The goal of this project is to investigate immunobiology and the relationship between HLA-D region gene sequences and human immune responsiveness to allergens. We will address such questions as: what is the molecular basis of the observed associations between HLA-D and immune responsiveness, and why are seemingly diverse autoimmune diseases and immune responses found associated with the same HLA-D specificity; how can a limited repertoire of HLA-D specificities (Ia specificities) permit recognition of an incalculably large number of antigens; what are the structural features of the Ia determinants and what is the relationship between Ia determinant and T-cell determinant? The model system selected for these studies is the human immune response to inhaled allergens of Lolium perenne (perennial rye grass), namely Lol p I, II and III, immune responsiveness to all of which is associated with HLA-DR3/Dw3. Studies will focus on whether there are any common structural features present (i) in the Ir gene(s) that control the immune response to this group of antigens, and (ii) in the Ia determinant(s) of these antigens. Human T-cell lines will be generated specific to the three antigens, and will be used in conjunction with monoclonal antibodies against various DQ and DR gene products, to narrow down the identification of the Ir gene locus. The Ir gene will be sequenced from 32 to 40 patients representing various sub-groups of the following four major groups: DR3+ responder; DR3+ nonresponder, DR3- responder; and DR3- nonresponder. Only the first exon (ca. 300 bp) of each gene will be sequenced, as the polymorphism is primarily found in this region. In addition, the use of allele-specific oligonucleotides (ASO's) will facilitate the identification of the genes, the use of the polymerase chain reaction (PCR) will facilitate the amplification and cloning of the gene segments of interest. By a combination of protein sequencing and cDNA cloning, complete amino acid sequences of the three allergens Lol p I, II and III and their variants will be determined. The T-cell determinants and Ia determinants of the three allergens will then be identified by a combination of methods including comparative tryptic peptide mapping, amphipathicity analyses of the proteins, synthesis of peptides and determination of their activities as Ia and T-cell determinants. The information obtained on Ir gene sequences and structure of Ia and T-cell determinants will be valuable in investigating structure-function correlations between specific epitopes and Ir gene products.
