Interleukin (IL)-27p28-induced STAT1 activation regulates Th17-cell differentiation and plasticity. ( a ) Western blot analysis of transcription factors in wild-type (WT) naive T cells after co-culturing with MΦs in the presence of cecal bacterial antigen (CBA). β-actin was used as a loading control. ( b ) Flow cytometry (FCM) analysis of IL-17 and interferon (IFN)-γ in STAT −/− naive T cells after in vitro differentiation with WT and Trif LPS2 MΦ in the presence of CBA. Representative results from five independent experiments are shown ( n =4 each, mean±s.e.m.). ( c ) Western blot analysis of transcription factors in WT naive T cells after co-culturing with WT MΦs and CBA in the presence of anti-IFNAR1 antibody. ( d ) FCM analysis of IL-17 and IFN-γ in STAT1 −/− naive T cells after co-culturing with Trif LPS2 MΦs in the presence of CBA and recombinant IL-27p28 (15 ng ml −1 ). Representative results from two independent experiments are shown ( n =4 each, mean±s.e.m.). ( e ) Western blot (top panel) and FCM (bottom panel) analysis of pSTAT1 in WT splenocytes after stimulation with IL-27p28 (15 ng ml −1 ) for the indicated period. For the FCM analysis, splenocytes were stained with CD3, CD4, and pSTAT1 after IL-27p28 stimulation. pSTAT1-positive cells in CD3 + CD4 + splenocytes are demonstrated. Results are representative of two independent experiments ( n =4 each, mean±s.e.m.). A full color version of this figure is available at the Mucosal Immunology journal online.
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