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.

从动态顺式蛋白质组中鉴定新的调节性T细胞功能调节剂 调节性T(Treg)细胞在胸腺或外周诱导，以抑制效应性T细胞，不仅 会导致自身免疫性疾病，但也会杀死肿瘤。因此，Treg细胞可以作为治疗的靶点。 各种免疫性疾病和癌症。尽管进行了密集的调查，但实现这一目标的方法 仍有待充分开发。在这里，我们的目标是确定新的因素来重新编程Treg免疫抑制功能。 为此，我们系统地研究了控制Treg的表观遗传和转录机制。 功能通过Treg主调节器Foxp3。Treg细胞也受环境信号的调节，包括T 细胞抗原受体(TCR)激动剂和白细胞介素2(IL-2)，通过转录机制作用于 调节Treg健身、归宿和抑制功能。我们假设反对核计划 协调解释环境线索，从而平衡Treg抑制功能。身份识别和 这些程序的特征将产生对Treg功能的机械性见解，从而产生新的方法 来调节Treg抑制活动。为了验证我们的假设，我们建议开发一种蛋白质组学方法来 调控Treg抑制的顺式调控元件上信号诱导的动态蛋白质组分 功能。在我们的初步实验中，我们采用了一种基于邻近生物素化的蛋白质组学方法来 鉴定Foxp3相关顺式调控元件在稳态和细胞接收后的蛋白质 TCR或IL-2刺激。这些实验揭示了可能参与的蛋白质的前所未有的细节。 Foxp3靶基因在静息状态或在TCR和IL-2刺激下的表达调节。作为证据 在概念上，这些结果证明了邻近生物素化的可行性，当与比较 蛋白质组学，无偏见地描述参与信号依赖的转录调控的核蛋白。 在这些初步结果的基础上，我们建议进一步定义顺式振动的时间动力学。 早、中、晚期TCR和IL-2刺激对Treg细胞蛋白质组的影响 信号转导。我们的深入比较分析将揭示Foxp3被招募和耗尽的蛋白质 靶细胞对TCR或IL-2刺激的反应。另一方面，我们将整合遗传扰动和 从信号依赖的动态顺式蛋白质组中识别Treg细胞功能的新调节因子的功能分析。 我们还将确定这些新因子通过调节调节Treg细胞功能的机制 它们对TCR或IL-2信号的反应。 总之，我们开发了一种比较蛋白质组学方法来原位分析顺式蛋白质组而不需要工程。 新的细胞或生物体。我们将确定Treg顺式蛋白质组对TCR和IL-1的时间动力学。 2刺激，并用基因组编辑的方法揭示它们在Treg免疫抑制功能中的作用。 这些因素可能成为改进基于Treg的自身免疫性疾病治疗的新靶点。