2b4 percp cy5

For surface staining, cells were incubated at room temperature with human Fc block (BD Biosciences, San Diego, CA, USA) and followed by staining with directly conjugated mAbs for 30 min at 4 °C. The mAbs used were anti-human CD3-BV605, CD4-V500, CD8-APC-H7, CD45RA-BV421, CCR7-PerCp-Cy5.5, PD-1-PE-Cy7, CD160-Alexa Fluor 488 (BD Biosciences), CD4-FITC, TIM-3-BV421, 2B4-PerCp-Cy5.5 (BioLegend, San Diego, CA, USA), and TIGIT-APC (eBioscience, San Diego, CA, USA). Data acquisition was performed on a LSR Fortessa flow cytometer (BD Biosciences). FlowJo Software (Tree Star, Ashland, OR, USA) was used in data analysis.

MHC class I tetramer was prepared in-house. Surface staining of T cells was achieved by addition of tetramer to whole blood, incubation for 10 minutes at room temperature, followed by addition of antibody co-stains for 20 minutes. Whole blood was preferred to thawed PBMCs for tetramer labelling due to greater consistency and signal intensity. Following lysis of erythrocytes using BD FACS Lysing solution (BD Biosciences), cells were either fixed using 2% formaldehyde (v/v) or permeabilized using the BD Cytofix/Cytoperm kit for intra-cellular staining. The following antibodies were used for surface and intra-cellular staining: CD3-PerCP, CD8-Horizon V500, CD8-APCH7, Ki-67-FITC, Bcl-2-PE, HLA-DR-Horizon V450, HLA-DR-PerCP, Perforin-FITC, Granzyme B-Horizon V450, CCR7-PE, CD27-Horizon V450, CD27-FITC, CD28-PECy7, CD28-PE (all BD Biosciences) and CD38-PECy7 (eBioscience), Granzyme B-PE (Caltag), Granzyme K-PE (Santa Cruz), CD45RA-FITC (Beckman Coulter), PD-1-PE and 2B4-PerCPCy5.5 (Biolegend). Intra-cellular cytokine staining with IFN-γ-FITC, TNF-α-APC, and IL-2-PerCpCy5.5 (all BD Biosciences) was undertaken after in vitro stimulation of PBMCs using peptide for 6 hours. Flow cytometry analysis was performed BD LSRII and BD FACSCanto flow cytometers. Flow cytometry data were analyzed using FlowJo software.