Fig. 5.

Co-culture of T cells with hepatoma cells and a stimulation of T cells with exogenous lactate promote an accumulation of FOXP3-expressing regulatory T cells. CD3⁺CD4⁺ T cells were sorted and purified from PBMCs using FACS, then activated in stimulating culture medium in the presence or absence of TGF-β1. At Day 4, lymphocytes were harvested, and FOXP3 expression was examined using flow cytometry. For the co-culture system, purified CD3⁺CD4⁺ T cells were seeded into the upper chambers of a transwell co-culture system a without or b with a glycolysis inhibitor, 2-DG, and the established lentiviral transduced SMMC-7721 cells were seeded into the lower chambers. c For the lactate stimulation assay, 20-mM sodium L-lactate was added to the stimulating culture medium mentioned above. A representative flow cytometry histogram profile from one repeat was shown for each group (left). Quantitative analyses of three independent experiments using PBMCs from three different healthy donors were summarized (right). *p < 0.05, **p < 0.01, ***p < 0.001