(A) Representative histogram of CTV dilution of naive CD4⁺ T cells stimulated with anti-mCD3 Ab and antigen-presenting cells (APCs) in the presence of WT or KO Tregs for 60 hours. (B) Frequencies of each cell division (n = 4). (C) Frequencies of initial body weights of recipient mice 25 days after transfer with 5 × 10⁵ Tcons alone, or together with 2.5 × 10⁵ WT (Tcon+WT Treg) or KO (Tcon+KO Treg) Tregs. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (Tcon+WT Treg vs. Tcon+KO Treg); ^#P ≤ 0.05 and ^##P ≤ 0.01 (Tcon vs. Tcon+WT Treg). (D–F) Representative images of colons (D), histology images of colon sections (E), and images of spleens (F) from recipient mice (original magnification, ×8). (G) Numbers of CD44⁺CD62L^– splenic CD4⁺ T cells (n = 3). (H and I) Representative FACS plots of IFN-γ expression (H) and frequencies of IFN-γ⁺ (I) in splenic CD4⁺ T cells. (J and K) Representative FACS plots of IL-17 expression (J) and frequencies of IL-17⁺ (K) in splenic CD4⁺ T cells (n = 4). (L) Schematic diagram of Treg transfer assay and images of recipient mice and spleens and LNs 19 days after the transfer. (M) Frequencies of CD44⁺CD62L^– cells in CD4⁺ and CD8⁺ T cells in spleens (left) and LNs (right) from recipient mice (n = 3–4). Data are representative of 2 independent experiments and are shown as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001; ^#P ≤ 0.05 and ^##P ≤ 0.01. Statistical significance was determined by 2-sided, unpaired t test (B and C) and 1-way ANOVA with Tukey’s multiple-comparison test (G, I, K, and M).