In the present study, we investigated the phenotype and plasticity of hepatic ILC2 in response to liver inflammation-induced cytokines and effector CD4 + T cells in order to identify mechanisms of ILC2 regulation in liver disease. Material and Methods Mice. C57BL/6 mice and OT-II mice were bred in the University Medical Center Hamburg-Eppendorf animal facility (Hamburg, Germany). Mouse experiments were conducted according to the German animal protection law and approved by the institutional review board (Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany; G44/15). Mice received humane care according to the national guidelines of the National Institutes of Health in Germany. Animal treatment. C57BL/6 mice received intraperitoneally recombinant murine (rm)IL-33 (0.3 μg; BioLegend, San Diego, CA) daily on four consecutive days. ConA (7 mg/kg; Sigma-Aldrich, St. Louis, MO) was injected intravenously into C57BL/6 mice that were analyzed 8 and 24 hours later. Heart blood was drawn from individual mice and liver injury was quantified by automated measurement of plasma activities of alanine transaminase (ALT) using a COBAS Mira System (Roche Diagnostic, Mannheim, Germany). Immunohistochemistry. Liver samples were fixed with 4% formalin and embedded in paraffin. 3 μm liver sections were cut, stained with H&E following standard protocols, and analyzed by light microscopy. Isolation of hepatic ILC2 and CD4 + T cells. Hepatic non-parenchymal cells were isolated from livers of naive and IL-33-treated mice by Percoll density gradient centrifugation. Single cell suspensions were stained with the Lineage Antibody Cocktail (APC; BD Pharmingen, Heidelberg, Germany) as well as anti-Sca-1 (Pacific Blue; D7; BioLegend) and anti-ST2 (PerCP-eFluor 710; RMST2-2; ThermoFisher Scientific, Waltham, MA) antibodies. Lineage-negative (lin −) cells were enriched by magnetic cell sorting and lin − Sca-1 + ST2 + ILC2 were purely isolated by FACS (BD FACSAriaTM III sorter, BD Biosciences). Ovalbumin (OVA)-specific CD4 + T cells were isolated from spleen and lymph nodes of OT-II mice. Therefore, CD4 + T cells were enriched from single cell suspensions using the CD4 + T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Subsequently, cells were stained with anti-TCRβ (PE-Cy7; H57-597) and anti-CD4 (BV421; GK1.5; both BioLegend) antibodies and TCRβ + CD4 + T cells were purified by FACS.

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