Probing the unique attributes of the naïve reservoir

探索天然储层的独特属性

基本信息

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
项目类别：
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

项目摘要

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.

静息的 CD4+ T 细胞形成一个储存库，能够在治疗停止时补充 HIV 病毒血症。静息 CD4+ T 细胞可分为幼稚亚型和记忆亚型。之前认为幼稚 T 细胞形成的病毒库的部分可以忽略不计，因为 HIV DNA 总量然而，我们已经证明，完整的 HIV DNA 相对来说比有缺陷的 HIV DNA 多。与记忆 T 细胞亚群相比，幼稚 T 细胞实际上，在携带 X4 热带病毒的慢性进展者中，幼稚 T 细胞是幼稚的。此外，它们的整合位点和原病毒序列往往是 HIV 病毒库的主要贡献者。幼稚细胞中的 HIV DNA 比记忆子集中的 HIV DNA 更加多样化，表明原病毒起源于来自许多不同感染事件的初始 T 细胞的寿命比记忆 T 细胞长得多，更高。增殖潜力和明显的清除阻力，这表明这种幼稚 T 细胞库是 CCR5 嗜性病毒仍能重新形成 HIV 病毒库。有点神秘，因为血液中的幼稚细胞通常不表达 CCR5 共同受体，但初步表达数据显示，淋巴组织中的幼稚细胞表现出不同的表达模式，包括偶尔的表达模式总体目标：我们将量化幼稚细胞对供体储存库的贡献。代表了一系列储层尺寸（目标 1）。我们利用了精英控制器所拥有的令人兴奋的发现。几乎不存在幼稚 T 细胞感染，而幼稚感染可作为慢性进展者的预后指标。我们将剖析初始 T 细胞的异质性，特别是在淋巴组织中，正如之前所研究的那样使用 RNA 测序对记忆 T 细胞进行研究（目标 2）。初始 T 细胞如何被感染设计和方法：在 O'Doherty 博士领导的目标 1 中，我们量化了使用复杂的测序分析幼稚 T 细胞对整个 HIV 病毒库的大小和多样性的贡献技术、独特的生物信息学方法（波尔森博士）和数学建模（祖拉科夫斯基博士）。在 Aim 2 中，由 Dr. 领导，研究幼稚 T 细胞补充储存库并逃避 CTL 清除的能力。 Levy，我们将 RNAseq 与抗体条形码结合起来，以识别具有升高水平的记忆和幼稚 T 细胞 CCR5。我们将通过感染淋巴细胞亚群和大量 T 细胞来探究幼稚细胞感染的机制。我们将确定病毒的表型趋向性，并监测是否恢复为初始表型。这些研究还将深入了解区分幼稚 T 细胞亚群的信号通路，我们提议的前提很大程度上是基于我们之前的工作：揭示了幼稚 T 细胞的作用，同时提供了关于为什么这些细胞具有这一提议可能会导致人们更加关注幼稚 T 细胞感染，因为这些细胞是非常适合隐藏原病毒，有必要将注意力集中在这种完全不同的细胞类型上。艾滋病病毒。