Regulation of Human PD-1 Gene Expression

程序性细胞死亡-1(PD-1)由Pdcd1基因编码，是一种表达的免疫抑制受体 免疫激活后在T细胞表面短暂表达，但长期在T细胞上高表达 暴露在抗原下的。通过PD-1持续的信号传递会导致T细胞耗竭，在这种状态下PD-1 表达维持在高水平，T细胞不再对抗原做出正常反应 挑战。抗体介导的PD-1-PDL1/2通路在多种癌症中的“检查点”阻断 结果在恢复T细胞免疫应答方面，在临床上取得了惊人的成功。PD-1已经被 在小鼠模型系统中进行了广泛的研究；然而，尽管它具有明显的治疗重要性，但几乎没有什么是 知道PD-1是如何在人类身上受到调控的！没有对顺位或跨监管机构进行系统检查 已经进行了人类PD-1基因的元件和调控其基因的表观遗传途径的作用 表达是有限的。此外，PD-1在TFH细胞上高表达，但仅有有限的信息涉及 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 在人的CD4、CD8和TFH T细胞中调节。阐明控制的分子和表观遗传程序 HPdcd1的表达将为操纵PD-1基因的表达提供新的理解、工具和靶点 可能被用于治疗感染、自身免疫和癌症。从报告的数据中可以清楚地看到，PD- 1基因调控是复杂的，取决于细胞类型和免疫环境/挑战。因此， 我们把这一应用的重点放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hPdcd1的表达。目标1将鉴定和确定hPdcd1‘S的功能 顺应性监管要素是接下来所有其他要素的关键。对于这些实验，我们将使用 识别，外周原始人类幼稚，体外激活，并记忆CD8和CD4T细胞，TFH细胞，AS 以及Jurkat细胞作为T细胞模型。抗原特异性的CD_4和CD_8 T细胞将从已鉴定的 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)，以探索顺式作用的机制。 元素正在发挥作用。目标2将定义负责hPdcd1调控的转录因子(TF)。 在这里，我们将使用基因组信息来确定推定因子的基序，并使用CRISPR/Cas9和慢病毒 在原代人类T细胞中表达系统敲除或外源表达因子并确定其 在调节hPdcd1中的作用。我们将使用芯片来定义因子绑定/占用。最后，我们将使用染色质 展示转录因子及其结合位点之间直接相互作用的构象捕获方法 HPdcd1启动子区域，并鉴定了增强子。本计划将共同确定基本的 HPdcd1被调控的机制，并最终将提供证据支持旨在 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.