IL-12 synergizes with IL-1β or IL-18 to induce the transition of Th17 cells to IFNγ-expressing progeny independent of TCR stimulation.

<p>Bulk Th1 cells (A) were activated as shown for 48 hours and the concentration of IFNγ in culture supernatants was determined by ELISA (B). Naïve CD4 T cells from <i>Il17f/Thy1.1</i> reporter mice were differentiated into Th17 cells and the Thy1.1hi fraction was enriched by FACS (C). Cells were then treated with indicated cytokine combination for 48 hours and ELISA was performed to measure the concentration of IFNγ in culture supernatants (D). To determine the role of STAT-4, naïve CD4 T cells from WT or <i>Stat4-/-</i> mice were cultured under Th17 polarizing conditions and their differentiation confirmed by intracellular cytokine staining (E). The number of IL-17A+ T cells was normalized based on intracellular cytokine staining then re-stimulated with indicated cytokine(s) for 48 hours and ELISA was performed to measure IFNγ concentration. Error bars represent means ± s.d. of triplicate determinations. Flow cytometry plots are gated on CD4+ cells and the numbers represent the percentages of cells in each quadrant. Data are representative of at least three independent experiments (*p<0.05, **p<0.01, **p<0.001 and **p<0.0001).