Rapid Analysis of Single T Cell Immunity Signatures in Tuberculosis

描述(由申请人提供)：当潜在的病理变化或阶段与预后的相关性与较大人群中的不同细胞状态相关时，就无法进行疾病阶段的分类和预后的阐明，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中的异质性，从而理解生物功能和疾病过程变化的基础(或确定其相关性)。在这里，我们建议开发一种快速检测少量血液中单个T细胞的抗原特异性反应的方法，该方法基于最先进的敏感和可靠的技术。我们的建议旨在开发一种测试，为结核病(TB)诊断人员提供关键能力，以区分稳定的潜在结核分枝杆菌感染(当无症状的受试者没有进展为疾病，不具有传染性，并且不需要治疗)和临床前疾病(当无症状的受试者正在发展疾病，仍然不具有传染性，需要早期治疗以阻止疾病的发展和大幅遏制感染的传播)。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记物研究方面的专业知识，结核病每年仍导致全球数百万人患病和死亡。我们的方法有望通过结合(I)使用人工抗原提呈细胞(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性指示物来产生单个T细胞功能状态的多参数测量。诱导基因的表达将通过单分子荧光原位杂交(SmFISH)的mRNA计数来检测。该研究计划包括四个目标，每个目标都集中在分析的一个特定方面：(1)读出：通过smFISH检测单个T细胞中的激活标记并在常规刺激后进行流式细胞术；(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出现疾病的微生物和临床体征和症状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。新的检测原理将可翻译用于诊断和 T细胞参与的任何病理的疾病分期，包括其他传染病、癌症、自身免疫和移植。 公共卫生相关性：对许多疾病的成功干预往往因对复杂的潜在病理认识不足而受到严重限制。特别是，当病理变化只能在较大人群中的少数细胞中识别时，对疾病阶段的分类和阐明预后的能力可能很差，甚至不可能。在这里，我们建议开发一种新的免疫学测试，可以从携带无症状结核分枝杆菌(导致结核病的细菌)感染的人中识别出那些进展为疾病并变得具有传染性的人。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，其中东南亚和撒哈拉以南非洲资源贫乏地区的新发病例最集中。尽管90%-95%的感染者没有患病，但感染者的数量足够高，每年在全球范围内导致800万新结核病病例和近200万死亡。现有的基于血液的临床免疫诊断方法可以识别结核病感染，但不能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，才能发现后者(据估计，到确诊时，一名活动性结核病患者已经感染了多达20名接触者)。因此，为了公共卫生的目的，在患者仍然没有症状和非传染性的情况下诊断活动性疾病是至关重要的。我们建议开发一种需要少量血液的新的快速检测方法，它可以检测区分相关群体的免疫细胞亚群。这项测试中的检测方法需要基本的流式细胞术，这是一项常规用于免疫病理学临床诊断的技术，目前在资源匮乏地区使用荧光显微镜的地区/外围一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能够识别和治疗早期结核病病例，从而阻止传播并帮助消除结核病。虽然目前的建议是针对结核病诊所的一组特定的最终用户，但建议的化验方法具有广泛的公共卫生意义，因为同样的原则和方法直接适用于任何传染病和非传染性疾病，这些疾病可以根据人体对特定疾病的识别或反应所涉及的单个免疫细胞的特性来表征。

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.