RA signaling inhibition reverses intestinal ILC2 adaptation and impairs anti-helminth immunity. (A) Experimental setup of RAi treatment and subsequent S. ratti infection in BALB/c Rag2^−/− and BALB/c Rag2^−/−Il2rg^−/− mice. (B) Representative flow cytometric plots of ILC2 and ILC3 populations isolated from the colon of BALB/c Rag2^−/− mice treated with either DMSO or RAi and infected with S. ratti. In S. ratti experiments, colon was analyzed for the RAi effect on ILC2s because the small intestine was needed for worm counting. Gating strategy is indicated in brackets and numbers indicate the percentage of cells in each gate. (C) Frequencies (upper panel) and absolute numbers (lower panel) of GATA-3⁺ ILC2s and RORγt⁺ ILC3s populations as well as their proportional shift (right panel) in the colon of BALB/c Rag2^−/− mice infected with S. ratti after 3 wk of DMSO or RAi treatment. (D and E) Representative histogram overlays (D) and MFI (E) of the surface marker expression in the two groups. (F) Worm burden in the small intestine of Rag2^−/− and Rag2^−/−Il2rg^−/− mice 6 d after infection with S. ratti. (G) Mast cell activity assessed as mMCPT-1 serum concentration in Rag2^−/− and Rag2^−/−Il2rg^−/− mice infected with S. ratti. Data are pooled from two (Rag2^−/−) or three (Rag2^−/−Il2rg^−/−) individually performed experiments with three to five mice per group. (H and I) Absolute numbers (H) and MFI (I) of IL-17RB and IL-33R of Lin⁻IL-13fm⁺ ILC2s isolated from different intestinal segments of naïve BALB/c IL-13^fm mice. Data represent at least two individually performed experiments with three mice per group. Symbols represent individual data points and bars indicate mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test (C–G) or one-way ANOVA with Tukey’s post-hoc test (H and I); *P < 0.05, **P < 0.01, ***P < 0.001.