a CEF viral antigens are presented on the cell surface membranes of HCT116 cells within a tumor spheroid. HCT116 tumor spheroids were loaded with CMVpp65 viral antigen conjugated to a BODIPY-488nm fluorochrome and treated with either Brefeldin (a protein transport inhibitor), DMSO (vehicle control), or an unlabeled CMV competitor peptide (CMV cold competitor). Spheroids were stained with a cell membrane dye (CellBrite) and a nuclear dye (Draq5) to identify the cellular localization of CMV-BODIPY peptides. Spheroids were imaged comprehensively in a large z-stack and then single planes were more closely scrutinized for CMV-BODIPY membrane localization. Blue arrows indicate membrane-localized CMV-BODIPY peptides and white arrows indicate cytoplasmic CMV-BODIPY peptides. Scale bar equals 60 µm. b Antigen-specific T-cell priming reveals pMHC-reactive memory T-cells. Human donor PBMCs were stimulated with either CMVpp65 or EBV-BMFL1 viral peptides and expanded for 7 days in culture. Flow cytometry analyses of CD3+CD8+ T-cell populations reveal binding of TCRs only to CMV tetramers and not EBV tetramers, indicating a CMV-reactive memory T-cell population had been selected and expanded. c Phenotyping of T-cells used for the spheroid-killing screen. PBMC cultures stimulated with CEF viral peptides and expanded in IL-2, then in IL-2, IL-4, and IL-7 were subjected to flow cytometry T-cell phenotyping at days 3, 6, and 9 post-stimulation. CD8+CD127+ T-cells indicate desirable memory effector T-cell population for screening.