Cd244 pe d594

PBMCs were stimulated with anti-CD3/CD28 (5 µg/mL, Ebioscience) for 5 h in the presence of anti-CD107a BV421 (BioLegend) and Golgiplug (BD Biosciences). CD107a expression was measured as a marker of degranulation on CD8⁺ T cells after stimulation. The cells were surface-stained with CD3-BV786, CD4-APC-Fire750, CD8-BV421, CD244-PE-D594, CD160-AF488, and intracellularly stained with TNF-α-BV711, IL-2-BV650 (BioLegend), or IFN-γ-AF700 (Ebioscience) antibodies. For Ki67, perforin, Granzyme B, T-bet, or Eomes staining, PBMCs were surface-stained with CD3-BV786, CD4-APC-Fire750, CD8-BV421, CD244-PE-D594, CD160-AF488, and intracellularly stained with Granzyme B-AF700, T-bet-BV421 (BD Biosciences), Ki67-BV711, perforin-APC (BioLegend), or Eomes-PE-CY7 (Ebioscience) antibodies. A fixable viability dye eFluor^® 506 (Ebioscience) was used to label dead cells.

PBMCs from healthy subjects were resuspended in PBS buffer and were incubated with directly conjugated antibodies for 30 min at 4°C. The cells were washed with 1× PBS before flow cytometry analysis. Antibodies used included anti-human CD3-BV786 or CD3-BUV737, CD8-BV510 or CD8-BUV395, CD160-AF488, CD45RA-AF700, CD28-APC, CD95-PE, CD57-BV421, PD-1-BV711, TIM-3-BV650 (BD Biosciences, San Diego, CA, USA), CD4-APC-Fire750, CD244-PE-D594, CCR7-BV421, HLA-DR-AF700, CD38-BV421, CD28-BV711, CD27-BV650, KLRG-1-APC-Fire750, CD95-PE-CY7 (BioLegend, San Diego, CA, USA), TIGIT-PE-Cy7, LAG-3-APC (Ebioscience, San Diego, CA, USA) and the corresponding isotype controls. Data were acquired with the LSR Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.5 (Tree Star, Ashland, OR, USA). More information about antibodies is listed in the Supplementary Material (Table S2).