Charles Edgar D, Orloff Michael I M, Nishiuchi Eiko, Marukian Svetlana, Rice Charles M, Dustin Lynn B
Rockefeller University, New York, New York.
Arthritis Rheum. 2013 Sep;65(9):2430-40. doi: 10.1002/art.38041.
Hepatitis C virus (HCV) is the most frequent cause of mixed cryoglobulinemia (MC), which is characterized by endothelial deposition of rheumatoid factor (RF)-containing immune complexes and end-organ vasculitis. MC is a lymphoproliferative disorder in which B cells express RF-like Ig, yet its precise antigenic stimulus is unknown. We have proposed that IgG-HCV immune complexes stimulate B cell expansion and somatic hypermutation (SHM)-induced affinity maturation in part via engagement of an RF-like B cell receptor. This study was undertaken to test the hypothesis that SHM augments RF activity.
RFs cloned from single B cells from 4 patients with HCV-associated MC (HCV-MC) were expressed as IgM, IgG, or IgG Fab. Selected Ig were reverted to germline. RF activity of somatically mutated Ig and germline-reverted Ig was determined by enzyme-linked immunosorbent assay.
Ig with SHM had RF activity, with the preference for binding being highest for IgG1, followed by IgG2 and IgG4, and lowest for IgG3, where there was no detectable binding. In contrast, reverted germline IgG exhibited markedly diminished RF activity. Competition with 1 μg/ml of protein A abrogated RF activity, suggesting specificity for IgG Fc. Swapping of mutated heavy-chain pairs and light-chain pairs also abrogated RF activity, suggesting that context-specific pairing of appropriate IgH and Igκ, in addition to SHM, is necessary for RF activity.
SHM significantly contributes to RF activity in HCV-MC patients, suggesting that autoreactivity in these patients arises through antigen-dependent SHM, as opposed to nondeletion of autoreactive germline Ig.
丙型肝炎病毒（HCV）是混合性冷球蛋白血症（MC）最常见的病因，其特征为含类风湿因子（RF）的免疫复合物在内皮沉积以及终末器官血管炎。MC是一种淋巴细胞增殖性疾病，其中B细胞表达RF样Ig，但其确切的抗原刺激尚不清楚。我们提出IgG-HCV免疫复合物部分通过RF样B细胞受体的结合刺激B细胞扩增和体细胞超突变（SHM）诱导的亲和力成熟。本研究旨在验证SHM增强RF活性这一假说。
从4例HCV相关MC（HCV-MC）患者的单个B细胞中克隆的RFs表达为IgM、IgG或IgG Fab。选择的Ig恢复为胚系状态。通过酶联免疫吸附测定法测定体细胞突变Ig和胚系恢复Ig的RF活性。
具有SHM的Ig具有RF活性，对IgG1的结合偏好最高，其次是IgG2和IgG4，对IgG3的结合偏好最低，IgG3无明显结合。相比之下，恢复的胚系IgG的RF活性明显降低。与1μg/ml蛋白A竞争可消除RF活性，提示对IgG Fc具有特异性。突变重链对和轻链对的交换也可消除RF活性，提示除SHM外，适当的IgH和Igκ的上下文特异性配对对于RF活性也是必需的。
SHM对HCV-MC患者的RF活性有显著贡献，提示这些患者的自身反应性是通过抗原依赖性SHM产生的，而非自身反应性胚系Ig的未缺失。
