ICAM-1 controls the development of ILC2. (A) Representative flow cytometric analysis of ICAM-1 expression on distinct ILC progenitor subsets in the BM and ILC2, as well as CD4⁺ T cells in lung from mice. (B) The mean fluorescence intensity (MFI) levels of ICAM-1 from A were shown (n = 4–7). (C) Representative flow cytometric analysis of iILC2, pregated on CD45⁺Lin⁻ in BM from WT and ICAM-1^−/− mice. (D) The frequencies and number of iILC2 from C (n = 12). (E) Quantification of iILC2 in BM from WT and ICAM-1^−/− mice after intraperitoneal injection with IL-33 or vehicle PBS (n = 6 per group). (F) The frequencies of CLP, α-LP, ChILP, and ILC2P among CD45⁺ cells in BM from WT and ICAM-1^−/− mice (n = 6 per phenotype). (G and H) CLPs from WT (CD45.1/2⁺) and ICAM-1^−/− (CD45.2⁺) mice were mixed at 1:1 ratio, followed by intravenously transferred into NCG (CD45.1⁺) mice (n = 6). 3 wk after transplantation, the levels of BM iILC2 (CD45⁺Lin⁻C-kit^lowSca1^highCD127⁺CD25⁺), lung ILC2 (CD45⁺Lin⁻CD90.2⁺CD25⁺) and BM B cells (CD19⁺B220⁺) from WT (CD45.1/2⁺) and ICAM-1^−/− (CD45.1⁻CD45.2⁺) was evaluated by flow cytometry (G). The ratio of ICAM-1^−/− to WT donor cells (ICAM-1^−/−/WT) among the indicated cell types, including BM iILC2, lung ILC2, B cells, spleen NK (CD45⁺Lin⁻NKP46⁺NK1.1⁺) cells, thymic CD4⁻CD8⁻ (double negative, DN) cells, CD4⁺CD8⁺ (double positive, DP) cells, CD4⁺CD8⁻ (single positive, CD4 SP), and CD4⁻CD8⁺ (single positive, CD8 SP) cells were shown (H). Dashed line indicated the expected result in the absence of genotype bias. (I) CLPs from ICAM-1^−/− or WT mice were co-cultured with OP9-DL1 cell line in the presence of IL-7 (20 ng/ml) and IL-33 (20 ng/ml) for 10 d, and the frequency of ILC2 (Lin⁻CD45⁺ICOS⁺CD25^low) was evaluated by flow cytometry and the number of ILC2 after co-culture was shown. (J) WT CLP cells were co-cultured with OP9-DL1 as in (I), in the presence of indicated blocking antibodies (5 µg/ml). The representative frequencies of ILC2s, pregated on CD45⁺Lin⁻cells and the number of ILC2 were shown. Data are representative of two (A, B, and E–H), three (I and J), or four (C and D) independent experiments. Error bars show mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by unpaired Student's t test. ns, not significant. Numbers within flow plots indicate percentage of cells gated, and arrows from outlined areas at top indicate gated cells analyzed below.