FIGURE 5.

IL-23 does not program commitment to the Th17 lineage. A, Naïve T cells were activated, primed with TGF-β+IL-6+IL-1β for the first round followed by 2 rounds of stimulation in IL-23 + IL-1β, were cultured for an additional round of stimulation in Th1 or Th2 conditions, or IL-23+IL-1β. Supernatants from anti-CD3 stimulated cells were tested for cytokine production using ELISA. B, Naïve CD4+ T cells were activated and cultured under Th1 or Th2 priming conditions for four rounds of stimulation. At the end of the fourth round of stimulation, supernatants from anti-CD3 stimulated cells were tested for cytokine production using ELISA. C, Cells stimulated and cultured as in (A) were stimulated with anti-CD3 for 4 hours and RNA was isolated for qPCR. D, Cells were stimulated and cultured for three rounds as in (A). After three days of culture in Th1, Th2 or IL-23+IL-1β conditions, RNA was isolated from control or switched cultures for qPCR. E, Naïve T cells were activated, primed with TGF-β+IL-6+IL-1β for the first round followed by 2 rounds of stimulation in IL-23 + IL-1β, were cultured for an additional round of stimulation in IL-23 + IL-1β or TGF-β + IL-2. The percentages of cells positive for Foxp3 or IL-17 intracellular staining are indicated with cells cultured for one week in TGF-β + IL-2 shown as a control for Foxp3 expression. F, Naïve CD4+ T cells were primed and cultured as in (A) and after the third round of culture, cells were enriched for IL-17-secreting cells by cytokine selection. Surface staining for IL-17 is shown pre- and post-sort. G, IL-17-high cells from (F) were cultured in Th1, Th2 or IL-23+IL-1β for an additional round of stimulation. Cells were stimulated for 4 hours and stained for intracellular IL-17 and IFN-γ. H, Supernatants from anti-CD3 stimulated cells cultured as in (G) were tested for cytokine production using ELISA.