Fig 1.
High glucose promotes iTreg differentiation.
a–c, Naïve CD4+ T cells were isolated from C57BL/6 mice and cultured in 96-well plates with plate-bound anti-CD3 and soluble anti-CD28, and no, active, or latent TGF-β for 72 hours. Cells were then stimulated with Cell Stimulation Cocktail for 4 hours and then stained with fluorescence-conjugated antibodies and analyzed by flow cytometry. a, Representative flow cytometry gating for CD4+ T cell populations. b, Percentages of CD4+ Foxp3+ T cells in all CD4+ cells. c, Percentages of CD4+ IFN-γ+ T cells in all CD4+ cells. N = 3, and data are presented as means ± S.E.M. * p<0.05 by Student’s t test.
Fig 2.
High glucose inhibits Th1 differentiation.
Naïve CD4+ T cells were isolated from C57BL/6 mice and cultured for 72 hours in 96-well plates with plate-bound anti-CD3, and soluble anti-CD28, anti-IL-4, IL-12, and IL-2. Cells were then stimulated with Cell Stimulation Cocktail for 4 hours before staining with fluorescence-conjugated antibodies and analyzed with flow cytometry. a, Representative flow cytometry gating for CD4+ T cell populations. b, Percentages of CD4+ T-bet+ IFN-γ+ T cells in all CD4+ cells. c, Percentages of CD4+ Foxp3+ T cells in all CD4+ cells. N = 3, and data are presented means ± S.E.M. * p<0.05 by Student’s t test.
Fig 3.
High glucose promotes Treg and inhibits Th1 differentiation under mixed cytokine conditions.
Naïve CD4+ T cells were isolated from C57BL/6 mice and cultured in 96-well plates with plate-bound anti-CD3, and soluble anti-CD28, IL-12, and latent TGF-β for 72 hours. Cells were then stimulated with Cell Stimulation Cocktail for 4 hours before staining with fluorescence-conjugated antibodies and analyzed with flow cytometry. a, Representative flow cytometry gating for T cell populations. b, Percentages of CD4+ Foxp3+ T cells in all CD4+ cells. c, Percentages of CD4+ T-bet+ T cells in all CD4+ cells. d, Percentages of CD4+ IFN-γ+ T cells in all CD4+ cells. N = 3, and data are presented means ± S.E.M. * p<0.05 by Student’s t test.
Fig 4.
High glucose increases lactate production that promotes iTreg differentiation.
a, Naïve CD4+ T cells were isolated from C57BL/6 mice and cultured in 96-well plates with plate-bound anti-CD3, soluble anti-CD28 and latent TGF-β, with or without lactate for 72 hours. Cells were then analyzed by flow cytometry for the percentage of Tregs (Foxp3+). b, Naïve CD4+ T cells were isolated from C57BL/6 mice and cultured in 96-well plates with plate-bound anti-CD3, soluble anti-CD28 and latent TGF-β for 24 hours. Culture supernatants were collected from each well, deproteinized, and assayed for total lactate level using a plate reader. Result represents pooled culture supernatants of 3 biological repeats. c, Naïve CD4+ T cells were cultured in 96-well plates with plate-bound anti-CD3, soluble anti-CD28, and latent TGF-β, with or without MCT1/2 inhibitor for 72 hours. Cells were then analyzed by flow cytometry for the percentage of Tregs (Foxp3+). N = 3, and data are presented means ± S.E.M. * p<0.05 by Student’s t test. d, Tregs were differentiated from naïve T cells for 3 days. Cells were then cultured in 3 (left panel) or 15 (right panel) mM lactate for 24 hours, and the concentration of the remaining lactate in the culture medium was measured by the lactate assay. e, Differentiated Tregs were cultured in 3 or 15 mM lactate for 24 hours, and whole cell extracts from these cells were analyzed by Western blotting for indicated markers. f–h, Naïve CD4+ T cells were cultured in 96-well plates with plate-bound anti-CD3, soluble anti-CD28, active TGF-β, and IL-6 for 3 days, under indicated glucose (g) or lactate (h) levels, and analyzed for Th17 differentiation. Representative gating strategy for Th17 flow cytometry are shown in f. i, Naïve CD4+ T cells were cultured in 96-well plates with plate-bound anti-CD3, soluble anti-CD28 and anti-IL-4, IL-2, and IL-12 for 3 days, under indicated lactate levels, and analyzed for Th1 differentiation. RPMI medium containing 5.5 mM glucose and 10% FBS was used as controls for lactate stimulation.