Early diagnosis and mechanistic studies of type 1 diabetes using single cell analysis

使用单细胞分析进行 1 型糖尿病的早期诊断和机制研究

基本信息

批准号：
9884757
负责人：
Luc Teyton
金额：
$ 78.9万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-15 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9884757
关键词：
Acute Advocate Antibodies Antigens Autoimmune Diseases Autoimmune Process Autoimmunity Autologous B-Lymphocytes Beta Cell Biological Assay Biological Markers Blood Blood Cells C-Peptide CD3 Antigens CD4 Positive T Lymphocytes CXCR3 gene Cells Charities Chronic Clinical Detection Diabetes Mellitus Diabetic mouse Diagnosis Disease Disease Progression Early Diagnosis Epidemic Epitopes Evolution Fasting Formulation Gene Expression Profiling General Population Genes Genetic Polymorphism Glycosylated Hemoglobin HLA-DQ Antigens HLA-DR Antigens Human Hyperglycemia Hypoglycemia Immunology Immunomodulators Immunotherapy In Vitro Inbred NOD Mice Incidence Injections Insulin Insulin-Dependent Diabetes Mellitus Islets of Langerhans Kidney Diseases Legal patent Life Life Expectancy Link Measures Mediating Medical Care Costs Monitor Mus Neuropathy Onset of illness Organ Pathogenicity Patients Peptide/MHC Complex Phase Population Populations at Risk Prediabetes syndrome Prevention Process Recombinants Replacement Therapy Research Design Retinal Diseases Risk Role Sampling Series Specificity T cell response T-Cell Activation T-Lymphocyte Techniques Technology Testing Therapeutic Intervention Time Translating Translations Vascular Diseases Western World Work alpha-beta T-Cell Receptor authority autoreactive T cell autoreactivity diabetic disease diagnosis humanized mouse insight insulin dependent diabetes mellitus onset insulin secretion islet man mouse model new therapeutic target non-diabetic novel therapeutics peripheral blood pre-clinical programmed cell death protein 1 programs single cell analysis single cell technology stem cells success transcriptome sequencing

Acute Advocate Antibodies Antigens Autoimmune Diseases Autoimmune Process Autoimmunity Autologous B-Lymphocytes Beta Cell Biological Assay Biological Markers Blood Blood Cells C-Peptide CD3 Antigens CD4 Positive T Lymphocytes CXCR3 gene Cells Charities Chronic Clinical Detection Diabetes Mellitus Diabetic mouse Diagnosis Disease Disease Progression Early Diagnosis Epidemic Epitopes Evolution Fasting Formulation Gene Expression Profiling General Population Genes Genetic Polymorphism Glycosylated Hemoglobin HLA-DQ Antigens HLA-DR Antigens Human Hyperglycemia Hypoglycemia Immunology Immunomodulators Immunotherapy In Vitro Inbred NOD Mice Incidence Injections Insulin Insulin-Dependent Diabetes Mellitus Islets of Langerhans Kidney Diseases Legal patent Life Life Expectancy Link Measures Mediating Medical Care Costs Monitor Mus Neuropathy Onset of illness Organ Pathogenicity Patients Peptide/MHC Complex Phase Population Populations at Risk Prediabetes syndrome Prevention Process Recombinants Replacement Therapy Research Design Retinal Diseases Risk Role Sampling Series Specificity T cell response T-Cell Activation T-Lymphocyte Techniques Technology Testing Therapeutic Intervention Time Translating Translations Vascular Diseases Western World Work alpha-beta T-Cell Receptor authority autoreactive T cell autoreactivity diabetic disease diagnosis humanized mouse insight insulin dependent diabetes mellitus onset insulin secretion islet man mouse model new therapeutic target non-diabetic novel therapeutics peripheral blood pre-clinical programmed cell death protein 1 programs single cell analysis single cell technology stem cells success transcriptome sequencing

