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.

项目摘要 尽管有效的抗逆转录病毒疗法（ART），HI​​V-1仍然存在于潜在储层终生中。虽然大多数艾滋病毒 - 1感染的细胞死于病毒细胞病毒作用或免疫化的HIV-1感染细胞，甚至具有活性的细胞 HIV-1表达，可以生存，持久和增殖。我们想检查HIV-1如何建立感染 HIV-1患者在病毒血症和持续抑制病毒抑制下。了解免疫细胞 HIV-1感染细胞的子集，免疫程序和细胞标记将识别治疗靶标。这 挑战是HIV-1感染细胞的异质性和稀有性：在经过艺术治疗的，几乎被抑制的个体中， 仅1-100/106（<0.01％）CD4+ T细胞具有诱导HIV-1。为此，我们从 病毒血症期间和使用单细胞ECITESEQ在病毒抑制期间的SABES队列，可捕获单细胞 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）。 我们已经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。这 进近可以实现免疫球体的多维，高分辨率，单细胞分析，免疫集 程序，细胞标记，T细胞克隆膨胀和HIV-1 RNA+细胞同时。我们的单细胞 Ecciteseq发现HIV-1 RNA+细胞更新了细胞毒性CD4+ T细胞基因。使用流式细胞仪 HIV-1 p24蛋白和颗粒酶B表达的测量，我们的基于RNA-seq的结果通过 一种基于蛋白质的正交方法，揭示了HIV-1通过隐藏颗粒+细胞毒性效应子来持续存在 内存CD4+ T细胞。根据我们的病毒样品结果，我们可以检查稀有的HIV-1 RNA+细胞 随着时间的推移，在抑制艺术下。使用单细胞Ecciteseq，我们发现目的地抑制作用，肿瘤 坏死因子（TNF）反应持续存在。此外，抗原和TNF反应推动了T的增殖 细胞克隆。另外，不同的抗原反应驱动不同的T细胞极化和增殖。共， 我们假设抗原刺激和TNF反应可以塑造T细胞极化，细胞敏感性 感染HIV-1感染，细胞存活和感染细胞的增殖，特别是颗粒状B细胞毒性CD4+ T细胞。我们的目标是了解为什么HIV-1感染的细胞毒性CD4+ T细胞可以优先生存，增殖， 并且坚持不懈，而不是其他T细胞子集。实现这一目标将确定驱动驱动的免疫计划 HIV-1持久性并鉴定HIV-1感染细胞的细胞标记，以进行更具体的治疗靶向。我们的 方法是结合尖端的单细胞eciteseq和正交验证，以检查HIV-1如何 RNA+ GRANZYME B+ CTL通过分析细胞亚群，免疫程序，在病毒抑制下持续存在并持续存在 在体内病毒抑制期间，稀有HIV-1 RNA+细胞的标记和增殖动力学在体内和 体外。总体而言，我们将了解为什么HIV-1优先在细胞毒性CD4+ T细胞中持续存在，识别上游 免疫驱动因素促进HIV-1感染的细胞毒性CD4+ T细胞的生存和增殖，并引导 针对HIV-1感染细胞的理论策略的发展。