Murine il 2 402 ml

T cells were seeded in primary T cell medium, i.e. RPMI1640 supplemented with 10% FCS (PAA Laboratories), 50 µM β-mercaptoethanol, 50 µg/ml each of penicillin and streptomycin, 1% non-essential amino acids and 1 mM sodium pyruvate (all from Life technologies). Bafilomycin A1 was obtained from Enzo Life Science. For Treg cell stimulation, 2 µg/mL anti-CD3 (145-2C11, BioLegend), 4 µg/mL anti-CD28 (37.51, Biolegend) and 10 ng/ml murine IL-2 (402-ML, R&D) were used. For in vitro Treg cell induction, 2 x 10⁵ FACS-sorted naive T cells (CD4⁺, CD62L⁺ and CD25^-) were cultured in 200 µl primary T cell medium for 5 days in the presence of 1 µg/mL plate-bound anti-CD3 (145-2C11, BioLegend), 2 µg/mL anti-CD28 (37.51, Biolegend), 10µg/ml anti-IL-4 (11B11, self-purified), 10µg/ml anti-IFNγ (XMG1.2, self-purified), 10 ng/ml murine IL-2 (402-ML, R&D), and 5 ng/mL TGF-β (R&D Systems) in a flat-bottom 96-well plate.