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.

1 程序性细胞死亡（PD-1），由 Pdcd1 编码，是表达于细胞表面的免疫抑制性受体 2个淋巴细胞。 PD-1 与其配体的结合会导致免疫效应子功能受到抑制， 3 随后抑制炎症反应。该信号通路对人类健康的影响 4 已通过 PD-1 抗体阻断在临床上成功治疗多种癌症得到证实 5 或其配体，导致免疫系统重振和连续的抗肿瘤反应。表面 6 PD-1 表达在称为滤泡辅助 T 细胞 (TFH) 的 CD4+ T 细胞子集中最高。 TFH细胞 7 对于最佳生发中心形成以及随后病原体和疫苗诱导的产生至关重要 B细胞产生8种抗体。因此，PD-1 是强大体液反应的关键组成部分。尽管 9 与人类健康有明确的联系，之前的大部分工作都集中在阐明其机制 10.周围鼠Pdcd1(mPdcd1)调节。令人惊讶的是，对于人类如何 11 Pdcd1 (hPdcd1) 受到调节！在这里，我们试图通过识别顺式、反式和 调节 TFH 细胞中 hPdcd1 的 12 条表观遗传途径。我们假设 TFH 细胞中 hPdcd1 的表达是 13 由旨在控制这些细胞上异常高水平的 PD-1 的新型调控机制控制 14 个细胞。这些发现可以识别出在其他方面可能功能失调的新元素和途径。 15 种免疫背景和疾病环境。在目标 1 中，我将识别顺式监管要素并对其进行分类 16 控制 TFH 细胞中 hPdcd1 的表达。我将从血液和废弃扁桃体中获取原代人类 TFH 细胞 17 组织，以及最近描述的模型来生成离体 TFH 样细胞。初步 ATAC-seq 数据 TFH 细胞中的 18 已定义了感兴趣的区域，将通过工程对其增强子功能进行询问 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 20 人类原代 TFH 细胞中这些区域的功能。为了进一步表征 hPdcd1 表观遗传 21 TFH 细胞中的景观，我将进行 ChIP-seq 进行共价组蛋白修饰，并进行 Hi-ChIP 来定义 3D 22增强子-启动子架构。在目标 2 中，我将确定调节的独特转录因子网络 23 hPdcd1 在 TFH 细胞中的表达。之前发表的工作与 RNA 和 ATAC-seq 数据集的集成 24 确定了可能在 hPdcd1 调节中发挥作用的候选转录因子。我将使用 ChIP-qPCR 来 25 确定 hPdcd1 基因座上的转录因子占据情况。随后，我将使用 CRISPR/Cas9 和 26个慢病毒表达系统在原代人细胞中分别敲除或外源表达因子， 27 并确定对 PD-1 表达的影响。共同挖掘 hPdcd1 的机制 28 分子水平的调控将有助于识别新的治疗靶点，这可能允许 29 在癌症、自身免疫和传染病治疗中精确调控 PD-1 表达。