Large-Scale Sequencing and Characterizing of Autoantibody Responses

主要项目将通过使用罗宾逊实验室最近开发的称为“抗体库捕获”（ARC）的DNA条形码技术来表征自身免疫性疾病患者的B细胞反应。该方法将DNA条形码与下一代测序相结合，以实现对单个浆母细胞或抗原特异性B细胞表达的成对重链（HC）和轻链（LC）免疫球蛋白基因的大规模表征。尽管存在用于分析抗体的方法，但是没有一种方法能够全面地表征参与主动免疫应答的抗体，然后生物信息学地鉴定那些最可能是功能性的，即，那些要么驱动疾病，要么作为触发致病性自身免疫反应的关键抗原的标识符。ARC生成的测序数据集的规模使得能够通过生物信息学生成基因组的“系统发育树”。 抗体库这些系统发育树指导亲和力成熟抗体的克隆家族的鉴定，从而合理选择关键抗体，然后可以表达关键抗体以直接分析其结合和功能特性。我们建议使用ARC对类风湿性关节炎（RA）和系统性红斑狼疮（SLE）中的自身抗体反应进行测序和全面剖析，并利用其他ACE项目和核心的资源，为了验证单克隆自身抗体通过形成促炎免疫复合物（IC）双重刺激免疫细胞（通过同时进行模式识别 受体和B细胞或Fc受体）。例如，我们假设RA相关的抗瓜氨酸抗体 蛋白抗体（ACPA）形成IC，双重刺激巨噬细胞产生TNF，和B SLE相关抗核抗体（ANA）结合核抗原， 从而形成双重刺激树突细胞产生IFN和B细胞产生ANA的IC。 在目标1中，我们将使用ARC对RA或SLE患者的抗体库进行测序，并鉴定与特定临床亚型或治疗反应相关的抗体谱。 在目标2中，我们将克隆和 表达来自RA或SLE个体的合理选择的、亲和力成熟的抗体，并阐明其 自身抗原靶标。 在目标3中，将表征目标2中鉴定的关键RA和SLE重组抗体 并揭示它们导致自身免疫性炎症的机制。成功将提供 深入了解自身抗体的作用和机制，他们有助于发病机制， RA和SLE，并可能导致新的诊断测试和治疗方法的发展。

The Principal Project will characterize B cell responses in patients with autoimmune disease by using a DNA-barcoding technology recently developed in the Robinson lab, termed 'antibody repertoire capture' (ARC). The approach couples DNA barcoding with next-generation sequencing to enable large-scale characterization of the paired heavy-chain (HC) and light-chain (LC) immunoglobulin genes expressed by single plasmablasts or antigen-specific B cells. Although methods exist for profiling antibodies, none are able to comprehensively characterize the, antibodies involved in an active immune response and to then bioinformatically identify those most likely to be functional i.e., those that either drive the disease or serve as identifiers of the key antigens that trigger pathogenic autoimmune responses. The scale of the sequencing datasets generated by ARC enables bioinformatic generation of "phylogenetic trees" of the antibody repertoire. These phylogenetic trees guide identification of clonal families of affinity-matured antibodies and thereby rational selection of key antibodies, which can then be expressed for direct analysis of their binding and functional properties. We propose to use ARC to sequence and comprehensively dissect the autoantibody responses in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and, leveraging resources from the other ACE Projects and Cores, to test the overarching hypothesis that monoclonal autoantibodies contribute to the pathogenesis of RA and SLE by forming proinflammatory immune complexes (ICs) that dual-stimulate immune cells (by simultaneously engaging a pattern recognition receptor and either the B-cell or the Fc receptor). For instance, we hypothesize that RA-associated anti-citrullinated proteins antibodies (ACPAs) form ICs that dual-stimulate macrophages to produce TNF, and B cells to produce ACPAs; and that SLE-associated anti-nuclear antibodies (ANAs) bind nuclear antigens and thereby form ICs that dual-stimulate dendritic cells to produce IFN, and B cells to produce ANAs. In Aim 1, we will use ARC to sequence the antibody repertoires in patients with RA or SLE and identify antibody profiles that are associated with specific clinical subtypes or response to therapy. In Aim 2, we will clone and express rationally selected, affinity-matured antibodies from individuals with RA or SLE, and elucidate their autoantigen targets. In Aim 3, will characterize key RA and SLE recombinant antibodies identified in Aim 2 and uncover mechanisnis by which they contribute to autoimmune inflammation. Success would provide insights into the role of autoantibodies and the mechanisms by which they contribute to the pathogenesis of RA and SLE, and could lead to development of novel diagnostic tests and therapeutic approaches.