TLR8 ligands reverse Treg-induced T-cell senescence. (A) The TLR8 ligands poly-G3 and ssRNA40, but not ligands for other TLRs, reversed markedly the ability of human CD4⁺CD25^hiFoxP3⁺ Treg cells to induce naive CD4⁺ T-cell senescence. CFSE-labeled naive CD4⁺ T cells were incubated alone or cocultured with Treg cells in anti-CD3–coated plates in the presence of the indicated TLR ligands for 5 days. The treated naive CD4⁺ T cells were purified by FACS sorting and the SA-β-Gal⁺ T-cell populations in the different groups were determined. Poly-T3 (3 μg/mL) served as a control. **P < .01 compared with the groups treated with medium or other TLR ligands. Data shown are representative of 3 independent experiments with similar results. (B) Poly-G3 restored the expression of CD27 and CD28 in naive CD4⁺ T cells induced by human CD4⁺CD25^hiFoxp3⁺ Treg cells. Naive CD4⁺ T cells were cultured with CD4⁺CD25^hiFoxP3⁺ Treg at a ratio of 1:1 in the presence of poly-G3 or control poly-T3 for 5 days. Treated naive CD4⁺ T cells were separated and CD27 or CD28 expression was analyzed by FACS.