The long-term objective of this study is to broaden our understanding of the structure-function relationships of the major histocompatibility complex (MHC) encoded class I molecules. This goal is of major importance because of the central role that class I molecules play as restricting elements in the immune response to virus and tumor antigens, and because of the recognition of these molecules as alloantigens on foreign tissue.
The specific Aims of this proposal are to: (1) generate a panel of cells expressing variant HLA-A2 molecules that differ from one another by single amino acid substitutions in either the alpha1 or alpha2 domains; (2) determine which regions of the alpha1 and alpha2 domains contribute to the epitopes recognized by antibodies specific for MHC class I molecules; (3) evaluate how the various regions of the alpha1 and alpha2 domains of the HLA-A2 contribute to the epitopes that are recognized by allogeneic CTL; (4) evaluate how the various regions of the alpha1 and alpha2 domains of HLA-A2 contribute to the epitopes that are recognized by influenza- specific, HLA-A2 restricted CTL; (5) determine which amino acid residues in the alpha1 and alpha2 domains of the HLA-A2 molecule are responsible for the binding of the matrix peptide derived from influenza virus; and (6) establish which regions of the alpha1 and alpha2 domains are important in the generation of an alloimmune response against class I molecules. These objectives will be met by first generating a panel of mutant HLA-A2 genes by site-directed mutagenesis. These genes will subsequently be expressed in a class I null cell line following their transfection by electroporation. RIA and FACS analysis will be utilized to quantitate the expression of genes on the cell surface and to analyze the effects of the substitutions on the serological epitopes recognized by murine mAb and human alloantisera. The contribution of the various substituted positions to the epitopes recognized by allogeneic and influenza virus-specific CTL will be investigated utilizing a standard 51Cr-release assay. The HLA-A2 restricted, influenza specific CTL will be generated against matrix peptide pulsed stimulator cells and will be tested on targets that are pulsed positions in the HLA-A2 molecule are important for peptide binding. Precursor frequency analysis will be used to determine the minimal molecular requirements for a class I molecule to be recognized as an alloantigen. Finally, the heterogeneity of the CTL clones responding to a given mutant HLA-A2 molecule will be determined by fine specificity analysis. Thus, these studies will provide critical information that will advance our understanding of how class I molecules function when the immune system responds during a viral infection or towards tumor cells, and they will also advance our understanding the phenomenon of graft rejection in transplant patients.
