Suppressive activity of Treg subsets.

<p>(<b>A</b>) Isolated CD4⁺CD25⁺ Tregs from CD28SA treated mice were analysed for IL-10 production using IL-10 Secretion Assay. FACS separated IL-10⁺ (solid black line) and IL-10⁻ CD4⁺CD25⁺ Tregs (dashed black line) were incubated with CFSE-labeled naïve CD4⁺CD25⁻ effector T-cells (T_eff), (ratio T_eff∶ T_reg 1∶1 to 81∶1) and irradiated APC in the presence of anti-CD3 for 3 days. Stimulated total CD4⁺CD25⁺ Tregs (solid grey line) and CD4⁺CD25⁺ Tregs from untreated mice (dashed grey line) were used as controls. (<b>B</b>) Bar graph of IL-10⁺ (white bar) and IL-10⁻ CD4⁺CD25⁺ Tregs (black bar) with statistical analysis. (<b>C</b> and <b>D</b>) like (<b>A</b> and <b>B</b>) but with FACS sorted Foxp3⁺ of unstimulated and CD28SA stimulated DEREG mice. (<b>E</b>) Suppression is IL-10 independent. Blocking anti-IL-10R mAb (10 µg/ml, grey line) was added to cultures with stimulated (solid line) and unstimulated total CD4⁺CD25⁺ Tregs (dashed line). Graphs show average numbers of cell division (acd, mean ± SD) from 3 wells assayed individually and results are representative of three independent experiments. * p<0.05, ** p<0.005, *** p<0.001, ns: no significant difference.