Integrative single cell and spatial transcriptomics of salivary glands in Sjogren's syndrome

摘要 干燥综合征（SS）是一种复杂的自身免疫性疾病，影响2- 310万美国人， 以不可逆的外分泌腺损伤为特征，通常导致口腔干燥， 眼睛严重的全身表现可能包括神经病变、淋巴瘤和临床显著疲劳 导致严重残疾和生活质量下降。虽然先天性和适应性的失调 免疫细胞无疑在外分泌腺功能障碍中起作用，为什么唾液腺导管细胞是靶向的 并且对这些结构周围的免疫细胞浸润形式的病灶知之甚少。已知的遗传风险变异 在SS中，已经定位到涉及抗原呈递、干扰素应答、T和B细胞活化 细胞迁移途径和自身抗体的产生，然而，这些功能性作用的细胞类型 SS中的基因是未知的。我们的中心假设是，唾液腺上皮细胞直接参与 异常局灶性免疫细胞浸润通过正常稳态途径的失调，这些 相互作用是由最近发现的SS遗传风险变体驱动的。该项目旨在定义基因 表达模式在存在或不存在免疫细胞灶的导管细胞之间不同，并决定 如果这些差异在风险和非风险SS基因型之间变化。我们将应用最近描述的综合 将基于10 X基因组学的单细胞RNA测序与空间转录组学数据相结合的方法， 允许在非常精确的组织形态学的背景下解释细胞表达水平。在目标1中， 我们将通过比较以下基因表达模式来确定参与SS的导管细胞的转录特征： 通过对25例SS患者的受试者内分析，发现导管细胞被免疫灶包围，而不是被免疫灶包围。 从25名年龄、性别和种族匹配的健康对照中获得的基线导管细胞表达模式将 也可以比较。在目标2中，我们将定义特定的唾液腺和免疫细胞亚群， 受已知SS相关表达数量性状基因座（eQTL）影响。使用生成的相同数据集 对于目标1，将比较基因eQTL中风险携带者和非风险变体之间的转录水平， 以前与SS。这些数据将识别特定的唾液腺（例如导管，腺泡， 肌上皮细胞）和免疫细胞（例如T、B、树突状细胞、自然杀伤细胞等）功能上受以下因素影响的子集 SS风险变体。我们将利用通过以下途径发展的广泛基础设施和多学科专业知识， 俄克拉荷马州舍格伦综合征研究翻译中心，以完成成功执行 这个项目这些研究将提供前所未有的深度和广度的新知识，关于细胞- 基因和途径的特定失调。这些知识对于开发新的治疗方法至关重要 它针对正确的基因、通路和细胞，阻断免疫系统与 外分泌腺是这种复杂疾病发病机制的基础。

ABSTRACT Sjögren’s syndrome (SS) is a complex autoimmune disorder that affects 2-3.1 million Americans and is distinguished by irreversible exocrine gland damage that often results in debilitating dryness in the mouth and eyes. Severe systemic manifestations may include neuropathies, lymphomas, and clinically significant fatigue leading to considerable disability and reduced quality of life. Although dysregulation of innate and adaptive immune cells undoubtedly plays a role in exocrine gland dysfunction, why salivary gland ductal cells are targeted and immune cell infiltrates form foci around these structures is poorly understood. Known genetic risk variants in SS have been mapped to loci involved in antigen presentation, interferon responses, T and B cell activation, cell migration pathways, and autoantibody production, however the cell types in which functional effects of these genes operate in SS are unknown. Our central hypothesis is that salivary gland epithelial cells contribute directly to aberrant focal immune cell infiltration through dysregulation of normal homeostatic pathways and that these interactions are driven by recently identified SS genetic risk variants. This project seeks to define gene expression patterns that differ between ductal cells with presence or absence of immune cell foci and determine if these differences vary between risk and non-risk SS genotypes. We will apply a recently described integrative approach that couples 10X Genomics-based single cell RNA-sequencing with Spatial Transcriptomics data, thus allowing cellular expression levels to be interpreted in the context of very precise tissue morphology. In Aim 1, we will define transcriptional signatures of ductal cells involved in SS by comparing gene expression patterns of ductal cells surrounded by immune foci with those that are not through intrasubject analyses of 25 SS patients. Baseline ductal cell expression patterns obtained for 25 age, gender and racially matched healthy controls will also be compared. In Aim 2, we will define specific salivary gland and immune cell subsets that are functionally affected by known SS-associated expression quantitative trait loci (eQTLs). Using the same datasets generated for Aim 1, transcript levels will be compared between carriers of risk and non-risk variants in eQTLs for genes previously associated with SS. These data will identify the specific salivary gland (e.g. ductal, acinar, myoepithelial) and immune cells (e.g. T, B, dendritic, natural killer, etc.) subsets that are functionally affected by SS risk variants. We will leverage the extensive infrastructure and multidisciplinary expertise developed through the Oklahoma Sjögren’s Syndrome Center of Research Translation to accomplish the successful execution of this project. These studies will provide unprecedented depth and breadth of new knowledge regarding cell- specific dysregulation of genes and pathways. This knowledge is essential for development of novel therapeutics that target the right genes, pathways and cells and block the interactions between the immune system and exocrine glands that are fundamental to the pathogenesis of this complex disease.