Figure 4.

Rapamycin induced IFN-γ producing-T-bet⁺Foxp3^lo cells, whereas 2DG induced IFN-γ nonproducing-T-bet⁺Foxp3^hi cells by different effects on fatty acid metabolism. CD45RA⁺ naïve and CD45RA^- memory CD4⁺ T cells from peripheral blood of healthy donors were stimulated with anti-CD3 Abs and anti-CD28 Abs or rapamycin (10 nM) or 2DG (3 mM) for 72 h. (A) Representative results of mTOR phosphorylation and T-bet expression measured by intracellular staining and flow cytometry. (B) Upper panel; Expression of Foxp3 and T-bet in CD45RA^- memory CD4⁺ T cells after anti-CD3 Abs and anti-CD28 Abs stimulation with or without agents for 72 h. Lower panel; Foxp3 expression in gated regions (Left: T-bet^-Foxp3⁺ cells in no stimulation or anti-CD3 Abs and anti-CD28 Abs stimulation, Middle: T-bet^-Foxp3⁺ cells in anti-CD3 Abs and anti-CD28 Abs stimulation or with rapamycin or 2DG, Right: T-bet⁺Foxp3⁺ cells in anti-CD3 Abs and anti-CD28 Abs stimulation or with rapamycin or 2DG). (C) Data are representative results of Foxp3 and IFN-γ expression (further treated with PMA, ionomycin and Breferdin for 1 h) estimated by intracellular staining (left panel) and bar graph of the percentage of IFN-γ⁺Foxp3⁺ cells (right panel). (D) Representative dot plots of expression of IL-10 and Foxp3 in CD45RA^- T-bet⁺Foxp3⁺ cells treated with anti-CD3 Abs and anti-CD28 Abs and rapamycin or 2DG and bar graph of the percentage of IL-10⁺ cells among T-bet⁺Foxp3⁺CD4⁺ cells (right panel). Pooled data (mean ± SD of MFI) of three independent experiments in (A–D). (E) The concentrations of glycerol, palmitic acid and stearic acid were measured by GC/MS. GC/MS data are mean ± SD of five independent experiments using cells from different healthy donors. (F, G) FASN, CPT1A, CPT2 and SREBF1 gene expression levels were determined by RT-PCR. These data are mean ± SD of four independent experiments using cells from different donors were shown. Data are mean ± SD. *p < 0.05. **p < 0.01. n.s., not significant.