Fig. 3

Treg-mediated glucose competition causes responder T-cell senescence and DNA damage. a Higher expression levels of glucose transporters (Glut1 and Glut3) in Treg cells than that of effector T cells analyzed by real-time PCR. Expression levels of each gene were normalized to β-actin expression and adjusted to the levels in CD4⁺CD25⁻ cells only (served as 1). Data shown are mean ± SD from four independent donors. b Human nTreg and tumor-derived γδ Treg cells have a high glucose uptake capacity determined by the flow cytometry after addition of 2-NBDG. Results shown are a representative of four independent experiments. c Co-culture of nTreg with naïve CD4⁺ T cells for 3 days significantly decreased glucose levels in the culture medium. **p < 0.01, compared with naive T cells in medium only group by unpaired t test. d Increased phosphorylation of AMPK was induced in naive CD4⁺ T cells treated with nTreg cells. The p-AMPK and total AMPK expression in treated T cells for 3 days was analyzed by the flow cytometry. e Low concentrations of glucose significantly promoted phosphorylation of AMPK in anti-CD3-activated naive CD4⁺ T cells for 3 days using the flow cytometry analysis. f, g Inhibition of glucose transport and glycolysis dramatically blocked Treg suppressive capacity on T-cell proliferation (in f) and responder T-cell senescence induction (in g). Treg cells were pretreated with inhibitors phloretin (2 μM) or 2-DG (1 mM) for 1 day, and then co-cultured with naive CD4⁺ T cells for 3 days. Data shown are mean ± SD from three independent experiments.*p < 0.05 and **p < 0.01, compared with the medium only group by paired t test. h Addition of high concentrations of glucose markedly rescued responder T-cell senescence induced by Treg cells determined with SA-β-Gal staining after 3-day co-culture. Data shown are mean ± SD from three independent experiments. **p < 0.01, compared with the Treg-treated group with normal concentration of glucose (11 mM) using paired t test. i High concentrations of glucose significantly prevented the DNA damage-associated molecule activation in responder T cells induced by nTreg cells for 3 days using the flow cytometry analyses