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: 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
• 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

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.

项目总结/摘要 联合抗逆转录病毒疗法（ART）的引入导致了寿命的显着改善， 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， 要求患者严格遵守药物治疗方案。HIV-1在感染后数天内建立稳定的储存库， 在病毒可以在外周血中检测到并可以持续复制之前， 细胞内的前病毒，被称为潜伏库。抗逆转录病毒疗法无法针对这些潜在的储库; 因此，需要治愈HIV-1。迄今为止，针对潜在储层的治疗方法 不成功，主要是因为对潜在的水库了解甚少。由于样品容易获取， 绝大多数关于潜伏病毒库的研究是用外周血进行的。然而，在这方面， 淋巴组织是病毒复制的主要场所，HIV-1已被证明在淋巴组织中持续存在。 血液中检测不到病毒载量的ART患者的组织。之前对潜在的 淋巴组织中的储存库受到只能靶向少数标记物或需要 细胞悬浮液的制备，这可能导致细胞表型的变化或细胞的损失，以及细胞的损失。 空间信息我们的团队以前开发了多路复用离子束成像， 离子质谱法在单个组织中以亚细胞分辨率显示多达40种蛋白质。通过组合 这种新的成像技术与空间转录组学，以评估免疫功能的淋巴组织，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 方法在恒河猴组织中，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，我假设应用新型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 在HIV-1附近的受感染细胞、细胞功能和组织结构的全面图片 水库在目标1中，我将使用原位杂交将MIBI扩展到HIV RNA和DNA的成像， 病毒RNA和DNA以及细胞蛋白质的同时定量，以充分表征受感染的细胞。在 目的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 以确定细胞积极响应病毒的微环境的特征。在目标3中，我 将比较病毒血症患者、病毒血症患者和健康个体之间的免疫特征， 揭示免疫微环境的特征，这些特征对ART后病毒的持续存在很重要。这项工作 将提高我们对淋巴组织中潜伏病毒库的理解，淋巴组织是HIV-1的关键屏障 根除这个奖学金将给我的科学培训，我准备博士后奖学金， 成为独立的科学家。