Multiscale Analysis of Immune Responses

免疫反应的多尺度分析

基本信息

批准号：
10927738
负责人：
Ronald N Germain
金额：
$ 191.06万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10927738
关键词：
Adjuvant Anti-CD40 Antigen Presentation Antigen-Presenting Cells Antigens Area Atlases B-Cell Activation B-Lymphocytes Bacteria Behavior Breast CD209 gene CD4 Positive T Lymphocytes CD8-Positive T-Lymphocytes Cell physiology Cells Color Communities Computer software Cytometry Data Dendritic Cells Detection Development Devices Dimensions Effector Cell Elements Event Experimental Models Flow Cytometry Follicular Lymphoma Freezing Generations Genes Goals Hand Human Image Imaging technology Immune Immune response Immune system Immunohistochemistry Immunologics Immunosuppression Immunotherapeutic agent Immunotherapy Interferons Intervention Intestines Laboratories Lectin Lung Lung Adenocarcinoma Lymphocyte Lymphocyte Subset Lymphoid Macrophage Malignant - descriptor Malignant Neoplasms Malignant neoplasm of ovary Maps Mediating Methods Microscopy Modeling Mus Mutation Myeloid Cells National Institute of Allergy and Infectious Disease Oligosaccharides Oncogenic Ontology Organ Outcome PARP inhibition Pancreas Pathology Pathway interactions Patients Pattern Phenotype Photons Population Positioning Attribute Productivity Property Proteins RNA Regulatory T-Lymphocyte Reporting Research Resolution Retina Running Sampling Signal Transduction Site Source Specific qualifier value Speed Spleen Stains Surface Immunoglobulins T-Cell Activation T-Cell Receptor T-Lymphocyte T-Lymphocyte Subsets Testing Thick Thymus Gland Tissue imaging Tissues Variant Visualization Work adaptive immune response analytical method autoreactivity beta catenin cell motility cell type checkpoint inhibition checkpoint therapy complex data detection method high dimensionality imaging modality imaging study immune system function improved in vivo innate immune function insight instrument intravital microscopy lymph nodes microbial products microwave electromagnetic radiation monocyte multiplexed imaging nano-string neoplastic cell neutrophil new technology novel optical imaging protein protein interaction responders and non-responders response technology development tool transcriptome sequencing transcriptomics tumor tumor immunology tumor microenvironment

