Fig. 7.

Involvement of mTORC1 activation in iTreg cells differentiated from Met-treated naïve CD4⁺ CD25⁻ cells. (A) The level of phosphorylated S6 protein (pS6) increased in Met-pretreated naïve CD4⁺ CD25⁻ T cells in response to different stimulations of TCR for 30 min, as indicated. Each lane includes lysates from 2 × 10⁵ cells. The results are representative of two independent experiments. (B) Foxp3 expression decreased in iTreg cells differentiated from Met-treated naïve CD4⁺ CD25⁻ cells. The presence of rapamycin or compound C during Met pretreatment restored Foxp3 expression. The histogram results are representative of three independent experiments, and the individual results are plotted as a bar graph. N = 3. (C) The level of pS6 protein increased in Ti-Treg cells of mice receiving Met. The histogram data were obtained from pooled spleens and TILs of five mice bearing MethA. The bar graph data were obtained from five individual mice. Both results are representative of three independent experiments. (D) Only Met (or Rot)-pretreatment of naïve CD4T cells before TCR stimulation resulted in a decrease in Foxp3 expression. Pre-treatment assay: CD4⁺ CD25⁻ T cells were treated with Met (or Rot) for 6 h and washed before TCR stimulation. Post-treatment assay: CD4⁺ CD25⁻ T cells were treated with culture medium for 6 h and then incubated with Met (or Rot) during TCR stimulation. Three days after TCR stimulation, Foxp3 expression was examined. The histogram results are representative of three independent experiments, and the individual data are plotted as a bar graph. *P < 0.05; **P < 0.01; ***P < 0.001.