Analyses of cell-surface TCRαβ and TCRγδ expression and intracellular TCRαβ expression of γδ T cells transduced with different CMV-TCRs. (A) Peripheral-blood–derived γδ T cells were transduced with 3 CMV-TCR complexes and with control retroviral vectors only expressing the marker genes. Double marker gene–positive cells were sorted and expanded, and the TCRαβ and TCRγδ cell-surface expression of the GFP⁺ NGF-R⁺ population was determined by FACS. The marker gene expression of the transduced γδ T cells is shown in the small dot-plots. (B left) Intracellular staining of TCRαβ complexes in γδ T cells transduced with control vector (filled histogram), original AV2BV2-TCR (black line), and original AV18BV13-TCR (gray line) with anti-TCRαβ mAb using saponin treatment. (B right) Intracellular staining of TCRαβ complexes in γδ T cells transduced with control vector (filled histogram), chimeric AV2BV13-TCR (black line), and chimeric AV18BV2-TCR (gray line) with anti-TCRαβ mAb using saponin treatment. (C) TCR β-chain mRNA from γδ T cells transduced with AV2BV2 (□), AV19BV21 (▪), or AV18BV13 (⊡) TCRs was quantified by RT-PCR and normalized to PBGD. Similar results were obtained for the TCR α-chain mRNA expression in these different TCR-transferred γδ T cells (data not shown). (D) Intracellular staining of TCR BV2 in γδ T cells transduced with control vector (filled histogram), original AV2BV2-TCR (black line), and the chimeric AV18BV2-TCR (gray line) with anti-BV2–specific mAb using saponin treatment.