Anti cd3ε clone 145 2c11

OT-I Rgs1^+/+ T cells were FACS sorted, and 1x10⁵ cells/well were distributed in a 96-U-bottom-well plate in 200μl cell culture media (RMPI 1640 + 2mM L-Ala/L-Glu, 1mM Sodium Pyruvate, 10mM HEPES, 1x MEM non-essential amino acids, 0.5mM 2-β-Mercaptothion, 10% FCS, 40 U/ml Penicillin, 40μg/ml Streptomycin (all Sigma-Aldrich, St. Louis, USA)) and stimulated with distinct cytokines and TCR-crosslinking mAbs. For TCR stimulation, plates were previously coated with anti-CD3ε and anti-CD28 mAb´s (anti-CD3ε: clone 145-2C11, anti-CD28: clone 37.51, both Biolegend, San Diego, USA) for 12h. The cells were treated with recombinant murine IL-2 (50 ng/ml, R&D Systems, Minneapolis, USA), IL-15 (50 ng/ml, Peprotech, London, United Kingdom), IL-33 (100 ng/ml, Peprotech, London, United Kingdom) and/or recombinant human TGFβ1 (50 ng/ml Peprotech, London, United Kingdom). The cells were incubated for 72h, at 37°C in 5% CO₂.

For the experiments described in the manuscript, soluble stimuli were used at the following concentrations: lipopolysaccharide (LPS, InvivoGen) 500 ng/ml, CpG (InvivoGen) 100 nM, IL-1ß (ThermoFisher) 200 ng/ml, anti-CD40 (clone 1C10, Biolegend) 1 µg/ml, PMA (Sigma) 10⁻⁷M, ionomycin (Sigma) 1 μg/ml. For plate coating, anti-CD3ε (clone 145-2C11, Biolegend) and anti-CD28 (clone 37.51, Biolegend) were used at a concentration of 2 µg/ml, at and anti-IgM F(ab’)₂ (Jackson Immunoresearch Laboratories) at 10 µg/ml. For the stimulation of in vitro cultured alveolar macrophages, cells were collected using 4 mM EDTA in PBS and counted. 1 × 10⁵ cells per well were plated in an untreated 96-well plate overnight and stimulated on the consecutive day with 400 ng/mL LPS and/or 100 µM luteolin (Sigma-Aldrich). At the end of the stimulation supernatant was carefully removed and frozen for ELISA analysis. Cells were detached using 4 mM EDTA in PBS, washed once and stained with 1:4000 SytoxRed in PBS 2% FCS before flow cytometric analysis.