Figure 4

Higher ICOS-L expression is critical for DCs from IL-10 KO mice to promote Th17, but not Th1, response in DC:CD4⁺ T-cell coculture. CD4 T cells isolated from Cm immunized mice were cocultured with freshly purified DCs from Cm-infected WT (DC(WT)) or IL-10 KO (DC(KO)) mice (DC:CD4⁺ T cells, 5 × 10⁵:2 × 10⁶) in the presence of killed Cm stimulation with anti ICOS-L mAbs or antibody isotype control. After 48-h cultures, cells were analyzed for surface CD3ε and CD4 and intracellular IL-17 and IFNγ as described in Materials and Methods. Cells were gated on CD3ε⁺CD4+ cells. The percentages of IL-17⁺CD4⁺ T cells or IFNγ⁺CD4⁺ T cells in total CD4⁺ T cells (CD3ε⁺CD4⁺) are indicated at the left upper corner. In separated cultures, coculture was carried for 72 h before supernatants were collected, and the supernatants were measured for IL-17 or IFNγ by ELISA. (A) Representative intracellular IL-17 staining in different culture conditions. (B) Summary of the percentages of IL-17⁺ CD4⁺ T cells in total CD4⁺ T cells. (C) IL-17A protein levels in the coculture supernatants of each condition. (D) Representative intracellular IFNγ staining in different culture conditions. (E) Summary of the percentages of IFNγ⁺ CD4⁺ T cells in total CD4⁺ T cells. (F) IFNγ protein levels in the coculture supernatants of each condition. Data are shown as mean ± SD (n = 4). *p < 0.05; **p < 0.01. Representative data of three independent experiments are shown.