Defining molecular pathways triggered by IL-10 and TGFb that drive HIV integration and persistence in Tfh cells in lymph nodes

定义由 IL-10 和 TGFb 触发的分子途径，驱动 HIV 整合并在淋巴结 Tfh 细胞中持续存在

• 批准号: 10762759
• 负责人: Susan Pereira Ribeiro
• 金额: $ 70.05万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-05 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10762759
• 关键词: Address; Aftercare; Animals; Anti-Inflammatory Agents; Antibodies; BCL1 Oncogene; Biology; CD4 Positive T Lymphocytes; Cells; Chromatin; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Data Analyses; Fine needle aspiration biopsy; Frequencies; Gene Targeting; Genes; Genetic Transcription; HIV; HIV Infections; Helper-Inducer T-Lymphocyte; Human; In Vitro; Individual; Infection; Interleukin-10; Interleukin-6; Interruption; Intervention; Lead; Lymph Node Tissue; Lymphoid Tissue; Macaca; Macaca mulatta; Maintenance; Methods; Modeling; Molecular; Morbidity - disease rate; Oligonucleotides; PD-1 blockade; PD-1 pathway; Pathogenesis; Pathway interactions; Pattern; Peripheral Blood Mononuclear Cell; Plasma; Play; Predisposition; Productivity; Protein Isoforms; Provirus Integration; Proviruses; RNA; Research Personnel; Retrospective Studies; Role; SIV; STAT3 gene; Sampling; Series; Signal Pathway; Signal Transduction; Site; Specimen; Suspensions; TGF Beta Signaling Pathway; Testing; Time; Tissue imaging; Tissues; Tonsil; Transforming Growth Factor beta; Viral; Viral Genes; Viral reservoir; Viremia; Virion; Work; antiretroviral therapy; biobank; chronic infection; cohort; confocal imaging; cytokine; immunoregulation; in vitro Model; in vivo; innovation; interdisciplinary approach; interest; interleukin-21; knockout gene; lymph nodes; mortality; multiple omics; novel; programmed cell death protein 1; single cell analysis; synergism; tool; transcription factor; transcriptomics; viral DNA; viral RNA; viral rebound; virology

Follicular helper T cells (Tfh) are believed to be major contributors to the viral reservoir that persists in HIV-infected individuals, even when on antiretroviral therapy. We will explore the novel hypothesis that cytokines IL-10 and TGF-b, which play important roles in lymph node biology and are upregulated post HIV infection, are critically important for the formation of such a reservoir and represent potential targets for intervention. Specifically, we hypothesize that IL-10/TGF-b promote the differentiation of HIV-susceptible Tfh cells, with reduced antiviral defenses, that favor integration of intact proviruses into open chromatin gene targets downstream of IL-10/TGF-b signaling pathways. We will examine multiple aspects of this model, including how IL-10 and TGF-b suppress the intrinsic antiviral machinery in Tfh cells and how chromatin accessibility in genes downstream of IL10/TGF-b signaling, promoted by STAT3/SMADs activation, allows infecting virions to integrate preferentially in these open loci. As a result of these mechanisms, infected Tfh cells provide a reservoir of actively transcribing intact proviruses, even under ART. In Aim 1, we will investigate these hypothesized roles of IL-10 and TGF-b in Tfh differentiation and HIV integration/transcription using biobanked samples from lymph nodes of HIV-infected individuals at different stages post HIV infection (i.e. Fiebig IV/V, untreated chronic infection, and ART-treated). In Aim 2, we will mechanistically validate our model, using tools such as CRISPR gene knockouts in ex vivo tonsil isolated CD4 T cells, to examine how the major pathways identified in Aim 1 contribute to the differentiation of Tfh cells, the suppression of anti-viral machinery, and the promotion of integration of intact proviruses into active transcription sites. Finally, in Aim 3, we will take advantage of lymph node specimens available from a cohort of rhesus macaques that previously controlled SIV replication post-ART interruption (VL<1000 cps/mL), subsequent to in vivo blockade of the IL-10 and PD-1 pathways. We will dissect mechanistically how this treatment led to SIV viral DNA decay in their LNs. This proposal has several innovative aspects. Additionally to the available biobanked human and macaque samples, we will use cutting-edge methods, including multiome, MIP-seq, multiplexed confocal imaging with vDNA/RNA scope, and spatial transcriptomics. The project builds on our expertise with HIV pathogenesis, Tfh biology, and the modulation of immune pathways through both in vitro models and in vivo interventions in rhesus macaques. Additionally, we have the support of strong Co-Investigators with expertise in virology (Vandekerckhove), tissue imaging (Petrovas), and data analysis, integration and interpretation (Kamaleswaran and Sekaly). With such multidisciplinary approaches, synergies across the Aims, and a highly collaborative group of established and early-stage investigators (proposing PI), we are confident that this project will lead to important discoveries about immune regulation in the LN milieu and its impact on the HIV tissue reservoir.

滤泡辅助性T细胞（Tfh）被认为是病毒库的主要贡献者， 艾滋病毒感染者，即使在抗逆转录病毒治疗。我们将探讨新的假设， IL-10和TGF-β在淋巴结生物学中起重要作用，并在HIV感染后上调， 对于这种储层的形成至关重要，并且代表了干预的潜在目标。 具体来说，我们假设IL-10/TGF-β促进HIV易感性Tfh细胞的分化， 降低抗病毒防御，有利于完整的前病毒整合到开放的染色质基因靶点中 IL-10/TGF-β信号通路的下游。我们将研究这个模型的多个方面，包括如何 IL-10和TGF-b抑制Tfh细胞中固有的抗病毒机制以及基因中染色质可及性的方式 在IL 10/TGF-β信号传导的下游，由STAT 3/SMADs激活促进，允许感染病毒体整合 优选在这些开放位点。作为这些机制的结果，感染的Tfh细胞提供了一个活跃的Tfh细胞库。 转录完整的前病毒，即使在ART下。 在目的1中，我们将研究IL-10和TGF-β在Tfh分化和HIV感染中的假设作用。 使用来自不同时间HIV感染个体的淋巴结的生物库样品进行整合/转录 HIV感染后阶段（即Fiebig IV/V、未经治疗的慢性感染和ART治疗）。在目标2中，我们将 使用诸如在离体扁桃体分离的CD 4中的CRISPR基因敲除的工具， T细胞，以检查Aim 1中鉴定的主要途径如何有助于Tfh细胞的分化， 抑制抗病毒机制和促进完整前病毒整合到活性转录中 网站.最后，在目标3中，我们将利用来自恒河猴队列的淋巴结标本 先前控制SIV复制的猕猴在ART中断后（VL<1000 cps/mL），在ART中断后（VL<1000 cps/mL）， IL-10和PD-1通路的体内阻断。我们将从机制上剖析这种治疗如何导致SIV病毒 DNA在它们的淋巴结中衰变。 这项建议有几个创新的方面。除了可用的生物库人类和 猕猴样本，我们将使用尖端的方法，包括multiome，MIP-seq，多重共聚焦成像 vDNA/RNA扫描仪和空间转录组学。该项目建立在我们对艾滋病毒发病机制的专业知识基础上， Tfh生物学，以及通过体外模型和体内干预对免疫途径的调节， 恒河猴此外，我们还得到了具有病毒学专业知识的强大合作研究者的支持 （Vandekerckhove）、组织成像（Petrovas）和数据分析、整合和解释（Kamaleswaran 和Sekaly）。有了这种多学科方法，目标之间的协同作用和高度协作的小组 我们有信心，这个项目将导致重要的 关于LN环境中的免疫调节及其对HIV组织储库的影响的发现。