Figure 2.

2A peptide and furin contribute to α- and β-chain maturation. (a) Proposed molecular events of expressed T-cell receptor (TCR). Diagrammatic illustration of gp100 TCR fused with V5 (between furin and foot-and-mouth disease virus (F2A) peptide) and hemagglutinin (HA) (in C terminus of TCR-β chain) tags. Translation continues up to the F2A peptide, which can initiate a ribosomal skip to generate two proteins; the TCR-α and -β chains as illustrated. The α-chain with furin site can be cleaved at the post-translational level as shown. (b) TCR products detected by western blotting. In upper panel, the cell lysates were prepared (3 days post-transduction) and 30 μg of lysates was subjected to SDS–polyacrylamide gel electrophoresis (PAGE) gels, the level of TCR expression was measured by immunoblotting with HA antibody. In lower panel, the level of TCR-α chain was detected by V5 antibody. Cells analyzed were SupT1 (left) Jurkat-T3 (middle) and primary human peripheral blood lymphocytes (PBLs) (right). The unprocessed TCR-α and -β chains are indicated in the image as α2Aβ. The bands of TCR-α or -β and α2Aβ were quantified using a CCD camera and image analysis software. (c) Furin accounts for the enhanced TCR expression in lentiviral construct. Lentiviral constructs harboring gp100 TCR modified with tags with or without furin are illustrated (left panel). PBL from melanoma patient was transduced with the same amount of lentiviral particles derived from above constructs, and expression of the TCR analyzed by tetramer staining. The images were overlaid, and histograms reflecting specific vector are as denoted.