Regulation of Human PD-1 Gene Expression

程序性细胞死亡-1（PD-1）由Pdcd 1基因编码，是一种免疫抑制受体， 免疫激活后在T细胞表面短暂表达，但长期在T细胞上高度表达 暴露于抗原。通过PD-1的持续信号传导导致T细胞耗竭，这是一种PD-1 表达维持在高水平，并且其中T细胞不再能够正常地应答抗原 挑战.在许多癌症中，抗体介导的PD-1-PDL 1/2途径的“检查点”阻断， 结果是T细胞免疫反应的重新激活，在临床上取得了惊人的成功。PD-1已经 在小鼠模型系统中进行了广泛的研究;然而，尽管其具有明显的治疗重要性， 了解PD-1在人类中的调节方式！没有系统地检查顺式或反式调节 已经进行了人PD-1基因的元件的研究，并研究了调节其表观遗传途径的作用。 表达有限。此外，PD-1在TFH细胞上高度表达，但关于PD-1的信息有限。 它在这种细胞类型中的调节是已知的。在这里，我们试图通过确定PD-1是如何被激活来填补这一知识空白。 在人CD 4、CD 8和TFH T细胞中调节。阐明控制癌症的分子和表观遗传程序 hPdcd 1的表达将提供新的理解、工具和靶点来操纵PD-1基因表达， 可能用于治疗感染、自身免疫和癌症。从报告的数据中可以清楚地看出，PD- 1基因调控是复杂的，并依赖于细胞类型和免疫环境/挑战。因此，我们认为， 我们的应用集中在阐明基本的转录和表观遗传调控机制上 控制人类T细胞中hPdcd 1表达的基因。目的1将鉴定和确定hPdcd 1的功能 顺式调节元件，因为这些是所有其他随后的关键。对于这些实验，我们将使用 去识别的外周原代人幼稚、离体活化和记忆性CD 8和CD 4 T细胞，TFH细胞， 以及Jurkat细胞作为模型T细胞系。抗原特异性CD 4和CD 8 T细胞将从去识别， HIV感染者代表长期暴露/耗尽的T细胞。我们将整合表观基因组数据 （ATAC-seq、ChIP-seq和亚硫酸氢盐测序）来探测顺式表达的机制。 元素正在发挥作用。目的2将确定负责hPdcd 1调控的转录因子（TF）。 在这里，我们将使用基因组信息来识别推定因子的基序，并使用CRISPR/Cas9和慢病毒技术。 在原代人T细胞中的表达系统，以敲除或外源表达因子，并确定其 调节hPdcd 1的作用。我们将使用ChIP来定义因子结合/占用。最后，我们将使用染色质 构象捕获方法，以证明TF与其结合位点之间的直接相互作用 hPdcd 1启动子区和鉴定的增强子。该计划将共同确定 hPdcd 1的调节机制，并最终提供证据支持旨在 通过操纵基因调控来控制这一途径。

Programmed cell death-1 (PD-1), encoded by the Pdcd1 gene, is an immune inhibitory receptor that is expressed transiently on the surface of T cells following immune activation but is highly expressed on T cells chronically exposed to antigen. Sustained signaling through PD-1 results in T cell exhaustion, a state in which PD-1 expression is maintained at a high level and where the T cells can no longer respond normally to antigenic challenge. Antibody mediated “checkpoint” blockade of the PD-1–PDL1/2 pathways in numerous cancers, which results in reinvigoration of T cell immune responses, has had astonishing success in the clinic. PD-1 has been extensively studied in mouse model systems; yet despite its clear therapeutic importance, nearly nothing is known about how PD-1 is regulated in humans! No systematic examination of the cis or trans-regulatory elements of the human PD-1 gene has been conducted and the role of epigenetic pathways regulating its expression is limited. Additionally, PD-1 is highly expressed on TFH cells, yet only limited information regarding its regulation in this cell type is known. Here we seek to fill this knowledge gap by determining how PD-1 is regulated in human CD4, CD8, and TFH T cells. Elucidating the molecular and epigenetic programs that control hPdcd1 expression will provide new understanding, tools, and targets to manipulate PD-1 gene expression that could potentially be used to treat infection, autoimmunity, and cancer. It is clear from the reported data that PD- 1 gene regulation is complex and dependent on the cell type and immune environment/challenge. Therefore, we have focused this application on elucidating the basic transcriptional and epigenetic regulatory mechanisms that control hPdcd1 expression in human T cells. Aim 1 will identify and determine the function of the hPdcd1’s cis-regulatory elements as these are the key to all else that follows. For these experiments, we will use deidentified, peripheral primary human naïve, ex vivo activated, and memory CD8 and CD4 T cells, TFH cells, as well as Jurkat cells as a model T cell line. Antigen-specific CD4 and CD8 T cells will be isolated from deidentified, HIV-infected individuals to represent chronically exposed/exhausted T cells. We will integrate epigenomic data (ATAC-seq, ChIP-seq, and bisulfite sequencing) of the above cells to probe the mechanisms by which the cis elements are functioning. Aim 2 will define the transcription factors (TFs) responsible for hPdcd1 regulation. Here we will use genomic information to identify motifs for putative factors and use CRISPR/Cas9 and lentiviral expression systems in primary human T cells to knockout or exogenously express factors and determine their role in regulating hPdcd1. We will use ChIP to define factor binding/occupancy. Lastly, we will use chromatin conformation capture methodologies to demonstrate direct interactions between TFs and their binding sites with the hPdcd1 promoter region and identified enhancers. Together this program will identify the fundamental mechanisms by which hPdcd1 is regulated and will ultimately provide evidence supporting therapies aimed at controlling this pathway through the manipulation of gene regulation.