Rapid Analysis of Single T Cell Immunity Signatures in Tuberculosis

结核病中单 T 细胞免疫特征的快速分析

基本信息

批准号：
8541693
负责人：
YURI BUSHKIN
金额：
$ 72.72万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-07 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8541693
关键词：
Address Africa South of the Sahara Antigen-Presenting Cells Antigens Appearance Area Autoimmunity Bacillus (bacterium)Bacteria Basic Science Biological Assay Biological Markers Biological Process Blood CD4 Positive T Lymphocytes CD8B1 gene Cell Count Cells Cellular Immunology Cessation of life Clinic Clinical Color Communicable Diseases Complex Coughing Detection Development Diagnosis Diagnostic Disease Disease Progression Early treatment Event Evolution Far East FarGo Flow Cytometry Fluorescent in Situ Hybridization Fostering Frequencies Gene Expression Genes HLA-A2 Antigen HLA-DR4 Antigen Heterogeneity Immune Immunity Immunoassay Immunologic Tests Immunological Diagnosis Individual Infection Interleukin-2 Intervention Laboratories Lead MHC Class I Genes MHC Class II Genes Malignant Neoplasms Measurement Measures Mediating Medical Memory Messenger RNA Methodology Methods Mycobacterium tuberculosis Pathology Patients Peptides Peripheral Peripheral Blood Mononuclear Cell Persons Population Process Production Property Public Health Reading Receptor Signaling Research Resources Sampling Signs and Symptoms Sneezing Specificity Staging Stream T cell response T-Cell Activation T-Cell Receptor T-Lymphocyte T-Lymphocyte Subsets TNF gene Techniques Technology Testing Time Translational Research Translations Transplantation Tuberculosis base clinical Diagnosis clinical practice complex biological systems cytokine disease diagnosis effective intervention enzyme linked immunospot assay fluorescence microscope immunopathology latent infection multidisciplinary mycobacterial novel outcome forecast peripheral blood pre-clinical prognostic public health relevance receptor response single cell analysis single molecule success successful intervention transmission process

项目摘要

DESCRIPTION (provided by applicant): Classifying disease stage and elucidating a prognosis, which allow the most effective medical intervention, are unattainable when the underlying pathological changes, or the correlates of stage and prognosis, are associated with heterogeneous cells states within a larger population. Single cell analyses can provide an unsurpassed means to measure and unravel heterogeneity in complex biological systems, and thereby to understand the basis for (or to identify correlates of) changes in biological function and disease processes. Here we propose to develop a rapid assay for detection of antigen-specific responses in single T cells from small amounts of blood, based on state-of-the-art techniques that are sensitive and robust. Our proposal aims at developing a test that provides tuberculosis (TB) diagnosticians with the critical ability to distinguish stable latent Mycobacterium tuberculosis infection (when the asymptomatic subject is not progressing to disease, is not infectious, and does not require treatment) from preclinical disease (when the asymptomatic subject is developing disease, is still not infectious, and requires early treatment to block progression of disease and drastically curb transmission of infection). Our multidisciplinary team includes expertise in development of novel single cell analysis methodology, cellular immunology, and biomarker research for TB, which still causes millions of cases of disease and death worldwide every year. Our assay is expected to yield multi-parameter measurements of single T cell functional states by integrating (i) use of artificial Ag-presenting cells (aAPC) to activate T cell receptor signaling and stimulation of gene expression, with (ii) measurement of inducible tell-tale markers of T cell activation and function by quantitative flow cytometry. Induced gene expression will be detected by mRNA enumeration using single molecule fluorescence in situ hybridization (smFISH). The research plan is articulated in four aims, each focused on the development of a specific aspect of the assay: (1) read-out: detection of activation markers in single T cells by smFISH and flow cytometry following conventional stimulation; (2) stimulation: response to aAPC assessed by detection of activation markers in single T cells; (3) response to infection-stage-specific Ag: association of single T cell responses with disease vs asymptomatic infection; (4) infection-stage-specific functional T cell signatures: multi-parameter characterization of single T cell responses and association with disease vs asymptomatic infection. The proposed plan should lead to recognizing and treating active TB prior to the appearance of microbiological and clinical signs and symptoms of disease. This is the current holy grail in TB diagnosis as it is considered to be critical to TB elimination efforts. The new assay principles will be translatable for diagnosis and disease staging of any pathology with T cell involvement, including other infectious diseases, cancer, autoimmunity, and transplantation.

描述（由申请人提供）：当潜在的病理变化或阶段和预后的相关性与较大群体中的异质细胞状态相关时，对疾病阶段进行分类并阐明预后是不可能实现最有效的医疗干预的。单细胞分析可以提供一种无与伦比的方法来测量和揭示复杂生物系统中的异质性，从而了解生物功能和疾病过程变化的基础（或识别其相关性）。在这里，我们建议基于灵敏且稳健的最先进技术，开发一种快速检测方法，用于检测少量血液中单个 T 细胞的抗原特异性反应。我们的提案旨在开发一种测试，为结核病 (TB) 诊断人员提供区分稳定潜伏结核分枝杆菌感染（当无症状受试者没有进展为疾病、不具有传染性且不需要治疗时）与临床前疾病的关键能力。当无症状受试者正在患病，但仍不具有传染性，需要早期治疗以阻止疾病进展并彻底遏制感染传播时）。我们的多学科团队包括开发新型单细胞分析方法、细胞免疫学和结核病生物标志物研究方面的专业知识，结核病每年仍然在全球造成数百万例疾病和死亡。我们的检测预计将通过整合 (i) 使用人工 Ag 呈递细胞 (aAPC) 来激活 T 细胞受体信号传导和刺激基因表达，以及 (ii) 诱导性抗原的测量，从而对单个 T 细胞功能状态进行多参数测量。通过定量流式细胞术检测 T 细胞激活和功能的标志物。将使用单分子荧光原位杂交 (smFISH) 通过 mRNA 计数来检测诱导的基因表达。该研究计划有四个目标，每个目标都侧重于该检测的特定方面的开发：（1）读出：在常规刺激后通过 smFISH 和流式细胞术检测单个 T 细胞中的激活标记； (2)刺激：通过检测单个T细胞中的激活标记来评估对aAPC的反应； (3) 对感染阶段特异性 Ag 的反应：单个 T 细胞反应与疾病与无症状感染的关联； (4) 感染阶段特异性功能性 T 细胞特征：单个 T 细胞反应的多参数表征以及与疾病与无症状感染的关联。拟议的计划应导致在疾病的微生物学和临床体征和症状出现之前识别和治疗活动性结核病。这是当前结核病诊断的圣杯，因为它被认为对消除结核病工作至关重要。新的检测原理将可用于诊断和涉及 T 细胞的任何病理学的疾病分期，包括其他传染病、癌症、自身免疫和移植。