Supplementary Figure 5: Type I interferon restrains mouse ILC2 cells.

(a,b) Sorted and expanded bone marrow-derived ILC2 from WT mice were stimulated for five days with IL‐7 and IL-33 (both at 10 ng/ml) only (black bars) or with IL‐7 and IL-33 (both at 10 ng/ml) in combination with IFN-β (500 U/ml; red bars). (a) IFN-β was added at different time points (0h, 24h, 48h and 72h) and left in the culture until supernatants were taken. (b) IFN-β was added at the beginning of the culture (0h) and at different time points (not removed, 24h, 48h and 72h) cells were washed and re-seeded with IL‐7 and IL-33 (both at 10 ng/ml) only until supernatants were taken. Five days after culture proliferation of cells was measured by alamarBlue assay and cytokine production was determined by ELISA. The percentages of reduction due to the addition of IFN-β are shown in brackets above the red bars. Data are representative of two independent experiments. (c) Sorted and expanded bone marrow-derived ILC2 from WT mice were stimulated for three days with medium only, or with IL‐7 and IL-33 (both at 10 ng/ml). IFN-β (25 or 500 U/ml) was added to some wells as indicated. GATA3 expression was determined by intracellular flow cytometry staining and histograms and the ratio of the GATA3 mean fluorescence intensities (MFI) to the MFI of the internal isotype control stainings are shown. Data are representative of three independent experiments. (d) Sorted and expanded bone marrow-derived ILC2 from WT mice were stimulated for five days with IL‐7 and IL-33 (both at 10 ng/ml) only (black bars) or with IL‐7 and IL-33 (both at 10 ng/ml) in combination with IFN-β (500 U/ml; red bars). IFN-β was added at the beginning of the culture (0h) and at different time points (24h, 48h and 72h) cells were washed and re-seeded with IL‐7 and IL-33 (both at 10 ng/ml) only until supernatants were taken. Five days after culture GATA3 expression was determined by intracellular flow cytometry staining and the ratio of the GATA3 MFI to the MFI of the internal isotype control stainings are shown. Data are representative of two independent experiments. The bars represent the mean ± standard deviation of triplicates of each cohort. The asterisks indicate statistically significant differences (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). RFU, relative fluorescence units.