Data Sheet 1_CD226+ B cells in primary Sjögren’s syndrome: a key player in clinical manifestations and disease pathogenesis.docx

IntroductionPrimary Sjögren’s syndrome (pSS) is a systemic autoimmune disorder characterized by lymphocytic infiltration of exocrine glands, leading to sicca symptoms and systemic complications. CD226, a co-stimulatory receptor implicated in the pathogenesis of multiple autoimmune diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis(RA), and pSS, regulates immune cell activation. However, the specific role of CD226+ B cells in pSS pathogenesis remains unclear. This study aims to elucidate the functional contribution of CD226+ B cells to pSS development and their clinical relevance.
MethodsThe percentages of CD226 on T cells, B cells, CD56+ NK cells and CD14+ monocytes in the peripheral blood(PB) of pSS patients and healthy controls (HCs) were detected by flow cytometry.Multicolor flow cytometry was employed to examine the distribution of CD226 in B cell subsets of pSS patients, as well as the expression levels of co-stimulatory molecules, activation and proliferation markers, immunoglobulins, and pro-inflammatory cytokines on both CD226+ B cells and CD226- B cells. Multicolor immunofluorescence staining was applied to detect the co-expression of B cells and CD226 in the salivary gland of pSS patients.Microarray analysis was conducted to analyze the transcriptomic profiles of sorted CD226+ CD19+ B cells and CD226- CD19+ B cells.
ResultsCD226 expression in the peripheral blood of pSS patients was significantly increased on T cells, CD19+ B cells and CD14+ monocytes, but significantly decreased on CD56+ NK cells.We identified a distinct CD226+CD19+ B cell subset that exhibited pathogenic features in pSS. CD226 was significantly upregulated on B cells in the peripheral blood and salivary glands of pSS patients.CD226+ CD19+ B cell showed a stronger correlation with clinical features, disease activity, and prognosis in pSS patients.The ROC curve demonstrated that CD226+ CD19+ B cell exhibited significant diagnostic capability to distinguish pSS patients from healthy controls and to differentiate disease activity.This subset also exhibited heightened activation and pro-inflammatory phenotypes.
DiscussionCD226+ B cells are expanded in pSS, strongly correlating with clinical manifestations and disease activity. These cells display enhanced effector profiles (activation, cytokine/immunoglobulin production) and demonstrate diagnostic utility. Our findings identify CD226+ B cell as a pathogenic driver in pSS, positioning CD226 as a promising novel therapeutic target and biomarker.

引言
原发性干燥综合征（Primary Sjögren’s syndrome, pSS）是一类以淋巴细胞浸润外分泌腺体为特征的全身性自身免疫性疾病，可引发干燥症状与全身性并发症。CD226作为一种共刺激受体，已被证实参与包括系统性红斑狼疮（systemic lupus erythematosus, SLE）、类风湿关节炎（rheumatoid arthritis, RA）及pSS在内的多种自身免疫性疾病的发病过程，可调控免疫细胞活化。然而，CD226阳性B细胞在pSS发病机制中的具体作用仍未明确。本研究旨在阐明CD226阳性B细胞对pSS发生发展的功能贡献及其临床相关性。

方法
本研究采用流式细胞术检测了pSS患者与健康对照（healthy controls, HCs）外周血（peripheral blood, PB）中T细胞、B细胞、CD56阳性自然杀伤（natural killer, NK）细胞及CD14阳性单核细胞表面CD226的表达百分比。通过多色流式细胞术，分析了pSS患者B细胞亚群中CD226的分布特征，并检测了CD226阳性与阴性B细胞表面共刺激分子、活化及增殖标志物、免疫球蛋白与促炎细胞因子的表达水平。采用多色免疫荧光染色技术，检测了pSS患者唾液腺组织中B细胞与CD226的共表达情况。通过微阵列分析，对分选得到的CD226阳性CD19阳性B细胞与CD226阴性CD19阳性B细胞进行转录组谱分析。

结果
pSS患者外周血中，T细胞、CD19阳性B细胞及CD14阳性单核细胞表面的CD226表达水平显著升高，而CD56阳性NK细胞表面的CD226表达则显著降低。本研究鉴定出一类独特的CD226阳性CD19阳性B细胞亚群，该亚群在pSS中表现出致病相关特征。pSS患者外周血与唾液腺组织中的B细胞表面CD226均显著上调。CD226阳性CD19阳性B细胞与pSS患者的临床特征、疾病活动度及预后均呈现显著相关性。受试者工作特征（Receiver Operating Characteristic, ROC）曲线分析显示，CD226阳性CD19阳性B细胞具备优异的诊断效能，可有效区分pSS患者与健康对照，并能鉴别疾病活动状态。该细胞亚群同时表现出增强的活化与促炎症表型。

讨论
CD226阳性B细胞在pSS患者体内发生扩增，且与临床表型及疾病活动度密切相关。这类细胞展现出增强的效应细胞功能特征，包括活化能力提升、细胞因子与免疫球蛋白产生增加，并具备潜在的诊断应用价值。本研究结果证实CD226阳性B细胞是pSS的致病驱动因素，提示CD226有望成为极具潜力的新型治疗靶点与生物标志物。