Epstein–Barr Virus Induces Activation-Induced Cytidine Deaminase Expression in T or NK Cells Leading to Mutagenesis and Development of Lymphoma
Authors: Arai Ayako , Horino Masato , Imadome Ken-Ichi , Imai Kohsuke , Komatsu Honami , Wang Ludan , Matsuda Go , Hamazaki Kasumi , Kurata Morito , Kurosu Tetsuya , Fujiwara Shigeyoshi , Miura Osamu 
Published: October 2019

Abstract

Introduction
Epstein–Barr virus (EBV) infects B cells and rarely T or NK cells, causing EBV-positive T/NK-cell lymphoproliferative neoplasms (EBV-T/NK-neoplasm), such as extranodal NK/T-cell lymphoma, aggressive NK-cell leukemia, and EBV-positive T- or NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs). EBV-T/NK-LPDs are fatal disorders presenting sustained inflammation, such as infectious mononucleosis-like symptoms, hypersensitivity to mosquito bites, or hydroa vacciniforme-like eruption accompanied by clonal proliferation of EBV-infected T or NK cells. EBV infects B cells making them immortal and resulting in B-cell lymphomas. However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of these EBV-induced malignancies has not been elucidated yet. Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of immunoglobulin genes. Deregulated AID expression acts as a genomic mutator leading to the development of B-cell lymphoma. In addition, EBV infection induces AID expression in B cells. These findings indicate that AID has a role in EBV-induced lymphomagenesis in B cells. However, of interest, AID transgenic mice developed T-cell lymphoma (J. Exp. Med., 197, p.1173, 2003). Recently, Nakamura et al. reported that AID expression was upregulated in the peripheral blood mononuclear cells (PBMCs) of EBV-T/NK-LPDs patients (Eur. J. Dermatol., 21, p.780, 2011). Therefore, we hypothesized that EBV infection induces AID expression in T or NK cells contributing to the tumor development.

Objectives
We designed this study to investigate AID expression and its contribution to EBV-T/NK-neoplasms development.

Materials and Methods
EBV-positive T- and NK-cell lines, SNT8, SNK6, SNT13, SNT15, and SNT16, were examined. The EBV-negative T-cell line HPB-ALL, Jurkat and human primary T cells derived from cord blood were used as the negative controls. Clinical samples were obtained from EBV-T/NK-LPDs patients who were diagnosed according to the previously described criteria (Blood, 119, p.673, 2012). To detect and isolate EBV-infected cells, T and NK cells were separated using magnetic beads from PBMCs. AID expression in tissue lesions were examined in clinical samples and xenograft models of EBV-T/NK-LPDs generated by transplantation of PBMCs from the EBV-T/NK-LPDs patients to NOD/Shi-scid, IL-2R γKO mice. For in vitro EBV infection, EBV was prepared from the culture medium of B95-8 cells and added to HPB-ALL or Jurkat cells (Proc. Natl. Acad. Sci., 100, pp.7836-40, 2003). To determine AID-induced mutation of the gene, DNA was extracted from SNT13, SNK6, or EBV-infected Jurkat cells, and c-myc gene was cloned by TA cloning, which was then sequenced. We compared the number of the mutation with that of Jurkat cells.

Results
We detected AID expression in EBV-positive T- or NK-cell lines by RT-PCR, western blotting, and immunological staining using confocal microscopy, whereas it was not detected in the control. Furthermore, we validated the results in EBV-infected T or NK cells derived from 12 EBV-T/NK-LPDs patients (infected cell types: CD4, 5; CD8, 3; and CD56, 4). Quantitative RT-PCR demonstrated that AID expression was upregulated in EBV-infected T or NK cells compared with the control. Expression was confirmed by immunological staining using confocal microscopy in 5 patients. We also detected AID expression by histopathological staining in EBV-infected cells in lesions of 4 patients and the EBV-T/NK-LPDs xenograft models. Subsequently, we examined the role of EBV for AID expression in T cells. It was demonstrated that AID expression was induced by in vitro EBV infection in the T-cell line HPB-ALL and primary T cells. Moreover, it was demonstrated by the luciferase assay that the viral protein LMP1 upregulated AID promoter activity in HPB-ALL cells. Finally, we found that SNT13, SNK6, and EBV-infected Jurkat cells had increased number of the mutation of c-myc gene compared with Jurkat cells.

Conclusions
We previously reported that EBV infection of T or NK cells enhances survival of the infected cells by the activation of NF-κB. The results of the present study suggest that EBV contributes to the development of EBV-T/NK-neoplasms through NF-κB-induced cell survival and AID-induced mutagenesis leading to clonal evolution and expansion.

Disclosures:
No relevant conflicts of interest to declare.