Epstein–Barr Virus Induces Activation-Induced Cytidine Deaminase Expression in T or NK Cells Leading to Mutagenesis and Development of Lymphoma

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.

APOBEC‐mediated DNA alterations: A possible new mechanism of carcinogenesis in EBV‐positive gastric cancer

“…28 In these cells, EBV-infection acts as a somatic mutator, leading to the activation of AID, an enzyme that mediates class switch recombination of immunoglobulin genes and induces cytosine deamination and somatic hypermutation. 29 These findings indicate that AID has a role in EBV-induced lymphomagenesis in B cells.…”

“…The mechanisms of tumorigenesis so far described for EBV‐induced tumors include the interference of EBV with cell‐cycle checkpoints (G1 as well as G2/M transitions and the assembly of the mitotic spindle), the inactivation of cell death pathways, when immortalized EBV‐infected B‐cells proliferate in a central neoplastic mechanism of B‐cell lymphomas as well as the genome‐wide reactivation of enhancers and promoters located near cryptic and otherwise repressed long terminal repeats of inactive human endogenous retroviruses . In these cells, EBV‐infection acts as a somatic mutator, leading to the activation of AID, an enzyme that mediates class switch recombination of immunoglobulin genes and induces cytosine deamination and somatic hypermutation . These findings indicate that AID has a role in EBV‐induced lymphomagenesis in B cells.…”

Evidence of lesions from Epstein-Barr virus infection in human breast cancer genomes

“…5C ). These deletions are typically caused by AID [50], which EBV induces prior to lymphomas [51, 52]. The deletions ( Fig 5C ) mainly target transcript variants of CTNNB1, which then deregulate WNT/CTNNB1/TCFL2/PDHB signaling, causing the Warburg effect.…”

APOBEC-mediated DNA alterations: a possible new mechanism of carcinogenesis in EBV-positive gastric cancer

“…The mechanisms of tumorigenesis so far described for EBV-induced tumors include the interference of EBV with cell-cycle checkpoints (G1 as well as G2/M transitions and the assembly of the mitotic spindle), the inactivation of cell death pathways, when immortalized EBV-infected B-cells proliferate in a central neoplastic mechanism of B-cell lymphomas [15] as well as the genome-wide reactivation of enhancers and promoters located near cryptic and otherwise repressed long terminal repeats of inactive human endogenous retroviruses [16]. In these cells, EBV-infection acts as a somatic mutator, leading to the activation of AID, an enzyme that mediates class switch recombination of immunoglobulin genes and induces cytosine deamination and somatic hypermutation [17]. These findings indicate that AID has a role in EBV-induced lymphomagenesis in B cells.…”