In vitro activated Tregs

CD4⁺CD25⁺ cells were purified by magnetic bead separation using negative selection for CD4⁺ and subsequent positive selection for CD25⁺ by incubating CD4⁺ enriched cells with PE-conjugated α-CD25 (PC61) followed by α-PE microbeads (CD4⁺CD25⁺ Regulatory T-cell Isolation Kit; Miltenyi Biotec, Bergisch Gladbach, Germany). CD4⁺ CD25⁺ separated Tregs were cultured for five days in 12-well plates in RPMI 1640 media (Biochrome, Berlin, Germany) supplemented with 200U/ml IL-2 (Sigma), 10% FCS (Linaris, Dossenheim, Germany), PenStrep (100U Penicillin, 100μg Streptomycin/ml; Sigma), 10mM Hepes (MP Biomedicals), 1mM Sodium Pyruvat (Sigma), 1x non-essential amino acids (Sigma) and 10μM β-Mercaptoethanol (Sigma). The plates were pre-coated with 10μg/ml α-CD3 (145-2C11) (BioXcell) and 1μg/ml α-CD28 (37.51) (BioLegend) in PBS overnight at 4°C.