A stable marker for specific T-cells: a TCR alpha/green fluorescent protein (GFP) fusionprotein reconstitutes a functionally active TCR complex

Abstract

The detection of antigen specific clonal T-cell populations in vivo during T-cell selection and an immune responses is often hampered due to the lack of suitable clonotype specific monoclonal antibodies. In order to determine the potential usefulness of green fluorescent protein (GFP) to follow specific T-cells in vivo, we decided to express and analyze the function of a T-cell receptor (TCR) α chain-GFP fusionprotein. The TCRα and β chain cDNAs of a Leishmania major-specific murine T helper 2 cell clone were cloned and inserted into the pHSE3′ expression vector. Simultaneously, a TCRα expression vector was constructed containing a C-terminal in frame fusion with the open reading frame of the enhanced GFP (EGFP). TCRα/TCRβ or TCRα-EGFP/TCRβ constructs were expressed in T-cell hybridoma cells 58α −β − which lack an endogenous TCR but still express CD3 components. The TCRα-EGFP fusionprotein was detected with the expected molecular weight by immunoprecipitation and Western Blot analysis. Surface staining of TCR components was detected in transfectants expressing the wild type TCR heterodimer and, with only a slight reduction in intensity, also in those expressing the TCR-EGFP complex. Hence, expression and transport to the outer cell membrane is possible despite the 27 kD C-terminal extension of the TCRα. Most importantly, the EGFP-tagged TCR was functional since the transfectants produced IL-2 in response to stimulation via their TCR. Thus, TCR-EGFP constructs represent attractive tools to study posttranslational regulation of TCR expression and ligand-induced TCR clustering as well as the fate of antigen specific T-cells during tolerance induction and immunity in transgenic mouse models.