Probing the unique attributes of the naïve reservoir

探索天然储层的独特属性

• 批准号: 10409284
• 负责人: DAVID N LEVY
• 金额: $ 83.11万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-12 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10409284
• 关键词: Antibodies; Attention; Back; Bar Codes; Bioinformatics; Biology; Blood; Blood Cells; CCR5 gene; CD4 Positive T Lymphocytes; Cells; Characteristics; Chronic; Clonal Expansion; DNA; Data; Disease Progression; Dissection; Doctor of Philosophy; Event; HIV; HIV Infections; Heterogeneity; Immune; In Vitro; Infection; Laboratories; Lead; Light; Longevity; Lymphoid; Lymphoid Tissue; Maintenance; Mature T-Lymphocyte; Memory; Methods; Monitor; Nature; Participant; Pattern; Peripheral Blood Mononuclear Cell; Phenotype; Play; Predisposition; Property; Proviruses; Research; Resistance; Rest; Role; Signal Pathway; Signal Transduction; Stains; Statistical Data Interpretation; Statistical Models; T cell differentiation; T memory cell; T-Lymphocyte; T-Lymphocyte Subsets; Techniques; Testing; Tissue Differentiation; Tonsil; Tropism; Up-Regulation; Viral; Viremia; Virus; Virus Diseases; Work; base; cell type; cohort; data curation; design; experimental study; in vivo; insight; integration site; lymph nodes; mathematical model; multiple omics; peripheral blood; pressure; prognostic indicator; receptor; single-cell RNA sequencing; stem; transcriptome sequencing

Resting CD4+ T cells form a reservoir capable of refueling HIV viremia when treatment is stopped. This is a major barrier to an HIV cure. Resting CD4+ T cells can be divided into naïve and memory subtypes. It was previously thought that naïve T cells formed a negligible portion of the reservoir, as the total amount of HIV DNA in the naïve pool is small. We have shown, however, that there is relatively more intact than defective HIV DNA in the naïve T cells compared to memory T cell subsets. In fact, in chronic progressors with X4 tropic virus, naïve are often the major contributor to the HIV reservoir. Moreover, the integration sites and proviral sequences of the HIV DNA in naïve cells are more diverse than in the memory subsets, indicating that the proviruses originated from many different infection events. Naïve T cells have a much longer lifespan than memory T cells, higher proliferative potential, and apparent resistance to clearance, which suggest that this naïve T cell reservoir is capable of repopulating the HIV reservoir. The formation of the naïve reservoir by CCR5-tropic viruses is still somewhat mysterious, as naïve cells in the blood do not usually express the CCR5 co-receptor, but preliminary data shows that naïve cells in lymphoid tissues show different expression patterns, including occasional expression of CCR5. Overall objective: We will quantify the contribution of naïve cells to the reservoir in donors that represent a spectrum of reservoir size (Aim 1). We exploit our exciting finding that elite controllers have nearly absent naïve T cell infection, while naïve infection serves as a prognostic indicator in chronic progressors. We will dissect the heterogeneous nature of naïve T cells, especially in lymphoid tissue, as has previously been done for memory T cells using RNA sequencing (Aim 2). We will elucidate important naïve biology and determine how naïve T cells become infected. Design and Methods: In Aim 1, led by Dr. O'Doherty, we quantify the contribution of naïve T cells to the entire HIV reservoir in both size and diversity using sophisticated sequencing techniques, unique bioinformatic approaches (Dr. Polson), and math modeling (Dr. Zurakowski). We will also study the ability of naïve T cells to replenish the reservoir and evade the CTL clearance. In Aim 2, led by Dr. Levy, we couple RNAseq with antibody barcoding to identify memory and naïve T cells with elevated levels of CCR5. We will probe the mechanism of naïve cell infection by infecting subset and bulk T cells from lymphoid tissue and blood. We will determine viral tropism phenotypically, and monitor for reversion to naïve phenotype ex vivo. These studies will also provide insights into the signaling pathways that distinguish naïve T cell subsets, and that determine their susceptibility to HIV. The premise of our proposal is largely based on our prior work that has shone a light on the role of naïve T cells and simultaneously provided insights into why these cells have previously been ignored. This proposal may lead to a greater focus on naïve T cell infection, as these cells are well-suited to hide proviruses. Focused attention on this entirely different cell type will be necessary to eradicate HIV.

静止的CD4+ T细胞形成一个水库，能够在治疗停止时为HIV病毒血症补充能量。这是一 艾滋病毒治愈的主要障碍。静息CD4+ T细胞可分为幼稚型和记忆型。这是 以前认为，幼稚T细胞形成的水库可以忽略不计的一部分，因为艾滋病毒DNA的总量， 在天真的游泳池是小的。然而，我们已经表明，相对而言，完整的HIV DNA比有缺陷的HIV DNA要多。 与记忆性T细胞亚群相比。事实上，在携带X4嗜性病毒的慢性进展者中， 往往是艾滋病病毒的主要来源。此外，整合位点和前病毒序列， 幼稚细胞中的HIV DNA比记忆亚群中的HIV DNA更多样化，表明前病毒起源于 许多不同的感染事件。幼稚T细胞的寿命比记忆T细胞长得多， 增殖潜力和明显的清除阻力，这表明这种幼稚T细胞库是 能够重新繁殖HIV病毒库。嗜CCR5病毒的幼稚储库的形成仍然是未知的。 这有点神秘，因为血液中的幼稚细胞通常不表达CCR5辅助受体，而是初步表达CCR5。 数据显示，淋巴组织中的幼稚细胞显示出不同的表达模式，包括偶尔的 CCR5的表达。总体目标：我们将量化幼稚细胞对供体库的贡献 代表了油藏规模的范围（目标1）。我们利用我们令人兴奋的发现， 几乎不存在幼稚T细胞感染，而幼稚感染作为慢性进展者的预后指标。 我们将剖析幼稚T细胞的异质性，特别是在淋巴组织中，如前所述。 使用RNA测序对记忆T细胞进行了研究（Aim 2）。我们将阐明重要的幼稚生物学，并确定 幼稚T细胞是如何被感染的设计和方法：在目标1中，由O'Doherty博士领导，我们量化了 使用复杂的测序，幼稚T细胞在大小和多样性方面对整个HIV库的贡献 技术，独特的生物信息学方法（波尔森博士），和数学建模（马奇科夫斯基博士）。我们还将 研究幼稚T细胞补充库和逃避CTL清除的能力。在目标2中，由博士领导。 Levy，我们将RNAseq与抗体条形码结合，以识别记忆和幼稚T细胞，其具有升高的免疫调节水平。 CCR 5。我们将通过感染来自淋巴细胞的亚群和大量T细胞来探索幼稚细胞感染的机制。 组织和血液我们将确定病毒的嗜性表型，并监测是否回复至初始表型 离体。这些研究还将为区分幼稚T细胞亚群的信号通路提供见解， 这决定了他们对艾滋病的易感性。我们建议的前提主要基于我们以前的工作， 揭示了幼稚T细胞的作用，同时提供了关于这些细胞为什么具有 以前被忽略了。这一提议可能会导致更多地关注幼稚T细胞感染，因为这些细胞是 非常适合隐藏前病毒集中注意力在这种完全不同的细胞类型将是必要的，以消除 艾滋病。