Multiparameter Flow Cytometry Analysis

Cells were labeled with fluorochrome-conjugated monoclonal Abs to the following antigens: anti–human CD3–PE (OKT3, Tonbo, Cytek Biosciences), CD4–PE–Cyanine7 (SK3, Tonbo, Cytek Biosciences), CD8–APC (Hit8a, Tonbo, Cytek Biosciences), CD34–Alexa Fluor 647 (581, BioLegend), CD69–PerCP–Cyanine5.5 (FN50, BioLegend), CD137–PE (4B4-1, Miltenyi Biotec), PD-1–BV650 (EH12.2H7, BioLegend), HLA-II–APC (Tü39, BioLegend), and anti–mouse TCRβ-APC (H57-597, Tonbo, Cytek Biosciences). Dead cells were stained with either DAPI or LIVE/DEAD Fixable Aqua (Invitrogen, Thermo Fisher Scientific). E6_29-38–HLA-A*02:01–PE tetramer was assembled and labeled by the NIH Tetramer Core Facility. Cells were stained with 1 μg/mL tetramer for 1 hour on ice. Ab staining for surface antigens was performed for 15 minutes on ice. For intracellular cytokine staining (ICS), T cells were cocultured with peptide-pulsed APCs for 4–6 hours in the presence of Brefeldin A. Cells were permeabilized and stained for intracellular cytokines with the Cytofix/Cytoperm Kit (BD Biosciences) after surface staining, according to manufacturer’s instructions. The following fluorochrome-conjugated Abs were used for cytokine staining: IFN-γ–APC (4S.B3, BioLegend), IL-2–FITC (MQ1-17H12, BioLegend), TNF-α–BV785 (Mab11, BioLegend), and Granzyme B–PE (QA16A02, BioLegend). Data were acquired with a FACSCelesta flow cytometer (BD Biosciences) and analyzed with FlowJo v10.7 software.