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.

项目摘要/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的快速恢复， 要求患者遵循严格的药物方案。 HIV-1在几天之内建立一个稳定的水库 在外周血中检测到病毒之前的传播，并可以持续为复制能力 细胞内部的病毒，称为潜在储层。艺术无法针对这些潜在储层。 因此，需要治愈HIV-1。迄今为止，针对潜在储层的治疗 未能成功，主要是因为潜在储层知之甚少。由于易于样本访问， 绝大多数对潜在病毒储存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已显示在淋巴样中持续存在 血液中无法检测到的病毒载荷的艺术患者组织。少数对潜伏的研究 淋巴组织中的储层受到只能针对几个标记或需要的方法的限制 细胞悬浮液的制备，这可能导致细胞表型的变化或细胞损失，以及丧失 空间信息。我们的小组以前已经开发了多路复用的离子束成像，该离子束成像 离子质谱法以在单个组织中的亚细胞分辨率下可视化多达40个蛋白质。通过组合 这项具有空间转录组学的新型成像技术可评估淋巴组织中的免疫特征，我们 将能够识别细胞和机制对于病毒持久性很重要。我们最近应用了 恒河猕猴组织中的方法，证明了SIV+猕猴的免疫抑制微环境 淋巴结。在这项工作的基础上，我假设应用新颖的成像和空间转录组 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附近感染细胞，细胞功能和组织结构的全面图片 水库。在AIM 1中，我将使用原位杂交将MIBI扩展到HIV RNA和DNA的成像，使得 同时定量病毒RNA，DNA和细胞蛋白完全表征感染细胞。在 AIM 2，我将评估病毒患者淋巴组织内的空间结构和卵泡异质性 确定细胞积极反应病毒的微环境的特征。在AIM 3中，我 将比较病毒性患者，恶毒患者和健康个体之间的免疫特征与 发现免疫微环境的特征对艺术后的病毒持久性很重要。这项工作 将提高我们对淋巴组织中潜在病毒储存的理解，这是HIV-1的关键障碍 根除。这项奖学金将为我提供科学培训，使我为博士后奖学金做好准备和 发展成独立科学家。