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.

由PDCD1编码的1个编程的细胞死亡（PD-1）是在表面表达的免疫抑制受体 2个淋巴细胞。 PD-1通过其配体的参与导致免疫效应功能抑制和 3随后的炎症反应减弱。这种信号通路对人类健康的影响已经 4通过临床成功治疗PD-1的抗体阻断的众多癌症证明了 5或它的配体，导致免疫系统重新振奋和连续的抗肿瘤反应。表面 6 PD-1表达在CD4+ T细胞的子集中最高，称为T卵泡辅助细胞（TFH）。 TFH细胞 7是最佳生发中心形成以及随后生成病原体和疫苗诱导的关键 B细胞8种抗体。因此，PD-1是强大的体液反应的关键组成部分。尽管有 9与人类健康的明确联系，以前的大多数工作都集中在阐明机制上 10周围的鼠PDCD1（MPDCD1）调节。令人惊讶的是，几乎一无所知 11 PDCD1（HPDCD1）受到调节！在这里，我们试图通过识别顺式，跨性别和 在TFH细胞中调节HPDCD1的12个表观遗传途径。我们假设TFH细胞中的HPDCD1表达是 13受新型调节机制的控制，旨在控制这些机制 14个细胞。这些发现可以识别出可能在其他中可能功能失调的新型元素和途径 15个免疫学环境和疾病环境。在AIM 1中，我将确定并对顺式调节元素进行分类 16控制TFH细胞中的HPDCD1表达。我将获得原代人TFH细胞形成血液并丢弃扁桃体 17组织以及最近描述的模型以生成离体TFH样细胞。初步的ATAC-SEQ数据 18在TFH细胞中，已定义了感兴趣的区域，该区域将通过工程询问增强子功能 19个启动子 - 重复构建体。此外，将使用停用的Cas9-Crispr技术来定义 20这些区域在原代人TFH细胞中的功能。为了进一步表征HPDCD1表观遗传学 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增强器促销架构。在AIM 2中，我将确定调节的唯一转录因子网络 TFH细胞中的23 HPDCD1表达。与RNA和ATAC-SEQ数据集的先前已发表工作的集成 24个确定的候选转录因子可能在HPDCD1调节中起作用。我将使用chip-qpcr到 25确定在HPDCD1基因座处的转录因子占用率。随后，我将使用CRISPR/CAS9和 26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。共同发掘了HPDCD1的机制 28分子水平的调节将有助于鉴定新的治疗靶标，这可以允许 29在癌症，自身免疫和传染病治疗中，精确操纵PD-1表达。