Adoptive transfer of regulatory T cells (Tregs) in the context of solid organ transplantation is an innovative therapeutic approach to facilitate long-term graft acceptance while minimizing the side effects of conventional immunosuppressants. Preclinical studies have shown that antigen-specific Tregs have remarkable efficacy in preventing allograft rejection. Reprogramming Tregs with chimeric antigen receptors (CAR) is a promising alternative for generating graft-specific Tregs in clinically relevant numbers. However, the clinical translation of adoptive immunotherapy with CAR-Tregs is hampered by the susceptibility of Tregs to pathogenic conversion in an IL-2-deprived or inflammatory environment, which impairs the expression of the critical transcription factor FOXP3.
 
To improve the resilience of CAR-Tregs, we engineered HLA-A*02 specific CAR vectors that confer Tregs IL-2 independence via the induction of membrane-associated IL-2 (mbIL-2). Moreover, we designed a CAR vector that co-expresses exogenous FOXP3 (FOXP3-CAR), leading to overexpression of canonical FOXP3 in edited cells. Both approaches increase the safety and efficacy of CAR-Treg cells. mbIL-2 CAR Tregs and FOXP3-CAR Tregs were more stable under pro-inflammatory conditions and displayed an unaltered suppressive phenotype. Furthermore, mbIL-2 CAR Tregs exhibit enhanced niche-filling capabilities and tacrolimus resistance in preclinical mouse models. Forced overexpression of  FOXP3 did not lead to dysfunctions, such as cell exhaustion, loss of Treg fitness, or abnormal cytokine secretion. In a humanized mouse model, FOXP3-CAR-Tregs showed excellent niche-filling capabilities as well as the ability to regulate an allospecific immune response and maintain immune tolerance.
 
Taken together, the overexpression of FOXP3 as well as the expression of membrane-associated IL-2 have the potential to enhance the efficacy and reliability of CAR-Treg cell products, promoting their clinical use in autoimmune and transplantation settings.