As a continuation of our multidisciplinary approach to define the immunopathogenesis of rheumatic fever, the present study will examine the hypothesis that susceptibility to rheumatic fever is encoded by genes inherited in association with the class II major histocompatibility genes (MHC). Rheumatic fever is associated with inheritance of class II MHC genes encoding for HLA-DR4 and -DR2. However, since most individuals with DR4 or DR2 are healthy, other factors must be considered in attempting to identify a putative disease susceptibility gene. Microheterogeneity of DR4 and DR2 specificities has recently been shown using HLA-D typing, electrophoretic analysis of immunoprecipitated DR antigen, or restriction endonuclease mapping. These techniques make it feasible to focus on the DR gene or the fine structure of the encoded class II molecule, rather than the polymorphic DR marker, to search for putative disease susceptibility gene/gene product. Lymphoblastoid lines will be prepared from B cells of rheumatic fever patients and race and sex-matched normal controls who are homozygous for HLA-DR2 or DR4. The cells will be labeled with 125I, lysed, and the 125I-labeled lysate incubated with a series of monoclonal antibodies specific for HLA-DR molecules. The precipitated HLA-DR immune complexes will be subjected to two-dimensional gel electrophoresis and autoradiographs of the gels will be compared. Because recent work has demonstrated that HLA-D typing identifies functional diversity of Class II antigens in serologically-defined DR-homozygous individuals, we will seek additional evidence for microheterogeneity or the lack thereof by performing HLA-D-typing using the mixed lymphocyte culture technique. The results of these studies will have direct impact on the hypothesis that electrophoretically distinct products of individual """"""""Ia-like"""""""" loci may represent genetic markers more relevant to HLA-DR-associated diseases than serologic specificities defined by conventional typing reagents. In addition, evidence of electrophoretically identical HLA-DR structures expressed by patients with rheumatic fever would provide the groundwork of the subsequent identification of putative immune response gene or gene-products at the molecular level.
