MOLECULAR MECHANISMS OF REGULATORY T CELL DEVELOPMENT

调节 T 细胞发育的分子机制

• 批准号: 6828596
• 负责人: Alexander Y Rudensky
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6828596
• 关键词: biomarker; cell differentiation; chimeric proteins; developmental immunology; gene expression; gene expression profiling; genetic regulation; genetically modified animals; green fluorescent proteins; helper T lymphocyte; immune tolerance /unresponsiveness; laboratory mouse; messenger RNA; organ culture; protein structure function; thymus; transcription factor

DESCRIPTION (provided by applicant): Naturally arising regulatory CD25+ CD4 T cells play an essential role in regulation of immune homeostasis. However, specific molecular mechanisms regulating their differentiation and activity have been largely unknown. We have found that forkhead transcription factor Foxp3 is specifically expressed in regulatory CD4 T cells and is required for their development and function. Although identification of Foxp3 as a critical molecular mechanism establishing regulatory T cell differentiation program is a major breakthrough, it remains unknown what are Foxp3 target genes essential for this program. In addition, our findings raise an important question as to why there is such a discrepancy in the severity and onset of the autoimmune syndrome in a genetic model of CD25+ CD4 regulatory T cell deficiency in Foxp3 deficient mice and in murine models relying on elimination of CD25+ CD4 T cells. We propose several possible non-mutually exclusive hypotheses to explain this apparent controversy: a) In addition to CD25+ CD4 T cells, Foxp3 is expressed by an unidentified subset(s) of CD25- CD4 or CD8 T cells with the regulatory function capable of providing some measure of protection from autoimmunity in mice subjected to acute depletion of CD25+ CD4 T cells; b) In the absence of Foxp3, T cells with an increased TCR affinity for self peptide/MHC class II complexes that normally develop into regulatory CD25+ CD4 T cells instead become pathogenic and largely responsible for the rapid disease progression; c) A subset of CD25+ CD4 T cells with autoreactive TCR lack Foxp3 and may represent a major population of autoreactive T cells normally controlled by Foxp3 expressing CD25+ CD4 regulatory T cells; d) Foxp3 expressed at a low level in nonregulatory CD4 T cells controls thresholds of activation, dependence on co-stimulation, or anergy induction. In this proposal we will address the aforementioned questions using genetic approaches including Foxp3 protein and mRNA reporter mice, conditional deletion and induced expression of Foxp3 gene in vivo and in vitro in combination with functional and molecular analyses. The following specific Aims will be pursued: 1. To investigate Foxp3 protein and mRNA expression and their regulation during thymic development (Aim 1a) and a role for Foxp3 in negative selection of T cells in the thymus and to evaluate autoreactivity of T cells with the Foxp3 deletion (Aim 1b). 2. To investigate Foxp3 protein and mRNA expression and to assess regulatory function of distinct Foxp3+ T cell subsets (Aim2a); to investigate Foxp3 protein and mRNA regulation in peripheral T cells and to explore, thereby, possible generation of Foxp3+ T cells in the periphery (Aim 2b); to test a requirement for Foxp3 expression in maintaining regulatory T cell function (Aim 2c). 3. To investigate potential cell-intrinsic role of Foxp3 in controlling activation, central and peripheral tolerance induction in non-regulatory CD25- CD4 T cells. 4. To identify molecular targets of the Foxp3 gene involved in regulation of regulatory T cell development and function.

描述（由申请人提供）：天然产生的调节性 CD25+ CD4 T 细胞在免疫稳态调节中发挥重要作用。然而，调节其分化和活性的具体分子机制在很大程度上尚不清楚。我们发现叉头转录因子 Foxp3 在调节性 CD4 T 细胞中特异性表达，并且是其发育和功能所必需的。尽管确定 Foxp3 作为建立调节性 T 细胞分化程序的关键分子机制是一项重大突破，但仍不清楚该程序所必需的 Foxp3 靶基因是什么。此外，我们的研究结果提出了一个重要问题，即为什么 Foxp3 缺陷小鼠的 CD25+ CD4 调节性 T 细胞缺陷遗传模型和依赖消除 CD25+ 的小鼠模型中自身免疫综合征的严重程度和发病情况存在如此差异CD4 T 细胞。我们提出了几种可能的非互斥假设来解释这一明显的争议： a) 除了 CD25+ CD4 T 细胞外，Foxp3 还由未识别的 CD25- CD4 或 CD8 T 细胞亚群表达，其调节功能能够提供对遭受 CD25+ CD4 T 细胞急性耗竭的小鼠进行某种程度的自身免疫保护； b) 在缺乏 Foxp3 的情况下，对自身肽/MHC II 类复合物的 TCR 亲和力增加的 T 细胞通常会发育成调节性 CD25+ CD4 T 细胞，而变得致病并在很大程度上负责疾病的快速进展； c) 具有自身反应性 TCR 的 CD25+ CD4 T 细胞亚群缺乏 Foxp3，可能代表通常由表达 Foxp3 的 CD25+ CD4 调节性 T 细胞控制的主要自身反应性 T 细胞群； d) 在非调节性 CD4 T 细胞中低水平表达的 Foxp3 控制激活阈值、对共刺激的依赖性或无反应性诱导。在本提案中，我们将使用遗传方法解决上述问题，包括 Foxp3 蛋白和 mRNA 报告小鼠、体内和体外 Foxp3 基因的条件删除和诱导表达，并结合功能和分子分析。将追求以下具体目标： 1. 研究 Foxp3 蛋白和 mRNA 表达及其在胸腺发育过程中的调节（目标 1a）以及 Foxp3 在胸腺 T 细胞阴性选择中的作用，并评估 T 细胞与胸腺发育的自身反应性。 Foxp3 删除（目标 1b）。 2. 研究Foxp3蛋白和mRNA表达并评估不同Foxp3+ T细胞亚群的调节功能（Aim2a）；研究外周 T 细胞中 Foxp3 蛋白和 mRNA 的调控，从而探索外周 Foxp3+ T 细胞的可能生成（目标 2b）；测试维持调节性 T 细胞功能对 Foxp3 表达的要求（目标 2c）。 3.研究Foxp3在控制非调节性CD25-CD4 T细胞的激活、中枢和外周耐受诱导中的潜在细胞内在作用。 4. 鉴定参与调节性T细胞发育和功能调节的Foxp3基因的分子靶点。