Stability and Plasticity of Regulatory T Cells

该项目的目标是确定调节性T细胞(Tregs)的可塑性和稳定性，以及 将效应细胞转化为功能性Treg细胞的可能性，作为细胞治疗的前奏。我们的假设是 激活的T细胞经历了一个瞬时Foxp3表达的阶段，这是一个关键的决策点，并且 稳定的FoxpS表达是Treg稳定性的关键决定因素。我们的方法是利用老鼠模型来 在体内和体外解决这些问题，并开发预测表型和分子标记 Treg稳定性，并使用该信息来定义人类Treg的稳定性，并从基因上操纵 以编程人类T细胞的分化途径。提出了以下具体目标。 1.调节性T细胞(Tregs)和效应性T细胞(Tefo. 在这个目标中，我们将使用TCR生成抗原特异的自然(胸腺)和适应性(外周)树 表达FoxpS和关键细胞因子(干扰素-v、IL-17和IL-10)的转基因小鼠和 不同形式的抗原暴露。纯化的Tregs将被转移到正常的接受者体内，并暴露在 有或没有炎症刺激的抗原，或转移到具有主动自身免疫反应的受者体内。 通过染色观察细胞FoxpS和细胞因子的表达及表面表型。 分类后的功能反应。Tregs将从健康志愿者和患有疾病的患者中分离 自身免疫性疾病(最初为T1D)，并在体外转化为效应器 激活性和炎症性刺激。一种新的表达PASS血统标志的转基因小鼠品系 Roxp3的表达将用于检测这些细胞的命运。效应细胞向树突状细胞的转化 使用相同的方法进行检查。 2.Treg稳定性和转化的生化基础。T细胞将在稳定的条件下进行检测 以及转化为表面表型的变化、Treg特异性基因的表达和TERG的甲基化 ：oxp3基因座。将分析瞬时FoxpS表达在小鼠和 人类细胞使用shRNA基因敲除和基因敲除小鼠。将努力保持稳定的 通过在人类Tregs中表达选定的基因来调节。

The objective of this project is to define the plasticity and stability of regulatory T cells (Tregs) and the possibility of converting effector cells to functional Tregs, as a prelude to cellular therapy. Our hypothesis is that activated T cells go through a phase of transient Foxp3 expression that is a critical decision point, and stable FoxpS expression is a key determinant of Treg stability. Our approach is to exploit mouse models to address these questions in vivo and in vitro and to develop predictive phenotypic and molecular markers for Treg stability, and use this information to define the stability of human Tregs and to genetically manipulate human T cells in order to program their differentiation pathway. The following Specific Aims are proposed. 1. Functional and phenotypic plasticity of regulatory T cells (Tregs) and effector T cells (TefO. In this Aim, we will generate antigen-specific natural (thymic) and adaptive (peripheral) Tregs using TCR transgenic mice expressing fluorescent reporters for FoxpS and key cytokines (IFN-v, IL-17 and IL-10) and different forms of antigen exposure. Purified Tregs will be transferred into normal recipients and exposed to antigen with and without inflammatory stimuli, or transferred into recipients with active autoimmune reactions. The cells will be followed for FoxpS and cytokine expression and surface phenotype by staining, and unctional responses after sorting. Tregs will be isolated from healthy volunteers and patients with autoimmune disease (initially T1D) and followed in vitro for conversion to effectors in the presence of activating and inflammatory stimuli. A novel transgenic mouse strain that expresses a lineage marker of past roxp3 expression will be used to examine the fates of these cells. The conversion of effector cells into Tregs be examined using the same approaches. 2.'Biochemical basis of Treg stability and conversion. T cells will be examined under conditions of stability and conversion for changes in surface phenotype, expression of Treg-specific genes, and methylation of the :oxp3 locus. The functional consequence of transient FoxpS expression will be analyzed in mouse and human cells using shRNA knockdowns and knockout mice. Attempts will be made to maintain the stability of regs by expressing selected genes in human Tregs.