Mechanisms of human PD-1 gene regulation in T follicular helper cells

滤泡辅助T细胞中人PD-1基因调控机制

• 批准号: 10647836
• 负责人: Michael Duane Powell
• 金额: $ 7.38万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10647836
• 关键词: 3-Dimensional; ASCL2 gene; ATAC-seq; Antibodies; Antibody Formation; Antigens; Antitumor Response; Architecture; Autoimmune Diseases; Automobile Driving; B-Lymphocytes; Biological Assay; Blood; CASP9 gene; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CRISPR/Cas technology; Cell Death; Cell Line; Cell Lineage; Cells; Cellular biology; ChIP-seq; Chimeric Proteins; Chromatin; Chronic; Classification; Clinical; Communicable Diseases; Conserved Sequence; Cytokine Signaling; Data; Data Set; Development; Disease; EP300 gene; Elements; Engineering; Enhancers; Epigenetic Process; Exhibits; Exposure to; Family; Future; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Goals; Health; Helper-Inducer T-Lymphocyte; Human; Immune; Immune response; Immune system; Immunologics; Immunosuppression; Inflammatory Response; Interleukin-12; Investigation; Jurkat Cells; Knock-out; Knowledge; Ligands; Lymphocyte; Lymphocyte Activation; Malignant Neoplasms; Modeling; Molecular; Mus; Nobel Prize; Nucleic Acid Regulatory Sequences; Pathway interactions; Population; Publishing; Regulation; Regulatory Element; Reporter; Sampling; Signal Pathway; Signal Transduction; Structure of germinal center of lymph node; Surface; System; T-Lymphocyte; T-Lymphocyte Subsets; Techniques; Testing; Therapeutic; Tissues; Tonsil; Transfection; Transforming Growth Factor beta; Vaccines; Work; activin A; cancer therapy; candidate identification; checkpoint therapy; cytokine; deactivated CRISPR-Cas9; design; exhaust; experimental study; genome sequencing; histone modification; human disease; insight; interest; new therapeutic target; novel; novel therapeutics; overexpression; pathogen; programmed cell death protein 1; promoter; receptor; response; success; targeted treatment; therapy design; transcription factor; transcriptome sequencing; vaccine-induced antibodies; vector; virtual; whole genome

1 Programed Cell death (PD-1), encoded by Pdcd1, is an immune inhibitory receptor expressed on the surface of 2 lymphocytes. Engagement of PD-1 by its ligands results in suppression of immune effector function and 3 subsequent dampening of inflammatory responses. The impact of this signaling pathway to human health has 4 been demonstrated by the clinically successful treatment of numerous cancers with antibody blockade of PD-1 5 or its ligand, resulting in reinvigoration of the immune system and successive anti-tumor responses. Surface 6 PD-1 expression is amongst its highest in a subset of CD4+ T cells called T follicular helper cells (TFH). TFH cells 7 are pivotal for optimal germinal center formation and subsequent generation of pathogen- and vaccine- induced 8 antibodies by B cells. As such, PD-1 resides as a key component of a robust humoral response. Despite the 9 clear connection to human health, the majority of previous work has centered on elucidating the mechanisms 10 surrounding murine Pdcd1 (mPdcd1) regulation. Surprisingly, there is almost nothing known about how human 11 Pdcd1 (hPdcd1) is regulated! Here we seek to close this gap in knowledge by identifying the cis-, trans-, and 12 epigenetic pathways that regulate hPdcd1 in TFH cells. We hypothesize that hPdcd1 expression in TFH cells is 13 controlled by novel regulatory mechanisms aimed at controlling the extraordinary high levels of PD-1 on these 14 cells. These findings could identify novel elements and pathways that might be dysfunctional in other 15 immunological contexts and disease settings. In Aim 1 I will identify and classify the cis-regulatory elements that 16 control hPdcd1 expression in TFH cells. I will acquire primary human TFH cells form blood and discarded tonsil 17 tissue, as well as from a recently described model to generate ex vivo TFH-like cells. Preliminary ATAC-seq data 18 in TFH cells has defined regions of interest that will be interrogated for enhancer functionality by engineering 19 promoter-reporter constructs. Additionally, deactivated Cas9-CRISPR technologies will be used to define the 20 functionality of these regions in primary human TFH cells. To further characterize the hPdcd1 epigenetic 21 landscape in TFH cells, I will conduct ChIP-seq for covalent histone modifications and Hi-ChIP to define the 3D 22 enhancer-promoter architecture. In Aim 2, I will determine the unique transcription factor network that regulates 23 hPdcd1 expression in TFH cells. Integration of previous published work with RNA- and ATAC-seq datasets has 24 identified candidate transcription factors that may play a role in hPdcd1 regulation. I will use ChIP-qPCR to 25 determine transcription factor occupancy at the hPdcd1 locus. Subsequently, I will use CRISPR/Cas9 and 26 lentiviral expression systems in primary human cells to knockout or exogenously express factors respectively, 27 and determine the resulting effect on PD-1 expression. Collectively, unearthing the mechanisms of hPdcd1 28 regulation at the molecular level will aid in the identification of novel therapeutic targets, that could allow for the 29 precise manipulation of PD-1 expression in the treatment of cancer, autoimmunity, and infectious disease.

