MOLECULAR MECHANISMS OF REGULATORY T CELL DEVELOPMENT

描述(由申请人提供)：自然产生的CD25+CD4T细胞在调节免疫动态平衡方面起着重要作用。然而，调控它们分化和活性的具体分子机制在很大程度上还不清楚。我们发现叉头转录因子Foxp3在调节性的CD4T细胞中特异表达，是其发育和功能所必需的。尽管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，但目前仍不清楚Foxp3对该程序至关重要的靶基因是什么。此外，我们的发现提出了一个重要的问题，即为什么在Foxp3缺陷小鼠中CD25+CD4调节性T细胞缺陷的遗传模型中，自身免疫综合征的严重程度和发病情况与依赖CD25+CD4T细胞消除的小鼠模型中存在如此大的差异。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CD25+CD4T细胞外，Foxp3是由一种未鉴定的CD25-CD4T细胞或CD8T细胞亚群(S)表达的，具有调节功能，能够在CD25+CD4T细胞急性耗尽的小鼠中提供一定程度的自身免疫保护；b)在没有Foxp3的情况下，T细胞对自体多肽/MHC II类复合体的TCR亲和力增加，正常情况下发展为CD25+CD4T细胞，但却成为致病的，并在很大程度上导致疾病的快速发展；C)具有自身反应性TCR的CD25+CD4T细胞亚群缺乏Foxp3，可能代表通常由表达CD25+CD4调节性T细胞的Foxp3控制的大部分自身反应性T细胞；d)在非调节性CD4T细胞中低水平表达Foxp3控制激活、对共刺激的依赖性或无能诱导的阈值。在本提案中，我们将利用遗传学方法，包括Foxp3蛋白和mRNA报告小鼠，体内和体外Foxp3基因的条件缺失和诱导表达，并结合功能和分子分析来解决上述问题。1.研究Foxp3蛋白和信使核糖核酸在胸腺发育过程中的表达及其调控(目标1a)，以及Foxp3在胸腺T细胞阴性选择中的作用，并评价缺失Foxp3的T细胞的自身反应性(目标1b)。2.研究不同Foxp3+T细胞亚群(Aim2a)的Foxp3蛋白和mRNA的表达及其调节功能；研究外周T细胞中Foxp3蛋白和mRNA的调节，从而探索外周Foxp3+T细胞的可能产生(Aim 2b)；检测维持调节性T细胞功能对Foxp3表达的要求(Aim 2c)。3.探讨Foxp3在调控非调节性CD25-CD4T细胞活化、中枢和外周耐受诱导中的潜在细胞内在作用。4.确定Foxp3基因参与调节T细胞发育和功能的分子靶点。

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.