Enhanced expression of introduced WT1-specific TCR and augmented functionality in WT1-siTCR–transduced CD8⁺ T cells. (A) An HLA-A*02:01-restricted HBZ_26–34-specific CTL clone (HBZ-1) was transduced with the WT1-siTCR or WT1-coTCR vector. Expression of the introduced WT1-specific and intrinsic HBZ-specific TCRs in TCR gene-modified HBZ-1 cells was examined using either HLA-A*24:02/WT1 tetramer or HLA-A*02:01/HBZ tetramer. A non–gene-modified HBZ-1 clone was used as a negative control. (B) HBZ-1 cells (▵), WT1-coTCR–transduced HBZ-1 cells (●), and WT1-siTCR–transduced HBZ-1 cells (○) were cocultured with HLA-A*02:01-positive T2 cells loaded with various concentrations of HBZ peptide for 3 hours. Thereafter, surface CD107a expression was analyzed as detailed in “Detection of CD107a and intracellular IFN-γ expression in WT1-TCR gene–transduced CD8⁺ T cells.” (C) IFN-γ production and degranulation of WT1-siTCR–transduced and WT1-coTCR–transduced CD8⁺ T cells in response to stimulation with WT1 peptide. Populations of WT1 tetramer-positive cells in WT1-coTCR– and WT1-siTCR–transduced CD8⁺ T cells before stimulation are shown in the upper column. The CD8⁺/WT1-tetramer⁺ cells shown with a broken line in each sample were analyzed for intracellular IFN-γ production and surface CD107a expression. One set of data obtained from experiments performed using CD8⁺ T cells from 2 different donors are representatively shown. (D) Cytotoxic activities of WT1-siTCR–transduced CD8⁺ T cells (○) and WT1-coTCR–transduced CD8⁺ T cells (●) against C1R-A*24:02 cells loaded with or without WT1 peptide and K562 cells transduced with or without HLA-A*24:02 gene were examined by standard 5-hour ⁵¹Cr-release assays at various effector/target (E/T) ratios.