MOLECULAR MECHANISMS OF REGULATORY T CELL DEVELOPMENT

描述（由申请人提供）：天然产生的调节性CD 25 + CD 4 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特定分子机制在很大程度上是未知的。我们发现叉头转录因子Foxp 3在调节性CD 4 T细胞中特异性表达，并且是其发育和功能所必需的。虽然确定Foxp 3作为建立调节性T细胞分化程序的关键分子机制是一个重大突破，但Foxp 3靶基因对该程序至关重要仍然未知。此外，我们的研究结果提出了一个重要的问题，为什么在Foxp 3缺陷小鼠的CD 25 + CD 4调节性T细胞缺陷的遗传模型和依赖于消除CD 25 + CD 4 T细胞的小鼠模型中，自身免疫综合征的严重程度和发病率存在如此大的差异。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CD 25 + CD 4 T细胞外，Foxp 3还由一种未鉴定的CD 25-CD 4或CD 8 T细胞亚群表达，其调节功能能够在经历CD 25 + CD 4 T细胞急性耗竭的小鼠中提供某种程度的自身免疫保护; B）在不存在Foxp 3的情况下，对自身肽/MHC II类复合物具有增加的TCR亲和力的T细胞（其通常发育成调节性CD 25 + CD 4 T细胞）反而变成致病性的，并且主要是疾病快速进展的原因; c）具有自身反应性TCR的CD 25 + CD 4 T细胞亚群缺乏Foxp 3，并且可以代表通常由表达Foxp 3的CD 25 + CD 4调节性T细胞控制的自身反应性T细胞的主要群体; d）在非调节性CD 4 T细胞中以低水平表达的Foxp 3控制激活阈值、对共刺激的依赖性或无反应性诱导。在本研究中，我们将利用基因学方法，包括Foxp 3蛋白和mRNA报告小鼠，Foxp 3基因在体内和体外的条件性缺失和诱导表达，并结合功能和分子分析来解决上述问题。具体目标如下：1。研究胸腺发育过程中Foxp 3蛋白和mRNA的表达及其调控（Aim 1a），Foxp 3在胸腺T细胞阴性选择中的作用，并评价Foxp 3缺失的T细胞的自身反应性（Aim 1b）。2.研究Foxp 3蛋白和mRNA表达，并评估不同Foxp 3 + T细胞亚群的调节功能（Aim 2a）;研究外周T细胞中Foxp 3蛋白和mRNA的调节，从而探索外周中Foxp 3 + T细胞的可能生成（Aim 2b）;检测维持调节性T细胞功能对Foxp 3表达的需求（Aim 2c）。3.研究Foxp 3在非调节性CD 25-CD 4 T细胞中控制活化、中枢和外周耐受诱导的潜在细胞内在作用。4.确定参与调节性T细胞发育和功能的Foxp 3基因的分子靶点。

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.