Multiscale Analysis of Immune Responses

免疫反应的多尺度分析

• 批准号: 10692027
• 负责人: Ronald N Germain
• 金额: $ 159.04万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10692027
• 关键词: Adjuvant; Antigen Presentation; Antigen-Presenting Cells; Antigens; Area; B-Cell Activation; B-Lymphocytes; Bacteria; Behavior; Breast; CD209 gene; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cell physiology; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Color; Communities; Cytometry; Data; Dendritic Cells; Detection; Development; Devices; Dimensions; Elements; Event; Experimental Models; Flow Cytometry; Follicular Lymphoma; Generations; Genes; Goals; Hand; Human; Image; Imaging technology; Immune; Immune response; Immune system; Immunohistochemistry; Immunologics; Immunotherapeutic agent; Immunotherapy; Intervention; Laboratories; Lectin; Lung; Lung Adenocarcinoma; Lymphocyte; Lymphocyte Subset; Lymphoid; Malignant - descriptor; Malignant Neoplasms; Mediating; Mesothelioma; Methods; Modeling; Mus; Mutation; Myeloid Cells; National Institute of Allergy and Infectious Disease; Oligonucleotides; Oligosaccharides; Oncogenic; Ontology; Organ; Pancreas; Pathology; Pathway interactions; Patients; Pattern; Phenotype; Photons; Property; Regulatory T-Lymphocyte; Reporting; Research; Sampling; Signal Transduction; Site; Source; Specific qualifier value; Stains; Surface Immunoglobulins; T-Cell Activation; T-Lymphocyte; T-Lymphocyte Subsets; Techniques; Thick; Tissue imaging; Tissues; To specify; Variant; Visualization; Work; adaptive immune response; analytical method; antibody conjugate; autoreactivity; beta catenin; cell motility; cell type; checkpoint therapy; complex data; high dimensionality; immune system function; in vivo; innate immune function; insight; instrument; intravital microscopy; macrophage; microbial; monocyte; multiplexed imaging; nano-string; neoplastic cell; neutrophil; new technology; novel; optical imaging; response; technology development; tool; transcriptome sequencing; transcriptomics; tumor; tumor microenvironment

To extend our Histo-cytometry we have moved in two directions. First, we have developed a method (IBEX) that permits performing rapid iterative 4-10 color fluorescent immunohistochemistry on sections to attain images with >60 markers and have developed novel methods (RAPID and SPACE) to computationally analyze the complex data emerging from this method. To examine tissue volumes rather than sections, we have developed a novel tissue clearing method called Ce3D and extended this to multiplex imaging in Ce3D-IBEX, achieving up to 25 parameters in tissues as thick as 300u. We have developed methods that permit detection of the oligo-conjugated antibodies used in the CITE-seq technique, facilitating direct correlation of the imaging data with RNA seq studies, while also providing an alternative to our iterative bleaching method that is especially suited to volume imaging. Finally, we have incorporated a sensitive enzymatic amplification step into these methods to permit imaging of severely fixed material from BSL4-level infected sources, while adapting all these tools to more rapid imaging using new instruments that incorporate computational clearing. These LBS-developed imaging technologies methods (Histo-cytometry, IBEX, Ce3D, Ce3D-IBEX) are now being employed in multiple distinct mouse tumor models (breast, pancreatic, lung) to explore the detailed spatial organization of the tumor micro-environment and the changes that occur with immunotherapeutic intervention. By examining multiple different tumors in different tissues, we are beginning to develop insights into what aspects of immune cell presence / spatial organization are unique to a particular malignancy and which represent common features across tumor types. Our methods provide a much more comprehensive analysis of the organization of tumor cells, stromal elements, and immune cells than conventional pathology of immunohistochemical methods, and are especially valuable given the disorganized mature of tumors such that single tissue sections or limited parameter analyses fail to reveal larger scale patterns or variations in different regions of the tumor that may be criterial for understanding the differential response among patients to immunotherapeutic interventions. Preliminary data show clear differences in the localization of distinct T cell subsets within various tumors, with CD8 T cells and CD4 T cells (both conventional cells and Tregs) often located quite differently from the CD8 T cells. These patterns also change with administration of checkpoint immunotherapy, and the phenotypic state of the cells is also dramatically altered when various forms of immunotherapy are employed. Similar asymmetric localization of myeloid cell subsets, such as dendritic cells, monocytes, TAM, and so on, is also evident and also changes dramatically when immunotherapy is used. We are now using sophisticated spatial analytic methods, some in hand and others still under development, to better understand these patterns and how they relate to whether immunotherapeutic treatment is effective or not. These studies are not just of material from mouse experimental models, but in the context of the NIAID-NCI Center for Advanced Tissue Imaging (CAT-I), include analysis of samples from humans with various malignancies, including but to limited to follicular lymphoma (FL), mesothelioma, and lung adenocarcinoma. The work on FL has revealed intriguing relationships between acellular matrix and malignant B cells as well as between mutationally added oligosaccharides on the B cell surface immunoglobulin of the malignant B cells and the lectin DC-SIGN expressed by unusual cells in the follicle. This study also included new methods for spatial analysis of tumor organization and for relating very high dimensional IF imaging to low dimension but larger area examined by conventional multiplex IF used in pathology laboratories. Additional study of tumor organization involved the application of a new technology for spatial transcriptomics. Employing the NanoString GTX device, we were able to identify tumor cells and not immune or parenchymal cells as the hosts for intratumor bacteria. This association of bacteria with tumor cells correlated with a high degree of expression of oncogenic b-catenin pathway genes in the tumor, suggesting that bacterial association with tumor cells contributes to their malignant properties. As part of the development of technology, we contributed to a community effort to specify the best practices in multiplex imaging and are engaged in work on ontologies for immune tissues and cells as well as generation of specified staining panels that will enable easier application of iterative multiplex imaging by more laboratories seeking to use these methods in their research efforts. We also reported on the latest developments in optical imaging as applied to the immune system.

