Objectives Recent evidences have revealed that anti-SSA/SSB antibodies, the major autoantibodies in Sjögren's syndrome (SS), are produced in salivary glands. This study aims to clarify overall of autoantibody production at lesion site, including anti-centromere antibody (ACA)-positive SS. Methods Antibodies of antibody-secreting cells in human salivary glands were produced as recombinant antibodies. The reactivity of these antibodies and their revertants were investigated by ELISA and newly developed antigen-binding beads assay, which can detect conformational epitopes. The target of uncharacterised antibodies was identified by immunoprecipitation and mass spectrometry. Autoantibody-secreting cells in salivary gland tissue were identified by immunohistochemistry using green fluorescent protein-autoantigen fusion proteins. Results A total of 256 lesion antibodies were generated, and 69 autoantibodies including 24 ACAs were identified among them. Beads assay could detect more autoantibodies than ELISA, suggesting autoantibodies target to antigens with native conformation. After somatic hypermutations were reverted, autoantibodies drastically decreased antigen reactivity. We showed that MIS12 complex, a novel target of ACA, and CENP-C are major targets of ACA produced in salivary glands by examining cloned antibodies and immunohistochemistry, whereas few anti-CENP-B antibodies were detected. The target profiling of serum ACA from 269 patients with SS, systemic sclerosis (SSc), primary biliary cirrhosis (PBC) and healthy controls revealed that ACA-positive patients have antibodies against various sites of centromere complex regardless of disease. Conclusion We showed direct evidences of antigen-driven maturation of anti-SSA/SSB antibody and ACA in SS lesion. ACA recognises centromere ‘complex’ rather than individual protein, and this feature is common among patients with SS, SSc and PBC.

中文翻译：

---

抗原驱动选择针对 SSA、SSB 和着丝粒“复合物”的抗体，包括人唾液腺中的新型抗原 MIS12 复合物

目的 最近的证据表明，抗 SSA/SSB 抗体是干燥综合征 (SS) 的主要自身抗体，是在唾液腺中产生的。本研究旨在阐明病变部位自身抗体产生的整体情况，包括抗着丝粒抗体 (ACA) 阳性 SS。方法制备人唾液腺抗体分泌细胞抗体作为重组抗体。这些抗体及其回复体的反应性通过 ELISA 和新开发的抗原结合珠试验进行了研究，该试验可以检测构象表位。通过免疫沉淀和质谱法鉴定未表征抗体的靶标。使用绿色荧光蛋白-自身抗原融合蛋白通过免疫组织化学鉴定唾液腺组织中的自身抗体分泌细胞。结果共产生256个病灶抗体，其中鉴定出自身抗体69个，其中ACA 24个。Beads 检测可以检测到比 ELISA 更多的自身抗体，表明自身抗体靶向具有天然构象的抗原。体细胞超突变恢复后，自身抗体显着降低了抗原反应性。通过检查克隆抗体和免疫组织化学，我们发现 MIS12 复合物（ACA 的新靶标）和 CENP-C 是唾液腺中产生的 ACA 的主要靶标，而检测到的抗 CENP-B 抗体很少。来自 269 名患有 SS、系统性硬化症 (SSc)、原发性胆汁性肝硬化 (PBC) 和健康对照的血清 ACA 的靶标分析显示，无论疾病如何，ACA 阳性患者都具有针对着丝粒复合体各个部位的抗体。结论 我们显示了 SS 病变中抗 SSA/SSB 抗体和 ACA 的抗原驱动成熟的直接证据。ACA 识别着丝粒“复合体”而不是单个蛋白质，这种特征在 SS、SSc 和 PBC 患者中很常见。