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

项目摘要 干燥病（SjD）是一种全身性自身免疫性疾病，影响美国400万人， 损害健康相关的生活质量，没有FDA批准的疾病改善治疗方法。当前试模 包括一小部分抗SSA抗体阳性（SSA+）SjD患者，不包括25%的SjD患者 抗SSA抗体阴性（SSA-）患者。SSA-SjD患者具有与SjD患者不同的临床表型。 SSA+患者。近年来，间充质基质细胞（MSCs）在SjD唾液腺中的作用已被证实 并且代表了这种疾病中的新的可靶向细胞类型。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MSCs在SjD发病机制中的作用。我们的长期目标是利用对SjD致病性的认识， 创造新的细胞靶向疗法本项目的目标是确定唾液中MSC的转录组学， 腺体组织我们还将确定唾液腺MSC附近的淋巴细胞亚群， 这些子集在对照和SjD腺体之间以及在SjD内，在SSA+和SSA-腺体之间有何不同。我们 假设SjD具有独特MSC亚型，它们将促进不同的局部细胞环境， 对照组和SSA+和SSA-受试者之间。这一假设的基本原理是基于我们的数据显示， IFNγ处理的MSC能够充当抗原呈递细胞并促进T细胞趋化性。的 中心假设将通过追求两个具体目标来检验。目标1将提供一个完整的理解， 与对照组相比，新细胞类型MSC的SjD转录表型。目标2将定义 SSA+和SSA-之间MSC的细胞分子框架和总体炎性细胞组成不同 科目为了实现这些目标，FISH结合基于信号的杂交链反应 扩增将用于进行空间基因组学和快速多重免疫荧光蛋白 唾液腺组织的剖析。MSC亚型、淋巴细胞与MSC的相互作用和分化淋巴细胞 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。该项目建立了一个跨学科的团队， 细胞生物技术，SjD和流变学，干细胞生物学，病理学和生物信息学，以创建一个地图集， MSC表型可用于增强SjD的干细胞疗法。建议的应用是创新的 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， 从传统的仅关注SSA+受试者，转向SSA+和SSA-唾液之间的差异 腺体这项研究意义重大，因为它定义了SjD MSC，一种潜在的新型治疗靶点， 因为它详细说明了SSA+和SSA-腺体组织之间的空间分布差异，这是迈向 了解这些临床上不同的表型之间的差异。

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.