TH17 in vitro differentiation. TH17 cells were generated as described in “Methods.” (A) Initial histogram of CD4⁺ T cells isolated after sorting. (B) Flow cytometric analysis of T cells differentiated for 6 days. Evaluation of T cells differentiated for 11 days: IL-17A (C,D), IFN-γ (D), and IL-17F (E). (B-E), plots derived from CD4⁺ gate. (F) Aliquots of cells from TH17 culture (day 11 in culture) and sorted naive CD4⁺ T cells were harvested, and RNA was extracted. RNA samples were subjected to quantitative PCR for RORγt and T-bet.