Cell therapy with autologous donor-specific regulatory T cells (Tregs) is a promising strategy to minimize immunosuppression in transplant recipients. Chimeric antigen receptor (CAR) technology has recently been used successfully to generate donor-specific Tregs and overcome the limitations of enrichment protocols based on repetitive stimulations with alloantigens. However, the ability of CAR-Treg therapy to control alloreactivity in immunocompetent recipients is unknown. We first analyzed the effect of donor-specific CAR Tregs on alloreactivity in naive, immunocompetent mice receiving skin allografts. Tregs expressing an irrelevant or anti-HLA-A2-specific CAR were administered to Bl/6 mice at the time of transplanting an HLA-A2 + Bl/6 skin graft.Donor-specific CAR-Tregs, but not irrelevant-CAR Tregs, significantly delayed skin rejection and diminished donor-specific antibodies (DSAs) and frequencies of DSAsecreting B cells. Donor-specific CAR-Treg-treated mice also had a weaker recall DSA response, but normal responses to an irrelevant antigen, demonstrating antigen-specific suppression. When donor-specific CAR Tregs were tested in HLA-A2-sensitized mice, they were unable to delay allograft rejection or diminish DSAs. The finding that donor-specific CAR-Tregs restrain de novo but not memory alloreactivity has important implications for their use as an adoptive cell therapy in transplantation. K E Y W O R D S alloantigen, B cell biology, basic (laboratory) research/science, cellular biology, cellular transplantation (non-islet), immunosuppression/immune modulation, T cell biology, tolerance, translational research/science | 1563 SICARD et Al.