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

• 批准号: 8706329
• 负责人: YURI BUSHKIN
• 金额: $ 37.96万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8706329
• 关键词: Rapid; Analysis; Single; Cell; Immunity

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. PUBLIC HEALTH RELEVANCE: Successful intervention in many diseases is often severely limited by insufficient understanding of the complex underlying pathology. In particular, the ability to classify the disease stage and to elucidate a prognosis may be poor or even impossible when pathological changes are identifiable only in a small number of cells within a larger population. Here we propose to develop a new immunological test that can identify, from among persons carrying an asymptomatic infection with Mycobacterium tuberculosis (the bacteria that cause tuberculosis), those individuals progressing toward disease and becoming infectious. Two billion people (about one-third of the world's population) are currently infected with the TB bacteria, with the highest concentration of new cases in resource-poor areas of South-East Asia and sub-Saharan Africa. Even though 90-95% of infected persons do not become sick, the number of infected individuals is high enough to give rise to 8 million new cases of TB and almost 2 million deaths each year worldwide. Existing blood-based clinical immunodiagnostic assays recognize TB infection but are not geared to distinguish between asymptomatic infection and active disease. The latter is currently identified only when patients excrete TB bacilli while coughing or sneezing (it is estimated that, by the time of diagnosis, a person with active TB has already infected up to twenty contacts). Thus, for public health purposes, it is critical to diagnose active disease when the patient is still asymptomatic and non-infectious. We propose to develop a new, rapid test requiring small amounts of blood that detects tell-tale immune cell subpopulations distinguishing the relevant groups. The detection method in this test requires basic flow cytometry, a technique routinely utilized in the clinical diagnosis of immunopathologies and fully accessible to district/peripheral level clinical laboratories that currently operate a fluorescence microscope in resource-poor regions. Translation of the proposed diagnostic methodology to clinical practice will allow identification and treatment of early cases of TB, thus impeding transmission and helping eliminate TB. While the present proposal is directed to a specific set of end-users in TB clinics, the proposed assay has broad public health relevance, because the same principles and methodologies are directly applicable to any infectious and non-infectious disease that can be characterized based on the properties of single immune cells involved in the body's recognition of or response to a particular condition.

描述（由申请人提供）：当潜在的病理改变或阶段和预后的相关性与更大人群中的异质细胞状态相关时，无法实现疾病分期和预后的分类，从而实现最有效的医疗干预。单细胞分析可以提供一种无与伦比的手段来测量和揭示复杂生物系统的异质性，从而了解生物功能和疾病过程变化的基础（或识别相关因素）。在这里，我们建议基于最先进的敏感和强大的技术，开发一种快速检测少量血液中单个T细胞抗原特异性反应的方法。我们的建议旨在开发一种测试，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（当无症状受试者未进展为疾病，不具有传染性，不需要治疗时）和临床前疾病（当无症状受试者正在发展疾病，仍不具有传染性时）的关键能力。并且需要早期治疗以阻止疾病的进展并大大遏制感染的传播)。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，结核病每年仍在全球造成数百万例疾病和死亡。我们的实验预计将通过整合(i)使用人工银呈递细胞（aAPC）来激活T细胞受体信号和刺激基因表达，以及（ii）通过定量流式细胞术测量T细胞激活和功能的诱导标记物，从而产生单个T细胞功能状态的多参数测量。诱导的基因表达将通过单分子荧光原位杂交（smFISH）的mRNA计数来检测。该研究计划有四个目标，每个目标都集中在检测的一个特定方面：(1)读出：在常规刺激后，通过smFISH和流式细胞术检测单个T细胞中的激活标记物；(2)刺激：通过检测单个T细胞的激活标记来评估对aAPC的反应；(3)对感染阶段特异性抗原的反应：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T细胞特征：单个T细胞反应的多参数表征及其与疾病与无症状感染的关联。拟议的计划应导致在微生物和临床症状和疾病症状出现之前识别和治疗活动性结核病。这是目前结核病诊断的圣杯，因为它被认为对消除结核病的努力至关重要。新的分析原理将翻译为诊断和