Determination of chromatin protein dynamics in CD8 T cells

CD8 T 细胞染色质蛋白动态测定

基本信息

批准号：
10735641
负责人：
Yongqiang Feng
金额：
$ 27.3万
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：
2023
资助国家：
美国
起止时间：
2023-06-23 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10735641
关键词：
Acetylation Antibodies Antigens Binding Bioinformatics Biological Biotinylation CD8-Positive T-Lymphocytes CRISPR screen Cell physiology Cells Cellular biology Cellular immunotherapy Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Coupled Cues Enhancers Epigenetic Process FOXP3 gene Gene Expression Gene Expression Regulation Genes Genetic Transcription Genomic Segment Goals Histone Acetylation Histone H3 Immune System Diseases Immune response Immunologics In Situ In Vitro Infiltration Interleukin-2 Investigation Knock-out Libraries Lysine Malignant Neoplasms Mass Spectrum Analysis Mediating Methods Mus Nuclear Nuclear Proteins Outcome Pathway interactions Physiological Play Process Protein Dynamics Proteins Proteomics Receptor Signaling Regulatory T-Lymphocyte Research Role Signal Pathway Signal Transduction System T cell differentiation T cell response T-Cell Development T-Cell Receptor T-Lymphocyte Testing Therapeutic Time Transcriptional Regulation Transducers Tumor Immunity Viral Work anti-tumor immune response cell type chromatin protein comparative cytokine epigenetic profiling epigenetic regulation experimental study extracellular genetic element genome editing immune function improved in vivo inducible gene expression innovation interest novel programs promoter receptor recruit response screening tumor

项目摘要

Determination of chromatin protein dynamics in CD8 T cells CD8 T cells are crucial for antiviral and antitumor immunity. How these cells respond to stimulations and how to manipulate their effector function for therapeutic purposes have been under intensively investigations. It is widely accepted that T cell antigen receptor (TCR) and co-receptor signaling drives almost all the key processes of CD8 T cell differentiation and function via transcriptional and epigenetic regulation of gene expression. Factors modulating TCR signal transduction or target gene expression presumably play important roles in dictating CD8 T cell differentiation and immunological functions. Although proteins directly mediating TCR and co-receptor signal transduction have been well defined, the nuclear proteins modulating target gene expression remain to be fully investigated. Unbiased approaches are required to comprehensively reveal the nuclear proteins regulating TCR and co-receptor dependent gene expression, potentially leading to the identification of novel factors governing CD8 T cell differentiation and function that determine antiviral and antitumor immune response. Recently CRISPR screening offers an unprecedented efficiency to systematically delineate the regulators of CD8 T cell differentiation and function. However, this method does not uncover the mechanistic linkage between candidate factors and signaling pathways and is also severely constrained by available experimental readouts. We reasoned that defining the dynamic proteins of the active gene promoters and enhancers in CD8 T cells upon TCR and co-receptor stimulation would be a solution, because proteins recruited to or depleted at these regions during this process would reveal novel key regulators. To determine the dynamic chromatin proteins at genomic regions of interest, we recently developed a comparative proteomics method using antibody-guided proximity biotinylation in a separate study. In preliminary experiments, we applied this method to mouse primary CD8 T cells and profiled the dynamic proteins at active enhancers and promoters marked by histone H3 lysine 27 acetylation (H3K27ac) upon TCR and co-stimulation, because this signaling appears to act on these genetic elements to control important functional genes in CD8 T cells (such as cytokines and Myc) by coordinating with the histone acetylation pathway. This experiment revealed known TCR signal transducers and a number of novel factors overrepresented or underrepresented. We then performed an in vivo pooled CRISPR screening by knocking out these dynamic proteins and discovered several positive and negative regulators of tumor infiltrating CD8 T cells. On the basis of these results, we propose to further define the chromatin protein dynamics in CD8 T cells and determine their roles in modulating inducible gene expression and CD8 T cell antitumor activity. Overall, we will employ a new proteomics method, developed by us, to determine signal dependent chromatin protein dynamics to unbiasedly identify novel regulators of CD8 T cell function. These factors may serve as new targets to improve CD8 T cell based immunotherapies.

CD8 T细胞中染色质蛋白动力学的测定 CD8 T细胞对于抗病毒和抗肿瘤免疫至关重要。这些细胞如何响应刺激以及如何响应为了治疗目的操纵其效应子功能一直在深入研究。这是广泛接受T细胞抗原受体（TCR）和共受体信号传导几乎所有关键过程通过转录和表观遗传学调节基因表达的CD8 T细胞分化和功能。因素调节TCR信号转导或靶基因表达可能在决定CD8中起重要作用 T细胞分化和免疫功能。尽管蛋白质直接介导TCR和共受体信号转导已很好地定义，调节靶基因表达的核蛋白仍然得到充分调查。需要无偏的方法来全面揭示核蛋白调节TCR和共受体依赖基因表达，可能导致新颖的鉴定控制CD8 T细胞分化和功能的因素，这些因素决定了抗病毒和抗肿瘤免疫反应。最近，CRISPR筛选提供了前所未有的效率，可以系统地描述 CD8 T细胞分化和功能。但是，此方法没有发现候选因素和信号通路，也受到可用的实验读数的严格限制。我们认为，定义活性基因启动子的动态蛋白和CD8 T细胞中的增强子在TCR和共受体刺激后，将是一种解决方案，因为在这些方面募集或耗尽了蛋白质在此过程中，区域将揭示新的关键调节剂。确定在感兴趣的基因组区域，我们最近使用抗体引导开发了一种比较蛋白质组学方法在另一项研究中接近生物素化。在初步实验中，我们将此方法应用于小鼠的主要 CD8 T细胞并在由组蛋白H3赖氨酸标记的活性增强子和启动子处介绍了动态蛋白 TCR和共刺激后，27乙酰化（H3K27AC），因为该信号似乎对这些遗传作用通过与组蛋白乙酰化途径。该实验揭示了已知的TCR信号传感器和许多新颖因素过多代表或代表性不足。然后，我们通过淘汰这些动态蛋白，发现肿瘤浸润的几个阳性和阴性调节剂 CD8 T细胞。根据这些结果，我们建议进一步定义CD8中的染色质蛋白动力学 T细胞并确定它们在调节诱导基因表达和CD8 T细胞抗肿瘤活性中的作用。总体而言，我们将采用我们开发的新蛋白质组学方法来确定信号依赖性染色质蛋白质动力学可公开鉴定CD8 T细胞功能的新型调节剂。这些因素可能是新的改善基于CD8 T细胞的免疫疗法的靶标。