Rapamycin facilitates differentiation of regulatory T cells via enhancement of oxidative phosphorylation

•Rapamycin promotes iTreg differentiation.•Rapamycin supports metabolic reprogramming during iTreg polarization.•Rapamycin inhibits glycolysis in polarizing iTregs via suppression of the mTOR pathway.•Rapamycin augments OXPHOS capacity, which is required for optimal induction of iTregs.•Rapamycin favors iTregs to utilize long-chain fatty acids as fuel for mitochondrial oxidative metabolism. We explored the interplay between energy metabolism and the impact of rapamycin (Rapa) on regulatory T cell (Treg) differentiation. Naïve CD4+ T cells were stimulated under Treg-polarizing conditions with or without Rapa. Rapa promoted Treg induction, as the expression of Foxp3 and Treg phenotypic markers were enhanced. Rapa disrupts glycolysis while favoring mitochondrial metabolism in induced Tregs (iTregs). Metabolic profiling showed reduced glycolytic metabolites in Rapa-treated iTregs, in line with the downregulation of glucose uptake and the expression of glycolytic enzymes. Conversely, Rapa increased the ratios of ATP/ADP and ATP/AMP, the production of mitochondrial ATP, and the expression of ATP5A. Treatment with oxidative phosphorylation inhibitors suppressed Foxp3 expression in Rapa-treated cells. Moreover, Rapa decreased oleic acid and palmitoleic acid levels and increased l-carnitine and acetylcarnitine levels and CPT1A expression in iTregs, indicative of augmented fatty acid oxidation. In conclusion, Rapa induces metabolic reprogramming in Tregs, affecting their differentiation.