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

滤泡辅助T细胞(TFH)被认为是持续存在的病毒库的主要贡献者 艾滋病毒感染者，即使在接受抗逆转录病毒治疗时也是如此。我们将探索一个新的假设，即细胞因子 IL-10和转化生长因子-β在淋巴生物学中发挥重要作用，并在HIV感染后上调。 对这种储集层的形成至关重要，是潜在的干预目标。 具体地说，我们假设IL-10/转化生长因子-b促进了艾滋病毒敏感的TFH细胞的分化， 抗病毒防御能力降低，有利于将完整的前病毒整合到开放的染色质基因靶标中 IL-10/转化生长因子-b信号通路下游。我们将研究此模型的多个方面，包括如何 IL-10和转化生长因子-β抑制TFH细胞固有的抗病毒机制以及染色质在基因中的可及性 在STAT3/Smads激活的推动下，IL10/转化生长因子-b信号的下游允许感染病毒粒子整合 尤其是在这些开放的基因座上。作为这些机制的结果，被感染的Tfh细胞提供了一个活跃的 转录完整的前病毒，即使在ART下也是如此。 在目标1中，我们将研究IL-10和转化生长因子-β在TFH分化和HIV中的这些假想作用 利用HIV感染者不同时期淋巴结生物库样本的整合/转录 艾滋病毒感染后的阶段(即FieBig IV/V、未经治疗的慢性感染和接受抗逆转录病毒治疗)。在目标2中，我们将 在体外分离的扁桃体中使用CRISPR基因敲除等工具，从机械上验证我们的模型 为了研究目标1中确定的主要途径如何促进TFH细胞的分化， 抑制抗病毒机制，促进完整前病毒整合到激活转录中 网站。最后，在目标3中，我们将利用从恒河猴队列中获得的淋巴结样本 之前控制SIV复制的猕猴在ART后中断(VL&lt；1000cps/mL)，在 体内阻断IL-10和PD-1通路。我们将机械地剖析这种治疗是如何导致SIV病毒的 DNA会在他们的LN中腐烂。 这项提议有几个创新方面。除了可用的生物库人类和 对于猕猴样本，我们将使用尖端方法，包括Multiome、MIP-Seq、多路共聚焦成像 VDNA/RNA范围和空间转录组。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，我们还得到了拥有病毒学专业知识的强大合作调查人员的支持 (Vandekerckhove)、组织成像(Petrovas)和数据分析、整合和解释(Kamaleswaran 和Sekaly)。有了这样的多学科方法，AIMS之间的协同效应，以及一个高度协作的小组 对于已建立的和早期阶段的调查人员(提议PI)，我们相信这个项目将导致重要的 关于LN环境中免疫调节的发现及其对HIV组织储存库的影响。

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.