Entry of Polarized Effector Cells into Quiescence Forces HIV Latency
Entry of Polarized Effector Cells into Quiescence Forces HIV Latency
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DOI：
10.1128/mbio.00337-19
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发表时间：
2019-03-01
期刊：
影响因子：
6.4
通讯作者：
Karn, Jonathan
中科院分区：
文献类型：
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作者：
Dobrowolski, Curtis;Valadkhan, Saba;Karn, Jonathan
潜伏的HIV储库是在HIV感染活化的效应CD 4 T细胞后产生的，然后转化为记忆表型。在这里，我们描述了一种离体方法，称为QUECEL（静止效应细胞潜伏期），有效地模拟这一过程，并允许大量的潜伏感染的CD 4（+）T细胞的生产。将初始CD 4（+）T细胞极化为四种主要的T细胞亚群（Th 1、Th 2、Th 17和Treg），随后用表达GFP/CD 8a的单轮报告病毒感染。纯化感染的细胞，并使用确定的细胞因子混合物（包括肿瘤生长因子β、白细胞介素-10（IL-10）和IL-8）迫使其静止，产生潜伏感染细胞的同质群体。流式细胞术和转录组测序（RNA-Seq）证明细胞保持正确的极化表型，并已退出细胞周期。在从静止到再活化的过渡期间富集的关键途径和基因组包括E2 F靶点、G（2）M检查点、雌激素应答晚期基因表达和c-myc靶点。潜伏期逆转剂（LRA）对HIV的再活化与使用体外转录测序（EDITS）测定的包膜检测在来自良好抑制的HIV患者样本的细胞中观察到的RNA诱导谱非常相似。由于可以回收潜伏感染细胞的同质群体，QUECEL模型具有出色的信噪比，并且在过去4年进行的众多实验中具有极高的一致性和可重复性。的易用性，效率和准确性的模拟生理条件下，使QUECEL模型一个强大的和可重复的工具来研究HIV潜伏期的分子机制。重要性目前的HIV潜伏期的原代细胞模型与患者细胞的再激活行为相关性很差。我们已经开发了一种新的模型，称为QUECEL，它产生了一个大的和同质的群体潜伏感染的CD 4（+）记忆细胞。通过纯化HIV感染的细胞并用确定的细胞因子混合物诱导细胞静止，我们消除了以前HIV潜伏期的原代细胞模型的最大问题：可变的感染水平，不明确的极化状态和细胞转录的低效关闭。在QUECEL模型中，通过广泛的试剂进行的潜伏期逆转与患者样品中的RNA诱导密切相关。这种可扩展和高度可重复的HIV潜伏期模型将允许详细分析控制HIV潜伏期和再激活的细胞机制。
The latent HIV reservoir is generated following HIV infection of activated effector CD4 T cells, which then transition to a memory phenotype. Here, we describe an ex vivo method, called QUECEL (quiescent effector cell latency), that mimics this process efficiently and allows production of large numbers of latently infected CD4(+) T cells. Naive CD4(+) T cells were polarized into the four major T cell subsets (Th1, Th2, Th17, and Treg) and subsequently infected with a single-round reporter virus which expressed GFP/CD8a. The infected cells were purified and coerced into quiescence using a defined cocktail of cytokines, including tumor growth factor beta, interleukin-10 (IL-10), and IL-8, producing a homogeneous population of latently infected cells. Flow cytometry and transcriptome sequencing (RNA-Seq) demonstrated that the cells maintained the correct polarization phenotypes and had withdrawn from the cell cycle. Key pathways and gene sets enriched during transition from quiescence to reactivation include E2F targets, G(2)M checkpoint, estrogen response late gene expression, and c-myc targets. Reactivation of HIV by latency-reversing agents (LRAs) closely mimics RNA induction profiles seen in cells from well-suppressed HIV patient samples using the envelope detection of in vitro transcription sequencing (EDITS) assay. Since homogeneous populations of latently infected cells can be recovered, the QUECEL model has an excellent signal-to-noise ratio and has been extremely consistent and reproducible in numerous experiments performed during the last 4 years. The ease, efficiency, and accuracy of the mimicking of physiological conditions make the QUECEL model a robust and reproducible tool to study the molecular mechanisms underlying HIV latency.IMPORTANCE Current primary cell models for HIV latency correlate poorly with the reactivation behavior of patient cells. We have developed a new model, called QUECEL, which generates a large and homogenous population of latently infected CD4(+) memory cells. By purifying HIV-infected cells and inducing cell quiescence with a defined cocktail of cytokines, we have eliminated the largest problems with previous primary cell models of HIV latency: variable infection levels, ill-defined polarization states, and inefficient shutdown of cellular transcription. Latency reversal in the QUECEL model by a wide range of agents correlates strongly with RNA induction in patient samples. This scalable and highly reproducible model of HIV latency will permit detailed analysis of cellular mechanisms controlling HIV latency and reactivation.