Interferon receptor signaling pathways regulating PD-L1 and PD-L2 expression. Homo sapiens

We studied the molecules involved in downstream signaling induced by interferons to regulate PD- L1 and PD-L2 expression using a shRNA screen, Western blot, mRNAexpression profiling, promoter truncation analysis and chromatin immunoprecipitation.These studies revealed that the interferon gamma-JAK1/2-STAT1-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter. PD-L2 responded equally to interferon beta and gamma, and shared regulation through IRF1 and alsoSTAT3, which bind to the PD-L2 promoter. Analysis of biopsies from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/2/3 and IRF1 in anti-PD-1 responding tumors. Therefore, these studies map the signaling pathway of interferon-inducible PD-1 ligand expression Overall design: Melanoma biopsies pre and on- anti-PD-1 treatment were sent for transcriptomic analysis by paired end RNAseq analysis

本研究通过短发卡RNA（short hairpin RNA, shRNA）筛选、蛋白质印迹法（Western Blot）、mRNA表达谱分析、启动子截短分析及染色质免疫共沉淀（Chromatin Immunoprecipitation）技术，探究了干扰素诱导的下游信号通路中参与调控PD-L1（programmed death-ligand 1）与PD-L2（programmed death-ligand 2）表达的分子机制。研究结果显示，干扰素γ（Interferon γ, IFN-γ）-JAK1/2-STAT1（Signal Transducer and Activator of Transcription 1）-IRF1（Interferon Regulatory Factor 1）信号轴主要调控PD-L1的表达，IRF1可结合PD-L1的启动子区域。PD-L2则可同时响应干扰素β（Interferon β, IFN-β）与干扰素γ，通过IRF1与STAT3（Signal Transducer and Activator of Transcription 3）共同介导其表达调控，二者均可结合PD-L2的启动子区域。对黑色素瘤（Melanoma）患者的活检组织进行分析证实，在对抗PD-1治疗产生应答的肿瘤中，干扰素特征基因集显著富集，且STAT1/2/3与IRF1的靶基因均呈现上调表达。综上，本研究明确了干扰素诱导型PD-1配体表达的信号通路调控网络。实验整体设计：收集接受抗PD-1治疗前及治疗期间的黑色素瘤活检组织，通过双端RNA测序（Paired-end RNA Sequencing）进行转录组分析。