Fitc anti human cd107a

Frequencies of peptide-specific CD8⁺ T cells were determined by tetramer staining using the PE/Cy7 anti-human CD8 mAb (#737661, Beckman Coulter, Brea, CA, USA) and 5 µg/mL of PE-labeled HLA:peptide tetramer (in-house production). Tetramers of the same HLA allotype containing irrelevant control peptides were used as negative control (Supplemental Table 2).
Functionality of peptide-specific T cells was analyzed by ICS using PE/Cy7 anti-human CD8, PacificBlue anti-human TNF (#502920), FITC anti-human CD107a (#328606, BioLegend, San Diego, CA, USA), and PE anti-human IFNγ (#506507, BD, Franklin Lakes, NJ, USA) antibodies as described previously^{30 (link)}.
Results of tetramer staining were considered positive if the frequency of peptide-specific CD8⁺ T cells was ≥0.1% of viable cells and at least three-fold higher than the frequency of peptide-specific cells in the negative control according to the harmonization guidelines for HLA-peptide multimer assays of the international Cancer Vaccine Consortium proficiency panel^{60 (link)}. The same evaluation criteria were applied for ICS. All samples were analyzed using a FACS-Canto^TM II cytometer and FlowJo software version 10.0.7 (BD).

Peptide-specific T cells were characterized by intracellular cytokine and cell surface marker staining. PBMCs were incubated with 10 μg/mL per peptide of EC or negative control peptide, 10 μg/mL Brefeldin A (Sigma-Aldrich), and a 1:500 dilution of GolgiStop (BD Biosciences) for 12 - 14 hours. Staining was performed using Cytofix/Cytoperm solution (BD Biosciences), APC/Cy7 anti-human CD4 (1:100 dilution, BioLegend, Cat# 300518, RRID: AB_314086), PE/Cy7 anti-human CD8 (1:400 dilution, Beckman Coulter, Cat# 737661, RRID: AB_1575980), Pacific Blue anti-human TNF (1:120 dilution, BioLegend, Cat# 502920, RRID: AB_528965), FITC anti-human CD107a (1:100 dilution, BioLegend, Cat# 328606, RRID: AB_1186036), and PE anti-human IFN-γ monoclonal antibodies (1:200 dilution, BioLegend, Cat# 506507, RRID: AB_315440). T cells exposed to phorbol myristate acetate (PMA, 5 μg/mL, Sigma-Aldrich) and ionomycin (1 μM, Sigma-Aldrich) served as positive controls. Viable cells were determined using Aqua live/dead (1:400 dilution, Invitrogen). All samples were analyzed on a FACS Canto II cytometer (BD Biosciences) and evaluated using FlowJo software version 10.0.8 (BD Biosciences). The gating strategy applied for the analyses of flow cytometry-acquired data is provided in fig. S14.