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 细胞特异性自身反应性耦合所致 免疫调节失败。由于活体个体无法接近胰腺，因此必须 大多数检查 T1D 免疫细胞功能的研究都来自外周血，这可能不是 准确地反映目标器官中发生的事件。此外，尽管 >150 个基因组区域相关 对于 T1D 风险，我们对这些基因座的子集和激活状态特异性表达知之甚少，并且 风险变异对免疫功能的影响。因此，需要进行研究来检验其分子基础 用于疾病相关组织（即胰腺和胰腺）中与 T1D 相关的免疫失调 引流淋巴结（pLN）。此外，虽然调节性 T 细胞 (Treg) 功能缺陷与 T1D 中观察到对 β 细胞抗原的耐受性丧失，但其潜在机制尚不完全清楚。我的 总体目标是确定 T1D 风险变异促进糖尿病免疫细胞的机制 使用高参数单细胞数据集的无监督和监督分析来识别基因的表型 以及当被操纵时将导致 Treg 功能增强的途径。 T1D 风险的丰富 DNA 调控区域内的变异意味着这些变异可能会影响候选基因的表达。而且， 许多已知的候选基因与 Treg 激活和功能相关。因此，我假设 免疫细胞中异常的候选基因表达和调控导致 T1D 耐受性丧失 促进 Treg 不稳定性，可以通过基因编辑进行机械研究。技术创新 这项研究在于将高维单细胞技术应用于所研究的组织中 T1D 发病机制所必需的。这项研究的理论创新在于有机会弥合多种 模式，从而通过整合转录组、表观基因组、 和蛋白质组谱。迄今为止，尚不存在以单细胞分辨率包含此信息的数据集 人体器官供体组织，因此我的目标是横断面评估免疫表型的遗传调控 在人体器官捐献者队列中。重要的是，我的初步数据表明表达 T1D 候选者的细胞子集 风险基因和 TH1 相关标记物在 T1D 患者的 pLN 中出现过多。目前，分子 该表型的基础尚不清楚。因此，我建议辨别 T1D 风险变异在以下方面的潜在作用： 通过进行单细胞 RNA 测序 (scRNA- seq）和 scATAC-seq。最后，虽然 T1D 候选基因被认为会影响 Treg 功能，但我建议 在抗原特定环境中对此进行建模，因为这些细胞可能代表更有效的细胞治疗产品 与多克隆 Tregs 相比。这项工作的意义在于有可能定义组织特异性 高分辨率的调节细胞和糖尿病细胞亚群，以及这些知识为转化提供信息 致力于开发针对 T1D 的优化途径靶点和细胞疗法。