PD-1是由Pdcd 1编码的一种免疫抑制受体，表达于细胞表面， 2个淋巴细胞。PD-1与其配体的结合导致免疫效应子功能的抑制， 3随后抑制炎症反应。这种信号通路对人类健康的影响 4已经通过使用PD-1的抗体阻断在临床上成功治疗许多癌症而得到证实 5或其配体，导致免疫系统的恢复活力和连续的抗肿瘤应答。表面 6 PD-1表达在称为T滤泡辅助细胞（TFH）的CD 4 + T细胞亚群中最高。TFH细胞 7是最佳生发中心形成和随后的病原体和疫苗诱导的世代的关键。 B细胞的8种抗体。因此，PD-1是强大的体液应答的关键组分。尽管 9与人类健康的明确联系，以前的大部分工作都集中在阐明机制上， 10周围鼠Pdcd 1（mPdcd 1）调节。令人惊讶的是，几乎没有人知道人类是如何 11 Pdcd 1（hPdcd 1）是受管制的！在这里，我们试图通过识别顺式，反式和 在TFH细胞中调节hPdcd 1的12种表观遗传途径。我们假设TFH细胞中hPdcd 1的表达是 13由新的调节机制控制，旨在控制这些细胞上异常高水平的PD-1。 14间牢房这些发现可以识别可能在其他组织中功能失调的新元素和途径。 15免疫学背景和疾病环境。在目标1中，我将鉴定并分类顺式调节元件， 16对照TFH细胞中的hPdcd 1表达。我将从血液和废弃的扁桃体中获得原代人TFH细胞 17组织，以及来自最近描述的模型以产生离体TFH样细胞。初步ATAC-seq数据 18在TFH细胞中具有确定的感兴趣区域，其将通过工程改造来询问增强子功能性 19个启动子-报告基因构建体。此外，失活的Cas9-CRISPR技术将用于定义 图20示出了这些区域在原代人TFH细胞中的功能性。进一步表征hPdcd 1表观遗传 在TFH细胞中的21个景观中，我将进行ChIP-seq用于共价组蛋白修饰和Hi-ChIP以定义3D 22增强子-启动子结构。在目标2中，我将确定调节转录因子的独特转录因子网络。 23 TFH细胞中的hPdcd 1表达。将先前发表的工作与RNA和ATAC-seq数据集相结合， 24个鉴定的候选转录因子可能在hPdcd 1调控中发挥作用。我将使用ChIP-qPCR 25确定在hPdcd 1基因座的转录因子占有率。随后，我将使用CRISPR/Cas9和 26种原代人细胞中的慢病毒表达系统，分别敲除或外源表达因子， 27，并确定对PD-1表达的影响。总的来说，揭示hPdcd 1的机制 在分子水平上的调节将有助于鉴定新的治疗靶点，这可以允许在分子水平上的调节。 29在癌症、自身免疫和感染性疾病的治疗中精确操纵PD-1表达。