PBMCs were incubated at 37°C in a 5% CO2 incubator, and BFA (1 μl/ml) was added to the cells 5 hours before staining. Antibodies were prepared, cells transferred to tubes then spun down at 350 g for 5 minutes at 4°C, and washed once in 500 μl FACS buffer. Samples were resuspended in 100 μl of diluted antibody against human cell-surface markers APC/H7-conjugated antihuman CD3 (Clone SK7, BD Pharmingen™) (1 : 200), BB515-conjugated antihuman CD4 (Clone PRA-T4, BD Horizon™) (1 : 200), and APC-conjugated antihuman CD279 (clone MIH4, BD Pharmingen™) (1 : 100); incubated for 30 minutes at 4°C; and washed twice in 500 μl FACS buffer. Supernatants were removed, and cells were resuspended in 100 μl of Cytofix/Cytoperm (BD Pharmingen™), then incubated for 40 minutes at 4°C. After washing, cells were suspended in diluted intracellular antibodies PerCP-Cy™5.5-conjugated antihuman IFN-γ (Clone B27, BD Pharmingen™) (1 : 100) and PE/Cy7-conjugated antihuman TNF-α (Clone MAb11, BioLegend) (1 : 100), incubated for 40 minutes at 4°C, then washed with 500 μl perm/wash buffer. After cell staining, cells were resuspended in 100 μl of 1% paraformaldehyde.

Data were collected using a BD Canto II flow cytometer (BD Biosciences), and at least 10,000 events were collected. The results were analyzed using FlowJo version 10.0.7 software (Tree Star, Ashland, OR, USA).

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