Stealth CAR-Tregs evade CD3 antibody therapy and skew immune responses toward regulation

Organ transplantation standard of care relies on lifelong immunosuppression, increasing infection and malignancy risks. An emerging approach using Chimeric Antigen Receptor Regulatory T cells (CAR-Tregs) targeting donor antigens, allows the suppression of alloimmune responses while preserving protective immunity. However, transplant recipients harbor a substantial pool of alloreactive memory T-cells, limiting CAR-Tregs tolerance-promoting effect. To address this challenge, our strategy combines selective depletion of activated T-cells, using non-mitogenic CD3 antibody therapy, with HLA-A2-targeted-CAR-Treg therapy. We established that T-cell receptor alpha chain (TRAC) deletion does not impair CAR-Treg antigen-specific activation, expansion, or their transcriptomic, phenotypic, epigenetic, and functional profiles. TRAC-/-CAR-Tregs exhibit robust protection from CD3 antibody depletion in circulation and peripheral tissues, whereas most TRAC+/+CAR-T cells are eliminated. Importantly, TRAC+/+CAR-T cells that escape CD3 antibody mediated depletion exhibit a transcriptomic signature consistent with T-cell exhaustion. Our findings demonstrate that CD3 antibody therapy provides an in vivo survival advantage to TRAC-/-CAR-Tregs, thereby shifting the immune balance toward regulation. This approach might enhance the translational potential of CAR-Treg therapy by reducing the required cell dose needed for clinical efficacy.