Identify novel modulators of regulatory T cell function from the dynamic cis-proteomes

从动态顺式蛋白质组中鉴定调节性 T 细胞功能的新型调节剂

• 批准号: 10288457
• 负责人: Yongqiang Feng
• 金额: $ 26.93万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-11 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10288457
• 关键词: Address; Agonist; Antibodies; Autoimmune Diseases; Binding; Biological Assay; Biotinylation; CD4 Positive T Lymphocytes; CRISPR screen; Cell physiology; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Coupled; Cues; Engineering; Epigenetic Process; Equilibrium; FOXP3 gene; Gene Expression; Genetic; Genetic Transcription; Goals; Harvest; Homing; Immune; Immune System Diseases; Immune Tolerance; In Situ; In Vitro; Interleukin-2; Intrinsic factor; Investigation; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Multiple Sclerosis; Nuclear; Nuclear Protein; Nuclear Proteins; Organism; Outcome; Proteins; Proteome; Proteomics; Protocols documentation; Receptor Signaling; Regulation; Regulatory Element; Regulatory T-Lymphocyte; Rest; Role; Side; Signal Transduction; Specific qualifier value; Streptavidin; T-Cell Receptor; T-Lymphocyte; Testing; Thymus Gland; Transcriptional Regulation; Transducers; base; cell type; comparative; effector T cell; experimental study; extracellular; fitness; genetic element; genome editing; improved; in vivo; insight; interest; mouse model; novel; programs; recruit; response; therapeutic target; tumor

Identify novel modulators of regulatory T cell function from the dynamic cis-proteomes Regulatory T (Treg) cells are induced in the thymus or periphery to suppress effector T cells that not only cause autoimmune diseases but also kill tumors. Therefore, Treg cells serve as a therapeutic target to treat a variety of immunological diseases and cancer. Despite intensive investigations, methods to achieve this goal remain to be fully developed. Here, we aim identify novel factors to reprogram Treg immune suppressive function. To this end, we systematically examined the epigenetic and transcriptional mechanisms governing Treg function through Treg master regulator Foxp3. Treg cells are also regulated by environmental cues, including T cell antigen receptor (TCR) agonists and Interleukin-2 (IL-2), that act through transcriptional mechanisms to modulate Treg fitness, homing, and suppressive function. We hypothesize that opposing nuclear programs coordinate to interpret environmental cues, thus balancing Treg suppressive function. Identification and characterization of these programs will produce mechanistic insights into Treg function, leading to novel methods to modulate Treg suppressive activity. To test our hypothesis, we propose to develop a proteomics method to profile signal-induced dynamic protein components at the cis-regulatory elements governing Treg suppressive function. In our preliminary experiments, we adapted a proximity biotinylation–based proteomics method to identify the proteins at Foxp3-associated cis-regulatory elements in the steady state and after cells receiving TCR or IL-2 stimulation. These experiments uncovered unprecedented details of proteins potentially involved in the regulation of Foxp3-target gene expression at the resting state or upon TCR and IL-2 stimulation. As a proof of concept, these results demonstrate the feasibility of proximity biotinylation, when coupled with comparative proteomics, in unbiasedly profiling the nuclear proteins involved in signal-dependent transcriptional regulation. On the basis of these preliminary results, we propose to further define the temporal dynamics of the cis- proteomes in Treg cells after cells receiving TCR and IL-2 stimulation at the early, middle, and later stages of signal transduction. Our in-depth comparative analysis will reveal the proteins recruited to and depleted at Foxp3 targets in response to TCR or IL-2 stimulation. On the other side, we will integrate genetic perturbations and functional assays to identify novel regulators of Treg cell function from signal-dependent dynamic cis-proteomes. We will also determine the mechanisms by which these novel factors control Treg cell function by modulating their response to TCR or IL-2 signaling. Overall, we develop a comparative proteomics method to profile cis-proteomes in situ without engineering new cells or organisms. We will determine the temporal dynamics of the Treg cis-proteomes upon TCR and IL- 2 stimulation and use genome editing approach to uncover their roles in Treg immune suppressive function. These factors may serve as new targets to improve Treg-based treatment of autoimmune diseases.

从动态顺式蛋白质体中识别调节T细胞功能的新型调节剂 调节性t（treg）细胞在胸腺或外围诱导以抑制效应T细胞，不仅是 引起自身免疫性疾病，但也会杀死肿瘤。因此，Treg细胞是治疗A的治疗靶标 各种免疫疾病和癌症。尽管进行了深入调查，但实现这一目标的方法 仍然有待充分发展。在这里，我们旨在确定重新编程Treg免疫抑制功能的新因素。 为此，我们系统地检查了有关Treg的表观遗传和转录机制 通过Treg Master调节器Foxp3功能。 Treg细胞还受环境线索的调节，包括T 细胞抗原受体（TCR）激动剂和白介素-2（IL-2），通过转录机制起作用 调节Treg健身，归巢和抑制功能。我们假设反对核计划 协调以解释环境线索，从而平衡Treg抑制功能。识别和 这些程序的表征将产生对Treg功能的机械见解，从而导致新方法 调节Treg抑制活性。为了检验我们的假设，我们建议开发一种蛋白质组学方法 剖面信号诱导的动态蛋白质成分在顺式调节元件上抑制了Treg 功能。在我们的初步实验中，我们改编了基于生物素化的蛋白质组学方法 在稳态和接收后的细胞中识别与FOXP3相关的顺式调节元件的蛋白质 TCR或IL-2模拟。这些实验发现了可能涉及的蛋白质的前所未有的细节 在静止状态或TCR和IL-2刺激下，FOXP3目标基因表达的调节。作为证明 概念，这些结果证明了接近生物素化的可行性，与比较 蛋白质组学无视与信号依赖性转录调控有关的核蛋白。 根据这些初步结果，我们建议进一步定义顺式的临时动力学。 在接受TCR和IL-2模拟的细胞在早期，中间和后期接受TCR和IL-2模拟后，Treg细胞中的蛋白质组织 信号转导。我们深入的比较分析将揭示在Foxp3上募集并耗尽的蛋白质 响应TCR或IL-2刺激的靶标。另一方面，我们将整合遗传扰动和 功能分析以从信号依赖性动态顺式蛋白质体中鉴定出Treg细胞功能的新型调节剂。 我们还将确定这些新因素通过调节控制Treg细胞功能的机制 它们对TCR或IL-2信号传导的响应。 总体而言，我们开发了一种比较蛋白质组学方法，以在没有工程的情况下介绍顺式蛋白质体 新细胞或组织。我们将确定TCR和IL-上Treg顺式蛋白质体的临时动力学 2模拟和使用基因组编辑方法来揭示其在Treg免疫抑制功能中的作用。 这些因素可能是改善基于TREG的自身免疫性疾病治疗的新目标。