Multi-modal single cell analysis for investigation of T1D pathogenesis

用于研究 T1D 发病机制的多模式单细胞分析

• 批准号: 10388620
• 负责人: Leeana D Peters
• 金额: $ 4.02万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2025-05-15
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10388620
• 关键词: Address; Adoptive Cell Transfers; Antigens; Autoantibodies; Autoimmune Diseases; Autoimmunity; Autologous; Beta Cell; Biological Assay; Blood specimen; C-Peptide; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; CRISPR/Cas technology; CTLA4 gene; Candidate Disease Gene; Cell Therapy; Cell physiology; Cells; Cellular Assay; Chromatin; Clinical Trials; Collection; Complex; Coupled; Cytometry; DNA; Data; Data Set; Development; Diabetes Mellitus; Disease; Disease Progression; Epigenetic Process; Eragrostis; Event; Failure; Family; Flow Cytometry; Gene Expression; Gene Expression Regulation; Gene Transfer; Gene-Modified; Genes; Genetic; Genetic Predisposition to Disease; Genomic Segment; Glean; Goals; Health; Human; IL2RA gene; Immune; Impairment; Incidence; Individual; Infusion procedures; Insulin; Insulin-Dependent Diabetes Mellitus; Investigation; Islets of Langerhans; Knock-out; Knowledge; Lymphocyte Subset; Mediating; Modality; Modeling; Molecular; Names; Nucleic Acid Regulatory Sequences; Organ; Organ Donor; Pancreas; Pathogenicity; Pathway interactions; Patients; Phenotype; Play; Population; Production; Proteomics; Regulator Genes; Regulatory T-Lymphocyte; Reporting; Research; Resolution; Resources; Risk; Role; Safety; Sampling; Structure of beta Cell of islet; Supervision; T-Cell Antigen Receptor Specificity; Technology; Testing; Therapeutic; Tissue Donors; Tissues; Transplantation; Transposase; United States; Untranslated RNA; Variant; Work; antigen-specific T cells; autoreactivity; base; biobank; cell type; chemokine receptor; chronic autoimmune disease; cohort; cytokine; data resource; design; diabetes pathogenesis; diabetes risk; diabetogenic; draining lymph node; effective therapy; effector T cell; epigenomics; genetic variant; genome wide association study; high dimensionality; immune activation; immune function; immunoregulation; improved; individualized medicine; innovation; insulin dependent diabetes mellitus onset; interleukin-21; islet; member; multimodality; peripheral blood; personalized medicine; preservation; prevent; receptor; risk variant; single cell analysis; single cell technology; single-cell RNA sequencing; targeted treatment; transcriptomics; treatment strategy

