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

项目摘要 了解体内携带HIV的CD 4 T细胞与不携带HIV的CD 4 T细胞的区别 可能会刺激预防性或治疗性抗病毒策略的发展。 不幸的是，由于HIV感染的CD 4 T细胞群的复杂性和关键的免疫缺陷， 技术障碍，检测和操纵血液中的这些稀有细胞， 来自HIV阳性个体的淋巴组织样品以前是不可行的。 因此，我们开发了一种称为PCR激活细胞分选的新技术 （PACS），允许识别和操作单个CD 4 T细胞， 通过细胞内HIV的存在微流体产生的油包水乳液 基因组DNA在拟议的调查中，我们将使用这种技术来排序单 来自HIV阳性个体的血液和淋巴组织的HIV感染的CD 4 T细胞， 通过全转录组深度测序进行基因表达谱分析。这 一种综合性的方法将允许发现HIV感染的CD 4 T细胞的新标志物 细胞，然后将在后续组织染色和细胞分选中进行验证 问题研究重要的是，获得表征良好的NIH临床中心HIV-1队列， 自发控制病毒的阳性研究参与者， 感染，并与病毒抑制抗逆转录病毒治疗（ART）将允许有针对性的 研究这些不同的临床亚组。比较这些亚组的结果将 允许HIV感染的CD 4 T细胞在不同程度的自然或免疫状态下的差异。 治疗诱导的病毒学抑制有待理解。通过这样阐明 这些研究可能导致艾滋病毒靶细胞选择和体内持续性的决定因素 开发新的艾滋病治疗策略。

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.