Barcode-enabled sequencing of plasmablast antibody repertoires in rheumatoid arthritis.
Barcode-enabled sequencing of plasmablast antibody repertoires in rheumatoid arthritis.
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DOI：
10.1002/art.38754
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发表时间：
2014-10
期刊：
影响因子：
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通讯作者：
Robinson WH
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文献类型：
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作者：
Tan YC;Kongpachith S;Blum LK;Ju CH;Lahey LJ;Lu DR;Cai X;Wagner CA;Lindstrom TM;Sokolove J;Robinson WH
类风湿性关节炎 (RA) 的一个标志是产生自身抗体，包括抗瓜氨酸蛋白抗体 (ACPA)。然而，这些自身抗体的具体靶点仍未完全确定。在免疫反应过程中，对刺激抗原具有特异性的 B 细胞被激活并分化为“浆母细胞”，然后释放到血液中。在这项研究中，我们对浆母细胞抗体库进行测序，以确定 RA 主动免疫反应的靶标。我们开发了一种新的 DNA 条形码方法，对 RA 中个体血液浆母细胞表达的同源重链和轻链抗体对进行测序。该方法使用通用 5' 接头，能够对抗体可变区进行全长测序并重组表达配对抗体链。对序列数据集进行生物信息分析，生成系统发育树，识别共享重链和轻链 VJ 序列的抗体克隆家族。表达了代表性抗体，并使用 CCP2 ELISA 和抗原微阵列表征了它们的结合特性。我们使用测序方法生成了代表 4 名抗 CCP+ RA 个体外周血浆母细胞抗体库的系统发育树，并重组表达了 14 种抗体，这些抗体要么是“单例”抗体，要么是克隆抗体家族的代表。 CCP2 ELISA 鉴定出四种 ACPA，抗原微阵列分析鉴定出差异靶向 α-烯醇酶、瓜氨酸纤维蛋白原和瓜氨酸组蛋白 2B 上表位的 ACPA。我们的数据提供证据表明，针对 α-烯醇化酶、瓜氨酸纤维蛋白原和瓜氨酸组蛋白 2B 的自身抗体是由 RA 中持续激活的 B 细胞反应产生的，因此可能有助于 RA 的发病机制。
A hallmark of rheumatoid arthritis (RA) is the production of autoantibodies, including anti-citrullinated protein antibodies (ACPAs). Nevertheless, the specific targets of these autoantibodies remain incompletely defined. During an immune response, B cells specific for the inciting antigen(s) are activated and differentiate into “plasmablasts”, which are released into the blood. In this study we sequence the plasmablast antibody repertoire to define the targets of the active immune response in RA. We developed a novel DNA barcoding method to sequence the cognate heavy- and light-chain pairs of antibodies expressed by individual blood plasmablasts in RA. The method uses a universal 5’ adapter that enables full-length sequencing of the antibodies’ variable regions and recombinant expression of the paired antibody chains. The sequence datasets were bioinformatically analyzed to generate phylogenetic trees that identify clonal families of antibodies sharing heavy- and light-chain VJ sequences. Representative antibodies were expressed, and their binding properties characterized using CCP2 ELISA and antigen microarrays. We used our sequencing method to generate phylogenetic trees representing the antibody repertoires of peripheral blood plasmablasts of 4 individuals with anti-CCP+ RA, and recombinantly expressed 14 antibodies that were either “singleton” antibodies or representative of clonal antibody families. CCP2 ELISA identified four ACPAs, and antigen microarray analysis identified ACPAs that differentially targeted epitopes on α-enolase, citrullinated fibrinogen, and citrullinated histone 2B. Our data provide evidence that autoantibodies targeting α-enolase, citrullinated fibrinogen, and citrullinated histone 2B are produced by the ongoing activated B cell response in, and thus may contribute to the pathogenesis of, RA.