ILC2s are a major in vivo source of CCL1. (A) Flow cytometric analysis of gut lamina propria (LP) cells. Lymphocytes were gated on GATA3⁺ cells and analyzed for CD4 and CCL1 expression. (B) ILC2s and CD4⁺ T cells were sorted from intestinal lamina propria cells of naive Rora^creRosa26-tdtomato^fl/fl mice. Cells were cultivated in the presence of PMA/ionomycin, and supernatants were collected after 48 h for CCL1-specific ELISA analysis. (C) Small intestinal tissue sections of C57BL/6 WT mice overexpressing IL-33 were stained with DAPI (blue), anti-CCL1 (green), and anti-KLRG1 (red, top) or anti-CD4 (red, bottom) antibodies and analyzed by confocal microscopy. Scale bars are 30 µm (left) or 10 µm (right). Pictures show results of one representative animal. (D) WT and Tie2^creRora^fl/sg mice were challenged with DNA vectors for systemic release of IL-25 or empty vectors (control). Livers were collected after 5 d, and the presence of Ccl1 transcripts was investigated by qPCR. (E) In vitro–expanded human ILC2s were restimulated with IL-2, IL-25, and IL-33, and supernatants were collected after 3 d for CCL1 measurements. CCL1 levels were compared with supernatants of feeder cells only. Each replicate represents one independent sorting experiment. (F) Sorted human ILC2s were expanded with or without neutralizing anti-CCL1 antibodies and cell number determined to investigate fold expansion. Each dot represents one independent sorting experiment. Paired t test was applied. All graphs show data of one representative experiment out of at least two independent experiments. Experimental groups consisted of at least three mice or donors. Data represent mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 by Mann–Whitney U tests or, if indicated, a paired t test.