(A and B) Comparison of the suppressive activity of young and aged Tregs by in vitro suppression assays (A); the composition of Foxp3⁺ Tregs was also assessed by flow cytometry (B) (n = 3 mice of 3 experiments; representative results are shown; means ± SD, **P < 0.01, ****P < 0.0001, by 2-way ANOVA followed by Holm-Šidák multiple-comparisons test). Tresp cell, responder T cell. (C) Schematic diagram of T cell–induced colitis. Rag1^–/– recipients received WT naive CD4⁺CD45RB^hi T cells (Tn) alone or in combination with young or aged CD4⁺CD25⁺ Tregs. (D) After transfer, the body weight loss was monitored to examine the suppressive ability of young and old Tregs (n = 10 mice per group of 2 experiments; means ± SEM, *P < 0.05 for young Tregs vs. no Tregs, P = 0.3682 for aged Tregs vs. no Tregs, *P < 0.05 for young Tregs vs. aged Tregs, by 2-way-ANOVA followed by Holm-Šidák test). (E) Percentages of Tregs recovered in periphery lymph nodes (PLN), spleens, and mesenteric lymph nodes (MLN) in recipient mice at the end of the experiments (n = 5 mice of 2 experiments; means ± SD, **P < 0.01, ****P < 0.0001, by 2-way ANOVA followed by Holm-Šidák multiple-comparisons test). (F) Schematic diagram of whole-body irradiation–induced senescence. WT CD45.1 mice were sublethally irradiated and transferred with or without young or aged CD4⁺CD25⁺ Tregs. (G) The naive T cell population (CD62L^hiCD44^loCD45.1⁺) (left) and p16^Ink4a mRNA expression (right) of host Tconv cells in the indicated group of mice were analyzed. (H) The percentage of transferred Tregs (CD45.2⁺) among host Tregs in the recipient mice (CD45.1⁺) was analyzed by flow cytometry (n = 3–5 mice of 3 experiments; means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test).