Epigenetic Plasticity Enables CNS-Trafficking of EBV-infected B Lymphocytes [ATAC-Seq]

Subpopulations of B-lymphocytes traffic to different sites and organs to provide diverse and tissue-specific functions. Here, we provide evidence that epigenetic differences confer a neuroinvasive phenotype. An EBV+ B cell lymphoma cell line (M14) with low frequency trafficking to the CNS was neuroadapted to generate a highly neuroinvasive B-cell population (MUN14). MUN14 B cells efficiently infiltrated the CNS within one week and produced neurological pathologies. We compared the gene expression profiles of viral and cellular genes using RNA-Seq and identified one viral (EBNA1) and several cellular gene candidates, including secreted phosphoprotein 1/osteopontin (SPP1/OPN), neuron navigator 3 (NAV3), CXCR4, and germinal center-associated signaling and motility protein (GCSAM)) that were selectively upregulated in MUN14. ATAC-Seq and ChIP-qPCR revealed that these gene expression changes correlated with epigenetic changes at gene regulatory elements. The neuroinvasive phenotype could be attenuated with a neutralizing antibody to OPN, confirming the functional role of this protein in trafficking EBV+ B cells to the CNS. These studies indicate that B-cell trafficking to the CNS can be acquired by epigenetic adaptations and provide a new model to study B-cell neuroinvasion associated CNS lymphoma and autoimmune disease of the CNS, including multiple sclerosis (MS).cell infection to investigate the underlying mechanisms that control EBV-induced B-cell immortalization. ATAC-seq revealed that over a third of accessible chromatin is altered with the most perturbed sites overlapping Ets-family, including PU.1 and RUNX1 transcription factors. EBV nuclear antigens (EBNAs) clustered with different gene categories and RNA-seq identified the transcriptional response of these gene. Focusing on EBNA1 revealed a selection of gene targets involved in nucleotide metabolism. Metabolomics indicated that adenosine and purine metabolism are significantly altered by EBV immortalization, and we validated that adenosine deaminase (ADA) is a direct and critical target of EBNA1 and the EBV-directed immortalization process. These findings reveal that purine metabolism and ADA inhibitors may be a useful therapeutic for EBV-driven lymphoid cancers ATAC-seq and RNA-seq in EBV+ B cell lymphoma cell line (M14) with low frequency trafficking to the CNS and highly neuroinvasive B-cell population (MUN14)

B淋巴细胞亚群可迁移至不同组织与器官，执行多样化的组织特异性功能。本研究证实，表观遗传差异可赋予细胞神经侵袭表型。我们以一株低频率向中枢神经系统（CNS）迁移的EB病毒（Epstein-Barr virus, EBV）阳性B细胞淋巴瘤细胞系（M14）为材料，通过神经适应手段构建出高度神经侵袭性的B细胞群体（MUN14）。MUN14 B细胞可在一周内高效浸润中枢神经系统，并引发神经病理损伤。我们通过RNA测序（RNA-Seq）对比病毒基因与细胞基因的表达谱，鉴定出在MUN14中选择性上调的1个病毒基因（EBNA1）及多个细胞基因候选靶点，包括分泌型磷蛋白1/骨桥蛋白（SPP1/OPN）、神经元导航蛋白3（NAV3）、CXCR4以及生发中心相关信号与运动蛋白（GCSAM）。转座酶可及性测序（ATAC-Seq）与染色质免疫沉淀定量PCR（ChIP-qPCR）结果显示，上述基因表达变化与基因调控元件的表观遗传改变密切相关。采用针对OPN的中和抗体可削弱该神经侵袭表型，证实了该蛋白在EBV阳性B细胞向中枢神经系统迁移过程中的功能性作用。本研究表明，B细胞向中枢神经系统的迁移可通过表观遗传适应获得，同时为研究B细胞神经侵袭相关的中枢神经系统淋巴瘤及中枢神经系统自身免疫性疾病（包括多发性硬化症（MS））提供了全新实验模型。本研究同时通过细胞感染模型，探究了调控EBV诱导B细胞永生化的潜在分子机制。ATAC测序（ATAC-Seq）结果显示，超过三分之一的可及染色质区域发生改变，其中受扰动最显著的位点与Ets家族转录因子（包括PU.1与RUNX1）的结合区域高度重合。EB病毒核抗原（EBNAs）可与不同基因类别聚类结合，RNA测序（RNA-Seq）鉴定出这些基因的转录响应谱。聚焦EBNA1的研究发现，其调控的基因靶点涉及核苷酸代谢通路。代谢组学分析显示，腺苷代谢与嘌呤代谢因EBV介导的B细胞永生化过程发生显著改变，我们验证了腺苷脱氨酶（ADA）是EBNA1及EBV诱导永生化过程的直接关键靶点。上述研究结果揭示，嘌呤代谢通路与腺苷脱氨酶抑制剂或可成为治疗EBV相关淋巴癌的潜在治疗策略。本研究同时在低频率向中枢神经系统迁移的EBV阳性B细胞淋巴瘤细胞系（M14）与高度神经侵袭性B细胞群体（MUN14）中开展了ATAC测序与RNA测序实验。