Improving matched TCR pairing by introducing cysteines in the constant domain of the αβTCR chains. (A) T cells transduced with vα21_wt and vβ21_wt TCR chains (○), or vα21_Cys and vβ21_Cys TCR chains (▵), were simultaneously stained with WT1_126-134 multimer-PE and the anti-vβ21–FITC antibody. MFI of the anti-vβ21–FITC signal was plotted against the MFI of the WT1_126-134 multimer-PE signal. (B) Transduced cells were sorted based on equivalent levels of low (left panel) or high (right panel) vβ21 TCR chain, (C) equivalent levels of WT1_126-134 multimer binding, or (D) equivalent levels of vα21TAG TCR and vβ21 TCR; expanded; and then reanalyzed for comparative expression of vβ21 (B-D), vα21-TAG (D), and binding of the WT1_126-134 multimer (B-D). (Mock-transduced T cells, thin line; wt-TCR transduced, dotted line; Cys-TCR transduced, dashed line.) (E) T cells mock transduced or transduced with vα21_wt and vβ21_wt TCR chains or vα21_Cys and vβ21_Cys TCR chains were stained for total TCR expression by an anti-αβTCR–PE antibody (MFI from 3 independent experiments were averaged; left). T cells transduced with vα21_wt and vβ21_wt TCR chains (▪) or vα21_Cys and vβ21_Cys TCR chains (□) were selected for lower (denoted as L: MFI 16) or higher (denoted as H: MFI 28) vβ21 TCR-chain expression and then were either not treated or stimulated with αCD3 (60 ng/mL) or T2 cells pulsed with irrelevant (p53_264-272) or relevant (WT1_126-134) 10⁻⁵M peptide. TCR-chain surface expression was calculated as percentage MFI of anti-αβTCR–PE (middle) or anti-vβ21–FITC (right) staining in the stimulated groups compared with control unstimulated cells. Error bars indicate SEM.