Project 2-Thymus

项目2-胸腺

• 批准号: 10211115
• 负责人: Todd Michael Brusko
• 金额: $ 46.75万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-14 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10211115
• 关键词: 3-Dimensional; Age; Anatomy; Asthma; Autoimmunity; Avidity; Benchmarking; Binding; Biologic Development; Biological Response Modifier Therapy; CD8-Positive T-Lymphocytes; Canis familiaris; Cell Therapy; Cells; Chromium; Clonal Deletion; Collection; Computer software; Coupled; Cryopreserved Tissue; Cytometry; Dendritic Cells; Deposition; Disease; Emigrations; Event; Expression Profiling; FOXP3 gene; Fluorescence Microscopy; Fluorescence-Activated Cell Sorting; Fluorescent in Situ Hybridization; Formalin; Gene Expression; Genetic Transcription; Histocompatibility; Human; Hypersensitivity; Image; Immune; Immune Tolerance; Immune response; Immunity; Immunofluorescence Immunologic; Immunohistochemistry; Immunologic Memory; Immunologics; Immunophenotyping; Investigation; Label; Life; Light; Lymphatic System; Magnetic Resonance Imaging; Maps; Measurement; Mediating; Medical; Microanatomy; Microscopy; Modernization; Modification; Molecular; Mus; Optics; Organ; Organ Donor; Pancreas; Paraffin; Paraffin Embedding; Pathology; Patients; Peripheral; Peripheral Blood Mononuclear Cell; Phase; Physiological; Polymers; Population; Procedures; Proteins; Protocols documentation; RNA; Race; Reagent; Recovery; Regulatory Element; Regulatory T-Lymphocyte; Reporting; Research Personnel; Resolution; Rodent Model; Role; Sampling; Secondary to; Source; Specimen; Stains; Standardization; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Technology; Temperature; Thinking; Three-Dimensional Image; Thymus Gland; Tissues; Training; Vaccines; Validation; base; cell type; central tolerance; confocal imaging; data de-identification; density; experience; hematopoietic cell transplantation; human disease; human imaging; imaging system; magnetic field; microscopic imaging; multiphoton imaging; preservation; reconstruction; scale up; secondary lymphoid organ; sex; single cell analysis; single-cell RNA sequencing; spectroscopic imaging; thymocyte

The thymus has long been considered an enigmatic and mysterious organ; having suffered to some extent from a widespread notion, especially amongst those with a background in medical training, that it has only of eminent importance in early life. Indeed, this line of thinking originates from its critical role in establishing central immune tolerance in early life subsequently followed by a waning period of involution. However, an emerging body of evidence has suggested this “dogma” is, in effect, a “dog” in that many of the notions related to thymic function in humans are, in fact, limited in terms of their confidence as to physiological activities. Much of our understanding of the lymphatic system and its role in immunity has been based on studies of rodent models that appear to differ in considerable ways and cannot be extrapolated directly to humans. On top of this, there are clear voids in terms of the relationship between anatomy and function as well as the influences of age, sex, and race on thymic function. Hence, with this disconnect, and given the importance of the immune response to so many aspects of human disease, we believe it begs the notion of studying the anatomy of the human thymus, from normal organ donors (i.e., unaffected by thymic or immunological pathology), in an attempt to eventually impact our understanding of human disease. To be clear, in terms of targeted organs, given the role of the thymus in establishing central tolerance, there is perhaps no organ more relevant that should be subject to such anatomical investigation. Specifically, we believe a biomolecular mapping of the human thymus is essential to identify where and how human T cell selection occurs, T cell receptor (TCR) reactivities/binding avidities that pass selection or are subject to clonal deletion, and the specific regulatory elements governing T lymphocyte development prior to emigration to secondary lymphoid organs. To achieve this, we posit the following specific aims: AIM I: Establish systematic, anatomical best practices/benchmarks for collection, preservation, validation and standardization of QC protocols (SOPs) for acquiring and handling organ donor grade human thymus; AIM II: Reconstruct the three-dimensional (3D) human thymus; AIM III: Immunophenotype human thymus cells by fluorescence activated cell sorting (FACS). As the thymus is uniquely involved in T lymphocyte development, thymocyte and dendritic cell (DC) populations from dispersed thymus and peripheral blood mononuclear cells from the same patient will be defined by FACS and the immune repertoires compared. Isolated cell subsets will serve as the source for RNA expression profiling (AIM IVB) to guide the selection of markers used in AIMs II and IV; and AIM IV; ultimately, to determine the molecular composition and gene expression within the normal 3D human thymus.

胸腺长期以来被认为是一个神秘莫测的器官， 从一个普遍的概念，特别是那些在医疗培训的背景，它只有 在早期生活中的重要性。事实上，这一思路源于其在建立 在生命早期的中枢免疫耐受，随后是退化的衰退期。但安 新出现的大量证据表明，这一“教条”实际上是一条“狗”，因为许多与之相关的概念 对人类胸腺功能的研究，事实上，在他们对生理活动的信心方面是有限的。多 我们对淋巴系统及其在免疫中的作用的理解是基于对啮齿动物的研究， 这些模型似乎在很大程度上有所不同，不能直接外推到人类身上。之上 这一点，在解剖和功能之间的关系以及 年龄性别种族对胸腺功能的影响因此，有了这种脱节，并考虑到免疫的重要性， 对人类疾病的许多方面的反应，我们相信它回避了研究人类疾病解剖学的概念。 人胸腺，来自正常器官供体（即，不受胸腺或免疫病理学影响）， 试图最终影响我们对人类疾病的理解。要明确的是，在目标器官方面， 考虑到胸腺在建立中枢耐受性中的作用，也许没有比胸腺更相关的器官了， 应该接受这样的解剖学研究具体地说，我们相信一个生物分子映射的 人胸腺对于鉴定人T细胞选择在何处以及如何发生是必不可少的，T细胞受体（TCR） 通过选择或进行克隆缺失的特异性反应性/结合亲合力，以及特异性调节因子， 在迁移到次级淋巴器官之前控制T淋巴细胞发育的元素。实现 为此，我们提出了以下具体目标：目标一：建立系统的、解剖学上的最佳实践/基准 收集、保存、验证和标准化用于采集和处理的QC方案（SOP） 器官供体级人胸腺; AIM II：重建三维（3D）人胸腺; AIM III： 通过荧光激活细胞分选（FACS）对人胸腺细胞进行免疫表型分析。就像胸腺 独特地参与T淋巴细胞发育、胸腺细胞和树突状细胞（DC）群体， 来自同一患者的胸腺和外周血单核细胞将通过FACS定义， 免疫系统比较分离的细胞亚群将作为RNA表达谱（AIM）的来源 IVB）指导选择AIM II和IV中使用的标记物;以及AIM IV;最终，确定 正常三维人体胸腺内的分子组成和基因表达。