CD8 T cell suppression of HIV latency establishment and maintenance in virally suppressed individuals

摘要： 目前的抗逆转录病毒疗法(Art)并不能根除HIV，正如病毒血症迅速反弹所表明的那样。 治疗中断。潜伏期化合物挽救潜伏感染细胞中病毒基因的表达 逆转剂(LRA)是一种减少经ART治疗的HIV感染者中的病毒库的策略(即， 电击和杀戮)。HIV潜伏期由导致病毒表达沉默的机制触发，包括 通过组蛋白甲基化和去乙酰化进行表观遗传DNA修饰，关键药物的有限可用性 转录因子和新生病毒转录本的低效延长。抗逆转录病毒治疗SIV的最新研究- 感染的猕猴表明，CD8+淋巴细胞耗尽之后是病毒的重新激活 产生，并增加对IL-15超级激动剂ALT803的LRA效应的敏感性。这些结果 强烈建议CD8+淋巴细胞在抑制HIV表达方面发挥重要作用，但尚未得到充分研究。 在这个方案中，我们假设CD8+T细胞通过促进HIV基因的表达来抑制HIV基因的表达 建立和维持艾滋病毒潜伏期，扭转这一影响可能导致显著 放大各种化合物的LRA效应，从而影响体内储存库的大小和稳定性。我们 将使用创新的方法来确定CD8+T细胞介导的HIV沉默机制。在目标1中，我们 将使用一种新的HIV潜伏期模型来表征CD8+T细胞在HIV建立中的作用 延迟。我们将量化HIV基因在CD4+T细胞亚群中的表达，并表征CD8+T细胞的影响 细胞对HIV潜伏期的建立。在目标2中，我们将描述CD8+T细胞在维持 艾滋病毒潜伏期。我们的体外模型将允许我们定义CD8+T细胞如何在细胞因子期间维持HIV潜伏期 驱动激活、增殖或分化。在目标3中，我们将设计一种组合反延迟策略 将消除大部分(如果不是全部)潜伏感染细胞。我们将使用高维流式细胞术来确定 与潜伏期逆转抑制和激活相关的表型改变。最终，我们会 CD8+淋巴细胞在对抗特定LRA(和LRA)逆转潜伏期中的作用 组合)作为开发有效干预措施的关键一步，在该干预措施中，潜伏感染细胞中的病毒转录 被抢救出来，以缩小水库的规模。这项提案中描述的实验将确定新的机制 可导致大多数(如果不是全部)潜伏感染的艾滋病毒重新激活和随后消除的艾滋病毒沉默 细胞。我们建议，这一总体目标可以通过循序渐进的科学方法来实现 包括体外和体外研究，这些研究将解决艾滋病毒持续存在的新定义的机制。我们相信 这项申请中提出的工作将使我们能够理解CD8+ 在ART治疗中，淋巴细胞抑制病毒转录并最终促进HIV潜伏期和持久性 艾滋病毒感染者，从而帮助设计根除艾滋病毒的新方法。

ABSTRACT: Current antiretroviral therapy (ART) does not eradicate HIV as shown by the rapid rebound of viremia upon treatment interruption. Rescuing viral gene expression in latently infected cells by compounds termed Latency Reversing Agents (LRA) is a strategy to reduce the virus reservoir in ART-treated HIV-infected individuals (i.e., shock & kill). HIV latency is triggered by mechanisms that lead to the silencing of virus expression including epigenetic DNA modification through histone methylation and deacetylation, limited availability of critical transcription factors and inefficient elongation of the nascent viral transcripts. Recent studies in ART-treated SIV- infected macaques demonstrated that depletion of CD8+ lymphocytes is followed by reactivation of virus production, and increased susceptibility to the LRA effect of the IL-15 superagonist ALT803. These results strongly suggest that CD8+ lymphocyte play an important yet understudied role in silencing HIV expression. In this proposal, we hypothesize that CD8+ T cells suppress HIV gene expression by promoting the establishment and maintenance of HIV latency, and that reversal of this effect may result in a significant amplification of the LRA effect of various compounds, thus impacting the reservoir size and stability in vivo. We will use innovative approaches to identify the CD8+ T cell mediated mechanisms of HIV silencing. In Aim 1, we will employ a novel model of HIV latency to characterize the role of CD8+ T cells in the establishment of HIV latency. We will quantify HIV gene expression in CD4+ T cell subsets and characterize the impact of CD8+ T cells on HIV latency establishment. In Aim 2, we will characterize the role of CD8+ T cells in the maintenance of HIV latency. Our in vitro model will allow us to define how CD8+ T cells maintain HIV latency during cytokine driven activation, proliferation or differentiation. In Aim 3, we will design a combinatorial anti-latency strategy that will eliminate most if not all latently infected cells. We will employ high dimensional flow cytometry to define alterations in phenotype associated with suppression and activation of latency reversal. Ultimately, we will characterize the role of CD8+ lymphocyte in counter-acting the latency reversal by specific LRAs (and LRA combinations) as a key step to develop effective interventions in which viral transcription in latently infected cells is rescued to reduce the reservoir size. The experiments described in this proposal will identify novel mechanisms of HIV silencing that can lead to the reactivation and subsequent elimination of most (if not all) latently infected cells. We propose that this overarching goal can be achieved through a step-wise scientific approach that includes in vitro and ex vivo studies that will address a newly-defined mechanism of HIV persistence. We believe that the work proposed in this application will allow us to understand the mechanisms by which CD8+ lymphocytes suppress virus transcription and ultimately promote HIV latency and persistence in ART-treated HIV-infected individuals, thus helping designing new approaches for HIV eradication.