T-CELL RECEPTOR CDR3 SEQUENCES AND RECOMBINANT T-CELL RECEPTORS

Background: The interaction of T cell receptors (TCRs) with peptide fr agments bound to major histocompatibility complex (MHC) molecules is c entral to the initiation and propagation of most immune responses, In order to understand and control the molecular interactions underlying T cell recognition of MHC/peptide complexes, recent efforts have focus ed on the production of recombinant soluble forms of the TCR heterodim er. Methods: TCRA variable (TCRAV) and TCRBV sequences used by human T cell clones were amplified by PCR, cloned and sequenced. The deduced amino acid sequences of the complementarity determining region (CDR) 3 loops of TCRs specific for the same allergenic epitope were compared. V region genes of a selected TCR were expressed as a single-chain (sc ) molecule in the periplasm of Escherichia coli. Results: Conserved am ino acid motifs specific for allergenic peptides of Bet v 1 and Phl p 1 were identified in CDR3 sequences of TCRs. A recombinant scTCR was p roduced. The ratio of insoluble to soluble material was 1:1. The recom binant protein was of the correct size and showed no signs of degradat ion. Conclusions: Conservation of amino acid motifs in CDR3 loops of T CRs specific for the same allergen fragments indicated that the three- dimensional structure of the CDR3 was determined by the presented pept ide. The recombinant scTCR will be used to identify ligands for the CD R3s from random peptide libraries to interfere with TCR binding to the MHC/peptide complex.