Isolation and Purification of Polyclonal CD8+ T Cells

CTL03.1 and N5.14 CD8+ T-cell clones specific for Melan-A and MUC1, respectively, were obtained and cultured as previously described (18 (link), 19 (link)). Polyclonal CD8+ T cells were obtained from fresh blood (Etablissement Français du Sang, ethics agreement CPDL-PLER-2018 021). Peripheral blood mononuclear cells were separated using Ficoll gradient (Eurobio, Les Ulis, France; Cat#CMSMSL01-01). Polyclonal CD8+ T cells were then sorted using EasySep Human CD8+ T Cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada; Cat#17953). Purity was assessed after sorting by flow cytometry following a 20-min staining at 4°C with CD3 and CD8 antibodies directly conjugated to fluorescein isothiocyanate (FITC, BD Biosciences, Pont de Claix, France; Cat#555339, RRID : AB_395745) and phycoerythrin (PE, BD Biosciences, Pont de Claix, France; Cat#555367, RRID : AB_395770), respectively. Polyclonal T cells were considered acceptable for further experiments when CD3+CD8+ population among viable cells represented over 90%.