Effector cells derived from naive precursors manifest less acquisition of effector function. Cryopreserved leukapheresis samples were thawed and rested overnight. CD8⁺ T-cell subsets were isolated then stimulated with allogeneic feeders, anti-CD3, and IL-2. Cells were transduced to express the 1G4-α-95:LY mutant TCR 48 hours after stimulation. The following data were obtained 10-16 days after stimulation. (A-B) Expression of CD45RO, CD62L, and CCR7. Gate frequencies are indicated. Data are representative of 6 patients. (C) EOMES expression by real-time RT-PCR. Expression is relative to ACTB then normalized to the average relative expression for each patient. Data from 5 patients are shown. (D-E) IFN-γ production from 16-hour coculture with NY-ESO-1⁺ HLA-A2⁺ tumor targets as determined by intracellular staining and supernatant concentrations, respectively. Coculture with 2361R (NY-ESO-1⁻ HLA-A2⁺) results in < 0.2 ng/mL of IFN-γ production in all groups. (F) Specific cell killing as determined by ⁵¹Cr release assay against the 624.38 tumor line.