Figure 1: IRF1 limits IRF4-driven IL-9 production dose-dependently.

(a–c) Naive CD44⁻CD62L⁺CD4⁺ T cells were isolated from WT, Irf4^−/−, Irf1^−/− or Stat1^−/− mice and then treated under Th9 (TGF-β and IL-4) or Th0 (without skewing cytokines) conditions with/without IFN-γ as indicated. (a,b) After resting and reculture under Th9 conditions for 2 days, intracellular flow cytometric analysis for IRF1 and IRF4 were performed. Bars give geometric mean of fluorescence intensity (MFI). (c) Cells were restimulated and then IL-9 and IFN-γ production was detected by flow cytometry, bars to the right give mean of IL-9⁺ T cells. (d,e) Irf4^−/− CD4⁺ T cells were isolated, activated under Th0 condition overnight and then spin-infected with retroviruses as indicated: IRF4-GFP-RV, control-Thy1.1-RV, and IRF1-Thy1.1-RV. Thereafter, cells were cultured under Th9 conditions for 2 days, rested for 3 days and recultured under Th9 conditions for additional 2 days. Four subsets (I–IV) were selected for analysis of IL-9 production. Bars to the right show fold induction of IL-9⁺ T cells relative to GFP⁻Thy.1.1⁻ cells (subset I). (e) Six subsets (a through f) expressing increasing levels of GFP and Thy1.1 were selected for analysis of IL-9 production (left panel). Dot plot to the left is representative for four independent experiments. Graph to the right shows fold induction of IL-9⁺ T cells relative to GFP⁻Thy1.1⁻ cells (subset a) combined from four independent experiments. Histogram and contour-plots are representative of two (a,b), three (c) or four (d,e) independent experiments. Bars show mean±s.d. from combined two (a,b), three (c) or four (d,e) experiments. *P<0.05, **P<0.005, ***P<0.001 (two-tailed Student’s t-test).