TISSUE-SPECIFIC ANTIGEN RECEPTOR REPERTOIRES IN RA

RA 中的组织特异性抗原受体库

• 批准号: 3804218
• 负责人: HARRY W SCHROEDER
• 金额: --
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3804218
• 关键词: B cell receptor; B lymphocyte; antibody formation; antibody specificity; autoantigens; clone cells; computer assisted sequence analysis; gene mutation; gene rearrangement; genetic library; genetic polymorphism; human subject; immunoglobulin genes; immunoglobulin idiotypes; leukocyte activation /transformation; molecular cloning; molecular pathology; nucleic acid probes; polymerase chain reaction; rheumatoid arthritis; rheumatoid factor; synovial fluid

Diseased synovium in severe, active rheumatoid arthritis contains lymphocyte germinal centers able to produce immunoglobulin at splenic levels. Studies of these immunoglobulins have focused on rheumatoid factor (RF) content and have shown that, in seropositive RA, 5-10% of the repertoire consists of both 1gM and 1gG anti-1gG Fc. There is strong circumstantial evidence that the resultant immune complexes are involved in the pathogenesis and progression of the disease. However, the three idiotypes which are most commonly expressed by 1gM RF paraproteins, e.g., Wa, Po and Bla, appear to be a minor component of synovia RF. Thus, the genetic composition of the RA synovial antibody repertoire - both non-RF and RF - remains an open question. During the course of this project, we will analyze the antibody repertoire expressed by synovial mononuclear cells in order to test the hypothesis that synovial immunoglobulin production is antigen-driven and, therefore, involved in the pathogenesis of the disease. Considerable support for this approach derives from murine models. Sequence analysis of antibody repertoires in the MRL/lpr mouse suggests that autoantibodies generated during nonspecific stimulation by LPS or secondary immunization appear to be polyclonal and reflect the germline repertoire; whereas in autoimmune states, highly self-reactive antibodies are clonally related and demonstrate all the characteristics of antigen-driven immunoglobulin production. Murine models also suggest that the physico-chemical properties of specific V germline elements may influence the manifestations of autoimmune disease, hence polymorphism or heteromorphism in the genomic repertoire may be an additional, independent factor in disease susceptibility. cDNA libraries from rheumatoid synovial cells will be generated and the antibody repertoire characterized at the nucleotide sequence level. Evidence of restriction in V region utilization in Cmu- and Ckappa-containing transcripts will be sought. Comparisons will be made between RF paraproteins, the germline compartment, EBV-transformed synovial cell lines from RA patients, and synovial repertoires at different stages of disease. Evidence of antigen-specific clonal expansion will be sought by analyzing the Vh-Dh-Jh joins of representative Cgamma- and Calpha-containing transcripts as well as the Vkappa-Jkappa cDNAs. Analysis of the extent and distribution of somatic mutation among these clones will provide additional evidence for or against antigen-driven selection. Combinatorial expression libraries will be generated to test for possible antigen specificity. Candidate germline autoreactive elements will be identified, their extent of polymorphism in the human population established, and their correlation, if any, to selected disease manifestations established. These studies should not only shed significant light into a poorly understood manifestation of rheumatic disease, but also serve as reference for future studies of abnormal immune responses.

严重的活动性类风湿关节炎的滑膜组织中含有 脾淋巴细胞增殖中心能产生免疫球蛋白 程度. 这些免疫球蛋白的研究集中在类风湿因子 (RF)含量，并已表明，在血清阳性RA，5-10%的 库由1gM和1gG抗1gG Fc组成。 有强有力 间接证据表明，所产生的免疫复合物参与了 疾病的发病机制和进展。 但三 最常由1gM RF副蛋白表达的独特型，例如， Wa、Po和Bla似乎是滑膜RF的次要组分。 因此 RA滑膜抗体库的遗传组成-非RF 而RF -仍然是一个悬而未决的问题。 在这个项目中，我们 将分析滑膜单核细胞表达的抗体库， 细胞，以测试假设，滑膜免疫球蛋白 产生是抗原驱动的，因此参与发病机制 的疾病。 这种方法得到了来自小鼠的大量支持。 模型 MRL/lpr小鼠抗体库的序列分析 表明在非特异性刺激过程中产生的自身抗体， LPS或二次免疫似乎是多克隆的，并反映了 生殖细胞库;而在自身免疫状态下， 抗体是克隆相关的，并表现出所有的特征， 抗原驱动的免疫球蛋白产生。 小鼠模型也表明， 特定V种系元件的物理化学性质可以 影响自身免疫性疾病的表现，因此多态性或 基因组库中的异型性可能是一个额外的，独立的， 疾病易感性的因素。 类风湿关节炎滑膜组织cDNA文库 将产生细胞，并在第一阶段表征抗体库。 核苷酸序列水平。 限制V区使用的证据 在Cmu-和Ckappa含有成绩单将被寻找。 比较将 在RF副蛋白、种系区室、EBV转化的 RA患者的滑膜细胞系，以及在不同的 疾病的阶段。 抗原特异性克隆扩增的证据将是 通过分析代表性的C γ-和C γ-的Vh-Dh-Jh连接来寻求 含有α的转录物以及Vkappa-Jkappa cDNA。 分析 这些克隆体中体细胞突变的程度和分布将 为支持或反对抗原驱动的选择提供了额外的证据。 将生成组合表达文库以测试可能的 抗原特异性 候选生殖系自身反应性元件将是 在人类群体中的多态性程度 已确定的，以及它们与选定疾病的相关性（如果有的话） 建立的表现。 这些研究不仅应该产生重大影响 光到一个不太清楚的风湿性疾病的表现，但也 作为今后研究异常免疫反应的参考。