Introduction: Regulatory T cells (Tregs) play an  important role in the maintenance of immune homeostasis and establishment of immune tolerance. Since Tregs do not secrete endogenous IL-2, they are especially dependent external  IL-2. IL-2 deficiency leads to lower Treg numbers, instability of the Treg phenotype and loss of immune regulation. After organ transplantation, patients are treated with calcineurin inhibitors (CNIs) which further limits available IL-2. Application of low dose IL-2 expands Tregs, but  also activates NK and CD8+ T cells. It was recently shown that graft-specific Tregs recognizing mismatched MHC I molecules via an chimeric antigen-receptor were far more potent in tolerance induction as compared to polyclonal Tregs.

Methods: We therefore aimed in amplifying transferred CAR-Treg function and stability by expression of a membrane-associated IL-2 (mbIL-2).

Results: mIL-2 Treg showed increased survival, better phenotypic stability and function as compared to CAR-Tregs currently used in clinical trials. They we also more stable under inflammatory conditions. In a preclinical humanized mouse model, we demonstrated that mbIL-2 CAR-Tregs showed a better survival within the Treg niche than control CAR Tregs and were even resistant to CNI therapy with no effect on other Treg so mainly acting in cis. mIL2 CAR Treg were more effective than CAR Treg in preventing the rejection of allogenic targets in humanized mice.

Conclusion: The functional and phenotypic improvements of membrane attached IL-2 on CAR-Tregs will be an important step to enhance CAR-Treg therapies currently being tested in clinical trials after kidney and liver transplantation.