Fig 2: Differentially expressed and tissue specific genes of ILCs in the lung and small intestine.a–c Differential gene expression of ILC subsets in the lung and the gut was analyzed and shown in Volcano plot. Statistical analysis was performed by using two-tailed unpaired Student’s t-test. Red dots represented the genes highly expressed in the lung, and blue dots represented the genes highly expressed in the gut. d–f GO analysis of differentially expressed genes in the lung and the gut. The orange bars showed the pathways that were enriched in the lung, and the green bars showed the pathways that were enriched in the small intestine. g Normalized relative expression (log(scaled UMI + 1)) levels of the selected tissue specific genes of ILCs displaying on a tSNE plot of Fig. 1b. Cxcr6, Stat5a and Rora were enriched in the intestine ILCs, and Anxa1, Ly6a and S100a4 were highly expressed in the lung ILCs. Selected tissue specific genes for ILC subsets are: Gpr55 for intestine ILC1s, Sell for lung ILC1s, Ccr4 for intestine ILC2s, Ccr2 for lung ILC2s, Ffar2 for intestine ILC3s, and Cd36 for lung ILC3s. Source data are provided as a Source Data file.

Fig 3: Maturation of ILC2s through lung-gut axis.a CCR2-RFP;CCR4-mNeonGreen mice were i.p. injected with IL-25 or IL-33. After 24 h, expression of mNeonGreen and red fluorescent protein (RFP) in lung or gut ILC2s was analyzed by flow cytometry. b Analysis of mNeonGreen and RPF expression of gut ILC2s from CCR2-RFP;CCR4-mNeonGreen mice after intraperitoneal administration of IL-33 for the indicated days. The cell frequency of indicated subgroups of gut ILC2s was calculated and shown as mean ± SD (lower panel). (n = 3 for each group). c Adoptive transfer of lung or gut ILC2s (5 × 104) from WT CD45.1 mice to NOD-PrkdcscidIL2rgtm1/Bcgen (B-NDG) mice by intravenous injection. Six days after of transfer, lung and gut ILC2s from the recipient mice were analyzed by flow cytometry. The cell frequency of ILC2s were calculated and shown as mean ± SD. (n = 3 for each group). d Adoptive transfer and development of ILC2Ps in the lung and gut. ILC2Ps (Lin-CD127+ST2+Sca1+KLRG1-) from BM of CD45.1 mice were isolated and intravenously injected into B-NDG mice. One week after transfer, the recipient mice were i.p. injected with 400 ng/mouse/day IL-33 for the indicated days. The cell frequency of ILC2s in the lung and gut were calculated and shown as mean ± SD. (n = 3 for each group). e Tracing of lung ILC2s. Solubilizing 4-OHT (2 µg/g mouse) were atomized and delivered into lung of Id2-Cre/ERT2;Rosa26-STOP-tdTomato mice by liquid aerosol devices as described in “Methods”. Expression of TdTomato in ILC2s were analyzed by flow cytometry two days after 4-OHT treatment (left panel). Two days after 4-OHT treatment, Id2-Cre/ERT2;Rosa26-STOP-tdTomato mice were i.p. injected with IL-33. The frequency of TdTomato+ ILC2s from the mice treated IL-33 with indicated days were calculated and shown as mean ± SD. The schematic diagrams in (c-e) were created with BioRender.com. f, g Depletion of ILC2s in the lung and gut. Ccr2-mNeonGreen-Cre;Rosa26-STOP-DTR mice were subjected to i.p. injection of 100 ng/mouse diphtheria (DT) every two days for six days. Cell frequency of ILC2s from the lung, gut and BM were analyzed by flow cytometry (f) and shown as mean ± SD (g). (n = 3 for each group). ***, P < 0.001 by Two-tailed unpaired Student’s t-test. NS, not significant (P > 0.05) by Two-tailed unpaired Student’s t-test. (P = 0.0003, 0.0007, 0.4971 for Lung, Gut, BM respectively by Two-tailed unpaired Student’s t-test). h The reconstitution of ILC2s in the lung and gut after ILC2 depletion. ILC2s were depleted as shown in (f) and the ILC2-depleted mice were subjected to the intraperitoneal injection of 400 ng/mouse IL-33 at day 0. After the indicated days, cell frequency of lung and gut ILC2s were analyzed by flow cytometry and shown as mean ± SD. (n = 3 for each group) Data are representative of at least three independent experiments. Source data are provided as a Source Data file.

