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

1程序性细胞死亡(PD-1)是一种表达于细胞表面的免疫抑制受体，由Pdcd1编码。 2淋巴细胞。PD-1与其配体结合导致免疫效应功能抑制和 3随后抑制炎症反应。这一信号通路对人类健康的影响 PD-1抗体阻断治疗多种癌症的临床成功证明了这一点。 5或其配体，导致免疫系统恢复活力和连续的抗肿瘤反应。表面 6 PD-1在一种被称为T滤泡辅助细胞(TFH)的CD4T细胞亚群中表达最高。TFH细胞 7对于最佳生发中心的形成和病原体和疫苗诱导的下一代至关重要 B细胞抗体8例。因此，PD-1是强大的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，以前的大部分工作都集中在阐明这些机制上。 10周边小鼠Pdcd1(MPdcd1)调控。令人惊讶的是，关于人类是如何 %11 Pdcd1(HPdcd1)受监管！在这里，我们试图通过识别顺式、反式和 调控TFH细胞hPdcd1的12条表观遗传通路。我们假设hPdcd1在TFH细胞中的表达是 13由新的调控机制控制，旨在控制这些细胞上极高水平的PD-1 14个细胞。这些发现可以确定在其他生物中可能功能失调的新元素和途径 15免疫学背景和疾病背景。在目标1中，我将确定和分类顺式监管要素，这些要素 16控制hPdcd1在TFH细胞中的表达。我将从血液和废弃的扁桃体中获取原代人类TFH细胞 17组织，以及最近描述的产生体外TFH样细胞的模型。初步ATAC-SEQ数据 18在TFH细胞中定义了感兴趣的区域，工程人员将询问这些区域以获得增强功能 19个启动子-报告基因构建。此外，停用的Cas9-CRISPR技术将用于定义 20这些区域在原代人TFH细胞中的功能。进一步鉴定hPdcd1表观遗传学 21在TFH细胞中，我将进行CHIP-SEQ进行共价组蛋白修饰和Hi-CHIP来定义3D 22增强子-启动子结构。在目标2中，我将确定调节 23 hPdcd1在TFH细胞中的表达。将以前发表的工作与RNA和ATAC-SEQ数据集集成在一起 24个可能在hPdcd1调控中发挥作用的候选转录因子。我将使用芯片-qPCR来 25确定hPdcd1基因座上的转录因子占有率。随后，我将使用CRISPR/Cas9和 26个慢病毒表达系统在原代人类细胞中分别敲除或外源表达因子， 27，并确定由此对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、自身免疫和传染病的治疗中精确操纵PD-1的表达。

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.