Spatial transcriptional phenotyping of Sjögren’s disease tissue-resident mesenchymal stromal cells and neighbors in labial salivary glands

项目总结 干燥症(SJD)是一种系统性自身免疫性疾病，在美国和 损害与健康相关的生活质量，没有FDA批准的疾病修改治疗可用。目前的试验 只包括一小部分抗SSA抗体阳性(SSA+)SJD患者，排除25%的SJD患者 抗SSA抗体阴性(SSA-)患者。SSA-SJD患者的临床表型与 SSA+患者。最近，间充质基质细胞(MSCs)被认为与SJD唾液腺有关。 并代表了这种疾病中一种新的靶向细胞类型。因此，迫切需要发展 新的靶向治疗，考虑到SSA+和SSA-SJD患者的异质性，并进一步 明确骨髓间充质干细胞在SJD发病中的作用。我们的长期目标是利用SJD致病的洞察力 创造新的细胞靶向疗法。这个项目的目标是确定唾液中间充质干细胞的转录组学。 腺体组织。我们还将测定唾液腺MSCs附近的淋巴细胞亚群，并确定 这些亚群在对照腺体和SJD腺体之间以及SJD内SSA+和SSA-腺体之间有何不同。我们 假设SJD有独特的MSC亚型，它们将促进不同于 对照组和SSA+和SSA-受试者之间。这一假设的理论基础是基于我们的数据显示 干扰素𝛾处理的骨髓间充质干细胞能够作为抗原提呈细胞，促进T细胞趋化。这个 核心假说将通过追求两个具体目标来检验。目标1将提供对 一种新的细胞类型MSC的SJD转录表型与对照相比。目标2将定义如何 SSA+和SSA-间充质干细胞的细胞分子结构和总炎性细胞组成不同。 研究对象。为了达到这些目的，FISH与基于信号的杂交链式反应相结合 扩增将用于空间基因组学和快速复合免疫荧光蛋白 唾液腺组织的侧写。间充质干细胞亚型、淋巴细胞与间充质干细胞的相互作用及分化淋巴细胞 对照、SSA+和SSA-受试者之间的组成将使用一系列空间分辨的 细胞状态和相互作用分析。这个项目建立了一个跨学科的团队，整合了来自 细胞生物技术、SJD和风湿学、干细胞生物学、病理学和生物信息学，以创建一份地图集 可用于增强SJD干细胞疗法的MSC表型。拟议的应用程序是创新的 因为它使用尖端技术来定义唾液腺组织的空间基因组学和蛋白质组学 从仅关注SSA+受试者的传统关注转向SSA+和SSA-唾液之间的差异 腺体。这项研究具有重要意义，因为它定义了SJD MSCs，一种潜在的新治疗靶点，以及 因为它详细描述了SSA+和SSA-腺体组织之间的空间分布差异，这是迈向 了解这些临床不同表型之间的差异。

PROJECT SUMMARY Sjögren’s disease (SjD), a systemic autoimmune disease that affects four million people in the United States and impairs health-related quality of life, has no FDA-approved disease-modifying treatments available. Current trials include only a narrow subset of SjD patients who are anti-SSA antibody positive (SSA+), excluding 25% of SjD patients who are anti-SSA antibody negative (SSA-). SSA- SjD patients have distinct clinical phenotypes from SSA+ patients. Recently, mesenchymal stromal cells (MSCs) have been implicated in SjD salivary gland pathobiology and represent a novel targetable cell type in this disease. Thus, there is a critical need to develop new targeted therapy that takes into account the heterogeneity of SSA+ and SSA- SjD patients and to further define the role of MSCs in the pathogenesis of SjD. Our long-term goal is to use SjD pathogenic insights to create new cell-targeted therapy. The goal of this project is to define the transcriptomics of MSCs within salivary gland tissue. We also will determine the lymphocyte subsets in proximity to salivary gland MSCs and determine how these subsets differ between control and SjD glands and, within SjD, between SSA+ and SSA- glands. We hypothesize that SjD has unique MSC subtypes and they will promote differential local cellular milieu from controls and between SSA+ and SSA- subjects. The rationale for this hypothesis is based on our data showing IFN𝛾-treated MSCs are capable of acting as antigen-presenting cells and promoting T-cell chemotaxis. The central hypothesis will be tested by pursuing two specific Aims. Aim 1 will provide an integral understanding of the SjD transcriptional phenotype of a novel cell type, the MSC, compared to controls. Aim 2 will define how the cellular molecular framework of MSCs and overall inflammatory cell composition differs between SSA+ and SSA- subjects. To accomplish these Aims, FISH combined with Hybridization Chain Reaction based signal amplification will be used to perform spatial genomics and rapid multiplexed immunofluorescence protein profiling of salivary gland tissue. MSC subtypes, lymphocyte interactions with MSCs, and differential lymphocyte composition between control, SSA+ and SSA- subjects will be determined using a series of spatially resolved cell state and interaction analyses. This project builds an interdisciplinary team integrating experts from single cell biotechnology, SjD and rheumatology, stem cell biology, pathology, and bioinformatics to create an atlas of MSC phenotypes that can be used to enhance SjD’s stem cell therapies. The proposed application is innovative because it uses cutting-edge technology to define spatial genomics and proteomics of salivary gland tissue and shifts from the traditional focus on SSA+ subjects alone, toward differences between SSA+ and SSA- salivary glands. This research is significant because it defines SjD MSCs, a potential novel therapeutic target, and because it details spatial profiling differences between SSA+ and SSA- gland tissue, a critical step toward understanding the difference between these clinically distinct phenotypes.