SINGLE-CELL TRANSCRIPTOMIC ANALYSIS OF HIV-INFECTED CD4 T CELLS IN BLOOD AND LYMPHOID TISSUE USING PCR-ACTIVATED CELL SORTING

使用 PCR 激活细胞分选对血液和淋巴组织中 HIV 感染的 CD4 T 细胞进行单细胞转录组分析

• 批准号: 9260642
• 负责人: Adam R. Abate
• 金额: $ 48.11万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-17 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9260642
• 关键词: Address; Anatomy; Antiviral Agents; Biological; Blood; CD4 Positive T Lymphocytes; Cell Separation; Cells; Chronic; Clinical; Coupled; DNA; Detection; Development; Emerging Technologies; Emulsions; Fluorescence-Activated Cell Sorting; Gene Expression; Gene Expression Profiling; Genetic; Genetic Transcription; Genomic DNA; HIV; HIV Infections; HIV Seropositivity; Immune; Immunobiology; Immunologic Deficiency Syndromes; Individual; Infection; Inflammation; Investigation; Lead; Link; Lymphoid Tissue; Microfluidics; Molecular Profiling; Oils; Participant; Pathogenicity; Pattern; Phenotype; Plasma; Population; Predisposition; Property; Recovery; Research; Sampling; Subgroup; Technology; Testing; Therapeutic; Time; Tissue Sample; Tissue Stains; Tissues; Translational Research; United States National Institutes of Health; Viral Proteins; Viremia; Virus; Water; Work; antiretroviral therapy; cohort; deep sequencing; design; follow-up; gene function; genetic signature; in vivo; lymph nodes; new technology; novel; novel marker; novel strategies; programs; prophylactic; transcriptome; transcriptome sequencing; transcriptomics; virology

Project Summary Understanding how the CD4 T cells that harbor HIV in vivo differ from those that do not may spur the development of prophylactic or therapeutic antiviral strategies. Unfortunately, due to the complexity of HIV-infected CD4 T cell populations and to key technical obstacles, the detection and manipulation of these rare cells in blood and lymphoid tissue samples from HIV-positive individuals has previously been infeasible. We have therefore developed a novel technology termed PCR-activated cell sorting (PACS) that allows identification and manipulation of individual CD4 T cells in microfluidically-generated water-in-oil emulsions by the presence of intracellular HIV genomic DNA. In the proposed investigations, we will use this technology to sort single HIV-infected CD4 T cells from blood and lymphoid tissue of HIV-positive individuals for gene expression profiling by whole transcriptome deep sequencing. This comprehensive approach will allow discovery of novel markers of HIV-infected CD4 T cell in vivo, which will then be validated in follow-up tissue staining and cell sorting studies. Importantly, access to well-characterized NIH Clinical Center cohorts of HIV- positive study participants with spontaneous control of the virus, with uncontrolled infection, and with virus suppression on antiretroviral therapy (ART) will allow targeted study of these distinct clinical subgroups. Comparing results from these subgroups will allow differences between HIV infected CD4 T cells under differing degrees of natural or treatment-induced virologic suppression to be understood. By thus elucidating the determinants of HIV target cell selection and persistence in vivo, these studies may lead to the development of novel HIV cure strategies.

项目摘要 了解体内含有HIV的CD4 T细胞与不相差的CD4 T细胞不同 可能刺激预防或治疗性抗病毒策略的发展。 不幸的是，由于HIV感染的CD4 T细胞群体的复杂性和关键 技术障碍，在血液中对这些稀有细胞的检测和操纵 来自HIV阳性个体的淋巴组织样品以前是不可行的。 因此，我们已经开发了一种新型技术，称为PCR激活的细胞分类 （PAC）允许识别和操纵单个CD4 T细胞 通过存在细胞内HIV的微氟生成的水乳液 基因组DNA。在拟议的调查中，我们将使用这项技术对单一进行分类 HIV感染的HIV感染的CD4 T细胞来自HIV阳性个体的血液和淋巴组织 通过整个转录组深度测序进行基因表达分析。这 全面的方法将允许发现HIV感染的CD4 T的新颖标记 体内细胞，然后将其在后续组织染色和细胞分类中进行验证 研究。重要的是，访问特征良好的NIH临床中心同类HIV- 积极的研究参与者对病毒的自发控制，不受控制 感染，以及抑制抗逆转录病毒疗法（ART）的病毒将允许有针对性 研究这些不同的临床亚组。比较这些亚组的结果将 在不同程度的天然或 治疗引起的病毒学抑制作用。通过阐明 HIV靶细胞选择和体内持久性的决定因素，这些研究可能会导致 发展新型HIV治疗策略。