PBMC Isolation and CD8+ T Cell Activation

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll-Paque density gradient centrifugation (GE Healthcare Bio-Sciences), as described previously (Chimote et al., 2018 (link)). CD8⁺ T cells were isolated from PBMCs by negative selection using the EasySep Human CD8⁺ T Cell Enrichment Kit (STEMCELL Technologies Inc.). The CD8⁺ T cells were maintained in RPMI 1640 medium supplemented with 10% human serum, penicillin (200 U/ml), streptomycin (200 μg/ml), 1 mM L-glutamine, and 10 mM Hepes. For all experiments, cells were activated in a cell culture dish pre-coated with mouse anti-human CD3 antibody (10 μg/ml) (BioLegend) and mouse anti-human CD28 antibody (10 μg/ml) (BioLegend) for 72 to 96 h, except where specified.