The major goal of this proposal is to contribute to a better understanding of how antigens, class II MHC molecules and T cells interact in the immune system. The approach will be to delineate the functional substructure of antigens and class II MHC molecules. For this purpose, the simple synthetic immunogen L- tyrosine-p-azobenzenearsonate (ABA-tyr) will be used as a model, against which we have now accumulated more than 50 murine T cell cloned lines, including I-A/k-, I-E/k-, and I-A/b- restricted lines from 3 inbred strains. 1. The component of ABA-tyr tentatively identified as comprising the structure that associates with Class II MHC molecules (agretope) is the aromatic core (see Progress Report). This inference will be tested by determining if phenylazotyrosine does indeed function as a universal """"""""carrier"""""""" for T cell epitopes. 2. Amino acid residues critical for functional restriction elements of class II molecules (histotopes) for ABA-tyr-specific T cell clones will be identified using site-specific mutagenesis and DNA transfection techniques. These studies have been initiated and look promising, although this approach clearly has interpretational constraints which limit its utility. 3. The capacity of ABA-tyr to interact in vitro with class II molecules will be assessed using affinity-purified class II proteins. It is hypothesized that ABA-tyr is immunogenic because it has a relatively high affinity for a functional site on class II. If a specific site exists, it should be possible to affinity label it, and an appropriately labelled ABA-tyr reagent will be used for this purpose. Identification of a binding site (candidate desetope) will be extremely useful for functional studies by the site-specific mutagenesis-DNA transfection approach. 4. Individual T cell clones manifest complex functional activities, including help, delayed hypersensitivity (DTH) and cytotoxicity, all of which are Ia-restricted and antigen-dependent. The mechanism of killing these L3T4+ cell will be investigated and their gene activation profiles with different types of antigen- presenting cells (spleen cells, resting B cells, B cell tumors, and transfected L cells) will be compared, using cDNA probes in RNA dot blot experiments. This should contribute to a better understanding of the role of the APC in the regulation of expression of manifold activities by individual T cells.
