Surface staining was performed for 30 min and with tetramer for 90 min at 4°C in PBS supplemented with 2% FCS and 0.01% azide (FACS buffer) using the following antibodies: anti-CD8α (53-6.7), CD127 (eBioSB/199), LAG-3 (C9B7W), and KLRG-1 (2F1; all eBioscience); anti–PD-1 (RMP1-30; BioLegend); gp276–286 tetramer (TCMetrix); and anti-CD4 (GK1.5), CD45.1 (A20), and CD45.2 (clone 104), obtained from or custom purified by Bio X Cell and coupled to Pacific blue, Alexa Fluor 647, or FITC using labeling reagents from Invitrogen. Cells were washed twice and fixed in PBS supplemented with 1% formaldehyde, 2% glucose, and 0.03% azide for 20 min. Then, cells were washed again and resuspended in FACS buffer. For intracellular cytokine staining, cells were fixed and permeabilized using the Cytofix/Cytoperm kit (BD) and stained with anti–IFN-γ (XMG1.2), TNF (MP6-XT22; both from eBioscience), or granzyme B (GB12; Invitrogen). T-bet and Eomes staining was performed with a transcription factor staining kit (eBioscience) and stained with Eomes (Dan11mag) and T-bet (eBio4B10; both from eBioscience).
For flow cytometry sorting, living cells were stained in 10% FCS RPMI media and sorted on a FACSAria instrument (BD).
For flow cytometry sorting, living cells were stained in 10% FCS RPMI media and sorted on a FACSAria instrument (BD).