In vivo development and reactivity of human autoreactive T cells

人类自身反应性 T 细胞的体内发育和反应性

基本信息

批准号：
10519104
负责人：
Remi J Creusot
金额：
$ 40.5万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-11-15 至 2024-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10519104
关键词：
Address Affinity Antigen Presentation Antigen-Presenting Cells Antigens Autoantigens Autoimmune Diseases Autoimmunity Autologous Behavior Beta Cell Blood Blood Circulation CD8-Positive T-Lymphocytes CD8B1 gene Cells Collection Development Environment Epitopes Evaluation Failure Fetal Liver Flow Cytometry Frequencies Gene Expression Profile Gene Expression Profiling Generations Haplotypes Hematopoietic Hematopoietic stem cells Human Image Immunofluorescence Immunologic Immunotherapy In Vitro Individual Infiltration Inflammation Insulin-Dependent Diabetes Mellitus Islets of Langerhans Lymphoid Tissue Measures Mediating Modeling Molecular Profiling Mus Outcome Patients Peptides Peripheral Phenotype Population Predisposition Process Regulatory T-Lymphocyte Reporter Reporting Research Rodent Rodent Model Skin Source Stress System T cell infiltration T-Cell Development T-Cell Receptor T-Lymphocyte Testing Thymic Tissue Thymus Gland Tissue Model Tissues Titrations Vitiligo Work autoreactive T cell autoreactivity cross reactivity fetal humanized mouse immunogenic in vivo in vivo Model insight intravenous injection islet melanocyte migration mouse model neoantigens novel novel therapeutic intervention peripheral tolerance promoter reconstitution research clinical testing response single-cell RNA sequencing transcriptome sequencing

项目摘要

Abstract Inadequate recognition of autoantigens during thymopoiesis can result in abnormally high frequency of autoreactive T cells, which can lead to autoimmune diseases when combined with defective mechanisms of peripheral tolerance in susceptible individuals. The process of thymic selection of autoreactive T cells in humans is largely not understood due to lack of in vivo models and could so far only be extrapolated from rodent studies. Analysis of autoantigen expression in the human thymus has provided an incomplete picture based on increasing evidence that this does not necessarily correlate with T cell deletion. As autoreactive T cells are also found circulating in `healthy' individuals, it is unclear to what extent incomplete thymic deletion (or regulatory T cell (Treg) selection) contributes to autoimmunity. Our proposed research is based on humanized mice reconstituted with fetal hematopoietic stem cells (HSCs) and thymic tissue, whereby HSCs can be transduced to express an autoreactive T cell receptor (TCR) and/or the actual epitope recognized by this TCR. The HSCs then develop into T cells in a normal human thymus in vivo, a fraction of which display the autoreactive TCR. Having established the model with T cells specific for a melanocyte autoantigen that fail to undergo thymic deletion unless the epitope is also introduced in HSCs, we will adapt this system to a collection of TCRs that have been identified in islet infiltrates of Type 1 diabetes patients. These TCRs were selected because it is yet unknown whether their specific epitopes are presented and recognized in a normal human thymus. Thus the model will help determine whether T cells bearing those TCRs escape thymic deletion through a number of mechanisms that will be tested (lack of expression, of cross- reactivity or of neoepitope formation). Introduction of the cognate epitopes in HSCs will allow us to evaluate their effect on developing autoreactive T cells (deletion versus positive selection of Tregs), and further explore the unique contribution of specific subsets of antigen-presenting cells (APCs) in this process, including their ability to relocate to the thymus from the bloodstream to influence thymic selection. Using recipient mice that are able to present antigens to human T cells and antigens that are cross-reactive between the two species, we will assess the conditions that are needed to activate autoreactive T cells to attack their target tissue. This work will feature immunofluorescence imaging, multi-parametric flow cytometry and single-cell RNA sequencing analysis to analyze human APC subsets that populate the thymus and peripheral lymphoid tissues and engage human autoreactive T cells, and to assess the phenotype and molecular profile of these autoreactive T cells based on different conditions of antigen encounter (thymus vs periphery, type of APC, etc). Overall, these studies will provide valuable insights into the generation of human autoreactive T cells causing autoimmune diseases, their reactivity to autoantigens in vivo, and will facilitate the development and evaluation of new therapeutic approaches targeting autoreactive T cells for deletion or tolerance.

抽象的胸腺生成过程中对自身抗原的识别不足可能导致异常高频率的自身反应性 T 细胞，当与缺陷机制相结合时，可能导致自身免疫性疾病易感个体的外周耐受性。人类自身反应性T细胞的胸腺选择过程由于缺乏体内模型，这一点在很大程度上尚不为人所知，迄今为止只能从啮齿动物研究中推断出来。对人类胸腺中自身抗原表达的分析提供了基于增加的不完整的图片有证据表明这并不一定与 T 细胞删除相关。由于还发现了自身反应性 T 细胞在“健康”个体中循环，目前尚不清楚不完全胸腺缺失（或调节性 T 细胞）的程度（Treg）选择）有助于自身免疫。我们提出的研究基于用胎儿造血干细胞 (HSC) 重组的人源化小鼠和胸腺组织，从而 HSC 可以被转导以表达自身反应性 T 细胞受体 (TCR) 和/或该 TCR 识别的实际表位。然后，HSC 在正常人胸腺中发育成 T 细胞 vivo，其中一部分显示出自身反应性 TCR。建立了针对特定 T 细胞的模型除非该表位也被引入 HSC 中，否则无法进行胸腺删除的黑素细胞自身抗原，我们将使该系统适应已在 1 型糖尿病的胰岛浸润中鉴定出的 TCR 集合患者。选择这些 TCR 是因为尚不清楚它们的特定表位是否存在并在正常人胸腺中被识别。因此，该模型将有助于确定 T 细胞是否携带这些 TCR 通过多种将要测试的机制（缺乏表达、交叉表达）来逃避胸腺缺失。反应性或新表位形成）。在 HSC 中引入同源表位将使我们能够评估它们的对发育中的自身反应性 T 细胞的影响（Treg 的删除与正选择），并进一步探索抗原呈递细胞 (APC) 的特定亚群在此过程中的独特贡献，包括它们的能力从血流转移到胸腺以影响胸腺选择。使用能够的受体小鼠为了向人类 T 细胞呈递抗原以及两个物种之间存在交叉反应的抗原，我们将评估激活自身反应性 T 细胞攻击其靶组织所需的条件。这项工作将采用免疫荧光成像、多参数流式细胞术和单细胞 RNA 测序分析以分析胸腺和外周淋巴组织中的人类 APC 亚群并参与人类自身反应性 T 细胞，并评估这些自身反应性 T 细胞的表型和分子特征 T 细胞基于不同的抗原遭遇条件（胸腺与外周血、APC 类型等）。总体而言，这些研究将为人类自身反应性 T 细胞的产生提供有价值的见解，从而导致自身免疫疾病，它们在体内对自身抗原的反应性，并将促进新药物的开发和评估针对自身反应性 T 细胞缺失或耐受的治疗方法。