The study of autoimmune disorders is greatly facilitated by characterizing the disease-associated autoantibodies which clearly contribute to the pathogenic process. Red blood cell (RBC) autoantibodies are an example of such pathologic autoantibodies as they are directly responsible for the autoimmune hemolytic anemia present in patients with B-cell malignancies an systemic lupus erythematosus (SLE). By determining the spectrum of their antigenic fine specificities and other structural properties, one can begin to ask questions regarding their clonality and cellular origin and ultimately explore the molecular basis for pathogenicity. The proposed research will utilize recently-described cellular and molecular biological approaches to isolate RBC autoreactive B-cells from patients which will be used for conventional B-cell immortalization and/or the preparation of M13 phage libraries displaying immunoglobulin Fab fragments on their surfaces. Specificallly, we will (1) establish the clonal expansion of B-cells from patients with autoimmune hemolytic anemia using a recently described system involving interleukins-4 and -10, anti-CD40, and the Ltk-cell line transfected with the human FcgammaRII/CDw32 receptor. The supernatants from these expanded B-cells will be screened for RBC autoreactivity by a solid phase ligand binding assay. B-cell expansions of interest will then be immortalized by conventional methods (e.g. Epstein Barr virus, somatic cell hybridization) and/or used to prepare M13 phage immunoglobulin display libraries. This approach will provide the means (i.e. expressed immunoglobulin-mRNA, cell line-derived antibodies, bacterially-expressed Fab molecules) to accomplish the following goals; (2) isolate and characterize the RBC autoantigenic structures by immunoprecipitation and immunoblotting techniques. In addition, autoreactive immunoglobulin derived from cell lines, phage display libraries, and/or patients red cell eluates, will provide the relevant material for the future screening of m13 peptide display libraries which may provide useful information regarding the corresponding autoantigen immunoglobulin-binding epitope(s); and (3) sequence the variable region genes encoding RBC autospecificities and examine the nature of the RBC autoimmune response with respect to B cell origin and the role of clonal selection by antigen. Collectively, these analyses will provide insight into the origin and fine specificity of pathogenic and non-pathogenic RBC autoantibodies. In addition, they will be fundamental to future investigations on the regulation of these autoreactive B cells and athe generation of therapeutic and diagnostic approaches.
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