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

在单细胞水平上了解 HIV-1 在细胞毒性 CD4 T 淋巴细胞中的持久性

• 批准号: 10700380
• 负责人: Ya-Chi Ho
• 金额: $ 85.69万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-11 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10700380
• 关键词: Affect; Antigen Presentation; Antigens; Benzoxazoles; Bioinformatics; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; Carboxylic Acids; Cell Survival; Cell secretion; Cells; Clonal Expansion; Clone Cells; Dimensions; Flow Cytometry; Frequencies; Genes; Genetic Transcription; Goals; Granzyme; HIV-1; Heterogeneity; Immune; In Vitro; Individual; Infection; Machine Learning; Malignant Neoplasms; Measurement; Persons; Predisposition; Proliferating; Proteins; RNA; Research; Resolution; Sampling; Shapes; Specificity; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; TNF gene; Testing; Time; Tumor Necrosis Factor Therapy; Up-Regulation; Validation; Viral; Viral Cytopathogenic Effect; Viremia; analysis pipeline; anticancer research; antiretroviral therapy; cancer cell; cohort; cytokine; cytotoxic; cytotoxic CD8 T cells; immune clearance; in vivo; inhibitor; memory CD4 T lymphocyte; multiple omics; perforin; polarized cell; preservation; prevent; programs; protein expression; response; therapeutic development; therapeutic target; tool; transcriptome; transcriptome sequencing

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.

项目概要 尽管抗逆转录病毒治疗 (ART) 有效，HIV-1 仍然存在于潜伏病毒库中。 1 感染的细胞会因病毒细胞病变效应或免疫清除而死亡，HIV-1 感染的细胞，甚至是那些具有活性的细胞 HIV-1 的表达能够存活、持续和增殖。我们想要研究 HIV-1 是如何建立感染的。 HIV-1 感染者在病毒血症期间并在病毒抑制下持续存在。 HIV-1感染细胞的亚群、免疫程序和细胞标记将确定治疗靶点。 挑战在于 HIV-1 感染细胞的异质性和稀有性：在接受 ART 治疗、病毒受到抑制的个体中， 只有 1–100/106 (<0.01%) CD4+ T 细胞含有可诱导的 HIV-1 为此，我们对来自 CD4+ T 细胞进行了分析。 使用单细胞 ECCITEseq 捕获单细胞病毒血症期间和病毒抑制后的 Sabes 队列 同一单细胞内的转录组、蛋白质表达、HIV-1 RNA 和 T 细胞受体序列 (TCR)。 我们建立了先进的单细胞生物信息学分析管道和机器学习工具。 该方法能够对免疫细胞亚群、免疫细胞进行多维、高分辨率、单细胞分析 同时进行程序、细胞标记、T 细胞克隆扩增和 HIV-1 RNA+ 细胞。 ECCITEseq 使用流式细胞术发现 HIV-1 RNA+ 细胞上调细胞毒性 CD4+ T 细胞基因。 HIV-1 p24 蛋白和颗粒酶 B 表达的测量，我们基于 RNA-seq 的结果得到了验证 基于蛋白质的正交方法，揭示了 HIV-1 通过隐藏在颗粒酶 B+ 细胞毒性效应子中而持续存在 记忆 CD4+ T 细胞 根据病毒血症样本的结果，我们可以检查罕见的 HIV-1 RNA+ 细胞。 随着时间的推移，在抑制性 ART 的作用下，使用单细胞 ECCITEseq，我们发现尽管有抑制性 ART，肿瘤仍然存在。 坏死因子 (TNF) 反应持续存在，此外，抗原和 TNF 反应可驱动 T 细胞增殖。 此外，不同的抗原反应会驱动不同的 T 细胞极化和增殖。 我们认为抗原刺激和 TNF 反应可以影响 T 细胞极化和细胞易感性 HIV-1 感染、细胞存活和受感染细胞增殖，特别是颗粒酶 B 细胞毒性 CD4+ 我们的目标是了解为什么 HIV-1 感染的细胞毒性 CD4+ T 细胞能够优先存活、增殖、 并持续存在，而不是其他 T 细胞亚群 实现这一目标将确定驱动免疫程序。 HIV-1 持久性并识别 HIV-1 感染细胞的细胞标记，以实现更具体的治疗目标。 方法是将尖端的单细胞 ECCITEseq 和正交验证相结合来检查 HIV-1 如何 RNA+ 颗粒酶 B+ CTL 通过分析细胞亚群、免疫程序、 体内病毒抑制过程中罕见的 HIV-1 RNA+ 细胞随时间的标记物和增殖动态 总体而言，我们将了解为什么 HIV-1 优先存在于细胞毒性 CD4+ T 细胞中，并识别上游。 免疫驱动因素促进 HIV-1 感染的细胞毒性 CD4+ T 细胞的存活和增殖，并指导 开发针对 HIV-1 感染细胞的治疗策略。