Notch signaling in intestine-specific adaptation of kidney ILC2s in vitro. (A) Violin plots depicting Notch signaling score in the scRNAseq data of kidney and SILP donors and recipient mice (see Fig. 4 A for experimental setup). (B and C) MFI (B) and representative histogram overlays (C) of surface marker expression of sorted kidney ILC2s after in vitro culture with IL-2 in the presence or absence of the Notch ligand DLL4 (2.5 µg/ml). Cells were sorted as CD45⁺Lin⁻CD127⁺IL-33R⁺KLRG1⁺ from the kidney of IL-33–treated C57BL/6 mice and cultured for 4 d in a 96-well plate (∼10,000 cells/well; n = 4 for each condition). (D) Cytokine quantification in the supernatant of the cultured kidney ILC2s stimulated with subthreshold IL-25 or IL-33 (1 ng/ml each). Cells were stimulated on day 3 for 24 h. (E and F) MFI (E) and representative histogram overlays (F) for surface marker expression of kidney ILC2s after culture with IL-2 in the presence or absence of RA (1 µM) and DLL4 (n = 4 for each condition). (G) Cytokine quantification in the supernatant of the cultivated kidney ILC2s stimulated as in E. Data are representative of three individual experiments with similar results. Symbols represent individual data points and bars indicate mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test (B and D) or one-way ANOVA with Tukey’s post-hoc test (E and G); *P < 0.05, **P < 0.01, ***P < 0.001.