Imaging of T-cell receptor fused to CD3ζ reveals enhanced expression and improved pairing in living cells.

T cell receptor (TCR) gene adoptive therapy is a promising clinical approach for the treatment of malignant tumors and viral diseases. However, the effectiveness of this strategy is hampered by the generation of mixed TCR heterodimers comprising both exogenous and endogenous TCR chains (i.e., mispairing of TCR chains). In the present study, we constructed genetically encoded reporters fused to a pair of fluorescent proteins [enhanced cyan fluorescent protein (ECFP)/enhanced yellow fluorescent protein (EYFP)] to monitor the expression of TCRαζβζ and pairing between TCRαζ and TCRβζ. We demonstrate that these reporters provide accurate images of TCRαζβζ expression, which is markedly stronger with evident microclusters accumulated at the plasma membrane compared to wild-type (wt)TCR. Using fluorescence resonance energy transfer (FRET) analysis, we demonstrate that, in addition to the improved pairing, the expression and assembly of TCRαζβζ are independent of endogenous CD3 subunits. These results suggest that the fusion genes, TCRαζ and TCRβζ, coupled to ECFP and EYFP, respectively can effectively monitor the expression and interaction in cells. Our data suggest a novel strategy with which it is possible to effectively express and pair TCRαζβζ, thus making TCR gene adoptive therapy more effective.