Anti ifn γ mab xmg1

Naive CD4⁺CD44⁻CD62L⁺ T cells were resuspended at 1 × 10⁶ cells/mL in RPMI 1640 supplemented with 10% HI-FCS, 50 μM β-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomyocin and stimulated with plate-bound 1 μg/mL anti-CD3ε mAb and 1 μg/mL anti-CD28 (37.51, BioLegend) at 200 μL/well in a 96-well plate. Cells were polarized as indicated using the following culture conditions: Th0, 5 μg/mL anti-IL-4 mAb (11B11, BioLegend) and 5 μg/mL anti-IFN-γ mAb (XMG1.2, BioLegend); Th1, 10 ng/mL murine IL-12 (PeproTech) and 5 μg/mL anti-IL-4 mAb; Th2, 10 ng/mL murine IL-4 (PeproTech) and 5 μg/mL anti-IFN-γ mAb; Th17, 5 μg/mL anti-IL-4 mAb, 5 μg/mL anti-IFN-γ mAb, 10 ng/mL human IL-23 (PeproTech), 1 ng/mL human TGF-β (PeproTech), and 5 ng/mL murine IL-6 (PeproTech); T_reg, 1 ng/mL human TGF-β, 5 μg/mL anti-IL-4 mAb, and 10 μg/mL anti-IFN-γ mAb. After 72 hr cells were analyzed by intracellular flow cytometry for CD4⁺ Th subset polarization.

Blood leukocytes were seeded in 96-well U-bottomed plates in complete RPMI medium in the presence of 0.7 µg/mL Brefeldin A (Biolegend), 1 µg/mL Monensin (Biolegend) and anti-CD107a mAb (LAMP-1) and stimulated with 10 μg/mL OVA_257–264 peptide or with 2 µg/mL Ionomycin (Sigma) plus 0.2 µg/mL phorbol-12-myristate-13-acetate (PMA, Sigma) for 5 h at 37 °C. At the end of incubation, cells were surface stained with fluorescent anti-CD3, anti-CD8, anti-CD44 (IM7), anti-CD62L (MEL-14), anti-PD-1, and Tim-3 (RMT3-23) mAbs (all from Biolegend). Cells were then permeabilized (Fixation/Permeabilization Concentrate, ThermoFisher Scientific) and stained with anti-IFNγ mAb (XMG1.2) or isotype control (Biolegend). In some experiments, to identify Tregs, spleen or tumor cell suspensions were surface stained with anti-CD4, anti-CD25 (PC61) and anti-GITR (DTA-1) mAbs, then permeabilized and stained with anti-Foxp3 mAb (FJK-16s) or matched isotype control (ThermoFisher Scientific, Waltham, MA, USA). Cells were then fixed with 1% paraformaldehyde and stored at 4 °C in the dark until acquisition by Gallios flow cytometer.