Murine il 36γ

Naive CD4⁺ T cells (CD4⁺CD25⁻) were purified from spleens by magnetic selection (Miltenyi Biotec) and subsequently sorted by fluorescence activated cell sorting (FACS) and cultured for 4 days in the presence of FACS-sorted CD45⁺MHCII⁺CD11c⁺ DCs at a 10:1 T/DC cell ratio (DCs were not irradiated unless specified). All cultures contained purified anti-CD3ε (2μg/ml; 145-2C11; eBioscience). For iT_reg cell induction, the cultures contained human TGFβ (5ng/ml) (Peprotech) and human IL-2 (20ng/ml) (Peprotech). The following cytokines were used in indicated experiments (100ng/ml): murine IL-1β (Peprotech), murine IL-18 (R&D), murine IL-33 (Peprotech), murine IL-36α, murine IL-36β and murine IL-36γ (R&D). Neutralizing Abs specific for IL-1β (B122), IL-2 (JES6-1A12), IL-4 (11B11), IL-5 (TRFK5), IL-6 (MP5-20F3), IL-9 (D9302C12), IL-12/23p40 (C17.8), IL-13 (ebio1316H), IL-21 (FFA21), IL-22 (IL22JOP), IL-25 (35B), TGFβ-1,2,3 (1D11) and IFNγ (XMG1.2) purchased from eBioscience, Biolegend and R&D were used (10μg/ml). In indicated experiments the STAT5 inhibitor (CAS 285986-31-4; EMD Millipore) and STAT6 inhibitor (AS 1517499; Axon Medchem) was used.

Antibodies were from eBioscience except for CD45, CD103, CD4 (Becton Dickinson). Dead cells were identified using the Aqua dead cell staining kit (Invitrogen). Murine IL-36γ was from R&D. ELISAs for IL-36γ (antibodies-online.com) and IL-22, CXCL1, and CXCL2 (eBioscience) were performed following the manufacturer’s instructions.