IFN-γ Intracellular Staining (ICS) after the standard six hours peptide exposure. All donors were exposed to both FluM158–66 and CMVpp65495–503 peptides and then analyzed for intracellular IFN-γ protein release. Panel A: FACS analysis was conducted on gated CD3 positive lymphocytes after selecting only viable cells (R1 and R2) and plotting CD8 positive (x-axis) against either mouse IgG1 isotype-stained (y-axis, upper row) or human anti-IFN-γ-stained (y-axis, lower row) lymphocytes. The figure shows one representative experiment of three carried out on each donor. Panel B: IFN-γ ICS results in 3 different donors. The calculation of the percentage of antigen-specific T cells was performed based on the CD8 and IFN-γ dot plot data according to the following formula: Percentage Ag-specific T-cells = Percentage Upper right quadrant /(Percentage Upper right quadrant + Percentage Lower right quadrant)* 100%.