The present grant proposes to use lymphoblastoid cell line (LCL) variants that have lost expression of different portions of the HLA-D region (of one or both D haplotypes) to permit both genetic mapping of HLA-D encoded determinants and studies of genetic complementation. Preliminary investigations have demonstrated the feasibility of creating loss variants of varying lengths of the HLA-D region (including DNA loss variants) and mapping of determinants within this region, using cloned primed lymphocyte typing (PLT) reagents. Such reagents are of key import both because they offer a highly productive approach for the definition of multiple determinants associated with single D haplotypes, and because they can be readily prepared against normal cells (or LCLs) in any given combination. We shall focus first on DR2+ and DR4+ haplotypes, since these antigens are found in association with a large number of different """"""""Dw antigenic clusters"""""""", defined with homozygous typing cells and PLT reagents. DNA probes will be used to determine the nature of the mutation underlying loss of antigen expression and to aid in construction of a genetic map of the D region, as well as to further our understanding of the genetic basis for the relationship between serologically and cellularly detected determinants of D region encoded molecules. Attempts will be made to generate serological reagents, including monoclonal antibodies against D region products. Molecules expressing DR determinants (and later other D region encoded molecules expressing serologically detected determinants) will be isolated and studied by peptide mapping to investigate whether some peptides are associated with the serologically detectable determinant and others with the cellularly defined determinant(s) within a single molecular entity. Finally, we shall perform studies on a molecule, B7/21, which is encoded by genes linked to HLA-DR but which is different from the presently defined D region encoded molecules. We expect that these various approaches will contribute to the generation of a coherent picture of the genetic structure of, and molecules encoded in, the D region and thus enhance continued progress in matching for transplantation and understanding of disease associations.
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