Polymorphic genetic differences between individuals contribute to the specificity of immune responsiveness. The original observation leading to the present studies has been the association of the DQI/DQ2 heterozygote state with high levels of anti-Ro/SSA autoantibodies which has since been confirmed in three studies of systemic lupus erythematosus. The goals of this project since last review have been to determine whether a gene complementation model could be confirmed at the DNA level and to establish a clinical resource composed of clinical data, serum, plasma, DNA, and transformed cell lines to test this hypothesis as well as a general model of disease heterogeneity. Substantial progress has been demonstrated. A restriction fragment length polymorphism study of the HLA-DQ-alpha, DQ- beta, and DR-beta genes has demonstrated that the combined DQl and DQ2 association with anti-Ro/SSA could be attributed to DQ-alpha and DQ-beta genes, respectively. This result establishes gene complementation at HLA-DQ as a mechanism for anti-Ro/SSA production in these patients. An additional separable effect at DQ-beta has also been described. Extension of this study to the T cell receptor beta gene has described restriction fragments which are closely associated with anti-Ro/SSA in the context of the identified histocompatibility alleles. In addition, this work has led to a theory of disease heterogeneity in systemic lupus erythematosus. In this model, once an individual is potentiated for the disease, the autoantibodies produced are strongly influenced by genetic factors including the DQ1/DQ2 heterozygous state for anti-Ro/SSA. Subsequently, the combination of autoantibodies influences the expression of disease found in individual patients. The plan for future experiments is to first test our recent findings in a larger patient group using analogous methods. In addition, the HLA-DQ genes associated with the anti-Ro/SSA response will be identified by allele-specific oligonucleotides and in selected groups DQ genes will be sequenced. Finally, two approaches will be explored to test the hypothesis that antigen presentation of Ro/SSA occurs through the defined DQ-alpha and DQ-beta elements, that the T cell recognizes this autoantigen-histocompatibility complex, and that autoantibody production results. First, in vitro cell culture techniques and an HLA-DQ transfected mouse cell line will be used to establish the HLA-DQ structural requirements. Second, a mouse model in which human grafts produce anti-Ro/SSA may also be suitable. In this model, immunization with small amounts of purified Ro/SSA antigen leads to a substantial rise of the human anti-Ro/SSA autoantibodies produced by these mice. Overall, these experiments will define the contribution to disease heterogeneity from HLA-DQ and will identify and test the molecular and structural features of DQ-alpha and DQ-beta which are important in gene complementation for anti- Ro/SSA.
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