Nib42

PBMCs (1 × 10⁵ cells per well) were either left non-stimulated or were stimulated with anti-CD3/CD28 mAb-conjugated beads (11131D; Gibco; 1 × 10⁵ beads per well) for 24 h, together with anti-IFN-γ neutralizing mAb (NIB42, 10 μg/ml; eBioscience) or isotype control (MOPC-21, 10 μg/ml) in a 96-well U-bottom plate. The non-adherent cells were transferred to a 96-well V-bottom plate by pipetting. The residual adherent cells were detached by incubation with the TryPLE enzyme (Gibco) at 37°C for 5 min and were then transferred to the same 96-well V-bottom plate. Cells were washed once with FACS buffer, surface-stained by incubation at 4°C for 1 h with various antibodies (anti-CD3-FITC [UCHT1, 1:100; Cytek], anti-CD19-BV650 [HIB19, 1:100; BD], anti-CD56-V450 [B159, 1:50; BD], anti-CD14-Spark NIR 685 [63D3, 1:100; BioLegend], anti-CD16-PE/Dazzle 594 [3G8, 1:100; BioLegend], anti-HLA-DR-APC/Fire 810 [L243, 1:100; BioLegend], anti-CD123-BV480 [9F5, 1:100; BD], anti-CD11c-Alexa Fluor 700 [Bu15, 1:1,000; BioLegend], anti-PD-1-BB700 [EH12.1, 1:100, BD], and anti-PD-L1-BV711 [29E.2A3, 1:100; BioLegend] or an isotype control [MPC-11; BioLegend]), washed, stained with 7-AAD (1:200), and acquired with an Aurora cytometer (Cytek). Data were analyzed with FlowJo software. The median fluorescence intensity (MFI) of PD-L1 was used as a readout.

Human 2+γ+, 2+γ-, 2-γ+ and 2-γ- cells were washed with R8 medium and plated into 96 well plates. Each well contained <500 sorted cells in 200 µl R8 supplemented with 1 ng/ml human IL-2 (eBioscience), 10 µg/ml anti-IL4 antibody (MP4-25D2, eBioscience), 10 µg/ml anti-IL-12 antibody (C8.6, eBioscience) and 10 µg/ml anti-IFNγ antibody (NIB42, eBioscience). 36 or 108 hours later, the cells were restimulated with PMA (25 ng/mL) and Ionomycin (1 µg/mL) (P/I) and stained by the ICS assay. Polyclonal P/I stimulation was used because antigen or SEB specificity was established by sorting, and P/I was a more efficient stimulus.