Phenotypic and mechanistic analysis of the in vivo HIV latent reservoir by single-cell technologies

项目概要 联合抗逆转录病毒疗法 (ART) 可抑制 HIV 复制并降低死亡率 在艾滋病毒感染者中。然而，ART 并不能消除潜伏的 HIV 病毒库，因此有效的病毒 抑制需要终生 ART 治疗。因此，开发一种方法来消除或实现无 ART 水库的控制是研究的重中之重。实现这一目标的一个挑战是我们的资源仍然有限 了解人们体内持续存在的潜伏感染细胞的表型和功能特性 患有艾滋病毒。表征体内潜伏感染细胞的一项挑战是无法直接对这些细胞进行表型分析 细胞，由于缺乏将它们与未感染细胞区分开的通用生物标志物。结果，唯一的办法就是 直接对潜伏细胞进行表型分析是在体外刺激大量患者来源的细胞，以便 诱导潜伏细胞表达病毒蛋白。尽管这允许识别，因此 通过FACS对重新激活的细胞进行表型分析，测得的表型与原始细胞不同 由于离体刺激改变了基因表达，因此潜伏感染细胞的表型。在这里，通过应用 基于伪时间的生物信息学方法，称为 PP-SLIDE，针对未刺激和刺激的配对组 通过高维单细胞分析方法（CyTOF、单细胞 RNAseq），我们推断潜伏感染细胞在原始刺激前状态的表型，并使用此 绘制体内潜在储库图的方法。在目标 1 中，我们将在 CyTOF 表型细胞上使用 PP-SLIDE 比较临床匹配的男性和女性血液和组织中存在的潜伏感染细胞。在 目标 2，我们将描述目标 1 中识别的标记以及从目标 1 中识别的标记的程度。 对单细胞 RNAseq 分析的细胞实施 PP-SLIDE 的无偏方法，富集储存细胞 携带 HIV 且具有基因完整且具有复制能力的 HIV 的细胞，因为这些细胞可能是最容易被感染的细胞。 对于无 ART 病毒控制而言，控制或消除非常重要。在目标 3 中，我们将描述以下机制： 让潜伏感染的细胞持续存在，重点关注抗原和稳态驱动的克隆的作用 CD4 T 细胞的扩增。通过将尖端的单细胞分析工具与高维数据相结合 分析方法来绘制体内潜伏细胞的“图谱”，我们的研究将为 储存细胞持续存在的特征揭示了是否有任何储存细胞特征与解剖学（血液）相关 与组织）或生物性别（男性与女性），并了解驱动水库维护的机制。 这些知识对于设计有针对性的方法以实现艾滋病毒的普遍治愈非常重要。

PROJECT SUMMARY Combination antiretroviral therapy (ART) can suppress HIV replication and lead to decreased mortality in HIV-infected individuals. However, ART does not eliminate the latent HIV reservoir, so effective viral suppression requires lifelong ART administration. Therefore, developing a way to eliminate or achieve ART-free control of the reservoir is a top research priority. One challenge to accomplishing this is that we still have limited understanding of the phenotypic and functional properties of the latently-infected cells that persist in people living with HIV. One challenge to characterizing in vivo latently-infected cells is the inability to directly phenotype these cells, due to the lack of a universal biomarker distinguishing them from uninfected cells. As a result, the only way to directly phenotype latent cells has been to stimulate a bulk population of patient-derived cells ex vivo in order to induce expression of viral proteins by the latent cells. Although this allows identification and therefore phenotyping of the reactivated cells by FACS, the measured phenotypes are different from the original phenotypes of the latently-infected cells since ex vivo stimulation alters gene expression. Here, by applying a pseudotime-based bioinformatics approach called PP-SLIDE on paired sets of unstimulated and stimulated patient cells deep-phenotyped by high-dimensional single-cell analytical approaches (CyTOF, single-cell RNAseq), we infer the phenotypes of latently-infected cells in their original pre-stimulation state, and use this approach to chart the in vivo latent reservoir. In Aim 1, we will use PP-SLIDE on CyTOF-phenotyped cells to compare the latently-infected cells present in the blood and tissues of clinically-matched men and women. In Aim 2, we will characterize the extent to which markers identified in Aim 1, as well as markers identified from an unbiased approach implementing PP-SLIDE on cells analyzed by single-cell RNAseq, enrich for reservoir cells harboring HIV with genetically-intact replication-competent HIV, for these are the cells that are likely the most important to control or eliminate for ART-free viral control. In Aim 3, we will characterize the mechanisms that allow the latently-infected cells to persist, focusing on the role of antigen- and homeostasis-driven clonal expansion of CD4+ T cells. By combining cutting-edge single-cell analysis tools with high-dimensional data analysis methods to map the “atlas” of in vivo latent cells, our studies will provide an unprecedented definition of the features of reservoir cells that persist, reveal whether any reservoir cell traits associate with anatomy (blood vs. tissues) or biological sex (men vs. women), and inform on the mechanisms driving reservoir maintenance. This knowledge that will be important for designing targeted methods to achieve a universal HIV cure.