HUMAN MONOCLONAL AUTOANTIBODIES FROM IMMUNE CYTOPENIAS

拟议的研究旨在确定和表征 人抗血小板和红细胞自身抗体 它们是在免疫性血小板减少症患者中产生的 紫癜性贫血(ITP)和温免疫性溶血性贫血(IHA)。这个 方法将是直接研究抗体的产生 ITP和ITP患者外周血和脾中的淋巴细胞 IHA患者通过丰富各自的靶细胞-免疫 B淋巴细胞。然后这些淋巴细胞亚群将被 受到各种文化条件的影响才能影响 抗血小板和抗红细胞抗体的体外合成 使用新鲜靶点的定量放射免疫分析检测 细胞和一种人类特异性的碘化单抗 免疫球蛋白。 这些浓缩的淋巴细胞培养物将用于融合 与人类来源的淋巴母细胞系或将是 由Epstein-Barr病毒转化为细胞 分泌人源性抗血小板和抗红细胞单抗的细胞株 抗体。将对产生免疫球蛋白的培养物进行筛选 用于与汇集的正常人类血小板、红细胞和 以确定抗体的特异性，并将 关于免疫球蛋白亚类和 抗体独特型。主要目标是生产一种 一组靶细胞特异性的人类单抗 代表所有四个免疫球蛋白亚类；其次，单克隆性 将获得其他重链类别的自身抗体。 一旦获得抗血小板和抗红细胞的单克隆体 抗体将被用作工具来识别和表征 各自的血细胞自身抗原参与了 ITP和IHA。这些单克隆自身抗体也将是 用于研究不同人的相对能力 免疫球蛋白重链亚型以及抗体 以不同的抗原特异性，介导Fc受体 依赖的人类效应细胞对各自的 人类体外靶细胞。

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.