展开

项目摘要

Project Summary The current project proposes an original framework to translate basic mouse immunology of the pre-clinical phase of type 1 diabetes (T1D) to human, using sophisticated single cell technologies. Over the past four years, we have dissected the antigen-specific CD4+ T cell response in the NOD mouse model using single cell gene profiling, TCR sequencing, and RNAseq to understand the process of activation of autoreactive T cells in target organs. After defining a unique program of activation for islet-specific CD4+ T cells, we isolated the same cells from peripheral blood of pre-diabetic mice. Our contention is that similar studies can now be done in human and help diagnose disease at a very early stage and follow disease evolution and monitor therapeutic intervention. In addition, the same approach will deliver important mechanistic insights in the role of CD4+ T cells in T1D onset and progression. Our work will be focused on two specific aims: SA #1: Expand the mouse studies to optimize their translational value. In the NOD mouse model, single cell analysis of islet CD4+ T cells has allowed the identification of recirculating autoreactive CD4+ T cells in the peripheral blood. Probing two antigen specificities with a series of pMHC tetramers, the profile of pathogenic cells was dissected by gene expression profiling, RNAseq, and TCR αβ pair sequencing. Using the same approach, further characterization of these cells will allow us to gain mechanistic insights and identify potential new therapeutic targets. We hypothesize that recirculating T cells from all stages of the disease process can be found in blood and analyzed by single cell technologies, and used to diagnose and follow disease evolution. SA #2: Analysis of activated CD4+ T cells from the peripheral blood of T1D patients. Using the same approaches and single cell technology, we will characterize a circulating CD4+HLA-DR+PD-1+CXCR3+ cell population that we have identified in T1D patients and not in controls. Antigen specificity will be examined using HLA-DQ tetramers, and functionally using T cell activation assays after TCR re-expression of sequenced αβ pairs as well as new humanized mouse models. A single antigen, insulin, and common mouse-human epitopes will be used for this translation before additional antigen reactivities are examined. The approach will be tested for its ability to measure the anti-islet autoreactivity in “at-risk”, “just-diagnosed”, and “established” T1D patients, and compared to the classic anti-islet antibody detection. The two aims will also evaluate the precise role of the “P9 switch” mode of T cell recognition in mice and human T1D, respectively, and potentially answer why this disease is linked to the single HLA class II β57 polymorphism. Hopefully, we will demonstrate that single cell technologies by interrogating rare cells in peripheral blood, have the power to diagnose T1D in its pre-clinical phase in at risk patients. Such a revolutionary approach would permit to monitor evolution and new therapies aimed at maintaining β cell mass.

项目摘要当前的项目提出了一个原始框架，以翻译临床前的基本鼠标免疫学使用复杂的单细胞技术，对人类的1型糖尿病（T1D）的阶段。在过去的四个几年，我们使用单个细胞在NOD小鼠模型中解剖了抗原特异性CD4+ T细胞响应基因分析，TCR测序和RNASEQ，以了解自动反应性T细胞激活的过程目标器官。在定义了胰岛特异性CD4+ T细胞的唯一激活程序后，我们分离了相同糖尿病前小鼠外周血的细胞。我们的论点是，现在可以在人类并在很早的阶段帮助诊断疾病，并遵循疾病进化并监测治疗干涉。此外，同样的方法将在CD4+ T的角色中提供重要的机械见解 T1D发作和进展中的细胞。我们的工作将集中在两个具体目标上：SA＃1：扩展鼠标研究以优化其翻译价值。在NOD小鼠模型中，胰岛CD4+ T的单细胞分析细胞已允许鉴定外周血中的自动反应性CD4+ T细胞。探测两种具有一系列PMHC四聚体的抗原规范，通过基因解剖致病细胞的特征表达分析，RNASEQ和TCRαβ对测序。使用相同的方法，进一步表征这些细胞将使我们能够获得机械洞察力并确定潜在的新治疗靶标。我们假设可以在血液和通过单细胞技术进行分析，用于诊断和遵循疾病进化。 SA＃2：分析来自T1D患者外周血的激活CD4+ T细胞。使用相同的方法和单一方法细胞技术，我们将表征循环的CD4+HLA-DR+PD-1+CXCR3+细胞种群在T1D患者中鉴定出来，而在对照组中则不确定。将使用HLA-DQ四聚体检查抗原特异性，并在TCR重新表达测序αβ对以及新的TCR后使用T细胞激活测定法人源化的小鼠模型。将使用单个抗原，胰岛素和常见的小鼠 - 人类表位检查额外的抗原反应率之前的翻译。该方法的能力将被测试测量“处于危险”，“刚诊断”和“既定”的T1D患者和“固定”患者的抗ISLET自动反应性以及与经典的抗ISLET抗体检测相比。这两个目标还将评估“ P9”的精确作用小鼠和人类T1D中T细胞识别的开关模式，并有可能回答为什么这疾病与单个HLA II类β57多态性有关。希望我们将通过询问外周血中的稀有细胞来证明单细胞技术在风险患者的临床前阶段诊断T1D的能力。这种革命性的方法会允许监测旨在维持β细胞量的新疗法。