The etiologies of chronic autoimmune cytopenias like idiopathic thrombocytopenic purpura (AITP) and warm-reactive hemolytic anemia (AIHA) are unknown. This proposal will test the hypothesis that autoantibodies produced in each of these diseases originate from limited repertoires of immunoglobulin variable region germline genes, and that such preferential genetic utilization will be reflected by the presence of frequently shared idiotypes among platelet or red cell reactive autoantibodies from populations of AITP or AIHA patients. The proposed experimental approach is to generate multiple anti-idiotypic reagents, using as immunogens AITP and AIHA autoantibodies generated by two different techniques. The first is to develop human monoclonal autoantibody-secreting cell lines by a novel system: Mice homoxygous for the scid (severe combined immunodefiency) mutation will be stably engrafted with AITP or AIHA patient lymphocytes and immunized with human platelets or red cells, which may activate engrafted human cells but not the genetically unresponsive scid mouse lymphocytes; engrafted and immunized scid mice will be used to generate human monoclonal autoantibody producing hybridoma cells lines. The second approach is direct purification of autoantibodies from AITP and AIHA patients, using antigen-affinity techniques to yield highly enriched polyclonal preparations of platelet or red cell reactive autoantibodies. Both monoclonal and polyclonal autoantibodies will be used to produce BALB/c mouse monoclonal idiotype-specific antibodies. These AITP and AIHA anti-idiotypes will be used in conjunction with extant anti-idiotypes to other human autoantibodies (RF and anti-DNA) to establish the pattern of idiotype expression among multiple AITP or AIHA patients, their family members, and random unrelated unaffected subjects. Additionally, antibody idiotype-positive proteins prepared from various donors will be analyzed for immunoglobulin isotype and binding to relevant autoantigens in vitro, providing data regarding the association of idiotype expression with frank autoimmune disease. Finally, both murine anti-idiotypic reagents and human autoreactive B cell lines generated by the proposed activities will be tools for characterizing specific immunoglobulin genes that encode autoantibodies in AITP and AIHA. These studies will provide important new information regarding genetic parameters in human autoimmune diseases.
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