Reversal of Treg-induced senescence by TLR8 ligand and MAPK inhibitors in vivo. (A) Colocalization of adoptively transferred naive CD8⁺ T cells and CD4⁺CD25^hiFoxP3⁺ Treg cells in Rag1^−/− mice. Naive CD8⁺ T cells (5 × 10⁶/mouse) and CD4⁺CD25^hiFoxP3⁺ Treg cells (5 × 10⁶/mouse) were injected intravenously into Rag1^−/− mice. Blood, lymph nodes (LN), and spleens (SP) were harvested from the mice on days 1 and 2 after injection. Cells were purified and analyzed for CD4⁺ and CD8⁺ T-cell populations using FACS with anti–human CD4 and CD8 Abs. (B) Increased SA-β-Gal⁺ cell populations were induced markedly in naive CD8⁺ T cells after cotransfer with CD4⁺CD25^hiFoxP3⁺ Treg cells, whereas cotransfer with CD4⁺CD25⁻ T cells did not induce increased senescent CD8⁺ T cells. (C) The purified CD8⁺ T cells cotransferred with CD4⁺CD25^hiFoxP3⁺ Treg cells had potent suppressive activity on the proliferation of responding CD4⁺ T cells. Naive CD8⁺ T cells (5 × 10⁶/mouse), CD4⁺CD25^hiFoxP3⁺ Treg cells (3 × 10⁶/mouse), and CD4⁺CD25⁻ T cells (3 × 10⁶/mouse) were preactivated with anti-CD3 Ab and adoptively cotransferred into Rag1^−/− mice. Blood, lymph nodes, and spleens were harvested at 12 days after injection. The transferred human CD8⁺ T cells were isolated for subsequent SA-β-Gal staining (B) and ³H-thymidine incorporation (C) assays. *P < .05 and **P < .01 compared with the groups cotransferred with CD4⁺CD25⁻ T cells or alone. (D-E) Pretreatment of CD4⁺CD25^hiFoxP3⁺ Treg cells with poly-G3 or naive T cells with ERK and p38 inhibitors before coinjection was able to block significantly the induction of senescence and reverse the suppressive activity in transferred naive CD8⁺ T cells. Naive CD8⁺ T cells were pretreated with ERK and p38 inhibitors and/or CD4⁺CD25^hiFoxp3⁺ Treg were pretreated with poly-G3 or poly-T3 (control) for 2 days before cotransfer. The transferred human CD8⁺ T cells in different organs were isolated at 12 days after injection for subsequent SA-β-Gal staining (D) and ³H-thymidine incorporation (E) assays. *P < .05 and **P < .01 compared with the medium-only and poly-T3 groups.