Presence of natural killer-cell clones with variable proliferative capacity in chronic active Epstein-Barr virus infection

Chronic active Epstein-Barr virus infection (CAEBV) is a syndrome that takes diverse clinical courses and is often associated with lymphoproliferative disorders of T/natural killer (NK)-cell lineage. We describe a patient with CAEBV associated with persistent pharyngeal ulcer, and with subsequent nasal T/NK-cell lymphoma in her neck lymph nodes and nasopharynx. Immunophenotyping of lymphoid cells showed that the lineage of Epstein-Barr virus (EBV)-positive cells in the patient was of NK-cell origin. By means of high-dose recombinant interleukin-2, we established an EBV-positive cell line of NK-cell lineage from her peripheral blood. Southern blot analysis for the number of terminal repeat sequences of EBV detected three NK-cell clones in the patient's lymph node. One of these clones was identical to the established cell line but was not observed in the pharyngeal ulcer, while the other two clones were present in the pharyngeal ulcer. These results suggest that the patient had expansion of the three NK-cell clones, one of which had proliferative capacity in vitro and was involved in the formation of the lymphoma. Moreover, the results suggest that the proliferative capacity of EBV-positive cells can be variable even in a single patient, and this variability may explain the clinical diversity in CAEBV.     

Adhesion of Epstein-Barr virus-positive natural killer cell lines to cultured endothelial cells stimulated with inflammatory cytokines

Chronic active Epstein-Barr virus (EBV) infection (CAEBV) is characterized by chronic recurrent infectious mononucleosis-like symptoms. Approximately one-fourth of CAEBV patients develop vascular lesions with infiltration of EBV-positive lymphoid cells. Furthermore, EBV-positive natural killer (NK)/T cell lymphomas often exhibit angiocentric or angiodestructive lesions. These suggest an affinity of EBV-positive NK/T cells to vascular components. In this study, we evaluated the expression of adhesion molecules and cytokines in EBV-positive NK lymphoma cell lines, SNK1 and SNK6, and examined the role of cytokines in the interaction between NK cell lines and endothelial cells. SNKs expressed intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) at much higher levels than those in EBV-negative T cell lines. SNKs produced the larger amount of tumour necrosis factor (TNF)-alpha, which caused increased expression of ICAM-1 and VCAM-1 in cultured human endothelial cells, than that from EBV-negative T cell lines. Furthermore, SNKs exhibited increased adhesion to cultured endothelial cells stimulated with TNF-alpha or interleukin (IL)-1beta, and the pretreatment of cytokine-stimulated endothelial cells with anti-VCAM-1-antibodies reduced cell adhesion. These indicate that the up-regulated expression of VCAM-1 on cytokine-stimulated endothelial cells would be important for the adhesion of EBV-positive NK cells and might initiate the vascular lesions.     

A probable identical Epstein-Barr virus clone-positive composite lymphoma with aggressive natural killer-cell leukemia and cytotoxic T-cell lymphoma

The patient was a 52-year old woman with a history of mosquito-bite hypersensitivity since childhood. In July 2011, she developed pyrexia, headaches, and nausea, and Epstein-Barr virus (EBV)-positive aggressive natural killer leukemia (ANKL) was diagnosed on the basis of both a peripheral blood and bone marrow examination. An inguinal lymph node biopsy, on the other hand, revealed EBV-positive cytotoxic T-cell lymphoma plus the presence of a small number of EBV-positive ANKL cells, and a diagnosis of EBV-positive composite lymphoma was made. Both the cytotoxic T-cell lymphoma and ANKL exhibited EBV terminal repeat (Southern blot analysis) monoclonal patterns, and they were almost the same size, approximately 9.0 kb. If it was the identical EBV clone, it is possible that EBV infected progenitor cells common to both NK cells and T cells, that the progenitor cells then differentiated into NK cells and T cells, a chronic active Epstein-Barr virus infection developed, and neoplastic transformation occurred. If it was not the identical EBV clone, fairly similar EBVs must have infected NK cells and T cells separately, and they then underwent neoplastic transformation. Because the mechanism by which EBV infects NK cells or T cells is still unknown, we concluded that this case is also important from the standpoint of elucidating it. We are currently in the process of conducting gene analyses to determine whether the fairly similar EBVs that infected the ANKL and cytotoxic T-cell lymphoma are the identical clone.     

