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

基本信息

批准号：
10480969
负责人：
CANDACE LIU
金额：
$ 3.94万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
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/10480969
关键词：
Antibodies B-Lymphocytes BLR1 gene Biological Assay Biological Markers Blood Blood Circulation CD4 Positive T Lymphocytes CD8-Positive T-Lymphocytes Cell Lineage Cell physiology Cell secretion Cells Collaborations DNA Data Development Ensure Environment 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 Lead Longevity Lymphoid Tissue Macaca Macaca mulatta Maintenance Measurement Measures Mentors Messenger RNA Metals Methods Multiplexed Ion Beam Imaging Oligonucleotide Probes Patients Pattern Pharmaceutical Preparations Phenotype Plasma 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 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 macrophage 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

项目摘要

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.

项目概要/摘要联合抗逆转录病毒疗法（ART）的引入使人们的寿命显着延长感染 HIV-1 的个体，但任何治疗中断都会导致病毒复制迅速恢复，要求患者严格遵守用药方案。 HIV-1 在数天内建立了稳定的储存库在病毒在外周血中被检测到并能够持续复制之前就已传播细胞内的原病毒，称为潜伏病毒库。 ART 无法针对这些潜在的病毒库；因此，需要治愈 HIV-1。迄今为止，针对潜在储存库的治疗方法已不成功，很大程度上是因为对潜在储层了解甚少。由于样品获取方便，绝大多数关于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场所，HIV-1 已被证明持续存在于淋巴组织中接受 ART 的患者血液中病毒载量无法检测到的组织。之前的一些潜在研究淋巴组织中的储库受到只能针对少数标记物或需要细胞悬液的制备，这可能导致细胞表型的变化或细胞的损失，以及细胞因子的损失空间信息。我们小组之前开发了多重离子束成像，它使用二次离子质谱法可在单个组织中以亚细胞分辨率可视化多达 40 种蛋白质。通过结合这种新颖的成像技术与空间转录组学一起评估淋巴组织的免疫特征，我们将能够识别对病毒持久性重要的细胞和机制。我们最近应用了这个恒河猴组织中的方法，证明了 SIV+ 猕猴中的免疫抑制微环境淋巴结。在这项工作的基础上，我假设应用新颖的成像和空间转录组学方法将揭示 HIV+ 患者淋巴组织中的免疫抑制表型。这项工作将产生 HIV-1 附近受感染细胞、细胞功能和组织结构的全面图景水库。在目标 1 中，我将使用原位杂交将 MIBI 扩展到 HIV RNA 和 DNA 成像，从而使同时定量病毒 RNA 和 DNA 以及细胞蛋白，以全面表征受感染的细胞。在目标 2，我将评估病毒血症患者淋巴组织内的空间结构和滤泡异质性识别细胞积极响应病毒的微环境特征。在目标 3 中，我将比较病毒血症患者、无病毒血症患者和健康个体之间的免疫特征揭示对 ART 后病毒持续存在很重要的免疫微环境特征。这部作品将提高我们对淋巴组织中潜伏病毒库的了解，这是 HIV-1 的关键屏障根除。该奖学金将为我提供科学培训，为我获得博士后奖学金做好准备发展成为一名独立科学家。