*Purpose: Donor-specific regulatory T-cell (Treg) therapy has emerged as a potent strategy to promote immune tolerance in experimental transplantation. However, a major stumbling block in the clinical use of donor-specific Tregs is their very limited number. Recently, the potential of chimeric antigen receptor (CAR) technology was investigated to redirect antigen-specificity of Tregs toward a relevant alloantigen. However, whether or not the type of CAR costimulatory domain (CSD) impacts the stability, persistence and function of CAR Tregs remains largely unexplored.

*Methods: Here, we used HLA-A2-targeted CAR Tregs incorporating either 4-1BB or CD28 CSD to study their effect on Treg biology and to assess their therapeutic potential in transplantation settings.

*Results: CAR Tregs elicited HLA-A2-specific suppression both in vitro and in vivo in a humanized mouse model where xenogeneic graft versus host disease (xeno-GVHD) was induced by the adoptive transfer of HLA-A2+ peripheral blood mononuclear cells. We found that the type of CSD significantly influenced the proliferative capacity and immunometabolism of CAR-Tregs, with ensuing effect on their stability (Foxp3 and Helios expression). Moreover, CAR-Treg activation and differentiation also greatly varied across CSD: 1- 41BB CSD steered CAR-Tregs toward effector differentiation. 2- Although activation through the CAR was not different between the two CSD at early time points in the culture, the type of CSD dramatically determined subsequent occurrence of CAR-Treg hyporesponsiveness to antigen stimulation. In vivo bioluminescent tracking of HLA-A2-targeted CAR Tregs demonstrated different homing capacities into lymphoid organs and sustainity according to CSD. Finally, both HLA-A2-targeted CAR Tregs were able to prevent xeno-GVHD, yet with different long-term efficacies.

*Conclusions: Together, our data provide new insights to optimize human CAR-Tregs engineering on the path toward clinical development.

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