The role of IL-10 and IFN-γ in the generation and function of the CD8+ NKT-like cells. (a) Cytokine levels in the cell culture media. Cultured CD8+ NKT-like cells and freshly isolated CD4+CD25+ Treg cells were stimulated with anti-CD3 (1.5 μg/ml) and splenic APCs. At 72 h of culturing, the culture supernatant was saved and used for measuring IL-10, IL-4 and IFN-γ using ELISA. Results are representative of two independent experiments. (b) Suppression activity of CD8+ NKT-like cells cultured from IFN-γ-/- and IL-10-/- mice. CD8+ NKT-like cells (Tr) cultured from knockout mice and wild-type B6 (WT) mice were co-cultured with naïve CD4+CD25- responder T cells (Tn) at different Tr/Tn ratios in the presence of splenic APCs and anti-CD3. The cultures were pulsed with 1 μCi/well of [3H]thymidine at 72 h and proliferation (cpm) was measured by [3H]thymidine incorporation in the last 16 h. Results are expressed as the mean of triplicate cultures. ANOVA p-values are <0.0004 for IFN-γ-/- and 0.001 for IL-10-/- when compared to wild-type mice. Error bars are standard deviation.