Characterizing the functionality of recombinant T-cell receptors in vitro:a pMHC tetramer based approach

The very low affinity of the T-cell receptor (TCR) for the peptide-major hi stocompatibility complex (pMHC) has made it very challenging to design assa ys for testing the functionality of these molecules on small scales, which in turn has severely hampered the progress in developing expression and ref olding methodologies for the TCR. We have now developed an ELISA assay for detecting pMHC binding to functional recombinant TCRs. It uses tetramers of biotinylated pMHCs bound to a neutravidin-horseradish peroxidase conjugate and detects the presence of functional TCR, bound in a productive orientat ion to an immobilized anti-Cp antibody. Specificity can be stringently demo nstrated by inhibition with monomeric pMHCs. The assay is very sensitive an d specific, and requires only very small amounts of protein. It has allowed us to study the unstable recombinant TCR P14, which we expressed and refol ded from Escherichia coli. The TCR P14 is directed against the most abundan t epitope of LCMV. We have confirmed the specificity of the interaction by BIAcore, and were able to determine the dissociation constant of the intera ction of the P14 TCR and of the gp33-pMHC as 6 mu M. This affinity ranks it among the tighter ones of TCR-pMHC interactions, and unusually low affinit y thus does not seem to be the cause of the modest protective power of thes e T-cells, compared to others elicited in the anti-LCMV response. This stra tegy of multimerizing one partner and immobilizing the other in both a nati ve form and productive orientation should be generally useful for character izing the weak interactions of cell-surface molecules. (C) 2000 Elsevier Sc ience B.V. All rights reserved.