Figure 4: IL-21 signalling and IL-10 production during T_H17-cell differentiation in Irf8^–/– mice.

Naive CD4⁺ T cells from WT and Irf8^–/– mice were prepared, and the cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of TGF-β plus IL-21 or IL-6 for 72 h. Intracellular staining for IL-17 and IFN-γ expression was performed and analysed by flow cytometry (a). The secretion of IL-21 protein in culture supernatants was analysed by ELISA (b). Data are from one experiment representative of three independent experiments. Error bars, s.d. Naive CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of TGF-β plus IL-6 or IL-21. At 96 h after stimulation, intracellular staining for IL-17 and IL-10 was performed and analysed after re-stimulation with PMA/ionomycin by flow cytometry (c). Naive CD4⁺ T cells from spleens and lymph nodes of WT and Irf8^–/– mice were prepared and the cells were stimulated under T_H0, T_H1, T_H2 and T_H17 polarizing conditions for 4 days. Then the cells were re-stimulated with PMA/ionomycin for 12 h and IL-10 protein secretion was analysed by ELISA (d). Data are from one experiment representative of three independent experiments. Error bars, s.d. Naive CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of TGF-β plus IL-6 or IL-21 for 72 h. IL-10 expression was analysed by RT–PCR (e). Data are from one experiment representative of three independent experiments. Error bars, s.d.