Lack of effect on Treg function of ex vivo deuterium labeling with [6, 6-²H₂] glucose. Purified Tregs were cultured in RPMI medium containing [6, 6-²H₂] glucose or natural glucose in the presence of sibling DC, IL-2, IL-15, and rapamycin. After 12 days, Tregs cultured in 2 culture conditions were tested for suppressive function, Foxp3 Treg-specific demethylation region analysis, and TGF-β cytokine production. (A,B) Cultured Tregs were tested at different ratios to self-Tconvs in the presence of DCs from the original sibling or an HLA-incompatible third party. (C) Foxp3 demethylation region was assessed by qPCR. Each bar represents mean + SD of triplicate measurements. TGF-β and TNF release of expanded Tregs. Sorted Tregs or Tconvs were restimulated with allogeneic or self-DCs or phorbol myristate acetate (PMA) and ionomycin (IO) for 24 hours. Culture supernatant was tested for TGF-β (D) or TNF (E) production by ELISA. *P < .05; ***P < .001.