Interleukin 2 (il 2)

Peripheral blood mononuclear cells in culture media were pre-treated with compounds (0.1% final DMSO concentration) for 45 minutes at 37°C, 5% CO₂ and then transferred into anti-CD3 (1 μg/mL, OKT3, eBioscience, San Diego, CA) coated plate containing soluble anti-CD28 (1 μg/mL, Fitzgerald, Acton, MA). Twenty four hours after stimulation, an aliquot of cell culture supernatant was collected and evaluated for IL-2 production (MesoScale Discovery, Rockville, MD) according to manufacturer’s instructions. The remaining culture was allowed to continue incubation for an additional 48 hours. To evaluate proliferation, Click-iT EdU Alexa Flour 647 Assay Kit (Invitrogen, Carlsbad, CA) was used according to manufacturer’s instructions, and proliferation was evaluated at 72 hours following stimulation. The cells were stained with flourochrome conjugated antibodies against CD3, CD4 and CD8 and analyzed in a BD Fortessa flow cytometer by acquiring 8–10,000 events. The data was analyzed by FACSDiva or FlowJo and reported as the percentage of EdU⁺CD3⁺CD4⁺ and EdU⁺CD3⁺CD8⁺ lymphocytes (Figure A in S2 Fig).

The BALF supernatant samples were analysed using MSD pro-inflammatory panel V-Plex including the following analytes: IFN-γ, IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-5, IL-6, KC/GRO and TNF-α (Meso Scale Diagnostics, Rockville, MD). The analysis was performed according to the manufacturer’s instruction.