T_Ecells derived from T_CMand T_EMin vitro are similar in phenotype and function. (A) Schematic of methods for deriving CMV-specific T_CM/E and T_EM/E. Purified T_CM, T_EM, and pp65-expressing vAPCs were generated from the same CMV-seropositive donor's PBMCs. (B) CD45RO and CD62L staining of T_CM (top) and T_EM (bottom) after sorting from PBMCs. (C) CD8 and pp65-tetramer staining of gated PBMCs (left panels) and of CMV-specific T_CM/E and T_EM/E at day 7 (middle panels) and 21 (right panels) after stimulation with vAPCs. Histogram quadrants are based on staining with isotype and negative tetramer controls, and percentage of double-positive cells is indicated. (D) pp65 tetramer and intracellular IFN-γ staining of T_CM/E and T_EM/E before infusion, after overnight coincubation with LCL-pp65. (E) Fold expansion of pp65-tetramer⁺ cells was determined by multiplying the total number of cells by the percentage pp65tet⁺ (determined as shown in panel C) found at days 0, 7, 14, and 21 of vAPC stimulation and 14 days after the first and second anti-CD3 (REM) stimulations; these values were then normalized to the input cell number (day 0). (F) Expression of CD62L, CD127, CD28, CCR7, and CD8 on the T_CM/E and T_EM/E cell products. (G) Cytotoxic activity of T_CM/E and T_EM/E cell products against auto-LCLs loaded with either an HLA-A2-restricted control peptide (cLCL) or CMV pp65 peptide (pp65LCL). Mean percentage of ⁵¹Cr release (± SD) of triplicate wells is depicted. (H) Cytokine production by T_CM/E and T_EM/E. Supernatants were collected after coincubating T cells overnight with CMV pp65 peptide-loaded auto-LCLs, and mean (± SD of triplicate wells) cytokine levels were determined using cytometric bead array.