Fig. 2

Recombinant interleukin (IL)-35 stimulation to CD14⁺ monocytes induced naïve CD4⁺ T cell activation in patients with Kawasaki disease (n = 18). CD14⁺ monocytes and CD4⁺ T cells were purified from peripheral bloods of Kwasaki disease patients. CD14⁺ monocytes were stimulated with recombinant human IL-35 (50 ng/ml) and 1 × lipopolysaccharide for 24 h. Direct contact and indirect contact co-culture system was set up between 10⁵ of CD14⁺ monocytes and 10⁵ of autologous CD4⁺ T cells. In the last 12 h of co-culture, phorbol 12-myristate 13-acetate (50 ng/ml), ionomycin (1 μg/ml), and Brefeldin A (10 μg/ml) were added. Cells were harvested 48 h post co-culture, and were stained with anti-CD4, anti-interferon-γ (IFN-γ), and anti-IL-17A for flow cytometry analysis. The isotype control was used for separation of positive and negative cells of IFN-γ and IL-17A. Typical flow dots analyses for (a) CD4⁺IFN-γ⁺ Th1 cells and (b) CD4⁺IL-17A⁺ Th17 cells in direct contact and indirect contact co-culture systems. (c) CD4⁺IFN-γ⁺ Th1 and (d) CD4⁺IL-17A⁺ Th17 percentage was elevated in direct contact co-culture system when compared with in indirect contact co-culture system or in CD4⁺ T cell cultured anlone. However, there was no significant difference of (c) Th1 or (d) Th17 percentage between CD4⁺ T cell cultured alone and CD14⁺/CD4⁺ indirect contact co-culture system. IL-35 stimulation to CD14⁺ monocytes down-regulated (c) Th1 and (d) Th17 percentage in direct contact co-culture system, but not in indirect contact co-culture system. Paired t test was used for comparison. Individual level of each subject was shown. The horizon line presented mean, and error bar presented standard deviation