Figure 3. IL-27 and IFN-γ suppress IL-17 induction and RORγt expression.

(A–D) Splenic lymphocytes (5×10⁶/ml) from estrogen- and placebo-treated male C57BL/6 mice were activated with IL-6+TGFβ+anti CD3 antibody in the presence or absence of (A) rIL-27 (placebo=3; estrogen =3; representative of two independent experiment) or (B) rIFN-γ for 48 hr (placebo=4; estrogen=6; representative of two independent experiment), and IL-17 levels determined by ELISA. (C, D) Splenic lymphocytes from estrogen- and placebo-treated male C57BL/6 mice were activated with IL-6+TGFβ+anti CD3 antibody in the presence or absence of (C) rIL-27 or (D) rIFN-γ (placebo=3; estrogen 3) for 48 hrs and the mean percent RORγt⁺ expression in splenocytes determined by flow cytometry. (E–G) Splenic lymphocytes from estrogen- and placebo-treated male C57BL/6 mice were activated with IL-6+TGFβ+anti CD3 antibody, and rIL-27 or rIFN-γ were added either together with IL-6+TGFβ+anti CD3 antibody stimulation or at the indicated time points. IL-17 levels were measured by ELISA after 72 or 96 hr (placebo=4; estrogen=6). (H) Splenocytes from estrogen- and placebo-treated male C57BL/6 mice were cultured in the presence of the JAK2 inhibitor AG490 for 48 hr and IL-17 levels analyzed (placebo=3; estrogen=5). (I) Splenocytes from estrogen- and placebo-treated male C57BL/6 mice were activated with IL-6+TGFβ+anti CD3 antibody in the presence or absence of AG490 and the percent RORγt⁺ cells determined after 24 hr (placebo=3; estrogen=4). Data are means ± SEM; * p<0.05, ** p<0.01, *** p<0.001; (A, B, E, and H), Tukey-Kramer test; (C, D, F and G), Student Newman-Keuls test.