Investigating the latent HIV-1 reservoir in lymphoid tissue using multiplexed imaging and spatial transcriptomics

使用多重成像和空间转录组学研究淋巴组织中的潜在 HIV-1 储存库

• 批准号: 10640882
• 负责人: CANDACE LIU
• 金额: $ 4.06万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-28 至 2024-03-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10640882
• 关键词: Antibodies; B-Lymphocytes; BLR1 gene; Biological Assay; Biological Markers; Blood; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cell Lineage; Cell physiology; Cell secretion; Cells; Circulation; Collaborations; DNA; Data; Development; Elements; Ensure; Environment; Exclusion; Fellowship; Follicular Dendritic Cells; Follow-Up Studies; Frequencies; Future; Genetic Transcription; Goals; Grant; Gut associated lymphoid tissue; HIV; HIV Core Protein p24; HIV Infections; HIV-1; Helper-Inducer T-Lymphocyte; Heterogeneity; Image; Immune; In Situ; In Situ Hybridization; Individual; Infection; Interleukin-10; Interruption; Label; Longevity; Lymphoid Tissue; Macaca; Macaca mulatta; Macrophage; Maintenance; Measurement; Measures; Mentors; Messenger RNA; Metals; Methods; Multiplexed Ion Beam Imaging; Oligonucleotide Probes; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Plasma; Postdoctoral Fellow; Proteins; Proteomics; Proviruses; Quality of life; RNA; Regimen; Reporting; Resolution; SIV; Sampling; Scientist; Site; Spectrometry, Mass, Secondary Ion; Structure; Suspensions; T-Lymphocyte; Tissue imaging; Tissues; Training; Viral; Viral Load result; Viral reservoir; Virus; Virus Diseases; Virus Latency; Virus Replication; Visualization; Work; antiretroviral therapy; cell killing; cell preparation; cytokine; detection limit; imaging platform; imaging study; improved; in situ imaging; innovation; insight; laser capture microdissection; latent HIV reservoir; lymph nodes; multiple omics; multiplexed imaging; novel; novel imaging technology; peripheral blood; phenotypic biomarker; public health relevance; spatial relationship; success; transcriptome sequencing; transcriptomics; transmission process; tumor-immune system interactions; viral DNA; viral RNA

PROJECT SUMMARY/ABSTRACT The introduction of combination antiretroviral therapy (ART) has led to a dramatic improvement in lifespan for individuals infected with HIV-1, but any interruption of treatment leads to rapid resumption of viral replication, requiring patients to follow a strict drug regimen. HIV-1 establishes a stable reservoir within days of transmission before the virus can be detected in the peripheral blood and can persist as replication-competent proviruses inside cells, known as the latent reservoir. ART is incapable of targeting these latent reservoirs; therefore, a cure for HIV-1 is needed. Treatments that target the latent reservoir have thus far been unsuccessful, largely because the latent reservoir is poorly understood. Due to ease of sample access, the vast majority of studies of the latent viral reservoir have been performed with peripheral blood. However, lymphoid tissue are the principal sites of viral replication and HIV-1 has been shown to persist in lymphoid tissue of patients on ART who have undetectable viral loads in the blood. The few prior studies of the latent reservoir in lymphoid tissue were limited by approaches that could only target a few markers or required the preparation of cell suspensions, which can lead to changes in cell phenotype or loss of cells, as well as loss of spatial information. Our group has previously developed Multiplexed Ion Beam Imaging, which uses secondary ion mass spectrometry to visualize up to 40 proteins at subcellular resolution in a single tissue. By combining this novel imaging technology with spatial transcriptomics to assess immune features in lymphoid tissue, we will be able to identify the cells and mechanisms important for viral persistence. We recently applied this method in rhesus macaque tissue, demonstrating an immunosuppressive microenvironment in SIV+ macaque lymph nodes. Building on this work, I hypothesize that applying novel imaging and spatial transcriptomic methods will reveal an immunosuppressive phenotype in lymphoid tissue of HIV+ patients. This work will yield a comprehensive picture of the infected cells, cell function, and tissue structure within the vicinity of the HIV-1 reservoir. In Aim 1, I will extend MIBI to the imaging of HIV RNA and DNA using in situ hybridization, enabling the simultaneous quantification of viral RNA and DNA and cellular protein to fully characterize infected cells. In Aim 2, I will assess the spatial structure and follicle heterogeneity within the lymphoid tissue of viremic patients to identify features of the microenvironment in which cells are actively responding against the virus. In Aim 3, I will compare the immune features between viremic patients, aviremic patients, and healthy individuals to uncover features of the immune microenvironment that are important to viral persistence after ART. This work will improve our understanding of the latent viral reservoir in lymphoid tissue, which is a critical barrier to HIV-1 eradication. This fellowship will give me the scientific training to prepare me for a postdoctoral fellowship and development into an independent scientist.

项目摘要/摘要 联合抗逆转录病毒疗法(ART)的引入大大延长了患者的寿命 感染HIV-1的人，但治疗的任何中断都会导致病毒复制的迅速恢复， 要求患者遵循严格的药物治疗方案。HIV-1在几天内建立了稳定的宿主 在病毒可以在外周血液中检测到之前进行传播，并可以作为复制能力持续存在 细胞内的前病毒，称为潜伏期。ART无法瞄准这些潜在的储集层； 因此，需要一种治愈艾滋病毒-1的方法。到目前为止，针对潜在储集层的治疗方法是 没有成功，很大程度上是因为人们对潜在储集层知之甚少。由于易于访问样本，因此 绝大多数对潜伏病毒库的研究都是用外周血进行的。然而， 淋巴组织是病毒复制的主要部位，HIV-1已被证明在淋巴组织中持续存在 接受抗逆转录病毒治疗的患者的组织，他们的血液中有无法检测到的病毒载量。关于潜伏期的少数前人研究 淋巴组织中的储存库受到只能针对少数标记物或需要 细胞悬浮液的制备，这可能导致细胞表型的改变或细胞的丢失，以及 空间信息。我们的团队之前开发了多路复用离子束成像，它使用二次 离子质谱仪在单个组织中以亚细胞分辨率可视化多达40种蛋白质。通过组合 这项新的成像技术结合空间转录技术来评估淋巴组织的免疫功能，我们 将能够识别对病毒持续存在重要的细胞和机制。我们最近应用了这个 方法在猕猴组织中，展示SIV+猕猴的免疫抑制微环境 淋巴结。在这项工作的基础上，我假设应用新的成像和空间转录 方法将揭示HIV+患者淋巴组织中的一种免疫抑制表型。这项工作将会取得成果 HIV-1附近受感染细胞、细胞功能和组织结构的综合图片 水库。在目标1中，我将使用原位杂交将MIBI扩展到艾滋病毒RNA和DNA的成像，使 病毒RNA、DNA和细胞蛋白的同时定量，以全面描述感染细胞的特征。在……里面 目的2、评估病毒症患者淋巴组织的空间结构和滤泡异质性。 以确定细胞对病毒做出积极反应的微环境的特征。在目标3中，我 将比较病毒血症患者、无血症患者和健康人之间的免疫特征，以 发现抗逆转录病毒治疗后免疫微环境的特征，这些特征对病毒的持久性很重要。这部作品 将提高我们对淋巴组织中潜伏的病毒库的理解，淋巴组织是HIV-1的关键屏障 根除。这一奖学金将给我科学培训，为我的博士后奖学金做准备，并 发展成为一名独立的科学家。