Expression Profile of EBV-Derived Micro-RNA in Systemic Chronic Active EBV Disease

Background and aim: Systemic chronic active EBV disease (sCAEBV), formerly named as chronic active EBV infection, is a rare and chemotherapy-resistant lymphoid neoplasm. EBV commonly infects B cells latently. In sCAEBV, however, EBV persistently infects T or NK cells leading to their clonal proliferation. It has been postulated that host immune deficiency may contribute to the establishment of infection of T and NK cells, but the details have not been clarified. EBV originally encodes 44 miRNAs: 4 miR-BHRFs and 40 miR-BARTs. Among them, miR-BARTs are expressed in EBV-positive tumors of all latent-infection patterns and have been reported to be involved in cell proliferation and maintenance of latent infection. In this study, we investigated the pathophysiology of sCAEBV focusing on these EBV-derived miRNAs. We studied the expression of EBV-derived miRNAs and their involvement in pathogenesis using sCAEBV patient samples for the first time in the world.

Methods: We used two EBV-positive T- (SNT15 and SNT16) and two NK- (SNK1 and SNK10) cell lines established from sCAEBV patients. sCAEBV was diagnosed according to the criteria which match the definition of the WHO classification of hematopoietic malignancies (Yonese et al, Blood advances, 2020). EBV-infected cells were isolated and obtained for assay from PBMCs of the patients as described in a previously published manuscript (Yoshimori et al, Blood advances, 2021). The expression of miR-BARTs was examined by RNA-sequencing using HiSeq2000® and by TaqMan® miRNA Assays. miRNA expression of the histopathological specimen was examined using miRNAscope™ in situ hybridization assay. The mutation of region encoding miR-BARTs was examined by NovaSeq6000® aligning to KC207813.1. The results were confirmed by Sanger sequencing. Knockdown assay of miR-BART7-3p was performed by the transfection of mirVana™ miRNA Inhibitor using Lonza Nucleofector® 2b. Gene expression was examined by RNA-sequencing using NovaSeq6000® and conducted Gene Ontology (GO) analysis using DAVID.

Results: We identified highly abundant miR-BARTs in the 4 cell lines and the EBV-infected T- or NK-cells from 12 sCAEBV patient's PBMCs (age of 17 to 47 y.o). The expression of miR-BHRFs was not detected in these cells. miR-BART7-3p, miR-BART6-3p, and miR-BART5-5p were the top three expressed among the EBV-derived miRNAs. The highest miR-BART expression among all samples was miR-BART7-3p. We also confirmed the expression of miR-BART7-3p and miR-BART5-5p by in situ hybridization in histological specimens of 3 patients which were observable. Two reports have demonstrated deletion in part of BART region in approximately 30% of sCAEBV cases (Okuno et al, Nature Microbiology, 2018. Wongwiwat et al, J. Virology, 2022). In contrast, no deletion was detected in the region encoding miR-BARTs of EBV obtained from 10 sCAEBV patients. Finally, we examined the role of miR-BART7-3p in sCAEBV. Inhibition of miR-BART7-3p by the inhibitor did not show significant effects on cell proliferation in SNT16 and SNK10 cells. However, GO analysis showed upregulation of immune activation-related genes after miR-BART7-3p inhibition. These results suggest that miR-BART7-3p may function as an immunosuppressor in sCAEBV.

Conclusion: We assume that miR-BART7-3p, a EBV-derived miRNA, plays a role in pathogenesis of sCAEBV by taking part in EBV persistent infection of T or NK cells.

Yoshimori:Nippon Shinyaku Co., Ltd.: Research Funding. Arai:AbbVie GK: Honoraria; Bristol-Myers Squibb K.K.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Eisai Co., Ltd.: Research Funding; Abbott Japan LLC: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Ono Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Nippon Shinyaku Co., Ltd.: Honoraria, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Shionogi & Co., Ltd.: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Sanofi K.K.: Honoraria; Pfizer Japan Inc.: Honoraria; Astellas Pharma Inc.: Honoraria.