Molecular analysis of T cell receptors: Mapping T cell recognition and repertoire in two human immune responses

The aim of this thesis was to characterise the T cell receptors (TCR) involved in two human immune responses, and their interaction with antigen in the context of major histocompatibility molecules. The TCR of a panel of human CD4+ house dust mite - specific T cell clones were analysed in the context of the peripheral TCR repertoire. The clones were found to use a restricted set of TCR variable (V) genes, although there was little conservation of junctional sequences. Several clones had identical TCR at the sequence level, despite the fact that they had been isolated in different years. Screening of amplified TCR transcripts from several years later suggested the presence in vivo of long lived dominant T cell clones in this response. The human T cell response to the influenza haemagglutinin peptide HA 307 - 319 in the context of the class 11 molecule DR1 was studied in two human T cell clones derived from different individuals. One of these clones, HA1.7, responds equally to HA 307 - 319/DR4, while the other, Cl-1, does not. Sequencing of amplified TCR transcripts revealed that the two clones express highly related TCRα chains, with a conserved junctional motif, but very different TCRβ chains. Modelling studies led to the prediction that a conserved glutamic acid residue (94E) in the TCRα junctional region might be critical to peptide recognition. After initial investigation of models for expression of TCR, mutagenesis studies of the HA1.7 TCRα chain were performed using transfection into the human T cell line JRT3T3.5, and the rat basophil line (RBL). In the RBL line, ligation of the transfected TCR leads to degranulation, measurable as serotonin release. TCRα mutants carrying either alanine or lysine at position 94 no longer recognised the HA 307 - 319 /DR1 complex. These mutants were not rescued by peptides carrying reciprocal charge changes