H ifnγ

Monocytes isolated from buffy coat were cultured in complete RPMI 1640. To induce M1 polarized activation, cells were stimulated with h-IFNγ (20 ng/mL, Miltenyi) and LPS (100 ng/mL, Enzo Life Science). After 24 h, the medium was collected, centrifuged at 1400 rpm, RT, 10 min, filtered, and stored at −20 °C in aliquots until use. In the same manner, aliquots collected from untreated monocytes (M0) and control RPMI 1640 medium containing the polarizing stimuli and left in the incubator for 24 h were stored.
MSTO-211H and BR-95 cells were plated in 100 mm dishes in complete RPMI 1640, until 60% of confluence, then culture medium was replaced with fresh medium containing 50% of control, M0, or M1 monocytes-conditioned medium. Cells were monitored for the following 48–72 h by optical microscope. To evaluate cell viability, cells were harvested, and proteins and total RNA were extracted and analyzed by Western Blot and RT-PCR, respectively.

Isolated human naive T cells were stimulated for 6 days in 96 well, flat bottomed plates (Corning) using 1 µg/mL of plate-bound anti-CD3 (BioLegend Cat# 317302, RRID:AB_571927) and 2 µg/mL anti-CD28 (BioLegend Cat# 302902, RRID:AB_314304). Cells were seeded at 100,000 naive T cells per well. Th1 cells were generated by addition of 10 ng/mL hIFNγ, 10 ng/mL IL-12 and 100 U/mL hIL-2 (all from Miltenyi). Th17 cells were generated using 10 ng/mL hIL-1β, 10 ng/mL hIL-6, 10 ng/mL hIL-23, 100 U/mL hIL-2, 10 ng/mL TGF-β (all from Miltenyi) with 10 µg/mL anti-IFNγ (BioLegend Cat# 502402, RRID:AB_315223). Cells were incubated at 37 °C, 5% CO₂. Cells were cultured in complete IMDM (ThermoFisher, with 10% FCS, Penicillin, streptomycin and glutamine).