Ad5f35- and LCL-stimulated PBMCs activate both Ad- and EBV-specific T cells. (Ai) Donor 4 and (Bi) donor 5 represent ELISPOT assays to assess the relative specificities of the CD4 and CD8 T cells expanded by 1 stimulation with Ad5f35GFP-transduced PBMCs, followed by 2 stimulations with Ad5f35GFP-transduced autologous LCLs. T cells were incubated with autologous LCL targets (auto) either alone or transduced with Ad5f35GFP (auto + Ad); negative controls were class I-mismatched LCLs alone (mis) or Ad5f35GFP transduced (mis + Ad). Results are expressed as SFCs per 5 × 10³ cells. Panels Aii, Aiii, Bii, and Biii display the ability of these expanded T cells to specifically kill autologous Ad-transduced targets in a standard chromium release assay using the same targets as described in the ELISPOT assay, and results are expressed as percent specific lysis. Results are expressed as percent specific lysis of autologous LCLs alone (♦), allogeneic LCLs alone (▪), Ad5f35GFP-transduced autologous LCLs (▴), or Ad5f35GFP-transduced allogeneic LCLs (×). Panels Aii and Bii show isolated CD4⁺ T-cell cytotoxicity assay results, and panels Aiii and Biii show CD8-isolated T cells.