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: 8541693
• 负责人: YURI BUSHKIN
• 金额: $ 72.72万
• 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/8541693
• 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; 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.

描述（由申请人提供）：当允许最有效的医疗干预措施的疾病阶段和阐明预后分类时，当潜在的病理变化或阶段和预后的相关性与较大人群中的异质细胞状态相关时，无法实现。单细胞分析可以提供一种无与伦比的方法来测量复杂的生物系统中的异质性，从而了解生物学功能和疾病过程变化的基础（或识别）相关性的基础。在这里，我们建议基于敏感且稳健的最新技术，开发一种快速测定，用于从少量血液中检测单个T细胞中抗原特异性反应。我们的建议旨在开发一项测试，该测试可提供结核病（TB）诊断医生，具有区分稳定的潜在潜伏分枝杆菌感染的关键能力 感染）。我们的多学科团队包括针对结核病的新型单细胞分析方法，细胞免疫学和生物标志物研究的开发专业知识，这些方法仍然每年造成数百万个疾病和死亡病例。预计我们的测定方法将通过（i）使用（i）使用人造AG呈递细胞（AAPC）来激活T细胞受体信号和基因表达刺激的多参数测量，并通过（ii）测量（ii）测量通过定量流式流式细计测量T细胞激活和功能的诱导型标记。使用单分子荧光原位杂交（Smfish），将通过mRNA枚举来检测诱导的基因表达。该研究计划在四个目标中阐明，每个目标都集中在分析的特定方面的发展：（1）读出：通过Smfish和常规刺激后通过Smfish和流式细胞仪检测单个T细胞中激活标记的检测； （2）刺激：通过检测单个T细胞中的激活标记评估的对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性T细胞特征：单参数表征单个T细胞反应以及与疾病与无症状感染的相关性。拟议的计划应导致在出现微生物和临床体征和疾病症状之前识别和治疗活跃的结核。这是当前在结核病诊断中的圣杯，因为它被认为对于消除结核病至关重要。新测定原则将用于诊断和 与T细胞受累的任何病理学的疾病分期，包括其他传染病，癌症，自身免疫性和移植。