Understanding HIV-1 persistence in cytotoxic CD4+ T lymphocytes at the single cell level

项目总结 尽管进行了有效的抗逆转录病毒治疗(ART)，但HIV-1终生存在于潜伏的宿主中。尽管大多数HIV病毒- 1-感染细胞死于病毒的细胞病变效应或免疫清除，艾滋病毒-1感染的细胞，即使是那些活跃的 HIV-1的表达，可以存活、持续和增殖。我们想要研究HIV-1是如何感染的 在病毒血症期间，在艾滋病毒-1携带者的病毒抑制下持续存在。了解免疫细胞 HIV-1感染细胞的亚群、免疫程序和细胞标记将确定治疗目标。这个 挑战在于HIV-1感染细胞的异质性和稀有性：在接受ART治疗、病毒抑制的个体中， 只有1-100/106(0.01%)的CD4+T细胞携带可诱导的HIV-1。为此，我们分析了来自 病毒血症期间和病毒抑制后使用单细胞ECCITEseq进行Sabes队列，该ECCITEseq捕获单细胞 同一细胞内的转录组、蛋白质表达、HIV-1RNA和T细胞受体序列(TCR)。 我们已经建立了先进的单细胞生物信息分析管道和机器学习工具。这 方法使免疫细胞亚群的多维、高分辨率、单细胞图谱成为可能 程序、细胞标志物、T细胞克隆性扩增，以及HIV-1RNA+细胞同时存在。我们的单细胞 ECCITEseq发现，HIV-1RNA+细胞上调了细胞毒CD4+T细胞基因。使用流式细胞仪 检测HIV-1p24蛋白和颗粒酶B的表达，我们基于RNA-SEQ的结果通过 基于蛋白质的正交法揭示HIV-1通过隐藏在颗粒酶B+细胞毒效应中而持续存在 记忆的CD4+T细胞。根据我们对病毒样本的结果，我们可以检测罕见的HIV-1RNA+细胞 随着时间的推移，在压抑的艺术下。利用单细胞ECCITEseq，我们发现尽管ART受到抑制，但肿瘤 肿瘤坏死因子(TNF)反应持续存在。此外，抗原和肿瘤坏死因子的反应推动T细胞的增殖 细胞克隆。此外，不同的抗原反应驱动不同的T细胞极化和增殖。总而言之， 我们假设抗原刺激和肿瘤坏死因子反应可以塑造T细胞极化、细胞敏感性 对HIV-1感染、细胞存活和感染细胞的增殖，特别是颗粒酶B细胞毒CD4+ T细胞。我们的目标是了解为什么感染HIV-1的细胞毒性CD4+T细胞能够优先存活，增殖， 与其他T细胞亚群相反，并持续存在。实现这一目标将确定推动 HIV-1持久性和识别HIV-1感染细胞的细胞标记物，以实现更具体的治疗靶点。我们的 方法是结合尖端的单细胞ECCITEseq和正交验证来检查HIV-1如何 RNA+颗粒酶B+CTL在病毒抑制下通过分析细胞亚群，免疫程序， 体内病毒抑制期间罕见HIV-1RNA+细胞的标志物和增殖动力学 在试管中。总体而言，我们将理解为什么HIV-1优先停留在细胞毒的CD4+T细胞中，确定上游 促进HIV-1感染的细胞毒性CD4+T细胞的存活和增殖的免疫驱动因素，并指导 制定针对HIV-1感染细胞的治疗策略。

PROJECT SUMMARY Despite effective antiretroviral therapy (ART), HIV-1 persists in the latent reservoir lifelong. Although most HIV- 1-infected cells die of viral cytopathic effects or immune clearance, HIV-1-infected cells, even those having active HIV-1 expression, can survive, persist, and proliferate. We want to examine how HIV-1 establishes infection during viremia and persists under viral suppression in people living with HIV-1. Understanding the immune cell subsets, immune programs, and cell markers of HIV-1-infected cells will identify therapeutic targets. The challenge is the heterogeneity and rarity of HIV-1-infected cells: in ART-treated, virally suppressed individuals, only 1–100/106 (<0.01%) CD4+ T cells harbor inducible HIV-1. To this end, we profiled CD4+ T cells from the Sabes cohort during viremia and after viral suppression using single-cell ECCITEseq, which captures single-cell transcriptome, protein expression, HIV-1 RNA, and T cell receptor sequence (TCR) within the same single cell. We have established advanced single-cell bioinformatic analysis pipelines and machine learning tools. This approach enables multi-dimensional, high-resolution, single-cell profiling of immune cell subsets, immune programs, cell markers, T cell clonal expansion, and HIV-1 RNA+ cells at the same time. Our single-cell ECCITEseq found that HIV-1 RNA+ cells upregulated cytotoxic CD4+ T cell genes. Using flow cytometric measurement of HIV-1 p24 protein and granzyme B expression, our RNA-seq based results were validated by a protein-based orthogonal approach, revealing that HIV-1 persists by hiding in granzyme B+ cytotoxic effector memory CD4+ T cells. Based on our results from viremic samples, we can examine the rare HIV-1 RNA+ cells under suppressive ART over time. Using single-cell ECCITEseq, we found that despite suppressive ART, tumor necrosis factor (TNF) responses persist. Furthermore, antigen and TNF responses drive the proliferation of T cell clones. In addition, different antigen responses drive distinct T cell polarization and proliferation. Altogether, we hypothesize that antigen stimulation and TNF responses can shape T cell polarization, cellular susceptibility to HIV-1 infection, cellular survival, and proliferation of the infected cells, particularly granzyme B cytotoxic CD4+ T cells. Our goal is to understand why HIV-1-infected cytotoxic CD4+ T cells can preferentially survive, proliferate, and persist, as opposed to other T cell subsets. Achieving this goal will identify immune programs that drive HIV-1 persistence and identify cell markers for HIV-1-infected cells for more specific therapeutic targeting. Our approach is to combine cutting-edge single-cell ECCITEseq and orthogonal validations to examine how HIV-1 RNA+ granzyme B+ CTLs survive and persist under viral suppression by profiling cell subsets, immune program, markers, and proliferation dynamics for the rare HIV-1 RNA+ cells over time during viral suppression in vivo and in vitro. Overall, we will understand why HIV-1 preferentially persists in cytotoxic CD4+ T cells, identify upstream immune drivers promoting the survival and proliferation of the HIV-1-infected cytotoxic CD4+ T cells, and guide the development of therapeutic strategies specific for HIV-1-infected cells.