Yamada Eiko, Dominick Kalie, Kulchar Rachel J, Twohig Joseph, Khavandgar Zohreh, Beach Margaret, Pelayo Eileen, Baer Alan, Perez Paola, Warner Blake M
Salivary Disorders Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, United States.
NIDCR Sjögren's Disease Clinic, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, United States.
Front Dent Med. 2025 Jun 12;6:1590516. doi: 10.3389/fdmed.2025.1590516. eCollection 2025.
AIM/INTRODUCTION: We aimed to achieve direct quantitative measurement of activated and therapeutically actionable pathways (e.g., Type-I interferon) in target organs of autoimmune disease using flow cytometry of human salivary glands. Sjögren's Disease (SjD) is a systemic autoimmune disorder characterized by lymphocytic inflammation and dysfunction of the lacrimal and salivary glands. Minor salivary glands are routinely biopsied and are used for the histopathological diagnosis of SjD. In this study, we optimized the dissociation, permeabilization, antibody panel, and analytical parameters to characterize both the immune and epithelial cells in the glands, and the activation status of a specified pathway by measuring intracellular phosphorylated proteins.
Fresh human MSG biopsies were dissociated into single-cell suspensions and permeabilized under optimized conditions. MSG suspensions were stained for cell surface and intracellular markers then analyzed using nine-color conventional flow cytometry, including two intracellular markers.
Our optimized dissociation and permeabilization protocols for human MSG preserved key cell surface markers. Our flow cytometry panel identified major immune cell populations and distinguished epithelial cells via cytokeratin-18. We demonstrate the protocol's utility showing differential interferon pathway activity in SjD vs. healthy MSG leukocytes and epithelial cells. We provide guidance on panel selection, analytical capabilities, and the impact of cell yield on resolution using conventional flow cytometry.
Our optimized protocol enables high-resolution characterization of immune and epithelial cell populations in human MSG, preserving key markers and capturing interferon pathway activity. Our protocol provides a robust framework for the direct study of immune heterogeneity and signaling dynamics in SjD at single cell resolution.
目的/引言：我们旨在通过对人类唾液腺进行流式细胞术，实现对自身免疫性疾病靶器官中活化且具有治疗可操作性的信号通路（如I型干扰素）进行直接定量测量。干燥综合征（SjD）是一种全身性自身免疫性疾病，其特征为泪腺和唾液腺的淋巴细胞炎症及功能障碍。小唾液腺通常会进行活检，并用于SjD的组织病理学诊断。在本研究中，我们优化了解离、通透处理、抗体组合及分析参数，以表征腺体中的免疫细胞和上皮细胞，并通过测量细胞内磷酸化蛋白来确定特定信号通路的激活状态。
将新鲜的人类小唾液腺活检组织解离成单细胞悬液，并在优化条件下进行通透处理。小唾液腺悬液用细胞表面和细胞内标志物进行染色，然后使用九色传统流式细胞术进行分析，其中包括两种细胞内标志物。
我们针对人类小唾液腺优化的解离和通透处理方案保留了关键的细胞表面标志物。我们的流式细胞术抗体组合鉴定出主要的免疫细胞群体，并通过细胞角蛋白-18区分上皮细胞。我们展示了该方案的实用性，表明在干燥综合征患者与健康人小唾液腺白细胞和上皮细胞中，干扰素信号通路活性存在差异。我们提供了关于抗体组合选择、分析能力以及细胞产量对使用传统流式细胞术分辨率影响的指导。
我们优化的方案能够对人类小唾液腺中的免疫细胞和上皮细胞群体进行高分辨率表征，保留关键标志物并捕捉干扰素信号通路活性。我们的方案为在单细胞分辨率下直接研究干燥综合征中的免疫异质性和信号转导动力学提供了一个强大的框架。
