Single-cell characterization and quantification of translation-competent viral reservoirs in treated and untreated HIV infection
Single-cell characterization and quantification of translation-competent viral reservoirs in treated and untreated HIV infection
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DOI：
10.1371/journal.ppat.1007619
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发表时间：
2019-02-01
期刊：
影响因子：
6.7
通讯作者：
Chomont, Nicolas
中科院分区：
文献类型：
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作者：
Pardons, Marion;Baxter, Amy E.;Chomont, Nicolas
在抗逆转录病毒治疗(ART)期间，艾滋病毒持续存在的细胞的表型特征在技术上仍然具有挑战性。我们开发了一种简单的基于流式细胞术的分析方法来量化和表征在未经治疗和治疗的HIV感染过程中产生HIV蛋白的感染细胞。通过在标准的细胞内染色方案中结合针对HIV衣壳的两种抗体，我们证明可以在HIV感染者的血液中以高特异性和敏感性检测到产生p24的细胞。在未经治疗的个体中，生产性感染细胞的频率与血浆病毒载量密切相关。感染细胞优先表现为过渡性记忆表型，并富含Th17、外周血Tfh和调节性T细胞亚群。这些细胞还优先表达活化标志(CD25、HLA-DR、Ki67)、免疫检查点分子(PD-1、LAG-3、TIGIT、TIM-3)以及整合素47和41。在ART病毒抑制的个体中，只有在刺激时才能检测到产生p24的细胞(中位数为4.3p24+细胞/10(6)细胞)。这些措施与其他评估持久储存库大小的方法相关，包括总的和整合的HIV DNA、TAT/REV诱导的限制性稀释试验(TILDA)和定量病毒生长试验(QVOA)。在ART抑制的个体中，产生p24的细胞优先表现为过渡性和效应性记忆表型，并表达免疫检查点分子(PD-1，TIGIT)和整合素41。值得注意的是，在未经治疗和ART抑制的个体中，超过70%的感染细胞都表达了41。总之，这些结果突出了治疗和未治疗感染中HIV感染细胞表型的广泛多样性，并表明需要针对多个不同表型的细胞储存库的策略，以对储存库的大小产生重大影响。作者摘要在抗逆转录病毒治疗期间，HIV持续存在于感染的CD4+T细胞的小池中。更好地描述这些细胞是制定根除艾滋病毒战略的先决条件。我们开发了一种新的检测方法，名为HIV-Flow，用于同时量化和表征接受抗逆转录病毒治疗的个体的储藏细胞。通过这项测试，我们发现在抑制ART的个人中，只有5个细胞/百万的中位数有能力产生HIV蛋白Gag。这些频率与其他旨在测量艾滋病毒宿主的化验结果相关联。重要的是，我们发现HIV储存库的表型是多样化的，有许多细胞亚群对持续感染的细胞池做出了贡献。尽管如此，我们确定了几个优先在表面或这些稀有的储藏细胞上表达的标记，包括免疫检查点分子和归巢受体。通过结合这些标记，我们确定了在HIV感染细胞中高度浓缩的离散细胞亚群。这一新的检测方法将有助于识别由细胞HIV储存库表达的标记。
The phenotypic characterization of the cells in which HIV persists during antiretroviral therapy (ART) remains technically challenging. We developed a simple flow cytometry-based assay to quantify and characterize infected cells producing HIV proteins during untreated and treated HIV infection. By combining two antibodies targeting the HIV capsid in a standard intracellular staining protocol, we demonstrate that p24-producing cells can be detected with high specificity and sensitivity in the blood from people living with HIV. In untreated individuals, the frequency of productively infected cells strongly correlated with plasma viral load. Infected cells preferentially displayed a transitional memory phenotype and were enriched in Th17, peripheral Tfh and regulatory T cells subsets. These cells also preferentially expressed activation markers (CD25, HLA-DR, Ki67), immune checkpoint molecules (PD-1, LAG-3, TIGIT, Tim-3) as well as the integrins 47 and 41. In virally suppressed individuals on ART, p24-producing cells were only detected upon stimulation (median frequency of 4.3 p24+ cells/10(6) cells). These measures correlated with other assays assessing the size of the persistent reservoir including total and integrated HIV DNA, Tat/rev Induced Limiting Dilution Assay (TILDA) and quantitative viral outgrowth assay (QVOA). In ART-suppressed individuals, p24-producing cells preferentially displayed a transitional and effector memory phenotype, and expressed immune checkpoint molecules (PD-1, TIGIT) as well as the integrin 41. Remarkably, 41 was expressed by more than 70% of infected cells both in untreated and ART-suppressed individuals. Altogether, these results highlight a broad diversity in the phenotypes of HIV-infected cells in treated and untreated infection and suggest that strategies targeting multiple and phenotypically distinct cellular reservoirs will be needed to exert a significant impact on the size of the reservoir.Author summary HIV persists in a small pool of infected CD4+ T cells during ART. A better characterization of these cells is a pre-requisite to the development of HIV eradication strategies. We developed a novel assay, named HIV-Flow, to simultaneously quantify and characterize reservoir cells in individuals receiving ART. With this assay, we found that a median of only 5 cells/million have the ability to produce the HIV protein Gag in individuals on suppressive ART. These frequencies correlated with other assays aimed at measuring HIV reservoirs. Importantly, we show that the HIV reservoir is phenotypically diverse, with numerous cell subsets contributing to the pool of persistently infected cells. Nonetheless, we identified several markers preferentially expressed at the surface or these rare reservoir cells, including immune checkpoint molecules and homing receptors. By combining these markers, we identified discrete cellular subsets highly enriched in HIV-infected cells. This novel assay will facilitate the identification of markers expressed by cellular HIV reservoirs.