Altered B cell receptor (BCR) signaling has been implicated in the pathogenesis of rheumatoid arthritis (RA). Here we aimed to identify signaling aberrations in autoantibody-positive and autoantibody-negative RA patients by performing a comprehensive analysis of the BCR signaling cascade in different B cell subsets. We first optimized phosphoflow cytometry for an in-depth analysis of BCR signaling across immunoglobulin isotypes in healthy donors. Subsequently, we compared BCR signaling in circulating B cell subsets from treatment-naïve, newly-diagnosed autoantibody-positive RA and autoantibody-negative RA patients and healthy controls (HCs). We observed subset-specific phosphorylation patterns of the BCR signalosome in circulating B cells from healthy donors. Compared with HCs, autoantibody-positive RA patients displayed enhanced responses to BCR stimulation for multiple signaling proteins, specifically in naïve and IgA memory B cells. Whereas in unstimulated healthy donor B cells, the phosphorylation status of individual signaling proteins showed only limited correlation, BCR stimulation enhanced the interconnectivity in phosphorylation within the BCR signalosome. However, this strong interconnectivity within the BCR signalosome in stimulated B cells from HCs was lost in RA, especially in autoantibody-positive RA patients. Finally, we observed strong correlations between SYK and BTK protein expression, and IgA and IgG anti-citrullinated protein antibody concentrations in serum from autoantibody-positive RA patients. Collectively, the isotype-specific analysis of multiple key components of the BCR signalosome identified aberrant BCR signaling responses in treatment-naïve autoantibody-positive RA patients, particularly in naïve B cells and IgA memory B cells. Our findings support differential involvement of dysregulated BCR signaling in the pathogenesis of autoantibody-positive and autoantibody-negative RA.

中文翻译：

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新诊断的自身抗体阳性类风湿性关节炎患者的循环初始 B 细胞和 IgA+ 记忆 B 细胞中异常的 B 细胞受体信号传导

B 细胞受体 (BCR) 信号传导的改变与类风湿性关节炎 (RA) 的发病机制有关。在这里，我们的目的是通过对不同 B 细胞亚群中的 BCR 信号级联进行全面分析，以确定自身抗体阳性和自身抗体阴性 RA 患者的信号传导异常。我们首先优化了磷酸流式细胞术，以深入分析健康供体中免疫球蛋白同种型的 BCR 信号传导。随后，我们比较了未经治疗、新诊断的自身抗体阳性 RA 和自身抗体阴性 RA 患者以及健康对照 (HC) 循环 B 细胞亚群中的 BCR 信号传导。我们观察到来自健康供体的循环 B 细胞中 BCR 信号体的子集特异性磷酸化模式。与 HC 相比，自身抗体阳性 RA 患者对多种信号蛋白的 BCR 刺激的反应增强，特别是在初始 B 细胞和 IgA 记忆 B 细胞中。尽管在未刺激的健康供体 B 细胞中，各个信号蛋白的磷酸化状态仅表现出有限的相关性，但 BCR 刺激增强了 BCR 信号体内磷酸化的互连性。然而，在 HC 刺激的 B 细胞中，BCR 信号体内的这种强互连性在 RA 中消失了，尤其是在自身抗体阳性的 RA 患者中。最后，我们观察到自身抗体阳性 RA 患者血清中 SYK 和 BTK 蛋白表达以及 IgA 和 IgG 抗瓜氨酸蛋白抗体浓度之间存在很强的相关性。总的来说，对 BCR 信号体多个关键成分的同种型特异性分析发现了初治自身抗体阳性 RA 患者中异常的 BCR 信号反应，特别是在初治 B 细胞和 IgA 记忆 B 细胞中。我们的研究结果支持失调的 BCR 信号在自身抗体阳性和自身抗体阴性 RA 发病机制中的不同参与。