MOLECULAR GENETICS OF AUTOANTIBODIES IN HUMANS

这项研究的长期目标是描绘分子和遗传 持续产生潜在致病性自身抗体的基础 自身免疫性疾病 这些“疾病特异性”自身抗体很高， IgM和IgG同种型的亲和性自身抗体，如IgG类风湿性 类风湿性关节炎（RA）中的RF因子和 系统性红斑狼疮（SLE）。 在上一个资助期间，我们的 对来自RA患者的IgG RF的分析显示， 患者是克隆相关的，并且由天然自身抗体产生。 最近的免疫耐受研究表明，自身反应性B细胞 或者通过细胞凋亡而缺失，或者功能失活（无反应性）。 然而，一些自身反应性B细胞在自身免疫性MRL中不耐受。 lpr/lpr小鼠，其具有缺陷的Fas介导的凋亡途径。 是 已知，在选择压力下，具有存活率的变异细胞， 优势可以以克隆的方式扩大。 我们假设某些疾病 RA和SLE中分泌特异性自身抗体的B细胞可能逃避耐受 调节，因为：1）在糖尿病相关的功能丧失突变 基因或凋亡抑制基因中的功能获得性突变（如 Bcl-2）在克隆B细胞水平上的表达，导致耐药 变体;或2）刺激相关基因中的功能获得性突变 (such作为B7），导致组成性激活的无反应性变体。 到 研究这些假设的具体目的是： 1)疾病特异性自身抗体的产生和表征- 分泌B细胞系和同种型特异性对照Ig分泌细胞系 从RA和SLE患者身上。 2)Fas介导的细胞凋亡途径的比较分析 细胞系 将对每例患者的细胞系进行Fas分析 细胞表面表达、可溶性Fas的分泌和凋亡 分别由Fas配体（FasL）或细胞毒性抗Fas 抗体的 3)比较分析Bcl-2在这些细胞系中的表达。 4)Fas非依赖性、表面Ig介导的免疫抑制剂的比较分析 这些细胞系中的凋亡途径; 5)B7共刺激分子在这些细胞中表达的比较分析 线 6)确定上述研究中识别的缺陷是否也 存在于新鲜分离的自身反应性B细胞中， 患者

The long term goal of this study is to delineate the molecular and genetic basis for sustained production of potentially pathogenic autoantibodies in autoimmune diseases. These "disease specific" autoantibodies are high affinity autoantibodies of IgM and IgG isotypes, such as IgG rheumatoid factors (RFs) in rheumatoid arthritis (RA) and IgG anti-DNA antibodies in systemic lupus erythematosus (SLE). During the last funding period, our analysis of IgG RFs from RA patients has revealed that the RFs in each patient are clonally related, and arise from natural autoantibodies. Recent studies of immunological tolerance show that self-reactive B cells either are deleted by apoptosis or are functionally inactivated (anergy). However, some self-reactive B cells are not tolerized in autoimmune MRL- lpr/lpr mice, which have a defective Fas-mediated apoptosis pathway. It is known that, under selection pressure, a variant cell with a survival advantage can expand in a clonal manner. We hypothesize that some disease specific autoantibody-secreting B cells in RA and SLE may escape tolerance regulation because of: 1) loss-of-function mutations in apoptosis-related genes or gain-of-function mutations in apoptosis suppressor genes (such as Bcl-2) at the clonal B cell level, resulting i tolerance-resistant variants; or 2) gain-of-function mutations in stimulation-related genes (such as B7), resulting in constitutively activated anergic variants. To examine these hypotheses, the specific aims are: 1) Generation and characterization of disease specific autoantibody- secreting B cell lines and isotype-specific control Ig-secreting cell lines from RA and SLE patients. 2) Comparative analysis of the Fas-mediated apoptosis pathway in these cell lines. Cell lines from each patient will be analyzed for Fas expression on the cell surface, secretion of soluble Fas, and apoptosis induced separately by either Fas ligand (FasL) or cytotoxic anti-Fas antibodies. 3) Comparative analysis of Bcl-2 expression in these cell lines. 4) Comparative analysis of the Fas-independent, surface Ig-mediated apoptosis pathway in these cell lines; 5) Comparative analysis of the B7 costimulator expression in these cell lines. 6) Determination if the defects identified in the above studies are also present in freshly isolated autoreactive B cells from corresponding patients.