αcd16 32

Peripheral blood mononuclear cells (PBMCs) were collected in 20 mM EDTA in Eppendorf tubes. PBMCs were spun for 8 minutes at 10,000 rpm at 20°C and serum was removed. RBCs were lysed with ACK Lysis buffer (GIBCO) for 8 minutes at room temperature (rt). If incomplete lysis occurred, cells were spun down for a second time at 12,000 rpm for 30 s and the ACK lysis step was repeated. Mononuclear cells were stained 1:100 with CB_101–109/H-2D^b-PE tetramer in the presence of Fc block 1:100 (αCD16/32, Tonbo), and monoclonal antibodies at 1:100 specific to CD45 (30-F11, Biolegend), CD8α (53–6.7, ebioscience), LFA-1 (clone H155–78, Biolegend), CD44 (clone IM7, BD), CD62L (MEL-14, Biolegend), PD-1 (J43, Invitrogen), KLRG1 (2F1, Biolegend), Tim-3 (RMT3–23, Biolegend), Lag-3 (C9B7W, Biolegend). CD19 (1D3, BD), F4/80 (T45–2342, BD), and CD11c (N418, BD) were used for a dump channel along with ghost dye BV510 at 1:500 (Tonbo). Antibodies were diluted in FACs buffer (PBS + 2.5% FBS) and cells were stained at room temperature in the dark for 60 minutes and then added to 100 μl of cell counting beads (Sigma Aldrich). Stained cells were fixed with 0.4% paraformaldehyde and stored overnight in the dark at 4°C prior to FACs acquisition using a Fortessa 1770 flow cytometer and Facs Diva software (BD Biosciences) and analyzed using FlowJo software (version 10).