The project proposes to use a battery of continuously growing cytotoxic T lymphocyte (CTL) clones and a panel of monoclonal antibodies for the molecular definition of the antigenic determinants on the human class I histocompatibility antigens HLA-A2 and HLA-B7. Murine CTL clones which recognize alloantigenic determinants on these antigens have been previously described, and the available panel will be extended. Specificity will be assessed using an extensive target cell panel expressing a variety of class I antigens, and by determining the pattern of inhibition of lysis by the monoclonal antibody panel. This approach will allow the relative localization of determinants recognized by both types of reagents. More precise structural localization will be determined by the construction of mutant HLA molecules. Genes encoding the HLA-A2 and HLA-B7 molecules will be manipulated by the reciprocal exchange of equivalent segments of DNA in order to create hybrid molecules. Species-specific determinants will be localized by similar reciprocal exchange with genes encoding H-2Dd and H-2Ld. Local alterations in molecular structure will be induced via insertion of small DNA fragments or base substitutions. After transfection of murine or human cell lines with the DNA, the effect of these genetic alterations on molecular structure will be assessed by radio immunoassay or cytotoxicity assay using the previously described reagents. Alterations in recognition pattern will be compared with the results of previously performed antibody binding and inhibition of lysis experiments to judge the extent of local alterations in structure. It should therefore be possible to define precisely the regions of the B7 and A2 molecules which are recognized by CTL and monoclonal antibodies. Such information is important in understanding the central role of these molecules as mediators of the human immune response to tissue grafts, tumors, and virally infected cells.
Showing the most recent 10 out of 81 publications
