Apc conjugate

CD4 and CD8 T cells from the peripheral blood lymphocytes (PBLs), draining lymph nodes, and TILs were stained for PD-1 (APC conjugate; BD Biosciences) and lymphocyte-activation gene 3 (LAG-3; FITC; Lifespan Biosciences, Seattle, WA) expression. The cells were also co-stained with CD4 or CD8 (PE or PE-Texas Red, PerCP-Cy5.5, respectively, BD Biosciences) for these analyses. For sorting, peripheral blood mononuclear cells were stained with CD3 (AF-700; BD Biosciences) and PD-1 (APC; BD Biosciences), and CD3+ cells were sorted based on PD-1 expression using the FACSAria (BD Biosciences). Sorted cells were stimulated in a 96-well plate for 3 hours at 37°C with PMA (20 ng/mL) and ionomycin (1 μg/mL) in the presence of Golgi-stop (BD Biosciences). Cells were then stained for CD4 (PE-Texas Red; BD Biosciences), CD8 (PerCP-Cy5.5; BD Biosciences), and intracellular IFN-γ (FITC; BD Biosciences) using the BD Cytofix/Cytoperm kit, in accord with the manufacturer’s recommended protocol. Flow cytometry was conducted using a FACSCalibur (BD Biosciences) or LSR II (BD Biosciences) and data were analyzed using FlowJo software (Tree Star, San Carlos, CA).