Fig 4: Postnatal development of lung ILC2s contributes to the proper function of gut ILC2s.a, b Analysis of lung and gut ILC2s from mice of indicated postnatal (P) days by flow cytometry and show as mean ± SD. c Expression of CCR2 and CCR4 on ILC2s from CCR2-RFP;CCR4-mNeonGreen mice at postnatal day 12 was analyzed by flow cytometry. d Transfer of lung ILC2s or gut ILC2s isolated from mice at postnatal day 12 to the B-NDG mice. Seven days after transfer, ILC2s from the lung and gut of B-NDG mice were analyzed by flow cytometry. e Treatment of HDM at early age affected the function of lung and gut ILC2s. Mice at postnatal day (P) 10 were i.n. treated with 10 µg HDM for 5 consecutive days, and lung or gut ILC2s were subsequently analyzed by flow cytometry (indicated as P15). Two weeks after first round of HDM treatment, mice were i.p. challenged with 400 ng/mouse/day IL-33 for two days and subjected to flow cytometry analysis (indicated as P30). f Postnatal mice were treated as (e), gut ILC2s from mice with IL-33 treatment at P30 were analyzed by bulk mRNA-sequencing. Blue dots, upregulated genes in HDM-treated (HDM) mice; red dots, upregulated genes in PBS-treated (Ctrl) mice. Representative DEGs are depicted. g Secretion cytokines of ILC2s from HDM-IL-33 treated mice or PBS treated mice (Ctrl) in (e) were analyzed by ELISA and show as mean ± SD. Data are representative of at least three independent experiments. **, P < 0.01 by Two-tailed unpaired Student’s t-test. (P = 0.0015, 0.0030, 0.0029, 0.0048 for IL-4 group; P = 0.0048, 0.0017, 0.0076, 0.0056 for IL-13 group). Source data are provided as a Source Data file.

Fig 5: CCR2 and CCR4 serve as tissue specific markers of ILC2s.a Violin plots showed ILC2 signature genes in the lung and the intestine. Ccr2 was highly expressed in lung ILC2s and Ccr4 is restricted to intestine ILC2s. Box plots indicate median (middle line), 25th, 75th percentile (box) of the data and the maximum and minimum values as endpoints for the whiskers. b Maturation trajectory of lung and intestine ILC2s were analyzed by Monocle pseudotime analysis. c, d Expression of CCR2 and CCR4 on ILC2s. CCR2-mNeonGreen reporter mice (c) and CCR4-mNeonGreen reporter mice (d) were generated as described in “Methods” and analyzed for the expression of CCR2 and CCR4 on ILC2s by flow cytometry. Lin-CD45+CD127+ lymphocytes were gated for analysis of KLRG1 and ST2. (Lin=CD3e,CD8a,CD19,CD11b,CD11c,Gr1,F4/80,Ter119). e, f CCR2-mNeonGreen and CCR4-mNeonGreen mice were intraperitoneally injected with 200 ng/mouse/day IL-25 for three constitutive days. Expression of CCR2-mNeonGreen (e) and CCR4-mNeonGreen (f) on ILC2s from respective mice were analyzed by flow cytometry. g, h CCR2-mNeonGreen mice (g) and CCR4-mNeonGreen mice (h) were analyzed for the expression of CCR2-mNeonGreen and CCR4-mNeonGreen on ILC2s after i.p. injection of 400 ng/mouse/day IL-33 for three constitutive days. (Gate strategies for ILC2s are: Lin-CD127+ST2+KLRG1+ for lung ILC2s, Lin-CD127+ST2-KLRG1+ for iILC2, Lin-CD127+KLRG1+ for gut ILC2s, Lin=CD3e,CD19,CD11b,CD11c,Gr1,F4/80,NK1.1). Data are representative at least three independent experiments. Source data are provided as a Source Data file.