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。

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.