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

自身免疫性疾病的研究极大地促进了对其特征的研究 与疾病相关的自身抗体显然有助于 致病过程。红细胞(RBC)自身抗体就是 这种病理性自身抗体直接导致了 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)。通过确定它们的光谱 抗原性良好的特异性和其他结构特性，人们可以开始 询问有关它们的克隆性和细胞来源的问题 最终探索致病的分子基础。 拟议的研究将利用最近描述的细胞和 分离RBC自身反应性B细胞的分子生物学方法 患者将用于传统的B细胞永生化和/或 免疫球蛋白Fab噬菌体M13文库的制备 它们表面上的碎片。具体来说，我们会 (1)建立慢性支气管炎患者B细胞的克隆扩增方法 使用最近描述的一种系统的自身免疫性溶血性贫血 白细胞介素4和10、抗CD40抗体及转染人角质形成细胞株 人FcGammaRII/CDw32受体。从这些扩大的上清液 通过固相配基筛选B细胞的红细胞自反应性 结合试验。然后，感兴趣的B细胞扩增将永垂不朽 常规方法(如爱泼斯坦-巴尔病毒、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(即表达免疫球蛋白-mRNA、细胞 线源抗体、细菌表达的Fab分子)来完成 以下目标； (2)分离和鉴定RBC自身抗原结构 免疫沉淀和免疫印迹技术。此外, 来源于细胞系的自身反应性免疫球蛋白，噬菌体展示 图书馆和/或患者的红细胞洗脱液将提供相关的 为将来筛选M13肽展示文库提供材料 可以提供关于相应自身抗原的有用信息 免疫球蛋白结合表位(S)； (3)对编码RBC自身特性的可变区基因进行测序 检查与B细胞相关的RBC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罚款的洞察 致病性和非致病性红细胞自身抗体的特异性。在……里面 此外，它们将是未来对 这些自身反应性B细胞的调节和治疗性B细胞的产生 和诊断方法。

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.