Blockage of ERK1/2 and p38 signaling in responder T cells prevents Treg-induced T-cell senescence. (A-B) Inhibition of ERK1/2 or p38 signaling pathways by specific pharmacologic inhibitors reversed the T-cell senescence induced by CD4⁺CD25^hiFoxP3⁺ Treg cells significantly, resulting in decreased SA-β-Gal expression (A) and restored CD27 and CD28 expression in Treg-treated naive T cells (B). CFSE-labeled naive CD4⁺ T cells were cocultured with Treg cells in anti-CD3–coated (2 μg/mL) plates in the presence or absence of different dosages of the inhibitors U0126, SB203580, or SP600125 (1, 5, or 10μM) for 5 days. The treated naive CD4⁺ T cells were purified by FACS and then stained with SA-β-Gal staining reagents or analyzed for CD27 and CD28 expression. *P < .05 and **P < .01 compared with the group not treated with inhibitor. (C) Pretreatment with inhibitors in naive CD4⁺ T cells, but not in Treg cells, decreased the SA-β-Gal⁺ CD4⁺ T-cell populations. Naive CD4⁺ T cells or CD4⁺CD25^hiFoxp3⁺ Treg cells were pretreated with each MAPK inhibitor (10μM) for 2 days and cocultured with untreated Treg cells or naive CD4⁺ T cells, respectively, for 5 days. The number of SA-β-Gal⁺–naive CD4⁺ T cells was then determined. *P < .05 compared with the group not treated with inhibitor. (D) Knockdown of ERK1/2 and p38 genes by shRNA in naive CD4⁺ T cells but not in CD4⁺CD25^hi FoxP3⁺ Treg cells reversed Treg-induced T-cell senescence dramatically. Naive CD4⁺ T cells or Treg cells were transfected with lentiviral shRNAs specific for ERK1/2 or p38 molecules. Transduced (green fluorescent protein–positive) naive CD4⁺ T cells or Treg cells were purified by FACS sorting and then cocultured with untransduced Treg cells or naive CD4⁺ T cells, respectively, for 5 days. The number of SA-β-Gal⁺–naive CD4⁺ T cells was then determined. *P < .05 and **P < .01 compared with the group transduced with control shRNA. Data shown are representative of 3 independent experiments with similar results.