Fig. 2. CD69⁺ Tregs inhibit effector T cell proliferation in an IL-10-dependent fashion.

a, c Overall 1 × 10⁶/ml freshly isolated Treg cell subsets or CD4⁺Foxp3⁻T cells from Foxp3^GFP knock-in mice were stimulated with or without 2 μg/ml anti-CD3/CD28 antibody for 24 h and the relative levels of Il-10 and TGF-β1 to β- actin in the cells were analyzed by real-time PCR (n = 3). b, d The levels of IL-10 and TGF-β1 in the supernatants of cultured cells as described in a and c were measured by ELISA. e, f The expression levels of GARP and mTGF-β1 in CD69⁺ Treg and CD69⁻Tregs were analyzed using flow cytometry with indicated antibodies. g 1 × 10⁶ /ml CD4⁺CD25⁻T cells were labeled with CFSE and co-cultured with CD4⁺CD25⁺CD69⁺ Tregs or CD4⁺CD25⁺CD69⁻Tregs from Il-10^+/+ or Il-10^−/− mice at a ratio of 1:1, 1:2, or 1:4 in the presence 1 μl anti-CD3/CD28 coated beads for three days. Then, the proliferation of CD4⁺ T cells was analyzed using flow cytometry. The cells were gated first on living lymphocytes and then CFSE⁺ T cells (n = 3). Date are representative images or expressed as the mean ± SD of three independent experiments, and Student’s t-test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant. 1, versus CD69⁻ Tregs from Il-10^+/+ mice; 2, versus CD69⁺ Tregs from Il-10^−/− mice