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: 8721333
• 负责人: YURI BUSHKIN
• 金额: $ 74.98万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 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/8721333
• 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; Interferons; 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

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）：当潜在的病理变化或阶段和预后的相关性与较大群体内的异质细胞状态相关时，分类疾病阶段和阐明预后（其允许最有效的医学干预）是无法实现的。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（或识别相关性）。在这里，我们建议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，该方法基于最先进的技术，具有灵敏度和鲁棒性。我们的建议旨在开发一种测试，为结核病（TB）诊断医生提供区分稳定潜伏结核分枝杆菌感染的关键能力（当无症状受试者未进展为疾病、无感染性且不需要治疗时）（当无症状受试者正在发展疾病时，仍然没有传染性，并且需要早期治疗以阻断疾病的进展并显著抑制感染的传播）。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。我们的测定预期通过整合（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，与（ii）通过定量流式细胞术测量T细胞活化和功能的可诱导指示标记物，来产生单个T细胞功能状态的多参数测量。将使用单分子荧光原位杂交（smFISH）通过mRNA计数检测诱导的基因表达。该研究计划阐述了四个目标，每个目标都集中在该测定的特定方面的开发上：（1）读出：在常规刺激后通过smFISH和流式细胞术检测单个T细胞中的活化标志物;（2）刺激：通过检测单个T细胞中的活化标志物评估对aAPC的响应;（3）对感染阶段特异性Ag的响应：单个T细胞应答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细胞应答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。这是目前结核病诊断的圣杯，因为它被认为是消除结核病工作的关键。新的检测原理将可用于诊断和 涉及T细胞的任何病理的疾病分期，包括其他感染性疾病、癌症、自身免疫和移植。 公共卫生关系：对许多疾病的成功干预往往受到对复杂的潜在病理学认识不足的严重限制。特别是，当病理变化仅在较大群体中的少量细胞中可识别时，对疾病阶段进行分类和阐明预后的能力可能很差或甚至不可能。在这里，我们建议开发一种新的免疫学测试，可以识别，从携带结核分枝杆菌（导致结核病的细菌）的无症状感染者中，那些个人向疾病发展，并成为传染性。目前有20亿人（约占世界人口的三分之一）感染了结核病细菌，新病例最集中在东南亚和撒哈拉以南非洲的资源贫乏地区。尽管90-95%的受感染者不会生病，但受感染者的数量高得足以在全世界每年引起800万新的结核病病例和近200万人死亡。现有的基于血液的临床免疫诊断测定识别TB感染，但不适于区分无症状感染和活动性疾病。目前，只有当患者在咳嗽或打喷嚏时排出结核杆菌时，才能识别后者（据估计，到诊断时，活动性结核病患者已经感染了多达20个接触者）。因此，出于公共卫生目的，在患者仍无症状且无传染性时诊断活动性疾病至关重要。我们建议开发一种新的，快速的测试，需要少量的血液，检测区分相关群体的免疫细胞亚群。本试验中的检测方法需要基本的流式细胞术，这是一种常规用于免疫病理学临床诊断的技术，目前在资源贫乏地区使用荧光显微镜的地区/周边级临床实验室完全可以使用。将拟议的诊断方法转化为临床实践将有助于识别和治疗结核病的早期病例，从而阻止传播并帮助消除结核病。虽然本提议针对TB诊所中的一组特定的最终用户，但是所提议的测定具有广泛的公共卫生相关性，因为相同的原理和方法直接适用于任何传染性和非传染性疾病，所述传染性和非传染性疾病可以基于参与身体对特定病症的识别或响应的单个免疫细胞的性质来表征。