MOLECULAR MECHANISMS OF REGULATORY T CELL DEVELOPMENT

描述（由申请人提供）：自然产生的调节性CD25+ CD4 T细胞在调节免疫稳态中起重要作用。然而，调节其分化和活性的具体分子机制在很大程度上是未知的。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，是其发育和功能所必需的。虽然Foxp3作为建立调节性T细胞分化程序的关键分子机制的鉴定是一项重大突破，但Foxp3在该程序中所必需的靶基因仍是未知的。此外，我们的研究结果提出了一个重要的问题，即为什么在Foxp3缺陷小鼠的CD25+ CD4调节性T细胞缺陷遗传模型和依赖CD25+ CD4 T细胞消除的小鼠模型中，自身免疫综合征的严重程度和发病存在如此大的差异。我们提出了几个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CD25- CD4或CD8 T细胞的一个未知亚群表达，这些细胞具有调节功能，能够在CD25+ CD4 T细胞急性耗竭的小鼠中提供一定程度的自身免疫保护；b)在缺乏Foxp3的情况下，TCR对自身肽/MHC II类复合物的亲和力增加的T细胞通常会发展成调节性CD25+ CD4 T细胞，而不是致病性的，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CD25+ CD4调节性T细胞Foxp3控制的主要自身反应性T细胞群体；d) Foxp3在非调节性CD4 T细胞中的低水平表达控制激活阈值、对共刺激的依赖或能量诱导。在本提案中，我们将利用遗传学方法解决上述问题，包括Foxp3蛋白和mRNA报告小鼠，Foxp3基因在体内和体外的条件缺失和诱导表达，结合功能和分子分析。将实现下列具体目标：研究Foxp3蛋白和mRNA的表达及其在胸腺发育过程中的调控（Aim 1a），以及Foxp3在胸腺T细胞负向选择中的作用，并评估Foxp3缺失T细胞的自身反应性（Aim 1b）。2. 研究Foxp3蛋白和mRNA的表达，评估不同Foxp3+ T细胞亚群（Aim2a）的调控功能；研究Foxp3蛋白和mRNA在外周T细胞中的调控，从而探索Foxp3+ T细胞在外周细胞中产生的可能性（Aim 2b）；以测试Foxp3表达对维持调节性T细胞功能的要求（Aim 2c）。3. 探讨Foxp3在控制非调节性CD25- CD4 T细胞活化、中枢和外周耐受诱导中的潜在细胞内在作用。4. 目的：确定参与调节性T细胞发育和功能调控的Foxp3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.