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

基本信息

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
项目类别：
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

项目摘要

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：近年来发现多种类型的 HIV 储存细胞可以在体内持续存在。抗逆转录病毒疗法（ART）的面貌包括那些“潜伏”的（转录-）HIV储存细胞。不活跃的）和转录活跃的储存细胞可能是主要的。正如分析治疗中断研究所示，这是导致 ART 中断后 HIV 反弹的因素。一些储存细胞在受到刺激后能够产生病毒蛋白（即诱导型储存细胞），并且这些细胞储存细胞对于反弹也可能很重要，因为它们富含基因组完整和复制能力不幸的是，缺乏对细胞表面表型的详细了解。这些 HIV 储存细胞阻碍了对 HIV 持久性生物学的充分了解，并阻碍了对 HIV 的研究。重要的是，之前描述储库表型的努力都集中在细胞表面。蛋白质和忽略的细胞表面聚糖，尽管事实上聚糖在调节多种关键的细胞过程和免疫功能，并已成为许多疾病中有用的生物标志物。我们最近证明，含有高水平岩藻糖基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+ 转录不活性细胞，并且这岩藻糖基化是由 HIV 感染直接诱导的，这些研究表明聚糖可能具有新颖的用途。在这项研究中，我们结合了新开发的单细胞表面的特定类型的储存细胞的生物标志物。糖组学 + 蛋白质组学技术与生物信息学分析相结合，用于分析 HIV 潜伏模型中的细胞和 ART 抑制的 HIV+ 个体，以便更好地了解这些细胞的构成，并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形象治疗性靶向生物标志物。在目标 1 中，我们将检验以下假设：细胞表面糖基化模式（包括岩藻糖基化）可区分这些研究将利用体外和体内的转录活性和非活性HIV储存细胞。我们最近建立的技术 CyTOF-Lec，将深度 CyTOF 表型分析与表面聚糖配对在目标 2 中，我们将检查体内诱导型储库的聚糖特征。为此，我们将采用我们最近建立和验证的 PP-SLIDE（预测前体单细胞）。使用距离估计的连接）方法来表征患者来源的聚糖+蛋白质特征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检验我们的中心假设，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 储存细胞和未感染的细胞，而且区分不同类型的储存细胞（例如，潜伏细胞、转录活性细胞和诱导细胞）。