Figure 1.
Organ-specific ILC subset distribution and phenotype of ILC2s. (A) Flow cytometric characterization and gating strategy of ILC subsets isolated from kidney, lung, and SILP of naïve C57BL/6 mice (blue = ILC2s, red = ILC3s, and yellow = ILC1s). Numbers indicate the percentage of events in the respective gates. (B) Frequencies and absolute numbers of total ILCs in kidney, lung, and SILP. (C) Frequencies of ILC subset distribution in the respective organs. Symbols represent individual data points and bars indicate mean ± SEM. (D) Principal component analysis of bulk RNAseq data from ILC2s purified from the indicated organs by flow cytometry. ILC2s from the kidneys and lungs of C57BL/6 mice were sorted as CD45+Lin−CD127+CD25+Sca-1+, whereas ILC2s from SILP were sorted as CD45+Lin−CD127+CD25+KLRG1+. Symbols represent individual ILC2 samples sorted from independent pools of mice (n = 3–4 samples with five mice per pool). (E) Sample distance plot based on all detected transcripts in the individual samples of the three organs. (F) Venn diagram of differentially expressed transcripts in each tissue compared with the other two tissues (false discovery rate < 0.1 and Log2FC > 1). Numbers indicate the up- (arrow up) or downregulated (arrow down) transcripts. Selected transcripts are specified for each organ with transcripts of interest marked in bold. (G) Flow cytometric analysis of Lin−GATA-3+ ILC2s from PBS- and IL-33–treated (400 ng i.p. injection on four consecutive days) wild type C57BL/6 mice (n = 3–5 mice per group). Analyses were performed at 2–3 wk after the first injection. Heat maps show geometric MFI of various surface markers of ILC2s in the indicated organs normalized to surface marker expression of kidney ILC2s. (H) UMAP clustering of the flow cytometry data from G. Lin−GATA-3+ ILC2s of the lung, kidney, and SILP were concatenated (40,000 events for each organ), and unbiased clustering was performed. Plots show combined and single-organ contribution of kidney (orange), lung (blue), and SILP (green) ILC2s to the UMAP clustering. (I) Absolute numbers of Lin−GATA-3+ ILC2s analyzed in G. (J and K) Representative flow cytometry plots (J) and absolute numbers (K) of ILC2s isolated from the kidney, lung, and SILP of PBS- (n = 8) and IL-33–treated (n = 10) wild type C57BL/6 mice. (L and M) Representative flow cytometry plots (L) and frequencies (M) of different cytokines produced by ILC2s of PBS- (n = 6) and IL-33–treated C57BL/6 mice (n = 5). Numbers in the flow cytometry plots indicate the percentage of events in the respective gates. Data in A, B, and G–I are representative of at least two independent experiments with similar results. Data in J and K are pooled from three and in L and M are pooled from two individually performed experiments. Symbols represent individual data points, and bars indicate mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Organ-specific ILC subset distribution and phenotype of ILC2s. (A) Flow cytometric characterization and gating strategy of ILC subsets isolated from kidney, lung, and SILP of naïve C57BL/6 mice (blue = ILC2s, red = ILC3s, and yellow = ILC1s). Numbers indicate the percentage of events in the respective gates. (B) Frequencies and absolute numbers of total ILCs in kidney, lung, and SILP. (C) Frequencies of ILC subset distribution in the respective organs. Symbols represent individual data points and bars indicate mean ± SEM. (D) Principal component analysis of bulk RNAseq data from ILC2s purified from the indicated organs by flow cytometry. ILC2s from the kidneys and lungs of C57BL/6 mice were sorted as CD45+Lin−CD127+CD25+Sca-1+, whereas ILC2s from SILP were sorted as CD45+Lin−CD127+CD25+KLRG1+. Symbols represent individual ILC2 samples sorted from independent pools of mice (n = 3–4 samples with five mice per pool). (E) Sample distance plot based on all detected transcripts in the individual samples of the three organs. (F) Venn diagram of differentially expressed transcripts in each tissue compared with the other two tissues (false discovery rate < 0.1 and Log2FC > 1). Numbers indicate the up- (arrow up) or downregulated (arrow down) transcripts. Selected transcripts are specified for each organ with transcripts of interest marked in bold. (G) Flow cytometric analysis of Lin−GATA-3+ ILC2s from PBS- and IL-33–treated (400 ng i.p. injection on four consecutive days) wild type C57BL/6 mice (n = 3–5 mice per group). Analyses were performed at 2–3 wk after the first injection. Heat maps show geometric MFI of various surface markers of ILC2s in the indicated organs normalized to surface marker expression of kidney ILC2s. (H) UMAP clustering of the flow cytometry data from G. Lin−GATA-3+ ILC2s of the lung, kidney, and SILP were concatenated (40,000 events for each organ), and unbiased clustering was performed. Plots show combined and single-organ contribution of kidney (orange), lung (blue), and SILP (green) ILC2s to the UMAP clustering. (I) Absolute numbers of Lin−GATA-3+ ILC2s analyzed in G. (J and K) Representative flow cytometry plots (J) and absolute numbers (K) of ILC2s isolated from the kidney, lung, and SILP of PBS- (n = 8) and IL-33–treated (n = 10) wild type C57BL/6 mice. (L and M) Representative flow cytometry plots (L) and frequencies (M) of different cytokines produced by ILC2s of PBS- (n = 6) and IL-33–treated C57BL/6 mice (n = 5). Numbers in the flow cytometry plots indicate the percentage of events in the respective gates. Data in A, B, and G–I are representative of at least two independent experiments with similar results. Data in J and K are pooled from three and in L and M are pooled from two individually performed experiments. Symbols represent individual data points, and bars indicate mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

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