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We analyzed the cDNA sequence data of complementarity determining regions (CDR3) of epitope mapped alphabeta TCR of T cell clones (TCC). The TCC were specific for the major timothy grass (Phleum pratense) pollen allergen Phl p 1 and were derived from the peripheral blood of seven unrelated grass pollen-allergic individuals. Each TCR recognized one of two immunodominant T cell epitopes, PP73 or PP103, of Phl p 1. Although a diversity of recombined V and J segments was observed, amino acid motifs as long as five residues were conserved among CDR3 loops of TCR from TCC of different atopic individuals specific for the same peptide. The conserved sequences could comprise as much as 60% of the CDR3. All amino acid residues of the motifs of the CDR3beta and most of the CDR3alpha of all TCR used in this study were encoded by randomly added nucleotides. This indicates that they were specifically selected for by the peptide bound to the MHC class II molecule. For one selected patient, a larger number of TCC, specific for PP103, was analyzed. The TCR repertoire was limited to three different TCR. The same MHC class II molecule, DRB1*1301, was identified to present PP103 to each of the three TCR.

Background: The interaction of T cell receptors (TCRs) with peptide fragments bound to major histocompatibility complex (MHC) molecules is central 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 focused on the production of recombinant soluble forms of the TCR heterodimer. 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 amino 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 produced. The ratio of insoluble to soluble material was 1:1. The recombinant protein was of the correct size and showed no signs of degradation. Conclusions: Conservation of amino acid motifs in CDR3 loops of TCRs specific for the same allergen fragments indicated that the three-dimensional structure of the CDR3 was determined by the presented peptide. The recombinant scTCR will be used to identify ligands for the CDR3s from random peptide libraries to interfere with TCR binding to the MHC/peptide complex.

We investigated the longevity of allergen-specific Th cells derived from patients suffering from either allergic rhinitis or atopic dermatitis. T cell clones (TCC) specific for seasonal and perennial allergens were raised. To determine whether these TCC were long-lived in vivo, PBMC and allergen-specific polyclonal T cell lines, collected and established inside a period of up to 4 years, were screened for the TCC of interest. For this purpose, a T cell tracing protocol was established in which oligonucleotides specific for the TCR β-chain hypervariable junctional region were used as tools to identify each particular TCC. Seven pollen-specific TCC and two house dust mite-specific TCC, with a Th2-like cytokine production pattern in vitro, were demonstrated to be long-lived memory T cells in vivo. Specificity of the tracing protocol was ascertained by TCR sequence analysis. We conclude that allergen-specific TCC can persist for years, evidence for which can be monitored in blood, but also in the target organ of the allergic disorder. The data indicate that in vitro-characterized, allergen-specific, long-lived TCC may well reflect a repertoire of T lymphocytes of pathogenetic importance in vivo.