Automated microarray platform for single-cell sorting and collection of lymphocytes following HIV reactivation.
Automated microarray platform for single-cell sorting and collection of lymphocytes following HIV reactivation.
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HIV重新激活后，用于单细胞分选和收集淋巴细胞的自动微阵列平台。
DOI：
10.1002/btm2.10551
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发表时间：
2023-09
影响因子：
7.4
通讯作者：
中科院分区：
文献类型：
--
作者：
治愈HIV感染者的一种有希望的策略是使用潜伏逆转剂（LRA）重新激活潜伏病毒，然后宿主清除受感染的储库细胞。然而，在感染的细胞内潜伏的前病毒的再活化是异质的并且通常是不完全的。这一事实限制了治疗HIV的策略，这些策略可能需要从所有细胞库中完全消除活病毒。出于这个原因，了解细胞库内HIV再活化的机制对于实现治疗成功至关重要。迫切需要能够在单细胞重新激活时对它们进行时间跟踪，然后对这些细胞进行分选和分子分析的方法。为此，微阵列适于在HIV长末端重复序列（LTR）启动子的控制下对表达mCherry的T淋巴细胞进行成像，以响应LRA（prostratin，iBET 151和SAHA）的应用。对prostratin、iBET 151和SAHA的反应分别为30.5%、11.2%和12.1%。该阵列能够随着时间的推移对大量单细胞（> 25，000）进行成像。mCherry荧光定量鉴定了具有不同再活化动力学的细胞亚群。在单细胞水平上观察到不同LRA之间在再活化时间、再活化后mCherry荧光增加速率和达到的峰值荧光方面存在显著异质性。在响应prostratin，T淋巴细胞亚群与慢和快的再活化动力学进行了鉴定。对作为快速或缓慢再活化剂的单个T淋巴细胞进行分选，并进行单细胞RNA测序。发现了与炎症、免疫激活、细胞和病毒转录因子相关的不同基因。
A promising strategy to cure HIV‐infected individuals is to use latency reversing agents (LRAs) to reactivate latent viruses, followed by host clearance of infected reservoir cells. However, reactivation of latent proviruses within infected cells is heterogeneous and often incomplete. This fact limits strategies to cure HIV which may require complete elimination of viable virus from all cellular reservoirs. For this reason, understanding the mechanism(s) of reactivation of HIV within cellular reservoirs is critical to achieve therapeutic success. Methodologies enabling temporal tracking of single cells as they reactivate followed by sorting and molecular analysis of those cells are urgently needed. To this end, microraft arrays were adapted to image T‐lymphocytes expressing mCherry under the control of the HIV long terminal repeat (LTR) promoter, in response to the application of LRAs (prostratin, iBET151, and SAHA). In response to prostratin, iBET151, and SAHA, 30.5%, 11.2%, and 12.1% percentage of cells, respectively. The arrays enabled large numbers of single cells (>25,000) to be imaged over time. mCherry fluorescence quantification identified cell subpopulations with differing reactivation kinetics. Significant heterogeneity was observed at the single‐cell level between different LRAs in terms of time to reactivation, rate of mCherry fluorescence increase upon reactivation, and peak fluorescence attained. In response to prostratin, subpopulations of T lymphocytes with slow and fast reactivation kinetics were identified. Single T‐lymphocytes that were either fast or slow reactivators were sorted, and single‐cell RNA‐sequencing was performed. Different genes associated with inflammation, immune activation, and cellular and viral transcription factors were found.
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影响因子：
7.3
作者：
Kang S;Tang H
通讯作者：
Tang H
影响因子：
6.7
作者：
Grau-Exposito, Judith;Luque-Ballesteros, Laura;Buzon, Maria J.
通讯作者：
Buzon, Maria J.
影响因子：
2.8
作者：
Hulver, Molly J.;Trautman, Julia P.;Miller, Heather B.
通讯作者：
Miller, Heather B.
影响因子：
30.3
作者：
Baxter AE;Niessl J;Fromentin R;Richard J;Porichis F;Charlebois R;Massanella M;Brassard N;Alsahafi N;Delgado GG;Routy JP;Walker BD;Finzi A;Chomont N;Kaufmann DE
通讯作者：
Kaufmann DE
影响因子：
6.1
作者：
Cortés-Llanos B;Wang Y;Sims CE;Allbritton NL
通讯作者：
Allbritton NL