T/NK cell type chronic active Epstein-Barr virus disease in adults: an underlying condition for Epstein-Barr virus-associated T/NK-cell lymphoma

A chronic infectious mononucleosis-like illness caused by Epstein-Barr virus (EBV) is called 'chronic active EBV disease', which is defined as an EBV-associated lymphoproliferative disease. This lymphoproliferative disease is rare and predominantly occurs in Japanese children. Between 1998 and 2010, seven adult-onset cases (aged 20-45 years, median 39 years) were identified, which initially presented with inflammatory diseases, including hepatitis, interstitial pneumonitis, uveitis, nephritis and hypersensitivity to mosquito bites. They showed an EBV viral load in the peripheral blood and evidence of EBV infection of T or natural killer (NK) cells. Five cases (71.4%) developed EBV-positive T/NK-cell lymphoma/leukaemia at a median of 5 years (range 1-7 years) after the diagnosis. Although l-asparaginase-containing chemotherapy was effective for the lymphomas, only allogeneic haematopoietic cell transplantation eradicated EBV-infected cells. This observation indicates that persistent EBV infection of T or NK cells defines a distinct disease entity, which provides an underlying condition for EBV-positive T/NK-cell lymphoma/leukaemia.     

A Case of Hypersensitivity to Mosquito Bite Associated with Epstein-Barr Viral Infection and Natural Killer Cell Lymphocytosis

Hypersensitivity to mosquito bites (HMB) is a disorder characterized by a necrotic skin reaction and generalized symptoms subsequent to mosquito bites. It has been suggested that HMB is associated with chronic Epstein-Barr virus (EBV) infection and natural killer cell leukemia/lymphoma. We describe here a Korean child who had HMB associated with chronic EBV infection and natural killer cell lymphocytosis. A 5-yr-old boy was suffered from necrotic skin lesions on the right ear lobe. Type A EB virus was detected from hlood cells and bone marrow biospy recognized hemophagocyrosis.     

Epstein-Barr virus-infected malignant T/NK-cell lymphoma in a patient with hypersensitivity to mosquito bites

We report an autopsy case of Epstein-Barr virus (EBV)-infected malignant lymphoma in a young male who had hypersensitivity to mosquito bites. The autopsy revealed multiple confluent lymphoma lesions in the lungs, and on the right leg irregular-shaped skin ulcers were seen. The left pleural effusion also contained a large number of lymphoma cells. The lymphoma cells were determined as T/NK-cell type cells by immunohistochemistry. EBV DNA was detected most intensively in the lungs and EBV-encoded small RNAs-positive lymphoma cells were also observed in the lungs at a high frequency. EBV latent membrane protein-1 expression and a high Ki-67 labeling indices were noted in the lymphoma cells of the lung lesions. These findings indicate that the development of the malignant lymphoma was associated with the proliferation of EBV-infected lymphoma cells, and the cells that infiltrated the whole the body, especially the lungs, caused the patient's death.     

Preferential expansion of Vgamma9-JgammaP/Vdelta2-Jdelta3 gammadelta T cells in nasal T-cell lymphoma and chronic active Epstein-Barr virus infection

We recently established an Epstein-Barr virus (EBV)-positive gammadelta T-cell line from a nasal T/natural killer (NK)-cell lymphoma (Nagata H, Konno A, Kimura N, Zhang Y, Kimura M, Demachi A, Sekine T, Yamamoto K, Shimizu N: Characterization of novel natural killer (NK)-cell and gammadelta T-cell lines established from primary lesions of nasal T/NK-cell lymphomas associated with the Epstein-Barr virus. Blood 2001, 97:708-713). Subsequently, we established two novel EBV-positive gammadelta T-cell lines from the peripheral blood of patients with chronic active EBV infection. Analysis of the terminal repeat of EBV showed that the three cell lines consisted of monoclonal populations, and flow cytometry showed that they had a common phenotype of gammadelta T cells: CD3(+) CD4(-) CD8(-) CD16(-) CD19(-) CD56(+) CD57(-) HLA-DR(+) T-cell receptor (TCR) alphabeta(-) TCR gammadelta(+). Analysis for the expression of TCR by flow cytometry showed that all three cell lines were Vgamma9(+)/Vdelta2(+), but negative for VgammaI, Vdelta1, or Vdelta3 TCR. Southern blot analysis for TCR genes showed that the three cell lines had a common rearrangement of Vgamma9-JgammaP and Jdelta3 genes. Polymerase chain reaction and sequence analysis of the junction between Vdelta and Jdelta genes revealed that the Jdelta3 genes were rearranged with the Vdelta2 genes. In contrast, none of the EBV-negative gammadelta T-cell lines, Molt-14, Peer, or Loucy, which were analyzed for controls, had Vgamma9 or Vdelta2 TCR, or a rearrangement of Jdelta3 genes. These results indicated that Vgamma9-JgammaP/Vdelta2-Jdelta3(+) gammadelta T cells were preferentially affected by EBV and expanded in patients with nasal gammadelta T-cell lymphoma and chronic active EBV infection. Jdelta3(+) gammadelta T cells are known to be a very minor population in gammadelta T cells of peripheral blood, whereas Vgamma9-JgammaP/Vdelta2-Jdelta1(+) cells are the major population. The close association of EBV with this particular gammadelta T-cell population may provide a key to the etiology of EBV-positive lymphoproliferative diseases.     

