The proposed studies are designed to identify and characterize human autoantibodies to platelets and red blood cells (rbc) which are produced in patients with immune thrombocytopenic purpura (ITP) and warm immune hemolytic anemia (IHA). The approach will be to directly study antibody-producing lymphocytes from the peripheral blood and spleens of ITP and IHA patients by enriching for the respective target cell-immune B lymphocytes. These lymphocyte subpopulations will then be subjected to a variety of culture conditions in order to effect in vitro synthesis of antiplatelet and anti-rbc antibodies as detected by a quantitative radioimmunoassay using fresh target cells and an iodinated monoclonal antibody specific for human IgG. These enriched lymphocyte cultures will be used in fusions with human-derived lymphoblastoid cell lines or will be transformed by Epstein-Barr virus in order to produce cell lines secreting human monoclonal anti-platelet and anti-rbc antibodies. Cultures producing immunoglobulin will be screened for reactivity with pooled, normal human platelets, rbc and lymphocytes in order to define antibody specificity and will be characterized with respect to immunoglobulin subclass and antibody idiotype. The primary goal is the production of a panel of target cell-specific human monoclonal antibodies which represent all four IgG subclasses; secondarily, monoclonal autoantibodies of other heavy chain classes will be obtained. Once obtained, the monoclonan anti-platelet and anti-rbc antibodies will be used as tools to identify and characterize the respective blood cell autoantigens which are involved in ITP and IHA. These monoclonal autoantibodies will also be employed in studies of the relative abilities of different immunoglobulin heavy chain isotypes, as well as of antibodies with different antigen specificities, to mediate Fc receptor dependent human effector cell functions against the respective human target cells in vitro.
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