Schematic diagram for generation of CMV-specific CD4⁺ and CD8⁺ T-cell lines. After stimulation of 20 × 10⁷ to 40 × 10⁷ PBMCs with CMV peptides and CMV lysate, IFN-γ-producing cells were isolated using the IFN-γ secretion assay and were expanded over 7 to 11 days with medium, IL-2, and autologous, irradiated PBMCs as feeder cells. In addition, autologous DCs were generated over 7 days, loaded with CMV peptide/lysate, and used as APCs for intracellular IFN-γ staining of expanded cells. Further characterization of the expanded cells includes tetramer staining, CFSE staining, expansion on restimulation, cytotoxicity assay, and assessment of alloreactivity.