Isolation of Cells, In Vitro Treg Suppression Assay, and Adoptive Transfer

In adoptive transfer studies, syngeneic Tregs were isolated using the CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec), as previously described,^16,55 from mice that were treated either with saline or IL233. Different numbers of cells (50×10³ or 100×10³) were intravenously injected into recipient mice 24 hours before surgery. Treg suppression activity was performed using the CFSE dilution assay as previously described,³¹ with the modification that CD4⁺CD25⁻ responder T cells were isolated from the spleens of B6.Thy1.1 congenic mice for unambiguous analysis by gating on Thy1.1⁺ cells. ILC2 were identified in flow cytometry as lineage negative Thy1.2⁺ST2⁺ cells in the lymphocyte or CD45⁺ gate, using fluorescence-labeled antibodies (Biolegend Inc.). ILC2 were isolated from the spleen of untreated mice by negative selection using biotinylated α-CD4, α-CD8, α-CD11c, α-CD11b, α-NK1.1, α-B220, α-Ter119, α-Gr-1 (BD Biosciences), and anti-biotin magnetic beads (Miltenyi Biotec). Cells were expanded for 2 weeks in complete media (RPMI 1640 +L-glutamine, 10% FBS, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, 10 mM HEPES, 25 mM glucose, 0.05 mM β-mercaptoethanol, 10 ml/L penicillin/streptomycin) as described.⁵⁶ The cultures were split at the ratio of 1:3 every third day, with the difference that instead of IL-2 and IL-33, 50 ng/ml of IL233 was used to induce the growth of ILC2. ILC2 were adoptively transferred (5×10⁵ cells per mouse, administered intravenously) into syngeneic recipient mice 24 hours before IRI. The control mice were injected with saline only. The purity of ILC2 and Tregs was >95% as measured by flow cytometry, using a five-color upgraded (Cytek Development) FACScan (BD Biosciences) equipped with CellQuest, and Rainbow software for data acquisition.