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

通过表征自身免疫性疾病的特征， 疾病相关的自身抗体，这显然有助于 致病过程 红细胞（RBC）自身抗体是一个例子， 这种病理性自身抗体，因为它们直接负责 自身免疫性溶血性贫血存在于B细胞恶性肿瘤患者中， 系统性红斑狼疮（SLE）。 通过测定它们的光谱 抗原的精细特异性和其他结构特性，人们可以开始 询问关于它们的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 分子生物学方法分离RBC自身反应性B细胞， 将用于常规B细胞永生化的患者和/或 M13噬菌体抗体库的制备 碎片在他们的表面。 具体来说，我们将 (1)建立B细胞的克隆扩增， 自身免疫性溶血性贫血使用最近描述的系统， 白细胞介素-4和白细胞介素-10、抗-CD40和转染有白细胞介素-4和白细胞介素-10、抗-CD40的Ltk细胞系 人Fc γ RII/CDw32受体。 这些扩增的上清液 将通过固相配体筛选B细胞的RBC自身反应性 结合分析 感兴趣的B细胞扩增然后将通过以下方式永生化： 常规方法（例如Epstein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 方法将提供方法（即表达的免疫球蛋白mRNA、细胞 细胞系来源的抗体、细菌表达的Fab分子）， 以下目标; (2)分离和表征RBC自身抗原结构， 免疫沉淀和免疫印迹技术。 此外，本发明还提供了一种方法， 来源于细胞系的自身反应性免疫球蛋白，噬菌体展示 库和/或患者红细胞洗脱液，将提供相关的 用于将来筛选M13肽展示文库的材料 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;和 (3)对编码RBC自身特异性的可变区基因进行测序， 检查红细胞自身免疫反应对B细胞的性质 起源和抗原克隆选择的作用。 总的来说，这些分析将提供深入了解的起源和罚款 病原性和非病原性RBC自身抗体的特异性。 在 此外，它们将是未来调查的基础。 这些自身反应性B细胞的调节和治疗性药物的产生 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.