Single-cell Analysis of Glycomic and Proteomic Features of the HIV Reservoir

项目概述：近年来，多种类型的HIV储库细胞可以持续存在， 抗逆转录病毒疗法（ART）的面貌。HIV储存细胞包括那些“潜伏”的细胞（转录上- 非活性）和转录活性的那些。转录活跃的储库细胞可能是一个主要的 如中断治疗的分析性研究所示，这是中断抗逆转录病毒治疗后艾滋病毒反弹的一个因素。此外，本发明还提供了一种方法， 一些储库细胞能够在刺激时产生病毒蛋白（即，可诱导的储库细胞），这些 储库细胞也可能对反弹很重要，因为它们富含基因组完整和复制， 有能力的HIV前病毒。不幸的是，缺乏对细胞表面表型的详细了解， 这些HIV储库细胞阻碍了对HIV持久性生物学的充分理解， 发展治愈。重要的是，以前表征储库表型的努力都集中在细胞表面 蛋白质和被忽视的细胞表面聚糖，尽管事实上，聚糖在调节多个 关键的细胞过程和免疫功能，并已在许多疾病中用作有用的生物标志物。 我们最近证明，外周血CD4 + T细胞携带高水平的岩藻糖基化碳水化合物， 富含HIV+转录活性细胞，缺乏HIV+转录失活细胞， 岩藻糖基化直接由HIV感染诱导。这些研究表明，聚糖可能具有作为新的 特定类型储层细胞的生物标志物。在这项研究中，我们联合收割机新开发的单细胞表面 糖组学+蛋白质组学技术，结合生物信息学分析，分析HIV潜伏期模型中的细胞 和ART抑制的HIV+个体，以便更好地了解这些细胞的组成， 目标是了解他们如何在面对ART时坚持下去，以及他们是否表达了一种独特的 治疗靶向生物标志物。 在目标1中，我们将检验细胞表面糖基化模式（包括岩藻糖基化）区分 转录活性和非活性HIV储库细胞在体外和体内。这些研究将利用 我们最近建立的技术CyTOF-Lec，将深入的CyTOF表型与表面聚糖配对， 在单细胞水平上进行表征。在目标2中，我们将研究体内可诱导储库的聚糖特征， 细胞为此，我们将调整我们最近建立和验证的PP-SLIDE（预测的前体单细胞 使用距离估计的连接）方法来表征患者来源的 可通过离体刺激诱导的储库细胞。 总之，这些分析将使我们能够测试我们的中心假设，即特定的细胞表面糖基化 包括岩藻糖基化在内的模式可以区分HIV储库细胞和未感染的细胞， 区分不同类型的储层单元（例如，潜伏的、转录活性的和可诱导的）。

PROJECT SUMMARY: It has emerged in recent years that multiple types of HIV reservoir cells can persist in the face of antiretroviral therapy (ART). HIV reservoir cells include those that are “latent” (transcriptionally- inactive) and those that are transcriptionally-active. The transcriptionally-active reservoir cells may be a major contributor to HIV rebound after ART interruption, as shown in analytic treatment interruption studies. In addition, some reservoir cells are able to produce viral proteins upon stimulation (i.e., inducible reservoir cells), and these reservoir cells may also be important for rebound as they are enriched for genome-intact and replication- competent HIV provirus. Unfortunately, the lack of a detailed understanding of the cell surface phenotypes of these HIV reservoir cells has precluded a full understanding of the biology of HIV persistence and hampered the development of a cure. Importantly, prior efforts to characterize reservoir phenotypes all focused on cell surface proteins and ignored cell surface glycans, despite the fact that glycans play a critical role in regulating multiple key cellular processes and immunological functions and have served as useful biomarkers in many diseases. We recently demonstrated that peripheral CD4+ T cells harboring high levels of fucosylated carbohydrates are enriched for HIV+ transcriptionally-active cells and deficient in HIV+ transcriptionally-inactive ones, and that this fucosylation is directly induced by HIV infection. These studies suggest that glycans may have utility as novel biomarkers of specific types of reservoir cells. In this study, we combine newly developed single-cell surface glycomic + proteomic technologies with bioinformatic analyses to analyze cells from an HIV latency model and ART-suppressed HIV+ individuals in order better understanding the makeup of these cells, with the ultimate goal of understand how they can persist in the face of ART and whether they express a unique profile of therapeutically targetable biomarkers. In Aim 1, we will test the hypothesis that cell-surface glycosylation patterns, including fucosylation, distinguish transcriptionally-active and inactive HIV reservoir cells in vitro and in vivo. These studies will take advantage of our recently established technique CyTOF-Lec, which pairs in-depth CyTOF phenotyping with surface glycan characterization at the single-cell level. In Aim 2, we will examine the glycan features of in vivo inducible reservoir cells. To do this, we will adapt our recently established and validated PP-SLIDE (predicted precursor single-cell linkage using distance estimation) approach to characterize the glycan + protein features of patient-derived reservoir cells inducible by ex vivo stimulation. Together, these analyses will allow us to test our central hypothesis that specific cell-surface glycosylation patterns, including fucosylation, can distinguish HIV reservoir cells from uninfected ones, and furthermore distinguish different types of reservoir cells (e.g., latent, transcriptionally-active, and inducible).