RED BLOOD CELL AUTOANTIBODIES-- B CELL ORIGIN AND ANTIGENIC TARGETS

红细胞自身抗体——B 细胞起源和抗原靶标

• 批准号: 6242488
• 负责人: Leslie Eric Silberstein
• 金额: $ 35.52万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6242488
• 关键词: B lymphocyte; antibody specificity; autoantibody; autoimmune hemolytic anemia; cell line; clinical research; enzyme linked immunosorbent assay; erythrocytes; genetic library; hematopoiesis; hemolytic anemia; human subject; human tissue; immune tolerance /unresponsiveness; immunogenetics; immunoglobulin genes; immunoglobulin structure; leukocyte activation /transformation; monoclonal antibody; tissue /cell culture; transfection; transforming virus; western blottings

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.

通过表征可以极大地促进自身免疫性疾病的研究 与疾病相关的自身抗体明显有助于 致病过程。 红细胞 (RBC) 自身抗体是一个例子 此类病理性自身抗体，因为它们直接负责 B 细胞恶性肿瘤患者存在自身免疫性溶血性贫血 系统性红斑狼疮（SLE）。 通过确定它们的频谱 抗原精细特异性和其他结构特性，我们可以开始 询问有关其克隆性和细胞起源的问题 最终探索致病性的分子基础。 拟议的研究将利用最近描述的细胞和 分子生物学方法从红细胞中分离出红细胞自身反应性 B 细胞 将用于常规 B 细胞永生化的患者和/或 展示免疫球蛋白Fab的M13噬菌体文库的制备 它们的表面上有碎片。 具体来说，我们将 (1) 建立来自患有以下疾病的患者的 B 细胞的克隆扩增： 使用最近描述的系统治疗自身免疫性溶血性贫血 白细胞介素-4和-10、抗CD40以及转染的Ltk细胞系 人类 FcgammaRII/CDw32 受体。 来自这些扩展的上清液 将通过固相配体筛选 B 细胞的红细胞自身反应性 结合测定。 感兴趣的 B 细胞扩增将通过以下方式永生化： 常规方法（例如 Epstein Barr 病毒、体细胞杂交） 和/或用于制备M13噬菌体免疫球蛋白展示文库。 这 方法将提供手段（即表达的免疫球蛋白-mRNA、细胞 细胞系衍生的抗体、细菌表达的 Fab 分子）以完成 以下目标； (2) 分离并表征红细胞自身抗原结构 免疫沉淀和免疫印迹技术。 此外， 源自细胞系的自身反应性免疫球蛋白，噬菌体展示 图书馆和/或患者红细胞洗脱物将提供相关 用于未来筛选 m13 肽展示文库的材料 可以提供有关相应自身抗原的有用信息 免疫球蛋白结合表位；和 (3) 对编码红细胞自身特异性的可变区基因进行测序，并 检查 RBC 相对于 B 细胞的自身免疫反应的性质 抗原克隆选择的起源和作用。 总的来说，这些分析将提供对起源和精细的洞察。 致病性和非致病性红细胞自身抗体的特异性。 在 此外，它们对于未来的调查至关重要 这些自身反应性 B 细胞的调节以及治疗药物的产生 和诊断方法。