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

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 spec ific T-cells in vivo, we decided to express and analyze the function of a T -cell receptor (TCR) alpha chain-GFP fusionprotein. The TCR alpha and beta chain cDNAs of a Leishmania major-specific murine T helper 2 cell clone wer e cloned and inserted into the pHSE3' expression vector. Simultaneously, a TCR alpha expression vector was constructed containing a C-terminal in fram e fusion with the open reading frame of the enhanced GFP (EGFP). TCR alpha /TCR beta or TCR alpha -EGFP/TCR beta constructs were expressed in T-cell h ybridoma cells 58 alpha (-) beta (-) which lack an endogenous TCR but still express CD3 components. The TCR alpha -EGFP fusionprotein was detected wit h the expected molecular weight by immunoprecipitation and Western Blot ana lysis. Surface staining of TCR components was detected in transfectants exp ressing 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-t erminal extension of the TCR alpha. Most importantly, the EGFP-tagged TCR w as functional since the transfectants produced IL-2 in response to stimulat ion 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 toleranc e induction and immunity in transgenic mouse models. (C) 2000 Elsevier Scie nce BN. All rights reserved.