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

基本信息

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
项目类别：
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

项目摘要

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调节性 T 细胞功能的新型调节剂调节性 T (Treg) 细胞在胸腺或外周被诱导以抑制效应 T 细胞，这不仅引起自身免疫性疾病，但也能杀死肿瘤，因此，Treg细胞可作为治疗靶点。尽管进行了大量的研究，但仍没有找到实现这一目标的方法。在此，我们的目标是确定重新编程 Treg 免疫抑制功能的新因素。为此，我们系统地研究了控制Treg的表观遗传和转录机制 Treg 主调节因子 Foxp3 的功能也受到环境因素的调节，包括 T。细胞抗原受体 (TCR) 激动剂和白细胞介素 2 (IL-2)，通过转录机制发挥作用调节 Treg 的适应性、归巢和抑制功能。协调解释环境线索，从而平衡 Treg 的识别和抑制功能。这些程序的表征将产生对 Treg 功能的机制见解，从而产生新的方法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节 Treg 抑制活性。在控制 Treg 抑制的顺式调节元件上分析信号诱导的动态蛋白成分在我们的初步实验中，我们采用了基于邻近生物素化的蛋白质组学方法。鉴定稳定状态下和细胞接收后 Foxp3 相关顺式调节元件的蛋白质这些实验揭示了可能参与 TCR 或 IL-2 刺激的蛋白质的未知细节。静息状态下或 TCR 和 IL-2 刺激下 Foxp3 靶基因表达的调节作为证据。从概念上讲，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，公正地分析参与信号依赖性转录调控的核蛋白。在这些初步结果的基础上，我们建议进一步定义顺式的时间动态细胞接受TCR和IL-2刺激后早期、中期和晚期Treg细胞中的蛋白质组我们深入的比较分析将揭示 Foxp3 中招募和耗尽的蛋白质。另一方面，我们将整合遗传扰动和响应 TCR 或 IL-2 刺激的目标。功能测定，从信号依赖性动态顺式蛋白质组中识别 Treg 细胞功能的新型调节因子。我们还将确定这些新因子通过调节 Treg 细胞功能的机制他们对 TCR 或 IL-2 信号传导的反应。总的来说，我们开发了一种比较蛋白质组学方法，无需工程即可原位分析顺式蛋白质组我们将确定 Treg 顺式蛋白质组对 TCR 和 IL- 的时间动态。 2刺激并利用基因组编辑方法揭示其在Treg免疫抑制功能中的作用。这些因素可能作为改善基于 Treg 的自身免疫性疾病治疗的新靶点。