Soluble ST2 suppressed IL-33-mediated morphological change and proliferation of lung ILC2. (A) Sorting strategy of naïve lung ILC2. After exclusion of doublets in live lung lymphocytes, ILC2 were isolated as a CD45.2+Lineage-CD127+CD25+c-Kit+Sca-1+ population by cell sorting. (B) Expression of ST2L on isolated ILC2 was analyzed with antibodies against T1/ST2 (anti-T1/ST2) and isotype control (Isotype) by flow cytometry. The numbers indicate the percentage of gated cells in the respective gating step. (C) Naïve lung ILC2 were pretreated with ST2-V5 or In-ST2-V5 (103.6 ng/ml, corresponding to 2.80 nM) for 1 h and then stimulated with IL-33 (10 ng/ml, corresponding to 0.56 nM) in medium containing IL-2 (20 ng/ml) for 96 h. PBS was added as a control for ST2-V5, In-ST2-V5, and IL-33. Cells were mounted on glass slides and stained with Diff-Quik. Scale bars, 10 μm. (D and E) CFSE-labeled lung ILC2 were pretreated and stimulated as in Panel C. CFSE dilution was analyzed by flow cytometry. (D) Profile of FSC/SSC in CFSE-labeled lung ILC2. (E) Cell division of CFSE-labeled lung ILC2. The solid-lined and black-filled histograms represent cells cultured for 0 and 96 h, respectively. Right- and left-side numbers indicate the percentage of single- and more-divided population, respectively. Results are representative of three independent experiments.