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

• 批准号: 9086041
• 负责人: YONGWON CHOI
• 金额: $ 15.38万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-15 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9086041
• 关键词: Adoption; Area; Arts; Bacteria; Biological Assay; CD8B1 gene; Cell Respiration; Cell Separation; Cell Survival; Cell physiology; Cells; Cellular Metabolic Process; Characteristics; Computing Methodologies; Coupled; Detection; Devices; Disease; Dyes; Encapsulated; Fatty Acids; Fluorocarbons; Future; Genes; Genetic Programming; Growth; Homeostasis; Immune; Immune response; Immunotherapy; Individual; Infection; Inflammatory; Lead; Lectin; Link; Malignant Neoplasms; Measures; Memory; Metabolic; Microfluidic Microchips; Microfluidics; Modeling; Molecular; Molecular Profiling; Oils; Pathway interactions; Phase; Physiological; Population; Population Dynamics; Proliferating; Role; Sorting - Cell Movement; Speed; Staging; Surface; System; T cell differentiation; T cell response; T-Cell Activation; T-Lymphocyte; T-Lymphocyte Subsets; TRAF6 gene; Techniques; Testing; Variant; Virus; Withdrawal; adapter protein; adaptive immunity; base; 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; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

 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（如记忆细胞）的分子特征对于了解适应性免疫应答至关重要。特别是，关于我们对代谢变化是否可以驱动T细胞分化（如果是，如何）的理解，或者更确切地说，如果这些变化只是对外部因素（例如，细胞因子）。此外，尚不清楚脂肪酸代谢机制的调节与决定从效应细胞向记忆细胞转变的关键细胞命运决定之间是否存在直接因果关系，以及个体细胞中的代谢状态是否与更大的群体动态相关。我们试图确定一组分子标记物，这将使未来的功能测试和确定这种至关重要的细胞类型的命运决定途径。为了克服单细胞分子分析和标准代谢测定的局限性，我们将开发一种新型的微流体装置，该装置将使快速单细胞代谢分析和分选与单细胞转录组分析相结合。利用该装置，我们将通过其代谢特征分离罕见的CD8 T细胞亚群，并确定每个亚群的分子标记。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。具体地，高速氟碳（FC）油 将开发液滴细胞封装微流体装置，其耦合到代谢功能染料探针和高含量检测器分选系统。该设备将针对速度、捕获效率、细胞活力、高速检测和分选进行优化。在目标2中，我们将使用单细胞RNASeq在应答程序期间鉴定CD8 T细胞亚群中的分子标志物。为了实现这一目标，我们将把微流体装置与选择的功能染料耦合，以表征单个细胞的代谢状态。将通过计算方法分析代谢读数的特征，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门控细胞，并且将通过单细胞RNASeq分析所得细胞亚群以鉴定每种亚型的分子标记物。这项研究的结果将导致未来的研究所确定的分子在T细胞反应动力学的作用。了解T细胞功能亚型的分子基础不仅会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。