Understanding and Reversing T Cell Dysfunction to Control and Eliminate Persistent HIV Reservoirs

项目总结 抗病毒细胞毒性CD8+T淋巴细胞(CTL)是控制病毒血症的关键，也是HIV的核心 治愈/缓解策略。然而，在对分子决定因素的理解上存在着根本的差距。 在未能控制病毒血症的个体中，HIV特异性CTL功能障碍的发病和持续存在， 恢复CTL功能的预防战略，以预防艾滋病毒和持久缓解。这样做的目的是 应用是定义HIV特异性CTL功能障碍的分子调控和生物标志物。基于Strong 初步证据，我们假设在失去病毒学控制之前，CTL功能障碍的启动 在抗逆转录病毒治疗过程中维持CTL功能障碍是由细胞内在机制介导的， 对其的确认将为抗击艾滋病毒顽固性的治疗干预提供信息。我们将定义监管 用多组学综合系统生物学方法在患者体内研究CTL功能障碍的机制 抗原特异性和单细胞分辨，并使用基因编辑验证恢复抗病毒CTL 功能。拟议的研究的基本原理是，对分子的更精确的理解 要推动以CTL为基础的艾滋病毒控制，需要管理持久和失败的CTL介导的艾滋病毒控制的机制 艾滋病毒治愈策略。目标1将定义CTL功能障碍在取得突破之前启动的机制 HIV控制员中的病毒血症。初步数据显示，抗病毒CTL功能的进行性丧失 在失去艾滋病毒控制的患者中出现病毒反弹。以此为目标的实验将阐明监管 通过评估表观遗传、转录和免疫功能的变化来控制CTL功能障碍的启动途径 在失去对艾滋病毒的控制之前使用纵向样本的转录后签名。调控途径 将被使用基因编辑进行机械验证而被扰乱。目标2将定义CTL 在药物抑制HIV的过程中维持功能障碍，以及功能可以在多大程度上 恢复了。初步证据表明，CTL功能障碍不能通过抗逆转录病毒治疗或免疫恢复 检查站封锁。这一目标的实验将确定功能障碍的分子机制。 在使用多组学方法进行抗逆转录病毒治疗期间，维持在未逃脱的艾滋病毒特异性CTL中，以及 确定通过抑制和编辑已识别的调节性基因可以在多大程度上恢复CTL功能 网络。这种方法是创新的，因为它结合了特定表位的受控患者内部比较 通过基因编辑按群体和单细胞转录、转录后和表观遗传学分析的CTL 以询问HIV特异性CTL功能障碍的分子机制。这个 拟议的研究具有重要意义，因为它将为发展和 评估旨在利用CTL根除或功能性治愈艾滋病毒感染的干预措施。

PROJECT SUMMARY Antiviral cytotoxic CD8+ T lymphocytes (CTLs) are critical for controlling viremia and are central to HIV cure/remission strategies. However, there is a fundamental gap in understanding the molecular determinants underlying the onset and persistence of HIV-specific CTL dysfunction in individuals who fail to control viremia, preventing strategies to restore CTL function for HIV prevention and durable remission. The objective of this application is to define molecular regulation and biomarkers of HIV-specific CTL dysfunction. Based upon strong preliminary evidence, we hypothesize that initiation of CTL dysfunction preceding loss of virologic control and maintenance of CTL dysfunction during antiretroviral therapy are mediated by cell-intrinsic mechanisms, identification of which will inform therapeutic interventions to combat HIV persistence. We will define regulatory mechanisms of CTL dysfunction using an integrative, multi-omics systems biology approach at intra-patient, antigen-specific, and single-cell resolution, and validate using gene editing for restoration of antiviral CTL function. The rationale of the proposed studies is that a more precise understanding of the molecular mechanisms governing durable versus failed CTL-mediated HIV control will be needed to advance CTL-based HIV cure strategies. Aim 1 will define mechanisms by which CTL dysfunction is initiated preceding breakthrough viremia in HIV controllers. Preliminary data demonstrate that a progressive loss of antiviral CTL function precedes viral rebound in patients who lose HIV control. Experiments in this aim will elucidate regulatory pathways governing the initiation of CTL dysfunction by evaluating changes in epigenetic, transcriptional, and post-transcriptional signatures using longitudinal specimens preceding loss of HIV control. Regulatory pathways will be disrupted using gene editing for mechanistic validation. Aim 2 will define mechanisms by which CTL dysfunction is maintained during pharmacologic HIV suppression and the extent to which function can be restored. Preliminary evidence indicates that CTL dysfunction is not restored by antiretroviral therapy or immune checkpoint blockade. Experiments in this aim will identify molecular mechanisms by which dysfunction is maintained in non-escaped HIV-specific CTLs during antiretroviral therapy using a multi-omics approach, and determine the extent to which CTL function can be restored by inhibition and gene editing of identified regulatory networks. This approach is innovative because it couples controlled intra-patient comparisons of epitope-specific CTLs by population and single-cell transcriptional, post-transcriptional, and epigenetic profiling with gene editing in primary cells to interrogate the molecular mechanisms that underlie HIV-specific CTL dysfunction. The proposed research is significant because it will provide the mechanistic groundwork for development and evaluation of interventions that aim to harness CTLs for eradication or functional cure of HIV infection.