ILC2s are resident in human skin. (A) Freshly isolated leukocytes from the human skin were stained with the combination of ILC surface markers, including lineage markers (CD3, CD4, CD8, CD14, CD19, CD56, CD11c, CD11b, FcεRI, TCR-γδ, TCR-αβ, and CD123), CD45, and IL-7Rα. Flow cytometric analysis of IL-7Rα and CRTH2 expression on lineage-negative CD45^hi and lineage-negative CD45^low cells (n = 15) is shown. (B) Expression of the indicated cell surface markers on Lin⁻CD45^hiIL-7Rα⁺CRTH2⁺ cultured ILC2 from the skin (n = 3). Black plots show the relevant marker; grey show the isotype control. Bars show the percentage of cells expressing the relevant marker. (C and D) CD45^hiIL-7Rα⁺CRTH2⁺ cells were sorted and cultured from blood and skin, and RORA (n = 8, where n is number of donors), RORC (n = 5), GATA3 (n = 3; C), and AREG (n = 3; D) gene expression relative to GAPDH was measured by RT-PCR. (E) Lin⁻CD45^hiIL-7Rα⁺CRTH2⁺ ILC2s were isolated from human skin and expression of the indicated homing receptors was analyzed by flow cytometry. Black plots show the relevant marker; grey show the isotype control. Bars show the percentage of cells expressing the relevant marker. *, P < 0.05; **, P < 0.01; ***, P < 0.001, unpaired Student’s t test (mean and SEM in C and D).