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

HIV 病毒库的糖组学和蛋白质组学特征的单细胞分析

• 批准号: 10481384
• 负责人: Mohamed Abdel Mohsen
• 金额: $ 29.84万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10481384
• 关键词: Abnormal Cell; Atlases; Back; Bioinformatics; Biological Markers; Biology; Blood; CD4 Positive T Lymphocytes; Carbohydrates; Cell Surface Proteins; Cell physiology; Cell surface; Cells; DNA; Data; Development; Disease; Frequencies; Genetic Transcription; Genome; Goals; HIV; HIV Infections; HIV antiretroviral; HIV therapy; Immunologics; In Vitro; Individual; Interruption; Link; Lymphoid Tissue; Maps; Membrane Proteins; Modeling; Patient observation; Patients; Pattern; Peripheral; Persons; Phenotype; Play; Polysaccharides; Process; Proteins; Proteomics; Proviruses; Reporter; Role; Sorting - Cell Movement; Specimen; Surface; T-Cell Activation; Techniques; Technology; Testing; Transcript; Validation; Viral Proteins; antiretroviral therapy; base; clinically relevant; design; glycosylation; immune function; in vivo; insight; novel; novel marker; single cell analysis; specific biomarkers; sugar; targeted biomarker; therapeutic target

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).

项目摘要：近年来出现了多种类型的艾滋病毒储存细胞可以持续存在 面对抗逆转录病毒治疗(ART)。HIV储存细胞包括那些“潜伏”的细胞(转录上- 不活跃)和转录活跃的那些。转录活跃的储藏细胞可能是主要的 在分析治疗中断研究中显示，ART中断后艾滋病毒反弹的贡献者。此外, 一些储藏细胞能够在刺激下产生病毒蛋白(即可诱导的储藏细胞)，而这些 储藏细胞可能对反弹也很重要，因为它们为基因组完整和复制而丰富。 有能力的艾滋病毒前病毒。不幸的是，缺乏对细胞表面表型的详细了解 这些艾滋病毒储存细胞阻碍了对艾滋病毒持久性生物学的全面了解，并阻碍了 一种治疗方法的发展。重要的是，先前研究储集层表型的工作都集中在细胞表面。 蛋白质和被忽视的细胞表面多聚糖，尽管多聚糖在调节多种 关键的细胞过程和免疫功能，在许多疾病中都是有用的生物标志物。 我们最近证实，含有高水平岩藻糖化碳水化合物的外周CD4+T细胞 富含HIV+转录活性细胞，而缺乏HIV+转录非活性细胞，这 岩藻糖基化是由HIV感染直接诱导的。这些研究表明，多聚糖可能具有新颖的用途 特定类型储集层细胞的生物标志物。在这项研究中，我们结合了新开发的单细胞表面 用生物信息学分析HIV潜伏模型细胞的糖链+蛋白质组学技术 以及被ART抑制的HIV+个体，以便更好地了解这些细胞的组成，最终 目标是了解他们如何在艺术面前坚持下去，以及他们是否表达了独特的 具有治疗靶向性的生物标志物。 在目标1中，我们将检验这样一个假设，即细胞表面糖基化模式，包括岩藻糖基化，区别于 体外和体内转录活性和非活性的艾滋病毒储存库细胞。这些研究将利用 我们最近建立的CytoF-Lec技术，它将深入的CyTOF表型与表面多糖配对 在单细胞水平上的表征。在目标2中，我们将检查体内可诱导储存库的葡聚糖特征 细胞。为了做到这一点，我们将采用我们最近建立和验证的PP-Slide(预测前体单细胞 用距离估计连锁)方法表征患者来源的多糖+蛋白特征 可通过体外刺激诱导的储藏细胞。 总之，这些分析将使我们能够检验我们的中心假设，即特定的细胞表面糖基化 模式，包括岩藻糖基化，可以区分艾滋病毒储存细胞和未感染的细胞，而且 区分不同类型的储藏细胞(例如，潜伏的、转录活跃的和可诱导的)。