Effects of PD-L1 silencing on global transcriptomic changes in tumor

Cancers evade anti-tumor immunity by up-regulation of immune checkpoint molecules, such as programmed cell death 1 ligand 1 (PD-L1), in response to stimuli, such as interferon-gamma (IFN?). Expression of PD-L1 within the tumor microenvironment inhibits the anti-tumor immune response by binding the immune checkpoint receptor PD-1 expressed on T cells. Immune checkpoint inhibitors that target the PD-1/PD-L1 pathway are less toxic than standard chemotherapy and produce durable tumor regression and overall survival benefits in several tumors. However, only a small group of patients respond to these therapies, and some of the responders develop acquired resistance. Understanding the effects of PD-L1 silencing on global gene expression is critical to identifying potential immune evasive mechanisms. In this study, we performed transcriptome analysis of PD-L1 knockdown cells and showed that silencing PD-L1 reduced the expression of only a few immunosuppressive genes. Our findings suggest that the identification of novel targets that can control a larger number of immunosuppressive molecules could lead to the development of more effective immunotherapies. Overall design: Tumor cell lines were transfected with an siRNA control or two different siRNAs targeting PD-L1. 48 hours after transfection, cells were stimulated with interferon gamma for 24 hours. RNA expression profiles were generated by RNA-seq, in duplicate, using Illumina NovaSeq6000.肿瘤细胞可通过上调免疫检查点分子（如程序性死亡蛋白1配体1（PD-L1））来逃避免疫抗肿瘤应答，以应对干扰素-γ（IFNγ）等刺激信号。肿瘤微环境中表达的PD-L1可通过结合T细胞表面表达的免疫检查点受体PD-1，抑制机体的抗肿瘤免疫应答。靶向PD-1/PD-L1通路的免疫检查点抑制剂相较于标准化疗毒性更低，且在多种肿瘤中可实现持久的肿瘤退缩并改善总生存期。然而，仅有少数患者可从此类治疗中获益，且部分应答患者会出现获得性耐药。阐明PD-L1沉默对全基因表达谱的影响，对识别潜在的肿瘤免疫逃逸机制至关重要。本研究通过对PD-L1敲低细胞进行转录组分析，发现PD-L1沉默仅能下调少数免疫抑制基因的表达。本研究结果提示，筛选可调控更多免疫抑制分子的新型靶点，有望开发出更为高效的肿瘤免疫治疗方案。实验设计：将肿瘤细胞系分别转染阴性对照小干扰RNA（siRNA）或两种靶向PD-L1的特异性siRNA；转染48小时后，用干扰素-γ刺激细胞24小时。随后采用Illumina NovaSeq6000平台进行双重复RNA测序，获取RNA表达谱数据。