly blood samples were collected to determined CAR-Treg engraftment and phenotype.
Results: The median graft survival for untreated or A2-CAR-Treg alone was 14 days. Treatment with ATG, ATG and A2-CAR-Tregs, or ATG and rapamycin all moderately extended median graft survival to 20-26 days. However, the combination of ATG, rapamycin and A2-CAR-Tregs resulted in significant prolongation, extending the median graft survival to 43 days. Circulating A2-CAR Tregs were detected in the peripheral blood of ATG and rapamycin treated mice, and they maintained FOXP3 and CAR expression. Mechanistically, when A2-CAR-Tregs were used in combination with ATG and rapamycin, there was a significant reduction in the frequency of OVA-specific CD8 T cells in the draining lymph nodes. Furthermore, A2-CAR-Tregs significantly reduced donor-specific antibody production by inhibiting T follicular helper cells and germinal center B cells.
Conclusion: These data show that A2-CAR-Tregs synergize with ATG and rapamycin and can be used clinically as a combination therapy for solid-organ transplantation. The findings have important implications for the application of A2-CAR Treg therapy in humans.