Transcriptomic profiling of peripheral blood NK cells of chronic HBV, HCV and HIV patients. Transcriptomic profiling of peripheral blood NK cells of chronic HBV, HCV and HIV patients

Background: NK cells during chronic viral infection have been well studied over the last decade. We performed an unbiased next-generation RNA-sequencing approach to identify commonalities or differences of the effect of HIV, HCV and HBV viremia on NK cell transcriptomes. Methods: Using cell sorting, we obtained CD3-CD56+ NK cells from blood of 6 HIV, 11 HCV, and 32 HBV infected and untreated patients. Library preparation and sequencing were done using Illumina mRNA-Seq Sample Prep Kit and the HiSeq 2000, HiSeq2500 or NextSeq 500, and further analysis by an in-house analytic pipeline. Results: In NK cells from HIV, HCV and HBV patients, transcriptome analysis identified 272, 53, and 56 differentially expressed genes, respectively (fold change >1.5, q-value 0.2). Interferon stimulated genes were induced in NK cells from HIV/HCV patients, but not during HBV infection. HIV viremia downregulated ribosome assembly genes in NK cells. In HBV, viral load and ALT variation had little effect on genes related to NK effector function. Conclusion: We compare, for the first time, NK cell transcripts of viremic HIV, HCV and HBV patients. We clearly demonstrate distinctive NK cell gene signatures in 3 different populations, suggestive for a different degree of functional alterations of the NK cell compartment as compared to healthy individuals. Overall design: We analyzed NK cell transcripts collected from the blood of well-characterized chronic HBV patients (n=32), chronic HCV patients (n=8), and HIV patients (n=6). Differential gene expression analysis, global module analysis, and unsupervised clustering analysis were performed by employing RNA-sequencing on blood NK cell transcriptomes.

研究背景：过去十年来，慢性病毒感染过程中的自然杀伤细胞（Natural Killer Cell, NK）已得到充分研究。本研究采用无偏倚下一代RNA测序（next-generation RNA sequencing）方法，旨在明确人类免疫缺陷病毒（Human Immunodeficiency Virus, HIV）、丙型肝炎病毒（Hepatitis C Virus, HCV）及乙型肝炎病毒（Hepatitis B Virus, HBV）病毒血症对NK细胞转录组的影响的共性与差异。
研究方法：本研究通过细胞分选技术，从6例未经治疗的HIV感染者、11例未经治疗的HCV感染者及32例未经治疗的HBV感染者的外周血中分离得到CD3⁻CD56⁺ NK细胞。采用Illumina mRNA测序文库制备试剂盒（Illumina mRNA-Seq Sample Prep Kit）及HiSeq 2000、HiSeq 2500或NextSeq 500测序平台完成文库构建与测序，并通过自研分析流程开展后续数据分析。
研究结果：对HIV、HCV及HBV感染者的NK细胞进行转录组分析，分别鉴定出272、53及56个差异表达基因（differentially expressed genes, DEGs，折叠变化>1.5，q值=0.2）。HIV/HCV感染者的NK细胞中可检测到干扰素刺激基因（interferon stimulated genes, ISGs）的诱导表达，而HBV感染者未出现该现象。HIV病毒血症可下调NK细胞中的核糖体组装基因表达。在HBV感染者中，病毒载量与丙氨酸氨基转移酶（Alanine Aminotransferase, ALT）水平变化对NK细胞效应功能相关基因的表达影响微弱。
研究结论：本研究首次对病毒血症状态下的HIV、HCV及HBV感染者的NK细胞转录本进行了比较分析。本研究明确证实三类感染者的NK细胞具有独特的基因特征谱，提示与健康个体相比，这三类人群的NK细胞群体功能改变程度存在差异。
总体实验设计：本研究分析了32例表型充分表征的慢性HBV感染者、8例慢性HCV感染者及6例HIV感染者外周血中分离得到的NK细胞转录本。通过对外周血NK细胞转录组进行RNA测序，开展了差异基因表达分析、全局模块分析及无监督聚类分析。