*Purpose: Regulatory T cells (Tregs) and chimeric antigen receptor (CAR) Tregs are a promising cell therapy to prevent or treat autoimmune diseases or allograft rejection. In conventional T cells, chronic stimulation due to repetitive antigen stimulation or CAR-mediated tonic signaling is associated with poor effector function and a diminished ability to clear infections or mediate anti-tumor immunity. In the context of cell therapy, exhaustion is thought to limited the longevity and efficacy of the infused T cell product. Whether or not Tregs may also be susceptible to exhaustion, and is so, if this could limit their therapeutic effect was unknown. Our objective was to determine if the concept of exhaustion is applicable in Tregs and if so, to define the phenotypic and functional consequences.

*Methods: Two methods known to induce exhaustion in conventional T cells were tested with human Tregs. Cells were repeatedly stimulated through the T cell receptor (weekly for 4 weeks), or genetically modified to over express a CAR known to mediate strong, antigen-independent signaling. Changes in the Treg phenotype, suppressive function, cytokine secretion, metabolic profile and epigenetic status were measured. Effects on the Treg transcriptome were measured in the CAR-expressing system. The functional impact of tonic signaling CARs on Tregs was tested in vivo in a graft-versus-host disease model.

*Results: Repetitive stimulation or expression of a tonic-signaling CAR resulted in Treg phenotypic changes which were typical of cellular exhaustion as described in T conventional T cells (e.g high Tim3, Lag-3, etc), but there was no loss of FOXP3 expression. Repetitive stimulation, but not tonic-CAR signaling, was associated with a decrease of in vitro suppressive function. However, tonic signaling CAR Tregs had significant changes in their transcriptome and altered expression of metabolic pathways, leading to enhanced usage of glycolysis. Moreover, tonic signaling CAR Tregs were dysfunctional in vivo in a graft-versus-host disease model.

*Conclusions: Tregs are susceptible to exhaustion; although it does not lead to lineage instability it leads to altered metabolism and poor in vivo function. Understanding how to limit Treg exhaustion will be an important step towards improving the success of Treg therapy.

« Back to 2020 American Transplant Congress