Stanford University, Stanford, California, and VA Palo Alto Health Care System, Palo Alto, California.
Arthritis Rheumatol. 2014 Oct;66(10):2706-15. doi: 10.1002/art.38754.
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. We undertook this study to 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 data sets 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 were characterized using anti-cyclic citrullinated peptide 2 (anti-CCP-2) enzyme-linked immunosorbent assay (ELISA) and antigen microarrays.
We used our sequencing method to generate phylogenetic trees representing the antibody repertoires of peripheral blood plasmablasts from 4 individuals with anti-CCP+ RA, and recombinantly expressed 14 antibodies that were either "singleton" antibodies or representative of clonal antibody families. Anti-CCP-2 ELISA identified 4 ACPAs, and antigen microarray analysis identified ACPAs that differentially targeted epitopes on α-enolase, citrullinated fibrinogen, and citrullinated histone H2B.
Our data provide evidence that autoantibodies targeting α-enolase, citrullinated fibrinogen, and citrullinated histone H2B are produced by the ongoing activated B cell response in, and thus may contribute to the pathogenesis of, RA.
类风湿关节炎（RA）的一个特征是产生自身抗体，包括抗瓜氨酸化蛋白抗体（ACPAs）。然而，这些自身抗体的具体靶标仍不完全明确。在免疫反应中，针对引发抗原的 B 细胞被激活并分化为浆母细胞，然后被释放到血液中。我们进行这项研究是为了对浆母细胞抗体库进行测序，以确定 RA 中活跃免疫反应的靶标。
我们开发了一种新的 DNA 条码方法，对 RA 患者血液浆母细胞中表达的抗体的重链和轻链对进行测序。该方法使用通用的 5' 接头，能够对抗体可变区进行全长测序，并对配对的抗体链进行重组表达。通过生物信息学分析对序列数据集进行分析，生成识别共享重链和轻链 VJ 序列的抗体克隆家族的系统发育树。代表性抗体被表达，并使用抗环瓜氨酸肽 2（抗-CCP-2）酶联免疫吸附试验（ELISA）和抗原微阵列来表征其结合特性。
我们使用我们的测序方法生成了代表 4 名抗 CCP+RA 患者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“单一”抗体，要么代表克隆抗体家族。抗-CCP-2 ELISA 鉴定出 4 种 ACPA，抗原微阵列分析鉴定出针对α-烯醇化酶、瓜氨酸化纤维蛋白原和瓜氨酸化组蛋白 H2B 的不同表位的 ACPA。
我们的数据提供了证据表明，针对α-烯醇化酶、瓜氨酸化纤维蛋白原和瓜氨酸化组蛋白 H2B 的自身抗体是由 RA 中持续激活的 B 细胞反应产生的，因此可能有助于其发病机制。
