T-bet negatively regulates Th1β development. (A) WT and Tbx21^-/- CD4⁺ T cells were cultured under Th1 (IL-12 + α-IL-4) or Th1β (IL-23 + αx-IL-4) conditions for 5 days and restimulated with α-CD3 for 18 hours. Cell-free supernatants were analyzed for IFN-γ or IL-17 using ELISA. Unstimulated populations did not produce detectable levels of cytokine. RNA was extracted from T cells activated for 18 hours, and duplicate samples were quantified by real-time PCR for IL-17A mRNA. Cycle number was normalized to β2-microglobulin mRNA and is represented as fold induction relative to unstimulated wild-type Th1 cells. ND indicates not detected. (B) WT CD4⁺ T cells from C57BL/6 mice were cultured with α-IL-4 alone (Unsk.) or under Th1 or Th1β conditions for 5 days as described in Figure 1A. Protein lysates from freshly isolated CD4⁺ T cells or differentiated cells were assayed for T-bet expression by immunoblot. (C) WT Th1β cells were cultured for 2 days and infected with a bicistronic retrovirus expressing EGFP alone (vector control) or T-bet and EGFP (T-bet RV). Cells were cultured for 3 more days, and EGFP-positive cells were sorted and restimulated with α-CD3 or left unstimulated for 18 hours. Cell-free supernatants were analyzed for IL-17 and IFN-γ production. Unstimulated cell populations did not produce detectable cytokine levels. ELISA data are represented as mean ± SE of replicate samples.