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

项目总结 联合抗逆转录病毒疗法(ART)可以抑制艾滋病毒复制并降低死亡率 在感染艾滋病毒的人身上。然而，抗逆转录病毒疗法并不能消除潜伏的艾滋病毒蓄积物，因此对病毒有效 镇压需要终生的艺术管理。因此，开发一种消除或实现无艺术的方法 对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，我们仍然有有限的 对潜伏感染细胞的表型和功能特性的理解 艾滋病毒携带者。表征体内潜伏感染细胞的一个挑战是无法直接对这些细胞表型 细胞，因为缺乏一个通用的生物标记物来区分它们和未感染的细胞。因此，唯一的办法是 直接表型潜伏细胞一直是为了在体外刺激大量患者来源的细胞 通过潜伏细胞诱导病毒蛋白的表达。尽管这允许身份识别，因此 用流式细胞仪对重新激活的细胞进行表型分析，测得的表型与原始表型不同 由于体外刺激改变了基因表达，潜伏感染细胞的表型。在这里，通过应用 一种基于伪时间的生物信息学方法PP-Slide 用高维单细胞分析方法(CyTOF、单细胞)对患者细胞进行深表型分析 RNAseq)，我们推断了潜伏感染细胞在其原始预刺激状态下的表型，并使用 绘制体内潜伏储集层的方法。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……里面 目标2，我们将描述在目标1中识别的标记以及从 在单细胞RNAseq分析的细胞上实现PP-SLORD的无偏方法，丰富了储集层细胞 携带有基因完整的复制能力的艾滋病毒的艾滋病毒，因为这些细胞很可能是 重要的是要控制或消除不含ART的病毒控制。在目标3中，我们将描述 让潜伏感染的细胞持续存在，专注于抗原和动态平衡驱动的克隆的作用 CD4T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 分析方法绘制体内潜伏细胞的图谱，我们的研究将提供前所未有的定义 持续存在的储藏细胞的特征，揭示了是否有储藏细胞的特征与解剖(血液)有关 或生物性别(男性与女性)，并告知推动水库维护的机制。 这些知识对于设计有针对性的方法以实现普遍的艾滋病毒治愈将是重要的。

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.