Fig. 3

ILC2 respond to C3a. C3ar1 mRNA expression in a fresh flow-sorted lung Lin− (CD11b, CD11c, Gr1, B220, CD19, TCRb, TCRgd, CD49b, CD4, CD8, FcER1) ICOS⁺IL-33R⁺ ILC2 from naive BALB/c and C3ar1^−/− mice, and b wt ILC2 cultured in IL-2 (10 ng/ml) alone or in addition to IL-33 (0.5 ng/ml) for 3 days. IL-13 in the supernatant of sorted lung ILC2 cultured in c IL-2 (10 ng/ml) or IL-2 with C3a (1 µg/ml) or d media, IL-2 + IL-33 or IL-2 + IL-33 + C3a after 3 days. e C3a in the supernatant of lung ICOS⁺IL-33R⁺ ILC2 cultured in IL-2 (10 ng/ml) containing 0, 0.5, or 1.0 ng/ml IL-33 for 3 days. Lung ICOS⁺IL-33R⁺ ILC2 cultured in IL-2 (10 ng/ml) or IL-2 in combination with IL-33 (0.5 ng/ml) for 3 days, and levels of f IL-13 and g GM-CSF in the supernatant was measured, and cells were harvested for h Il10 mRNA determination. Rag1^−/− mice exposed to PBS, C3a (1 µg), IL-33 (0.5 µg) or C3a + IL-33 i.n. on days 0, 2, and 4, and lungs were analyzed on day 6 for i numbers and j frequency of IL-13⁺ ILCs (Lin^-CD45⁺IL-33R⁺IL-13⁺), as well as k IL-13 median fluorescence intensity (MFI) in ILC2. Data represent means + SEM. Data are representative of two to three independent experiments with four replicate wells or 3–5 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001