Anti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA). Although AMPA-IgG cross-reactivity to multiple post-translational modifications (PTMs) is evident, it is unknown whether the first responding B cells, expressing IgM, display similar characteristics or if cross-reactivity is crucially dependent on somatic hypermutation (SHM). We now studied the reactivity of (germline) AMPA-IgM to further understand the breach of B cell tolerance and to identify if cross-reactivity depends on extensive SHM. Moreover, we investigated whether AMPA-IgM can efficiently recruit immune effector mechanisms. Polyclonal AMPA-IgM were isolated from RA patients and assessed for cross-reactivity towards PTM antigens. AMPA-IgM B cell receptor sequences were obtained by single cell isolation using antigen-specific tetramers. Subsequently, pentameric monoclonal AMPA-IgM, their germline counterparts and monomeric IgG variants were generated. The antibodies were analysed on a panel of PTM antigens and tested for complement activation. Pentameric monoclonal and polyclonal AMPA-IgM displayed cross-reactivity to multiple antigens and different PTMs. PTM antigen recognition was still present, although reduced, after reverting the IgM into germline. Valency of AMPA-IgM was crucial for antigen recognition as PTM-reactivity significantly decreased when AMPA-IgM were expressed as IgG. Furthermore, AMPA-IgM was 15- to 30-fold more potent in complement-activation compared to AMPA-IgG. We provide first evidence that AMPA-IgM are cross-reactive towards different PTMs, indicating that PTM (cross-)reactivity is not confined to IgG and does not necessarily depend on extensive somatic hypermutation. Moreover, our data indicate that a diverse set of PTM antigens could be involved in the initial tolerance breach in RA and suggest that AMPA-IgM can induce complement-activation and thereby inflammation.

中文翻译：

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尽管突变负荷有限，但 IgM 抗修饰蛋白抗体在类风湿性关节炎中的交叉反应性

针对瓜氨酸化、乙酰化和/或氨基甲酰化自身抗原的抗修饰蛋白抗体 (AMPA) 是类风湿性关节炎 (RA) 的标志。尽管 AMPA-IgG 与多种翻译后修饰 (PTM) 的交叉反应性很明显，但尚不清楚表达 IgM 的第一个响应 B 细胞是否表现出相似的特征，或者交叉反应性是否主要依赖于体细胞超突变 (SHM)。我们现在研究了（种系）AMPA-IgM 的反应性，以进一步了解 B 细胞耐受性的破坏，并确定交叉反应性是否取决于广泛的 SHM。此外，我们研究了 AMPA-IgM 是否可以有效地招募免疫效应机制。从 RA 患者中分离出多克隆 AMPA-IgM，并评估对 PTM 抗原的交叉反应性。AMPA-IgM B 细胞受体序列是通过使用抗原特异性四聚体的单细胞分离获得的。随后，产生了五聚体单克隆 AMPA-IgM、它们的生殖系对应物和单体 IgG 变体。在一组 PTM 抗原上分析抗体并测试补体激活。五聚体单克隆和多克隆 AMPA-IgM 显示出对多种抗原和不同 PTM 的交叉反应性。在将 IgM 恢复为种系后，PTM 抗原识别仍然存在，尽管有所减少。AMPA-IgM 的价态对于抗原识别至关重要，因为当 AMPA-IgM 表达为 IgG 时，PTM 反应性显着降低。此外，与 AMPA-IgG 相比，AMPA-IgM 在补体激活方面的效力要高 15 到 30 倍。我们提供了第一个证据表明 AMPA-IgM 对不同的 PTM 有交叉反应，表明 PTM（交叉）反应性不限于 IgG，也不一定依赖于广泛的体细胞超突变。此外，我们的数据表明，一系列不同的 PTM 抗原可能参与 RA 的初始耐受性破坏，并表明 AMPA-IgM 可以诱导补体激活，从而诱导炎症。