Antigen-receptor stimulation induces T-bet expression in Th1β cells, which is required for its progression to an IFN-γ secreting state. (A) WT C57BL/6 Th1 and Th1β cells were cultured for 5 days and left unstimulated or restimulated with α-CD3 with or without 15 μg/mL α-IFN-γ for 4 hours. Protein lysates were analyzed for T-bet expression by immunoblot (B) WT and Tbx21^-/- Th1β cells were cultured for 5 days and left unstimulated or were restimulated with α-CD3 for 24 hours. Cell-free supernatants were collected on day 6 and measured for IFN-γ or IL-17 secretion using ELISA. In parallel, 5-day differentiated cells were replated on α-CD3-coated plates and IL-23 for 5 more days as in Figure 1C. After the culture period, live cells were washed and left unstimulated or restimulated with α-CD3 for 24 hours. Cell-free supernatants were collected on day 11. Supernatants from both time points were analyzed for IL-17 and IFN-γ production using ELISA. (C) Tbx21^-/- Th1β cells were cultured as in panel B with or without the indicated doses of IFN-γ during the second week of culture. Cells were left unstimulated or were restimulated with α-CD3 for 24 hours, and cell-free supernatants on days 6 and 11 were analyzed for IL-17 production using ELISA. Unstimulated cell populations did not produce detectable cytokine levels (B-C). ELISA data are represented as mean ± SE of replicate samples.