Peripheral blood mononuclear cells (PBMCs) were isolated from 300 ml whole blood donations using Lymphoprep (Stemcell Technologies) density gradient centrifugation. CD8 T cells were depleted by positive selection using the EasySep™ Human CD8 Positive Selection Kit (Stemcell Technologies) and EasySep magnet (Stemcell Technologies). Depletion of CD8 T cells was con rmed by staining an aliquot of 50,000 CD8 depleted PBMC with anti-CD3-FITC (clone: OKT3), anti-CD4-PE (clone: OKT4) and anti-CD8-APC (clone: HIT8a) uorescent antibodies and analysis with an Accuri C6 ow cytometer and software (BD Biosciences).
Antigen reactivity of CD8 T cell depleted PBMC to the peptide pools was determined where indicated by IFN-g ELISpot assay (see below), and cells were subsequently stimulated with the various antigen peptide pools (see below) at 1 µg/ml each peptide. After 48 hours of antigen stimulation, an aliquot of ~50,000 CD8 depleted PBMC of each condition was stained for anti-CD4-APC (clone: OKT4) and anti-CD69-PE (clone: FN50) and analyzed using the Accuri C6 ow cytometer and software (BD Biosciences). All antibodies for ow cytometry were obtained from BioLegend.
Antigen reactivity of CD8 T cell depleted PBMC to the peptide pools was determined where indicated by IFN-g ELISpot assay (see below), and cells were subsequently stimulated with the various antigen peptide pools (see below) at 1 µg/ml each peptide. After 48 hours of antigen stimulation, an aliquot of ~50,000 CD8 depleted PBMC of each condition was stained for anti-CD4-APC (clone: OKT4) and anti-CD69-PE (clone: FN50) and analyzed using the Accuri C6 ow cytometer and software (BD Biosciences). All antibodies for ow cytometry were obtained from BioLegend.