The recognition of antigen by T cells involves the interaction between the T cell receptor and an antigenic complex of self-major histocompatibility complex protein and peptide derived from foreign protein. The complex is of very low affinity. Surface plasmon resonance techniques (BIAcore) will be used to analyze the relationship between the kinetics of T cell receptor interactions with MHC-peptide ligands, and the biological response of T cells and thymocytes. Soluble TCR and MHC-peptide complexes for three different antigenic systems will be determined. Agonists and antagonists of various strengths have been defined in these systems. These ligands have also been related to positive and negative selection in the thymus. There is a relationship between the kinetics and the biological response but there is a special effect on binding of strong agonists. This appears to be dimerization of the TCR, which has profound implications for T cell/thymocyte activation, since it massively increases the stability of an agonist-TCR complex. The factors causing this effect, and the type of ligands that cause it will be determined. Standard biochemical assays will be used to determine if the TCR dimerizes in response to agonists. Fluorescence resonance energy transfer will be used as a probe for dimerization, and then as a probe to find which parts of the TCRs interact with each other. Using data from this, and from refolding of alpha and beta-chains of TCR from different T cells, the region involved in dimerization will be identified, and then dissected it in detail using mutagenesis. Single Molecule Fluorescence Correlation Spectroscopy will be used as a tool to analyze TCR-ligand interactions. This new technique has the potential to show detailed stoichiometry of the interactions in a non- invasive manner.
