Identifying Rare Subtypes of CD8 T-cells Using Single Cell Reactors

使用单细胞反应器鉴定 CD8 T 细胞的稀有亚型

基本信息

批准号：
9262845
负责人：
YONGWON CHOI
金额：
$ 27.46万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-15 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9262845
关键词：
Adaptor Signaling Protein Adoption Area Bacteria Biological Assay CD8-Positive T-Lymphocytes CD8B1 gene Cell Respiration Cell Separation Cell Survival Cell physiology Cells Cellular Metabolic Process Characteristics Computer Analysis Computing Methodologies Coupled Detection Devices Disease Dyes Encapsulated Fatty Acids Fluorocarbons Future Genes Genetic Growth Factor Homeostasis Immune Immune response Immunotherapy Individual Infection Inflammatory Lectin Link Malignant Neoplasms Measures Memory Metabolic Microfluidic Microchips Microfluidics Modeling Molecular Molecular Profiling Oils Pathway interactions Phase Physiological Population Population Dynamics Proliferating Role Secondary to Sorting - Cell Movement Speed Surface System T cell differentiation T cell response T-Lymphocyte T-Lymphocyte Subsets TRAF6 gene Techniques Testing Variant Virus Withdrawal adaptive immune response cell mediated immune response cell type cytokine detector fatty acid metabolism interest metabolic profile molecular marker novel precursor cell programs public health relevance receptor response secondary infection single cell analysis transcription factor transcriptome transcriptome sequencing transcriptomics

项目摘要

DESCRIPTION (provided by applicant): Understanding the molecular characteristics of immune cells, such as memory cells, is critical for understanding adaptive immune response. In particular, gaps remain concerning our understanding of whether metabolic changes can drive T cell differentiation (and if so, how), or rather, if these changes are simply by-products of responses to external factors (e.g., cytokines) encountered during the course of differentiation. Further, it remains unclear whether there exists a direct causal relationship between modulation of the fatty acid metabolizing machinery and the critical cell fate decision(s) dictating the transition from an effector to a memory cell, and whether metabolic states in individual cells can be correlated with larger population dynamics. We seek to identify a panel of molecular markers will allow future functional testing and identification of fate decision pathways for this criticaly important cell type. To overcome the limitations of single cell molecular profiling and standard metabolic assays, we will develop a novel microfluidics device that will enable rapid single cell metabolic profiling and sorting coupled to single cell transcriptome profiling. Utilizing this devie, we will isolate rare subpopulations of CD8 T cells by their metabolic signatures and identify molecular markers for each subpopulation. In Aim 1, we will develop a microfluidics system for rapid single cell metabolic profiling and sorting. Specifically, a high-speed fluorocarbon (FC) oil droplet cell encapsulating microfluidics device coupled to metabolic functional dye probes and high-content detector sorting system will be developed. The device will be optimized for speed, capture efficiency, cell viability, high-speed detection, and sorting. In Aim 2, we will identify molecular markers in sub-population of CD8 T cells during response program using single cell RNASeq. To achieve this aim, we will couple the microfluidics device with select functional dyes to characterize individual cell's metabolic states. The profiles of the metabolic readouts will be analyzed by computational methods to identify distinct subpopulations. These metabolic signatures will be used to gate the cells by the microfluidics device in a sorting mode and the resulting subpopulations of cells will be analyzed by single cell RNASeq to identify molecular markers for each subtype. The results of this study will lead to future studies on the role of identified molecules in T cell response dynamics. Understanding the molecular basis of T cell functional subtypes will not only enhance understanding of our body's response to diseases, it will lead to translational applications in areas such as cancer immune-therapy.

描述（由申请人提供）：了解免疫细胞（例如记忆细胞）的分子特征对于了解适应性免疫反应至关重要。特别是，我们对代谢变化是否可以驱动 T 细胞分化的理解仍然存在差距（如果是的话，或者更确切地说，如果这些变化只是分化过程中遇到的外部因素（例如细胞因子）反应的副产品，那么脂肪酸代谢的调节之间是否存在直接因果关系仍不清楚。机械和关键细胞命运决定决定从效应细胞到记忆细胞的转变，以及单个细胞的代谢状态是否可以与更大的群体动态相关。我们寻求鉴定一组分子标记，这将允许未来的功能测试和命运鉴定。为了克服单细胞分子分析和标准代谢分析的局限性，我们将开发一种新型微流体装置，利用该装置能够快速进行单细胞代谢分析和分类。在目标 1 中，我们将开发一种用于快速单细胞代谢分析和分选的微流体系统，具体来说是一种高速碳氟化合物 (FC) 油。在目标 2 中，我们将开发与代谢功能染料探针和高内涵检测器分选系统相结合的液滴细胞封装微流体装置，该装置将针对速度、捕获效率、细胞活力、高速检测和分选进行优化。使用单细胞 RNASeq 识别 CD8 T 细胞亚群中的分子标记为了实现这一目标，我们将微流体装置与选定的功能染料结合起来，以表征单个细胞的代谢状态。读数将通过计算方法进行分析，以识别不同的亚群。这些代谢特征将用于通过微流体装置以分选模式对细胞进行门控，并且将通过单细胞 RNASeq 分析所得的细胞亚群，以识别每个亚型的分子标记。这项研究的结果将引导未来对已识别分子在 T 细胞反应动力学中的作用的研究，了解 T 细胞功能亚型的分子基础不仅将增强对我们身体对疾病反应的理解，还将导致癌症免疫治疗等领域的转化应用。