Large-Scale Characterization of Autoantibody Responses in Rheumatoid Arthritis

描述(申请人提供)：类风湿性关节炎(RA)是一种自身免疫性滑膜炎，影响0.5%的世界人口，但关键的自身抗原靶点仍不清楚。自身抗体的产生，如抗瓜氨酸蛋白抗体(ACPA)，是类风湿关节炎的一个标志。然而，关键的ACPA和其他RA相关自身抗体以哪些抗原为靶标在很大程度上仍不清楚。同样未知的是这些ACPA是如何发展的，不同的RA患者之间的ACPA谱系有多相似，以及它们是否以及如何在RA的发病机制中起作用。到目前为止，研究领域缺乏手段来全面表征与特定疾病相关的自身抗体，然后合理地将它们筛选出重要的抗体--即那些要么驱动疾病，要么作为触发致病T细胞反应的关键抗原的识别符。我们现在已经开发出“抗体库捕获”技术，这是一种高通量的方法，使我们能够做到这一点。利用下一代测序的能力，我们开发了一种新的方法来对单个抗体表达细胞产生的所有cDNA进行条形码，从而高通量地分析由单个B细胞、浆母细胞或浆细胞表达的成对的重链和轻链免疫球蛋白基因。我们推测，我们可以通过定义RA患者血液中的浆母细胞和抗原分类记忆B细胞以及滑膜中的浆母细胞和浆细胞的抗体库来阐明与RA相关的致病自身抗体反应。然后，我们将对我们获得的抗体序列进行生物信息分析，以生成抗体库的进化树，从而识别和克隆可能是关键自身抗体的亲和力成熟的抗体。我们将确定这些重组的、亲和力成熟的自身抗体所针对的抗原，调查它们的特定序列是如何发展的，分析它们的结合和免疫刺激特性，并评估它们的致病性。这一建议的成功将阐明自身抗体反应的发展，并确定RA靶向的关键自身抗原，这一发现可能导致开发新的RA诊断和治疗方法。

DESCRIPTION (provided by applicant): Rheumatoid arthritis (RA) is an autoimmune synovitis that affects 0.5% of the world population, yet the key autoantigen targets remain unknown. Production of autoantibodies, such as the anti-citrullinated protein antibodies (ACPAs), is a hallmark of RA. However, which antigens the critical ACPAs and other RA- associated autoantibodies target remains largely unknown. Also unknown is how these ACPAs develop, how similar the ACPA repertoires are between different individuals with RA, and whether and how they contribute to the pathogenesis of RA. So far, the research field has lacked the means to comprehensively characterize the autoantibodies associated with a given disease and to then rationally winnow them to those that are important-that is, those that either drive the disease or serve as identifiers of the key antigens that trigger the pathogenic T-cell response. We have now developed "antibody repertoire capture" technology, a high- throughput method that allows us to do just that. Harnessing the power of next-generation sequencing, we have developed a novel method for barcoding all the cDNAs generated from individual antibody-expressing cells, thereby enabling high-throughput analysis of the paired heavy- and light-chain immunoglobulin genes expressed by single B cells, plasmablasts, or plasma cells. We hypothesize that we can elucidate the pathogenic autoantibody responses associated with RA by defining the antibody repertoire of plasmablasts and antigen-sorted memory B cells in the blood, and of plasmablasts and plasma cells in the synovium, of individuals with RA. We will then bioinformatically analyze the antibody sequences we obtain to generate evolutionary trees of the antibody repertoires and thereby identify and clone the affinity-matured antibodies that are likely the key autoantibodies. We will identify the antigens targeted by these recombinant, affinity-matured autoantibodies, investigate how their specific sequences develop, dissect their binding and immunostimulatory properties, and assess their pathogenicity. Success of this proposal would shed light on the development of the autoantibody response and identify the key autoantigens targeted in RA, findings that could lead to the development of new diagnostics and therapies for RA.