IL-23-cultured CD4⁺ T cells transiently secrete IL-17 and progress to a Th1 cytokine-secreting profile. (A) CD4⁺ T cells from C57BL/6 mice were activated with α-CD3 and α-CD28 and cultured with α-IL-4. IL-12 or IL-23 was added to the culture to generate Th1 or Th1β cells, respectively. Cells were cultured for 3 days and expanded in the presence of IL-12 or IL-23 for 2 additional days. After 5 days, cells were left unstimulated or were restimulated with α-CD3 for 24 hours, and cell-free supernatants were analyzed for IL-17 and IFN-γ secretion using ELISA. (B) Th1β cells were cultured for 5 days as in panel A. Cells were replated with media alone or restimulated with α-CD3 and α-CD28 for 5 hours in the presence of Brefeldin A. Cells were fixed, permeabilized, and stained with PE-α-IL-17 and FITC-α-IFN-γ antibodies and analyzed for intracellular cytokine staining by flow cytometry. Numbers indicate percentage of cells within each region. (C) Th1 or Th1β cells differentiated for 5 days (week 1) in panel A were cultured for 5 additional days (week 2) with or without plate-bound α-CD3. Th1 and Th1β cultures were maintained in media containing IL-12 or IL-23, respectively. At day 10, live cells were cultured in the presence or absence of plate-bound α-CD3 for 24 hours, and supernatants were analyzed for IFN-γ and IL-17 production using ELISA. Unstimulated cultures at day 10 did not produce detectable levels of cytokine. (D) Five-day Th1β cultures differentiated in the presence or absence of α-IFN-γ (10 μg/mL) were restimulated as in panel A, and cell-free supernatants were measured for IL-17 quantities using ELISA (week 1). In parallel, 5-day differentiated cells were cultured for 5 more days (week 2) in the presence of plate-bound α-CD3 and maintained in media containing IL-23 with or without α-IFN-γ as indicated in the figure. Two-week cultures were restimulated as in panel C, and IL-17 levels from cell-free supernatants were measured using ELISA. ELISA data are represented as mean ± SE of replicate samples.