Key Points Anti-NET/cit-H2B RA-rmAb immunoreactivity is dependent on somatic hypermutation. SHM in the HC and/or LC is necessary for the anti-NET/cit-H2B immunoreactivity. Fab–N-linked–glycosylation introduced by SHM can influence NET–Ag binding. We previously showed that anti–neutrophil extracellular trap (NET) rheumatoid arthritis (RA)-rmAbs derived from CD19+ B cells within RA human synovial tissues frequently react against NETs. In this study, we aimed to characterize the importance of affinity maturation via somatic hypermutation (SHM) within the Ig variable H (VH) and variable L (VL) chains and Fab–N-linked glycosylation in RA synovial B cell clones reactive to NETs and NET-derived Ags such as citrullinated histones. Selected anti-NET RA-rmAbs derived from synovial RA CD19+ B cells were subjected to overlap-PCR to generate germline (GL; VH and VL reverted into GL), hybrid clones (VH/VL region reverted into GL), and N-glycosylation mutants (N→Q) and analyzed for anti-NETs and citrullinated histones (cit-H2B) immunoreactivity. Anti-NET/cit-H2B immunoreactivity of selected RA-rmAbs was abrogated in the VH and VL GL counterpart. In RA B cell hybrid clone RA015/11.88 and RA056/11.23.2, NET and/or cit-H2B immunoreactivity was solely dependent on SHM in the IgVH region whereas RA B cell hybrid clone RA015/11.91 required affinity maturation of both VH and VL for efficient binding to cit-H2B. In 7/80 RA-rmAb, SHM resulted in ex novo N-glycosylation sites in VH and/or VL regions. Removal of Fab-linked glycans in RA056/11.23.2 in the N-mutant counterpart resulted in 90% reduction in immunoreactivity to cit-H2B. Thus, SHM in the IgVH and/or VL regions of RA synovial B cells is necessary for the immunoreactivity to NET-Ags. Fab–N-linked–glycosylation introduction sites are observed in a minority of anti-NET B cell clones but can strongly influence NET-Ag binding.

中文翻译：

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H 和 L 链亲和力成熟和/或 Fab N-糖基化影响类风湿性关节炎滑膜 B 细胞克隆中对中性粒细胞胞外陷阱抗原的免疫反应性

要点 Anti-NET/cit-H2B RA-rmAb 免疫反应性取决于体细胞超突变。HC 和/或 LC 中的 SHM 是抗 NET/cit-H2B 免疫反应所必需的。SHM 引入的 Fab-N-连接糖基化可以影响 NET-Ag 结合。我们之前表明，来自 RA 人体滑膜组织内 CD19+ B 细胞的抗中性粒细胞胞外陷阱 (NET) 类风湿性关节炎 (RA)-rmAb 经常对 NET 产生反应。在这项研究中，我们旨在通过 Ig 可变 H (VH) 和可变 L (VL) 链中的体细胞超突变 (SHM) 和对 NET 有反应的 RA 滑膜 B 细胞克隆中的 Fab-N 连接糖基化来表征亲和力成熟的重要性和 NET 衍生的 Ags，例如瓜氨酸化组蛋白。对来自滑膜 RA CD19+ B 细胞的选定抗 NET RA-rmAb 进行重叠 PCR 以产生种系（GL；VH 和 VL 恢复为 GL）、杂交克隆（VH/VL 区域恢复为 GL）和 N-糖基化突变体（N→Q）并分析抗 NET 和瓜氨酸化组蛋白 (cit-H2B) 免疫反应性。所选 RA-rmAb 的抗 NET/cit-H2B 免疫反应性在 VH 和 VL GL 对应物中被废除。在 RA B 细胞杂交克隆 RA015/11.88 和 RA056/11.23.2 中，NET 和/或 cit-H2B 免疫反应性仅依赖于 IgVH 区域中的 SHM，而 RA B 细胞杂交克隆 RA015/11.91 需要 VH 和 VL 的亲和力成熟有效结合 cit-H2B。在 7/80 RA-rmAb 中，SHM 在 VH 和/或 VL 区域产生新的 N-糖基化位点。在 N 突变对应物中去除 RA056/11.23.2 中的 Fab 连接聚糖导致对 cit-H2B 的免疫反应性降低 90%。因此，RA 滑膜 B 细胞的 IgVH 和/或 VL 区域中的 SHM 对于 NET-Ag 的免疫反应性是必需的。在少数抗 NET B 细胞克隆中观察到 Fab-N-连接的-糖基化引入位点，但可以强烈影响 NET-Ag 结合。