项目摘要

To extend our Histo-cytometry we have moved in two directions over the past several years. 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 have now extended this approach to multiplex imaging in a new technology called Ce3D-IBEX, achieving up to 25 parameters in tissues as thick as 300u. Most recently, we have improved the pipeline for Ce3D-IBEX, increasing the speed of sample processing using a special microwave and demonstrating that the method is suitable for mouse and humans samples from diverse tissues including lung, intestine, lymph node, spleen, retina, and various tumors, whether fixed frozen or FFPE. 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. With respect to the problem of autofluorescence in many types of samples we have developed a new software approach called CASPER that computationally removes the autofluorescence if the user has not collected such data for all channels throughout the imaging run. We are also working on new methods for detecting protein-protein interactions at subcellular resolution in our tissue imaging studies, testing various methods that enable a combination of RNA and protein detection in the same sample, and finishing a pipeline for a new type of correlative microscopy that combined 2P dynamic imaging with Ce3D-IBEX to enable many more cell types to be studied dynamically and the relationship of their dynamic and interactive behavior to be mapped to their subsequent cell state. These LBS-developed imaging technologies methods (Histo-cytometry, IBEX, Ce3D, Ce3D-IBEX, RAPID, SPACE, CASPER) 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 changes dramatically when immunotherapy is used. Recent studies have shown that combining checkpoint inhibition blockade with agonistic anti-CD40 converts regulatory T cells into IFN- producing Tbet+ effector cells within the tumor, representing a novel way to both reduce immunosuppression and augment effector function. We have also developed a novel method that permits identification of T cell receptor-activated T cells in the polyclonal populations within an inflamed tissue or tumor and the identification of the relevant antigen presenting cell. This method will be especially valuable in human cancer studies. 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), ovarian cancer, 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. Other uses of IBEX in the field of cancer immunology include a study of responders and non-responders to a combination of checkpoint therapy and PARP inhibition, with preliminary finings suggesting a possible pre-treatment signature that correlates with outcome. 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. We are completing a collaborative study connected to the Human Cell Atlas that involves creating an in-depth spatial map of the human thymus using both transcriptomic and multiplex imaging methods. Related methods are being employed to map the mouse thymus across development. 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，我们在过去几年中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，现在将这种方法扩展到了一种称为CE3D-IBEX的新技术中的多重成像，在组织中达到300U的组织中最多可实现25个参数。最近，我们改善了CE3D-IBEX的管道，使用特殊的微波炉提高了样品加工的速度，并证明该方法适用于来自不同组织的小鼠和人类样品，包括肺，肠道，淋巴结，脾脏，视网膜和各种肿瘤，无论是固定的FROZEN或FFPE。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。关于在许多类型的样本中自动荧光问题的问题，我们开发了一种称为Casper的新软件方法，该方法在整个成像运行过程中没有为所有通道收集此类数据，从而在计算上删除自动荧光。我们还正在研究组织成像研究中亚细胞分辨率下检测蛋白质蛋白质相互作用的新方法，测试各种方法，可以在同一样品中结合RNA和蛋白质检测的组合，并为新型的相关显微镜与新型相关性显微镜的互动与CE3D-EB的相互作用结合在一起，以使其与CE3D-EB的相互作用结合在一起，以使其与CE3D-ib的相互作用结合在一起，以使其与CE3D-ib的相互作用结合起来，以使其与他们的动态相互作用，以使他们的动态化合物与他们的动态相互作用，以使他们的动态化合物与他们的动态相互作用，以使他们的动态成像与他们的相互作用进行了研究，以使他们的动态化合物与他们的相互作用进行了研究，以使其与CE3D-ib的动态相互作用，以使他们的动态成像与他们的相互作用相互作用。映射到其随后的细胞状态。这些LBS开发的成像技术方法（组织仪，IBEX，CE3D，CE3D-IBEX，快速，空间，Casper）现在被用于多种不同的小鼠肿瘤模型（乳腺，胰腺，胰腺，肺）中，以探索肿瘤微环境的详细空间组织，并在肿瘤微型环境中详细的空间组织，并在不受段会上发生了变化。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。在使用免疫疗法时，也很明显地对髓样细胞亚群（例如树突状细胞，单核细胞，TAM等）类似的不对称定位也显而易见。最近的研究表明，将检查点抑制作用与激动性抗CD40结合在一起，将调节性T细胞转化为肿瘤内的IFN-TBET+效应细胞，这是一种新颖的方法，是一种减少免疫抑制和增强效应子功能的新方法。我们还开发了一种新的方法，该方法允许鉴定发炎组织或肿瘤中多克隆种群中T细胞受体激活的T细胞，并鉴定出相关的抗原呈现细胞。该方法在人类癌症研究中特别有价值。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品，包括但仅限于卵泡淋巴瘤（FL），卵巢癌，卵巢癌和肺癌。 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。 IBEX在癌症免疫学领域的其他用途包括对检查点疗法和PARP抑制的反应者的研究和无反应者，初步罚款表明可能具有与结果相关的预处理签名。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。我们正在完成一项与人类细胞地图集有关的协作研究，该研究涉及使用转录组和多重成像方法创建人类胸腺的深入空间图。正在采用相关方法来绘制跨发育过程中的小鼠胸腺。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。

项目成果

期刊论文数量（20）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

SAP-controlled T-B cell interactions underlie germinal centre formation.

DOI：
10.1038/nature07345
发表时间：
2008-10-09
期刊：
NATURE
影响因子：
64.8
作者：
Qi, Hai;Cannons, Jennifer L.;Klauschen, Frederick;Schwartzberg, Pamela L.;Germain, Ronald N.
通讯作者：
Germain, Ronald N.

A spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread.