IFNγ, IL-17 and double-positive IFNγ/IL-17 cytokines in joint tissues are reduced in IL-23p19−/−mice after subcutaneous immunization. Cells were obtained from ankle joints minced, treated with collagenase, and stained with specific antibodies. (A) Joint cells (%) and (B) number of cells identified by flow cytometry. Data represent mean ± standard error of the mean (SEM, n =4 mice). *P <0.05. (C) Representative flow cytometry plots of gated CD4+ T cells expressing IFNγ and IL-17 from joint tissues of wild type (WT) and IL-23p19−/− mice immunized either by the intraperitoneal (i.p.; left two panels) or subcutaneous (s.c.; right two panels) route with rG1 in adjuvant. For intracellular cytokine staining, cells were stimulated with Phorbol 12-myristate 13-acetate and ionomycin for 4 hours. Cells were surface stained for CD4, permeabilized and stained for IFNγ and IL-17 with specific antibodies. (D) Bar graphs showing mean ± SEM of percentage of gated CD4+ T cells in WT and IL-23p19−/− mice immunized by the i.p. (left panel) and s.c. (right panel) routes expressing IFNγ and IL-17. Data presented as mean ± SEM (n =4) from two independent experiments. *P <0.05. IFN, interferon; IL, interleukin.