Mechanisms controlling Foxp3 expression and regulatory T cell homeostasis

描述（由申请人提供）：功能性免疫系统必须在有效消除外来病原体和尽量减少对宿主的附带损害之间保持微妙的平衡。调节性T细胞（Regulatory T cells，Tcells）是T淋巴细胞的一个亚群，是免疫系统的抑制因子，在维持免疫稳态中起重要作用。Treg功能缺陷导致人类和小鼠的自身免疫性疾病和免疫病理学。Treg生物学领域的里程碑之一是转录因子Foxp 3作为Treg发育和功能的重要调节因子的鉴定。该项目的目标是阐明控制Foxp 3表达的分子机制，这将决定如何产生和维持调节性T细胞。通过比较不同物种的Foxp 3基因组序列，Zheng博士及其同事确定了三个进化上保守的内含子区域，他们将其命名为CNS 1，2和3（保守的非编码序列）。他们假设这些中枢神经系统区域在调节Foxp 3表达中发挥重要作用，并继续敲除小鼠中的单个Foxp 3中枢神经系统区域。三只CNS敲除小鼠中的每一只都在其Treg群体中显示出不同的缺陷，这表明它们在Foxp 3表达的控制中发挥着不同的作用。CNS 3是胸腺发育所必需的，而CNS 1参与TGF-β依赖性外周转化。最有趣的是，CNS 2在维持Foxp 3表达和稳定Treg谱系中起作用。在目前的提案中，Zheng博士的团队将跟进Foxp 3 CNS敲除小鼠的初步特征，以详细研究CNS区域如何在细胞和分子水平上控制调节性T细胞稳态。首先，他们将使用生理学上更相关的小鼠模型来研究Foxp 3 CNS 2在维持天然Treg谱系稳定性中的作用，并进行TCR库研究以检查在不存在CNS 2的情况下Treg亚群的通量（Aim 1）。第二，他们将探索Foxp 3 CNS区域在由抗DEC 205嵌合抗体体内诱导的抗原特异性Tclase的产生和维持中的作用（Aim 2）。最后，他们计划使用改良的染色质免疫沉淀试验来鉴定与调节Foxp 3表达的CNS区域结合的蛋白质（Aim 3）。本文提出的研究将为控制Treg稳态的机制提供更多的见解，并可能产生新的途径来操纵Treg以减弱或增强免疫应答的幅度，用于治疗自身免疫性疾病和癌症。

DESCRIPTION (provided by applicant): A functional immune system has to maintain a delicate balance between effective elimination of foreign pathogens and minimizing collateral damage to the host. Regulatory T cells (Tregs), a subset of T lymphocytes, function as suppressor of the immune system and play a critical role in maintaining immune homeostasis. Defects in Treg function lead to autoimmune diseases and immunopathology in humans and mice. One of the milestones in Treg biology field was the identification of transcription factor Foxp3 as an essential regulator of Treg development and function. The goal of this project is to elucidate the molecular mechanisms that control Foxp3 expression, which will determine how regulatory T cells are generated and maintained. By comparing Foxp3 genomic sequences of different species, Dr. Zheng and colleagues identified three intronic regions that are evolutionarily conserved, which they named CNS1, 2, and 3 (Conserved Non- coding Sequence). They hypothesized that these CNS regions could play an important role in regulating Foxp3 expression, and proceeded to knockout individual Foxp3 CNS regions in mice. Each of the three CNS knockout mice showed different defects in their Treg population, suggesting they play different roles in control of Foxp3 expression. CNS3 is required for thymic development of Tregs, whereas CNS1 is involved in TGF-beta dependent peripheral conversion of Tregs. Most interestingly, CNS2 plays a role in maintaining Foxp3 expression and stabilizing Treg lineage. In the current proposal, Dr. Zheng's group will follow up on the initial characterization of the Foxp3 CNS knockout mice to perform a detailed study on how the CNS regions control regulatory T cell homeostasis at both cellular and molecular levels. First, they will use more physiologically relevant mouse models to investigate the role of Foxp3 CNS2 in maintenance of natural Treg lineage stability and perform TCR repertoire study to examine the flux of Treg subsets in the absence of CNS2 (Aim 1). Second, they will explore the role of Foxp3 CNS regions in generation and maintenance of antigen-specific Tregs induced in vivo by anti-DEC205 chimeric antibody (Aim 2). Lastly, they plan to use a modified chromatin immunoprecipitation assay to identify proteins bound to CNS regions that regulate Foxp3 expression (Aim 3). The study proposed here will provide more insight on mechanisms controlling Treg homeostasis, and potentially generate new avenues to manipulate Tregs to attenuate or augment the amplitude of immune response for treatment of autoimmune diseases and cancer.