Figure 1.

NLV-specific human TCR RA14 displays on the surface of CHO-K1 cells. a, RA14 variable and constant regions were cloned in-frame with the mouse IgH leader sequence (LS), a T2A peptide for cleavage, and the PDGFR transmembrane domain (TM) with either the α-chain (α/β-TM) or the β-chain (β/α-TM) in the first position. The cassettes were then cloned into a pcDNA3 mammalian expression vector. b, display of functional RA14 TCR was detected with a dual-staining approach, in which an anti-Vβ6-5 antibody-PE conjugate was used to detect expression of the TCR β-chain, whereas a peptide/A2 tetramer conjugated to APC was used to assess ligand binding. c, plasmids encoding the TCR in both chain orientations and with the wildtype (wt) or engineered disulfide (ds) constant regions were transfected, stained 2 days later, and assayed for APC and PE signal via flow cytometry. Rainbow dots depict staining using tetramer presenting the NLV peptide from the CMV pp65 protein, and the gray dots depict staining with tetramer presenting the control peptide KLV. Control transfections without plasmid and with a plasmid lacking the α-chain are also shown.