为了扩展我们的历史仪表仪，我们已经向两个方向移动。首先，我们开发了一种方法（IBEX），该方法允许在切片上执行快速迭代4-10彩色荧光免疫组织化学，以获得> 60个标记的图像，并开发了新颖的方法（快速和空间）来分析从该方法中出现的复杂数据。为了检查组织体积而不是切片，我们开发了一种称为CE3D的新型组织清除方法，并将其扩展到CE3D-IBEX中的多重成像，在组织中最多可实现25个参数，高达300U。我们开发了允许检测Cite-Seq技术中使用的寡聚偶联抗体的方法，从而促进了成像数据与RNA SEQ研究的直接相关性，同时还为我们的迭代漂白方法提供了一种替代方法，该方法特别适合于体积成像。最后，我们将一个敏感的酶扩增步骤结合到这些方法中，以允许从BSL4级感染的来源对严格的固定材料进行成像，同时使用结合计算清除的新仪器将所有这些工具调整为更快的成像。 这些LBS开发的成像技术方法（Histo-Cymitmertry，IBEX，CE3D，CE3D-IBEX）现在被用于多种不同的小鼠肿瘤模型（乳腺，胰腺，肺，肺）中，以探索肿瘤微环境的详细空间组织，以及因免疫疗法介入而发生的变化。通过检查不同组织中的多个不同肿瘤，我们开始洞悉免疫细胞存在 /空间组织的哪些方面在特定的恶性肿瘤中是独有的，哪些代表肿瘤类型的共同特征。与免疫组织化学方法的常规病理相比，我们的方法对肿瘤细胞的组织，基质元素和免疫细胞的组织提供了更全面的分析，鉴于肿瘤的成熟成熟的成熟，因此单个组织切片或有限的参数分析可能无法揭示出较大范围的响应或差异范围的偏差，因此特别有价值，并且尤其是有价值的。干预措施。初步数据表明，在各种肿瘤中不同T细胞亚群的定位明显差异，CD8 T细胞和CD4 T细胞（常规细胞和Tregs）通常与CD8 T细胞差异很大。这些模式也随检查点免疫疗法的给药而改变，当采用各种形式的免疫疗法时，细胞的表型状态也会发生巨大改变。髓样细胞亚群（例如树突状细胞，单核细胞，TAM等）的类似不对称定位也很明显，并且在使用免疫疗法时也会发生巨大变化。现在，我们正在使用复杂的空间分析方法，其中一些方法仍在开发中，以更好地理解这些模式，以及它们如何与免疫治疗是否有效。这些研究不仅是小鼠实验模型的材料，而且是NIAID-NCI晚期组织成像中心（CAT-I）的背景，包括分析来自各种恶性肿瘤的人类的样本，包括但限于卵泡淋巴瘤（FL），间皮瘤，间皮瘤和肺腺癌。 FL上的工作揭示了细胞基质基质和恶性B细胞之间的有趣关系，以及在卵泡中不寻常细胞表达的恶性B细胞的B细胞表面免疫球蛋白上添加了寡糖的突变型寡糖。这项研究还包括用于肿瘤组织空间分析的新方法，以及如果成像与低维度相关，但如果在病理实验室中使用，则通过常规多路复用检查更大的面积。 肿瘤组织的其他研究涉及将新技术应用于空间转录组学。使用纳米弦GTX设备，我们能够鉴定肿瘤细胞，而不是免疫或实质细胞作为肿瘤内细菌的宿主。这种细菌与肿瘤细胞的缔合与肿瘤中致癌性B-catenin途径基因的高度表达相关，这表明细菌与肿瘤细胞的关联有助于其恶性特性。 作为技术开发的一部分，我们为社区努力做出了贡献，以指定多重成像中的最佳实践，并从事免疫组织和细胞的本体学工作，以及生成指定的染色面板，可以通过更多实验室在其研究工作中使用这些方法来更轻松地应用迭代的多重成像。我们还报告了适用于免疫系统的光学成像的最新发展。