Lytic activity, IFN-γ release, and tetramer binding in IE4-MVA IVS cultures. IVS using IE4-MVA was performed with HLA A^*0201 donors 009, 010, and 011. (A) Cytotoxic activity detected after IVS is shown for each donor. ▦ indicates background lysis to autologous LCLs loaded with p53_149-157; ♦ indicates lysis of autologous LCLs pulsed with HLA A^*0201 IE1_316-324 peptide. (B, left panel) IE1_316-324 tetramer binding frequencies in CD8⁺ T cells from donor PBMCs (□) and IE4-MVA IVS cultures (▪). CD8⁺ T-cell binding to HIV pol_464-472, used as control, was subtracted. (B, right panel) Percentages of CD8⁺ with IFN-γ release after incubation with IE1_316-324 peptide in fresh PBMCs (□) or IE4-MVA IVS cultures (▪) were detected using ICC. Percentage of CD8⁺ T cells with IFN-γ release to p53_149-157 was subtracted. (C) Donor 009 tetramer binding FACS plots are shown. Tetramers used were conjugated with APCs, and with PE for plot ii. Numbers on the upper-right quadrant indicate CD8⁺ T-cell tetramer binding percentages to (i) pp65_495-503 tetramer after pp65/pp150-MVA IVS, (ii) IE1_316-324 tetramer after IE4-MVA IVS, (iii) pp65_495-503 tetramer, (iv) IE1_316-324 tetramer, and (v) HIV pol_464-472 control tetramer after combined pp65/pp150-MVA and IE4-MVA IVS.