Autoantibody production is a hallmark of rheumatoid arthritis (RA). Anti-citrullinated protein antibodies (ACPA) are highly disease-specific, and their presence is associated with more severe disease and poor prognosis compared to ACPA-negative patients. However, the immune cell composition associated with antibody-positive/negative disease is incompletely defined. Mass cytometry (MC) is a high-dimensional technique offering new possibilities in the determination of the immune cell composition in rheumatic diseases. Here, we set up a broad phenotyping panel to study the immune cell profile of early untreated RA to investigate if specific immune cell subsets are associated with ACPA+ versus ACPA− RA. Freshly obtained PBMCs of early, untreated RA patients (8 ACPA+ and 7 ACPA−) were analysed using a 36-marker MC panel, including markers related to various immune lineages. Data were processed using Cytosplore for dimensional reduction (HSNE) and clustering. Groups were compared using Cytofast. A second validation cohort of cryopreserved PBMCs obtained from early RA patients (27 ACPA+ and 20 ACPA−) was used to confirm MC data by flow cytometry (FC). FC data were processed and analysed using both an unsupervised analysis pipeline and through manual gating. MC indicated no differences when comparing major immune lineages (i.e. monocytes, T and B cells), but highlighted two innate subsets: CD62L+ basophils (p = 0.33) and a subset of CD16− NK cells (p = 0.063). Although the NK cell subset did not replicate by FC, FC replication confirmed the difference in CD62L+ basophil frequency when comparing ACPA+ to ACPA− patients (mean 0.32% vs. 0.13%; p = 0.01). Although no differences in major lineages were found between early ACPA+ and ACPA− RA, this study identified the reduced presence of activated basophils in ACPA-negative disease as compared to ACPA-positive disease and thereby provides the first evidence for a connection between activated basophils and ACPA status.

中文翻译：

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使用质谱流式细胞术对早期未治疗的类风湿性关节炎进行免疫分析，揭示了一个与 ACPA 状态呈负相关的活化嗜碱性粒细胞亚群

自身抗体的产生是类风湿性关节炎 (RA) 的标志。抗瓜氨酸蛋白抗体 (ACPA) 具有高度的疾病特异性，与 ACPA 阴性患者相比，它们的存在与更严重的疾病和较差的预后相关。然而，与抗体阳性/阴性疾病相关的免疫细胞组成尚未完全确定。质谱流式细胞术 (MC) 是一种高维技术，为确定风湿性疾病中的免疫细胞组成提供了新的可能性。在这里，我们建立了一个广泛的表型小组来研究早期未经治疗的 RA 的免疫细胞谱，以研究特定的免疫细胞亚群是否与 ACPA+ 与 ACPA-RA 相关。使用 36 标记 MC 面板分析了新获得的早期未治疗 RA 患者（8 个 ACPA+ 和 7 个 ACPA-）的 PBMC，包括与各种免疫谱系相关的标记。使用 Cytosplore 处理数据以进行降维 (HSNE) 和聚类。使用 Cytofast 比较各组。从早期 RA 患者（27 个 ACPA+ 和 20 个 ACPA-）获得的冷冻保存的 PBMC 的第二个验证队列用于通过流式细胞术 (FC) 确认 MC 数据。FC 数据使用无监督分析管道和手动门控进行处理和分析。MC 在比较主要免疫谱系（即单核细胞、T 和 B 细胞）时没有显示差异，但突出了两个先天亚群：CD62L+ 嗜碱性粒细胞（p = 0.33）和 CD16- NK 细胞亚群（p = 0.063）。虽然 NK 细胞亚群没有通过 FC 复制，但在比较 ACPA+ 和 ACPA- 患者时，FC 复制证实了 CD62L+ 嗜碱性粒细胞频率的差异（平均 0.32% 对 0.13%；p = 0.01）。