Analysis of CCR8 expression on ILC2s. (A–F) Flow cytometric analyses of CCR8 surface expression using fluorophore-coupled recombinant CCL1 (CCL1-AF647) or a human anti-CCR8 antibody (F). (A) ILC2s (Lin⁻Thy1⁺KLRG1⁺ICOS⁺ST2⁺) or Th2 (CD3⁺CD4⁺ST2⁺) cells in lung or the ileal/colonic lamina propria of naive WT and Ccr8^−/− mice were analyzed by flow cytometry. (B) WT mice were infected with N. brasiliensis, and lung lineage⁺ lymphocytes (lin⁺) and ILC2s (Lin⁻Thy1⁺KLRG1⁺ST2⁺) were analyzed after 9 d. A Mann–Whitney U test was applied. (C) Lung cells of WT and Ccr8^−/− mice challenged for 3 d with DNA vectors encoding Il25 or Il33 or empty vectors (control) were analyzed for CCR8 on ILC2s (Lin⁻Thy1⁺KLRG1⁺ICOS⁺), eosinophils (CD11b⁺SiglecF⁺CD11c⁻SSC^hi), and Th2 cells (Lin⁺Thy1⁺ST2⁺). (D) To investigate CCR8 expression on ILC2 subsets, WT mice were treated with Il25 (to induce iILC2s) or Il33 (to induce nILC2s) vectors for 5 d, and iILC2s (Lin⁻CD127⁺KLRG1^hiST2⁻) or nILC2s (Lin⁻CD127⁺KLRG1^intST2⁺) from lungs and blood were analyzed and compared by flow cytometry. (E) Murine sorted and in vitro–expanded ILC2s were analyzed and compared with a fluorescence minus one (FMO) control. (F) ILC2s (Lin⁻CD127⁺CD161⁺CRTH2⁺) and lineage⁺ (Lin⁺) lymphocytes in freshly isolated human PBMCs were analyzed. An unpaired t test was applied. All results are representative of two or more independent experiments. A and C–E show representative histograms of one animal of two to four animals in total per experimental group (A, C, and D) or four independent sorting experiments (E). Bar graphs represent five mice (B) or three donors (F). Data represent mean ± SEM. **, P ≤ 0.01; ***, P ≤ 0.001 by Mann–Whitney U tests or unpaired t test.