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.

长期以来，胸腺一直被认为是一个神秘而神秘的器官；在某种程度上遭受了 来自一种普遍的观念，特别是在那些有医学培训背景的人中，它只有 在早期生活中具有突出的重要性。事实上，这种思路源于它在建立 中枢免疫耐受在生命早期，随后是退化期。然而，一个 新出现的大量证据表明，在许多相关的概念中，这种“教条”实际上是一种“狗”。 事实上，人类的胸腺功能在对生理活动的信心方面是有限的。大有可为 我们对淋巴系统及其在免疫中的作用的了解建立在对啮齿动物的研究基础上 这些模型似乎有很大的不同，不能直接推论到人类身上。在……顶端 这在解剖和功能的关系以及影响方面都有明显的空白 年龄、性别和种族对胸腺功能的影响。因此，在这种脱节的情况下，鉴于免疫的重要性 对人类疾病的如此多方面的反应，我们认为它回避了研究人的 人胸腺，来自正常器官捐赠者(即，不受胸腺或免疫病理影响)，在 试图最终影响我们对人类疾病的理解。需要明确的是，在靶器官方面， 鉴于胸腺在建立中枢耐受中的作用，也许没有比胸腺更相关的器官了 应该接受这样的解剖学调查。具体地说，我们认为生物分子图谱 人类胸腺是识别人类T细胞选择发生在哪里以及如何发生的关键，T细胞受体(TCR) 通过选择或受到克隆删除的反应性/结合亲和力，以及特定的调控 T淋巴细胞在向次级淋巴器官移居前发育的调控因素。要实现 为此，我们提出了以下具体目标：目标一：建立系统的解剖学最佳做法/基准 用于收集、保存、验证和标准化用于获取和处理的质量控制协议(SOP 器官捐赠级人胸腺；目的II：重建三维(3D)人胸腺；目的III： 用荧光活化细胞分选法对人胸腺细胞进行免疫表型分析。就像胸腺 独一无二地参与T淋巴细胞发育，胸腺细胞和树突状细胞(DC)群体从分散 来自同一患者的胸腺和外周血单核细胞将由FACS和 比较了免疫曲目。分离的细胞亚群将作为RNA表达谱(AIM)的来源 四)指导选择AIMS II和AIM IV中使用的标记；以及AIM IV；最终确定 正常3d人体胸腺内的分子组成和基因表达。