Probing the unique attributes of the naïve reservoir

探索天然储层的独特属性

• 批准号: 10663946
• 负责人: DAVID N LEVY
• 金额: $ 79.38万
• 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/10663946
• 关键词: Antibodies; Attention; 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; Light; Longevity; Lymphoid; Lymphoid Tissue; Maintenance; Mature T-Lymphocyte; Memory; Methods; Monitor; Nature; Participant; Pattern; Peripheral Blood Mononuclear Cell; Phenotype; Play; Predisposition; Property; Provirus Integration; Proviruses; Research; Resistance; Rest; Role; Signal Pathway; Signal Transduction; Sorting; Stains; Statistical Data Interpretation; Statistical Models; T cell differentiation; T memory cell; T-Lymphocyte; T-Lymphocyte Subsets; Techniques; Testing; Tonsil; Tropism; Up-Regulation; Viral; Viremia; Virus; Virus Diseases; Work; 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.

停止治疗时，静止的CD4+ T细胞形成能够加毒HIV病毒血症的储层。这是一个 艾滋病毒治愈的主要障碍。静止的CD4+ T细胞可以分为幼稚和记忆亚型。是 以前认为幼稚的T细胞形成了储层中可忽略的部分，因为HIV DNA的总量 在幼稚的游泳池里很小。但是，我们已经表明，与缺陷HIV DNA相对完整 在幼稚的T细胞中，与记忆T细胞子集相比。实际上，在患有X4热带病毒的慢性进度者中 通常是艾滋病毒水库的主要贡献者。此外，集成站点和配置序列 幼稚细胞中的HIV DNA比记忆子集更长，表明该病毒起源于 来自许多不同的感染事件。幼稚的T细胞的寿命比记忆T细胞更长，更高 增殖的潜力和对清除的明显抗性，这表明这是T细胞储层是 能够重现HIV水库。 CCR5冰球病毒的幼稚储层形成仍然是 有些神秘，因为血液中的幼稚细胞通常不会表达CCR5共同受体，而是初步的 数据表明，淋巴组织中的幼稚细胞显示出不同的表达模式，包括偶尔 CCR5的表达。总体目标：我们将量化幼稚细胞对供体中储层的贡献 这代表了一系列储层大小（AIM 1）。我们利用精英控制者拥有的令人兴奋的发现 几乎没有天真的T细胞感染，而幼稚的感染是慢性进度者的预后指标。 我们将剖析幼稚的T细胞的异质性，尤其是在淋巴组织中，就像以前 使用RNA测序为记忆T细胞完成（AIM 2）。我们将阐明重要的幼稚生物学并确定 幼稚的T细胞如何感染。设计和方法：在O'Doherty博士领导的AIM 1中，我们量化了 幼稚的T细胞使用复杂的测序在大小和多样性方面对整个HIV储层的贡献 技术，独特的生物信息学方法（Polson博士）和数学建模（Zurakowski博士）。我们也会 研究幼稚的T细胞复制储层并逃避CTL清除率的能力。在AIM 2中，由博士领导。 征税，我们将RNASEQ与抗体条形码搭配，以识别具有升高水平的记忆和幼稚的T细胞 CCR5。我们将通过感染的亚群和大量T细胞探测幼稚细胞感染的机制 组织和血液。我们将通过表观上确定病毒疗法，并监测反向幼稚的表型 前体。这些研究还将提供有关区分幼稚T细胞子集的信号通路的见解 这决定了他们对艾滋病毒的敏感性。我们提案的前提在很大程度上基于我们先前的工作 Shony阐明了幼稚的T细胞的作用，并简单地提供了这些细胞为什么具有的见解 以前被忽略。该建议可能会导致对幼稚的T细胞感染更加关注，因为这些细胞是 非常适合隐藏原病毒。将注意力集中在这种完全不同的细胞类型上是必需的 艾滋病病毒。