Département de microbiologie, infectiologie et immunologie, Faculté de médecine, Université de Montréal, Montréal, QC, Canada.
CHUM-Research Centre, Montréal, QC, Canada.
Methods Mol Biol. 2022;2407:81-89. doi: 10.1007/978-1-0716-1871-4_7.
Antiretroviral therapy (ART) has transformed the deadly human immunodeficiency virus type I (HIV-1) epidemic into a manageable chronic condition. Current ART is not curative and treatment interruption leads to viral rebound in people living with HIV-1 (PLWH). The main cause of viral rebound is the persistence of HIV reservoirs in long-lived memory CD4 T cells. Accurate techniques to identify and quantify viral reservoirs are required to monitor therapeutic approaches designed to cure HIV infection. Th17-polarized CD4 T cells are located at mucosal sites of HIV entry and are preferentially targeted for infection and viral reservoir persistence. They constitute an important reservoir in both blood and colon. In this chapter we describe a step-by-step flow cytometry-based protocol to isolate a fraction of Th17-enriched cells from PBMC based on their expression of the Th17 surface marker CCR6. The isolation of memory CCR6CD4 T cells allows subsequent PCR/RT-PCR-based HIV DNA/RNA quantifications, as well as their culture for quantitative viral outgrowth assays (QVOA). This method can be adapted for the isolation of CCR6CD4 T cells from peripheral tissues, such as the colon.
抗逆转录病毒疗法 (ART) 将致命的人类免疫缺陷病毒 I 型 (HIV-1) 流行转变为可控制的慢性疾病。目前的 ART 并不能治愈疾病，治疗中断会导致 HIV-1 感染者 (PLWH) 病毒反弹。病毒反弹的主要原因是 HIV 储库在长寿记忆 CD4 T 细胞中的持续存在。为了监测旨在治愈 HIV 感染的治疗方法，需要准确的技术来识别和量化病毒储库。Th17 极化的 CD4 T 细胞位于 HIV 进入的粘膜部位，并且优先被感染和病毒储库持续存在。它们在血液和结肠中都是一个重要的储库。在本章中，我们描述了一种基于流式细胞术的逐步方案，基于 Th17 表面标志物 CCR6 的表达，从 PBMC 中分离富含 Th17 的细胞。记忆 CCR6CD4 T 细胞的分离允许随后进行基于 PCR/RT-PCR 的 HIV DNA/RNA 定量，以及进行定量病毒扩增测定 (QVOA) 的培养。该方法可适用于从结肠等外周组织中分离 CCR6CD4 T 细胞。
