FIG. 3. In vitro functional capacity of CD4⁺CD25⁺ T-cells. A: We used 6-week-old (n = 22), 8-week-old (n = 5), and diabetic NOD (n = 17) mice. Cocultures were performed by incubating CD4⁺CD25⁺ and CD4⁺CD25⁻ T-cells from the same mouse at a 1:1 ratio with APCs and anti-CD3 antibody. A significant difference was observed when comparing mean values between 6-week-old and diabetic NOD mice (P < 0.0001) and between 6- and 8-week-old NOD mice (P < 0.042). The addition of anti–TGF-β antibody, 2G.7, reversed the suppression in a dose-dependent manner (no antibody: 60.9 ± 2.6% inhibition; + 2G.7 10 μg/ml: 32.8 ± 3.6% inhibition, P < 0.005; + 2G.7 50 μg/ml: 4.5 ± 1.2% inhibition, P < 0.001). B: Suppression of CD4⁺CD25⁻ T-cell proliferation by increasing proportion of CD4⁺CD25⁺ T-cells. C: In vitro inhibition of CD4⁺CD25⁻ T-cells by CD4⁺CD25⁺ T-cells is cell contact–dependent. The proliferation of 5,6-carboxyfluorescein diacetate-succinimidyl ester (CFSE)-labeled CD4⁺CD25⁻ T-cells from 6-week-old NOD mice was analyzed by immunofluorescence after being incubated 4 days with young NOD CD4⁺CD25⁺ T-cells separated or not separated by a transwell membrane. The results were representative of 5–8 experiments. wk, week. FIG. 3. In vitro functional capacity of CD4+CD25+ T-cells. A: We used 6-week-old (n = 22), 8-week-old (n = 5), and diabetic NOD (n = 17) mice. Cocultures were performed by incubating CD4+CD25+ and CD4+CD25− T-cells from the same mouse at a 1:1 ratio with APCs and anti-CD3 antibody. A significant difference was observed when comparing mean values between 6-week-old and diabetic NOD mice (P < 0.0001) and between 6- and 8-week-old NOD mice (P < 0.042). The addition of anti–TGF-β antibody, 2G.7, reversed the suppression in a dose-dependent manner (no antibody: 60.9 ± 2.6% inhibition; + 2G.7 10 μg/ml: 32.8 ± 3.6% inhibition, P < 0.005; + 2G.7 50 μg/ml: 4.5 ± 1.2% inhibition, P < 0.001). B: Suppression of CD4+CD25− T-cell proliferation by increasing proportion of CD4+CD25+ T-cells. C: In vitro inhibition of CD4+CD25− T-cells by CD4+CD25+ T-cells is cell contact–dependent. The proliferation of 5,6-carboxyfluorescein diacetate-succinimidyl ester (CFSE)-labeled CD4+CD25− T-cells from 6-week-old NOD mice was analyzed by immunofluorescence after being incubated 4 days with young NOD CD4+CD25+ T-cells separated or not separated by a transwell membrane. The results were representative of 5–8 experiments. wk, week. More