Cell-cycle–regulatory molecules and phosphorylated activation of MAPKs are involved in Treg-induced senescence. (A) Cell-cycle–regulatory molecules p21, p16, and p53 are involved in human Treg-induced T-cell senescence. (B) CD4⁺CD25^hiFoxP3⁺ Treg cells induced phosphorylation of ERK and p38 in senescent CD4⁺ T cells. Results from Western blotting analyses of phosphorylated activation of MAPKs are shown in the left panel. Phosphorylated ERK and p38 protein levels shown in the right histogram were analyzed quantitatively and compared against the β-actin expression level with a densitometer. Results shown in the histogram are means ± SD from 3 independent experiments. *P < .05 and **P < .01 compared with the medium and CD4⁺CD25⁻ control groups. (C) ERK and p38 signaling regulated cell-cycle–regulatory molecule p21 expression in senescent CD4⁺ T cells. The left panel shows the Western blot analysis results. The right panel shows p21 expression analyzed quantitatively and compared with β-actin expression with a densitometer. Results shown in the histogram are means ± SD from 3 independent experiments. **P < .01 compared with the group not treated with inhibitor. CFSE-labeled naive CD4⁺ T cells were cocultured with CD4⁺CD25^hiFoxp3⁺ Treg cells at a ratio of 5:1 in anti-CD3 Ab (2 μg/mL)–precoated plates in the presence or absence of different inhibitors (10μM) SB203580 (p38 inhibitor), U0126 (ERK1/2 inhibitor), or SP600125 (JNK inhibitor) for 0, 1, 3, and 5 days. Cocultured naive CD4⁺ T cells were purified by FACS and then lysates were prepared for Western blot analyses. Data shown in panel C are the results from day 3 cocultured CD4⁺ T cells. Data shown in panels A through C are representative of 3 independent experiments with similar results.