Membrane-bound IL-2 improves the expansion, survival, and phenotype of CAR Tregs and confers resistance to calcineurin inhibitors

Abstract

BackgroundRegulatory T cells (Tregs) play an important role in the maintenance of immune homeostasis and the establishment of immune tolerance. Since Tregs do not secrete endogenous IL-2, they are especially dependent on 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 a chimeric antigen receptor were far more potent than polyclonal Tregs in the regulation of immune responses after solid organ transplantation in a humanized mouse model.MethodsTherefore, our aim was to enhance the function and stability of transferred CAR-Tregs via expression of membrane-associated IL-2 (mbIL-2).ResultsmbIL-2 promoted higher survival, phenotypic stability, and function among CAR-Tregs than observed in clinical trials. The cells were also more stable under inflammatory conditions. In a preclinical humanized mouse model, we demonstrated that mbIL-2 CAR Tregs survive better in the Treg niche than control CAR Tregs and are even resistant to CNI therapy without affecting other Tregs, thus acting mainly in cis.DiscussionThe functional and phenotypic improvements observed after membrane-attached IL-2 expression in CAR-Tregs will be important step for enhancing CAR-Treg therapies currently being tested in clinical trials for use after kidney and liver transplantation as well as in autoimmune diseases.

Gene editing ofCD3 epsilongene to redirect regulatory T cells for adoptive T cell transfer

“…Engineered CD3ε -TruC Tregs may also benefit from other modifications to improve their functionality 9 . In patients immunosuppression, Tregs could be enhanced by gene editing to induce drug resistance 52,53 or providing artificial IL-2 cytokine signals 54,55 . Here, the minimized transgene size enables easier co-delivery of therapeutic transgenes.…”

Eras of designer Tregs: Harnessing synthetic biology for immune suppression

“…To address the issue off‐target effects of native IL‐2, many groups have tested IL‐2 engineering strategies or muteins 145,146 to selectively boost Tregs. In a xenoGVHD model, A2‐CAR Tregs engineered with membrane‐bound IL‐2 showed enhanced persistence and stability in vivo 147 . An alternative approach is to utilize an orthogonal receptor‐ligand system, in which an exogenously administered IL‐2 mutein selectively binds an orthogonal receptor expressed on T cells 148 .…”

“…Although evidence suggests that tacrolimus and Treg therapy can function together, several studies have demonstrated that IL‐2 engineering can be used to overcome any consequences of IL‐2 depletion during tacrolimus treatment. A2‐CAR Tregs expressing a membrane‐bound IL‐2 were better able to survive and function compared to standard A2‐CAR Tregs when coadministered with multiple injections of tacrolimus 147 . Similarly, an orthogonal IL‐2 system enhanced the survival and proliferation of Tregs in mice treated with tacrolimus and helped maintain heart allograft tolerance 149 .…”

“…In a xenoGVHD model, A2-CAR Tregs engineered with membrane-bound IL-2 showed enhanced persistence and stability in vivo. 147 An alternative approach is to utilize an orthogonal receptor-ligand system, in which an exogenously administered IL-2 mutein selectively binds an orthogonal receptor expressed on T cells. 148 Tregs expressing this orthogonal IL-2 receptor have demonstrated enhanced proliferation and survival in vivo, leading to improved heart allograft survival, when the ligand was simultaneously administered.…”

“…A2-CAR Tregs expressing a membrane-bound IL-2 were better able to survive and function compared to standard A2-CAR Tregs when coadministered with multiple injections of tacrolimus. 147 Similarly, an orthogonal IL-2 system enhanced the survival and proliferation of Tregs in mice treated with tacrolimus and helped maintain heart allograft tolerance. 149 Therefore, Treg engineering can be used to overcome the negative effects of immunosuppressive drugs.…”