*Purpose: A growing body of evidence recognizes the balance between graft-reactive effector T cells and graft-protective regulatory T cells (Tregs) as the ultimate determinant of long-term allograft survival. Adoptive Treg cell therapy requires large numbers of clinically competent Tregs that must be obtained after ex vivo expansion. We have shown that PI3K/ mTOR signaling regulates the metabolic switch that controls the balance between glycolysis-driven effector and Treg cells, which rely more on mitochondrial oxidative phosphorylation (OXPHOS). Recent data has emerged in support of the fundamental role of Wnt/β-catenin pathway in the functional fitness of T cells. In this study, we investigated the metabolic effect of the Wnt/β-catenin signaling inhibitor FH535 in Treg cells and the potential interplay with the PI3K/mTOR pathway.

*Methods: Sorted CD25⁺ Treg cells were placed in culture with different doses of the β-catenin inhibitor FH535, the mTOR pathway inhibitor Rapamycin, alone or in combination. Multi-parametric FACS analyses were used to monitor the expansion rates, mitochondrial function and integrity, and suppressor function of the cells. The bioenergetics profiles of the drug-treated cells, including OXPHOS and glycolytic rates, were assessed on an extracellular flux analyzer.

*Results: Addition of FH535 to the Treg cell culture induced a rapid but temporary increase of cell expansion with a concurrent inhibition of their suppressor function. These effects were linked to substantial metabolic changes, including higher glycolytic rates and impaired mitochondrial respiration. Simultaneous inhibition of the mTOR pathway with Rapamycin prevented the effects of FH535. However, the sequential addition of FH535 followed by Rapamycin increased between 20 and 50% the levels of Treg cell expansion and promoted higher suppressor activity when compared to Rapamycin alone. These effects were linked to the metabolic changes consistent with glycolysis enhancement similar to that induced by FH535 alone, and OXPHOS increase similar to the Rapamycin-induced alone.

*Conclusions: Dual metabolic targeting of Treg cells ex vivo with FH535 and Rapamycin may represent a promising new approach to increase the efficiency of functional Treg expansion for clinical use.

« Back to 2019 American Transplant Congress