Type 1 diabetes (T1D) is a chronic autoimmune disease which results from β-cell specific autoreactivity coupled with failures in immunoregulation. The inaccessibility of the pancreas from living individuals mandates that the majority of studies examining immune cell function in T1D be derived from peripheral blood, which may not accurately reflect events occurring in the target organ. Moreover, although >150 genomic regions are associated with T1D risk, little is known regarding subset- and activation state-specific expression of these loci, and the effect of risk variants on immune function. Thus, there exists a need for studies examining the molecular basis for T1D-associated immune dysregulation in disease-relevant tissues, namely, the pancreas and pancreatic draining lymph nodes (pLN). Additionally, while deficits in regulatory T cell (Treg) function are implicated in the loss of tolerance to β-cell antigens seen in T1D, the underlying mechanisms are incompletely understood. My overall goal is to identify the mechanisms by which T1D risk variants contribute to diabetogenic immune cell phenotypes using unsupervised and supervised analysis of high parameter single-cell datasets to identify genes and pathways which, when manipulated, will result in enhanced Treg function. The enrichment of T1D risk variants within DNA regulatory regions implies these variants may impact candidate gene expression. Moreover, many known candidate genes are associated with Treg activation and function. Therefore, I hypothesize that aberrant candidate gene expression and regulation in immune cells contributes to loss of tolerance in T1D by promoting Treg instability that can be studied mechanistically through gene-editing. The technical innovation of this research lies in the application of high-dimensional single cell technologies in understudied tissues that are essential to T1D pathogenesis. The theoretical innovation of this research lies in the opportunity to bridge multiple modalities and thereby, characterize key immune cell subsets by integrating their transcriptomic, epigenomic, and proteomic profiles. To date, a dataset comprising this information at single cell resolution does not exist for human organ donor tissue, thus I aim to assess the genetic regulation of immune phenotypes cross-sectionally in a human organ donor cohort. Importantly, my preliminary data indicate cell subsets expressing T1D candidate risk genes and TH1-associated markers are overrepresented in the pLN of T1D patients. Currently, the molecular basis for this phenotype is unclear. Therefore, I propose to discern the potential role of T1D risk variants in promoting proinflammatory over regulatory T cell phenotypes by performing single cell RNA-sequencing (scRNA- seq) and scATAC-seq. Lastly, while T1D candidate genes are thought to impact Treg function, I propose to model this in an antigen specific context, as these cells likely represent a more efficacious cell therapy product as compared to polyclonal Tregs. The significance of this work lies in the potential for defining tissue specific regulatory and diabetogenic cell subsets at high resolution, as well as for this knowledge to inform translational efforts developing optimized pathway targets and cellular therapies for T1D.

1型糖尿病（T1D）是一种慢性自身免疫性疾病，由β细胞特异性自动反应性耦合而产生 免疫调节失败。胰腺来自活个人的胰腺的无法访问性要求 大多数研究检查T1D中免疫细胞功能的研究是从外周血得出的，这可能不是 准确地反映了目标器官中发生的事件。此外，尽管> 150个基因组区域是相关的 对于T1D风险，关于这些基因座的子集和激活状态特异性表达，几乎没有人知道 风险变异对免疫功能的影响。那存在研究分子基础的研究 对于与疾病相关的时机中T1D相关的免疫失调，即胰腺和胰腺 排水淋巴结（PLN）。另外，尽管调节T细胞（Treg）功能的缺陷与 在T1D中看到的对β细胞抗原的耐受性丧失，基本机制尚不完全理解。我的 总体目标是确定T1D风险变体有助于糖尿病免疫细胞的机制 使用无监督和监督的高参数单细胞数据集的表型来识别基因 和操纵后将导致Treg功能增强的途径。 T1D风险的丰富 DNA调节区域内的变体意味着这些变异可能会影响候选基因表达。而且， 许多已知的候选基因与Treg的激活和功能有关。因此，我假设 免疫细胞中异常候选基因表达和调节导致T1D在T1D中的耐受性丧失 促进可以通过基因编辑机械研究的Treg不稳定性。技术创新 这项研究在于在理解的组织中应用高维单细胞技术 对于T1D发病机理必不可少。这项研究的理论创新在于有机会桥接多个 模态，从而通过整合其转录组表观基因组学来表征关键免疫细胞子集 和蛋白质组学曲线。迄今为止，在单元分辨率上完成此信息的数据集尚不存在 人体器官供体组织，因此我旨在在横截面上评估免疫表型的遗传调节 重要的是，我的初步数据表明表达T1D候选的细胞子集 在T1D患者的PLN中，风险基因和Th1相关标记的代表性过高。目前，分子 这种表型的基础尚不清楚。因此，我建议辨别T1D风险变体在 通过进行单细胞RNA测序（SCRNA- seq）和scatac-seq。最后，尽管认为T1D候选基因会影响Treg功能，但我建议 在抗原特定的情况下对此进行建模，因为这些细胞可能代表了更有效的细胞治疗产物 与多克隆Treg相比。这项工作的重要性在于定义特定组织的潜力 高分辨率的调节和糖尿病细胞子集以及这些知识以告知翻译 为T1D开发了优化的途径靶标和细胞疗法的努力。