Single cell transcriptomics of HIV persistence and latency

摘要 HIV-1感染的抗逆转录病毒疗法(ART)抑制病毒复制，保存和改善 CD4T细胞计数和防止疾病进展。然而，抗逆转录病毒治疗不是治愈的，而且 中断治疗会持续释放病毒复发。病毒携带者的持久性遵循ART 是治愈艾滋病毒的主要障碍。为激活和消除艾滋病毒而提出的“电击和杀戮”战略 储蓄者目前并不知道支持潜伏期的T细胞状态或谱系。一 潜伏期研究中最大的挑战之一是区分潜伏期细胞和非潜伏期细胞，这是 本质上很难做到不扰乱细胞的潜伏状态。在目前的方法下，蜂窝 需要激活才能计数潜伏细胞，这就扰乱了我们想要研究的状态。至 克服了潜伏期研究的这一主要障碍，我们开发了一个HIV诱导的血统追踪模型 (柄)在人源化的小鼠身上，不可逆转地在基因上标记受感染的细胞。当与单个单元格组合时 在感染艾滋病毒、接受ART治疗的动物中，RNA测序(ScRNAseq)方法的结果是一个新兴的 与HIV感染和潜伏期相关的转录状态的基因组分辨率视图。初步研究 本文开始提供了前所未有的HIV感染的CD4T的单细胞基因组分类 急性感染和早期抗逆转录病毒治疗期间的细胞谱系和状态。在这项提案中，我们 探索人源化小鼠持续感染人CD4T细胞的单细胞多组学研究 它对寡克隆性TCR激活而不是稳态增殖有反应。T细胞的系统生物学将 被用来解剖HIV的新型小动物模型中的潜伏宿主，以了解宿主是如何 在不同的单元状态下生成和维护。基因组分析可用于鉴定药物或生物 可推动细胞主动表达HIV并独立于细胞激活的干预措施 州政府。这些可以被用来开发旨在增强表达和渐进性的治疗策略 潜伏储集层的衰变。我们假设单细胞多组学方法将阐明 发育不同的T细胞谱系和转录状态蕴藏着艾滋病毒的储存库，而且每一个 CLUSTER可能会显示与HIV持久性相关的独特基因程序。反转表示 与艾滋病毒持久性相关的因素可能会重新激活储存库。单细胞多组学可能揭开新的面纱 有针对性的策略，从不同的T细胞状态清除艾滋病毒。这项拟议的研究利用了一个团队 在HIV免疫发病机制、人源化小鼠、单细胞基因组学方面的专业知识，以深入研究人类T细胞表型 新的小动物模型中的细胞储存库。

Summary Antiretroviral therapy (ART) in HIV-1 infection suppresses viral replication, preserves and improves the CD4+ T cell count and prevents disease progression. However, treatment with ART is not curative, and interruption of therapy consistently unleashes viral relapse. The persistence of a viral reservoir following ART is the major obstacle to an HIV cure. Proposed “shock and kill” strategies to activate and eliminate the HIV reservoir are currently not informed by a knowledge of the T cell states or lineages that support latency. One of the greatest challenges in latency research is to distinguish latent cells from non-latent cells, which is inherently difficult to do without perturbing the latent state of the cells. With current approaches, cellular activation is required to enumerate latent cells, which disrupts the very state that we would like to study. To overcome this major roadblock to latency studies, we have developed an HIV-induced lineage tracing model (HILT) in humanized mouse that irreversibly genetically marks infected cells. When combined with single cell RNA sequencing (scRNAseq) approaches in HIV-infected, ART-treated animals, the result is an emerging genomic resolution view of transcriptional states associated with HIV infection and latency. Preliminary studies presented here begin to provide an unprecedented, single cell genomic classification of HIV-infected CD4 T cell lineages and states during acute infection and during early antiretroviral treatment. In this proposal, we explore single cell multi-omics of persistently infected human CD4 T cells in humanized mice and examine how it responds to oligoclonal TCR activation versus homeostatic proliferation. The systems biology of T cells will be used to dissect latent reservoirs in novel small animal models for HIV to understand how a reservoir is generated and maintained in distinct cell states. Genomic analysis may be used to identify drugs or biologic interventions that can push cells towards active HIV expression and are independent of cellular activation state. These could be used to develop cure strategies aimed at enhancing expression and the progressive decay of the latent reservoir. We hypothesize that a single cell multi-omics approach will elucidate developmentally diverse T cell lineages and transcriptional states that harbor HIV reservoirs, and that each cluster may display unique gene programs associated with HIV persistence. Reversing the expression of factors associated with HIV persistence may reactivate the reservoir. Single cell multi-omics may unveil new targeted strategies to purge HIV from different T cell states. The proposed study leverages a team with expertise in HIV immunopathogenesis, humanized mice, single cell genomics, to deeply phenotype human T cell reservoirs in novel small animal models.