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。

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.