Lymphocytes were identified by forward and side scatter, and cell identity was assessed using fluorochrome-conjugated antibodies anti-CD3 (clone SK7; eBioscience), anti-CD4+ (RPA-T4; BD), anti-CD8 (RPA-T8; BD), anti-CD161 (DX12; BD), and anti-TCR Vα7.2 (3C10, BioLegend). Viable cells were gated using Live/Dead Yellow or Live/Dead Aqua viability dyes (Invitrogen) according to the manufacturer’s instructions. Cells were stained for 20 minutes in the dark at room temperature, washed, and fixed in PBS containing 2% formaldehyde. All samples were acquired on LSRII or LSRFortessa flow cytometers (BD). Cell division was assessed by labeling PBMCs with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes) for 10 minutes at 37°C. Staining was quenched by the addition of FBS for 5 minutes on ice. Cells were then washed and cultured as described.
For detection of intracellular cytokines, cells were stimulated with 50ng/mL soluble anti-CD3 (HIT3a; BD) and 3μg/mL soluble anti-CD28 (CD28.2; BD) for 24 hours at 37°C and 5% CO2, in the presence of brefeldin A (GolgiPlug; BD) for the final 6 hours of stimulation. After stimulation, cells were washed, stained with viability dye and antibodies to surface antigens, then fixed and permeabilized using the Cytofix/Cytoperm kit (BD) and stained intracellularly with fluorochrome-conjugated antibody to IFNγ (B27, BD).
For detection of intracellular cytokines, cells were stimulated with 50ng/mL soluble anti-CD3 (HIT3a; BD) and 3μg/mL soluble anti-CD28 (CD28.2; BD) for 24 hours at 37°C and 5% CO2, in the presence of brefeldin A (GolgiPlug; BD) for the final 6 hours of stimulation. After stimulation, cells were washed, stained with viability dye and antibodies to surface antigens, then fixed and permeabilized using the Cytofix/Cytoperm kit (BD) and stained intracellularly with fluorochrome-conjugated antibody to IFNγ (B27, BD).