Rapid determination of Epstein-Barr virus latent or lytic infection in single human cells using in situ hybridization.

Epstein-Barr (EBV) virus is associated with malignancies such as lymphoma and carcinoma. Infection of cells with EBV may result in either lytic infection with production of viral particles, characterized by the presence of linear DNA forms, or latent infection, characterized by either episomal or integrated DNA forms. To examine whether the different lytic and latent EBV DNA forms can reliably be distinguished in single human cells, in situ hybridization was performed in EBV-positive cell lines. Immunocytochemistry and Southern blot analysis were performed supplementary to in situ hybridization. In latent infection, three in situ hybridization patterns were observed: large-disperse (episomal), small-punctate (integrated) and combined (both), signal types 1, 2 and 3 respectively. These were associated with expression of latent membrane protein 1, but not with Z fragment of Epstein-Barr replication activator or viral capsid antigen. In lytic infection, three additional in situ hybridization patterns were observed: nuclear membrane associated, bubble (filling up the nucleus) and spillover (covering the lysed cells) signals types 4, 5 and 6 respectively. Signal types 4 and 5 were associated with expression of latent membrane protein 1 and Z fragment of Epstein-Barr replication activator but not viral capsid antigen, whereas type 6 was associated with expression of viral capsid antigen only. Southern blot analysis confirmed these results; however, low copy numbers of integrated virus were often missed by Southern blot, confirming that in situ hybridization is more sensitive in determining the presence of all types of EBV DNA. In situ hybridization may prove useful in rapidly screening large series of tissue microarrays and other clinical specimens for the presence of lytic or latent EBV.     

Inconsistent association of Epstein-Barr virus with CD56 (NCAM)-positive angiocentric lymphoma occuring in sites other than the upper and lower respiratory tract

We previously described nine cases of angiocentric lymphoma of a possible natural killer (NK)-cell lineage with a surface CD3− CD56+ phenotype occurring in sites other than the upper and lower respiratory tract. This study was performed to investigate the association of Epstein-Barr virus (EBV) with these lymphomas, using the polymerase chain reaction (PCR) for the presence of EBV-DNA, in situ hybridization (ISH) for EBV-encoded small RNAs (EBERs) and immunohistology for EBV-determined nuclear antigen-2 (EBNA-2) and latent membrane protein-1 (LMP-1) in paraffin sections. PCR and ISH produced almost identical results, and EBERs were identified in the nuclei of the lymphoma cells of three cases, two of which exhibited LMP-1 in the cytoplasm of tumour cells without EBNA-2 expression. Molecular genetic analysis revealed EBV to be incorporated into these three EBER-positive cases either clonally or biclonally. It was revealed by re-evaluation of their morphology with the established EBV status on each case that, in contrast to the rather variable and irregular cellular composition of the EBV- positive tumours, the EBV-negative tumours stood out because of their remarkably uniform 'blastoid' appearance, and could be grouped as blastic NK-cell lymphoma. The relationship of the EBV-positive cases with nasal NK-cell tumours has yet to be clarified.     

Aggressive natural killer cell lymphoma presenting as an anterior mediastinal mass in a patient with acquired immunodeficiency syndrome

We report a case of aggressive natural killer cell lymphoma presenting as an anterior mediastinal mass in an African-American man with acquired immunodeficiency syndrome. Histologically, the anterior mediastinal mass showed a diffuse dense infiltrate of atypical intermediate-sized and large lymphoid cells, as well as scattered immunoblasts with angiocentric and angiodestructive growth and extensive zonal necrosis. Similar lymphoid infiltrates were present in the patient's lungs, spleen, and bone marrow, accompanied by extensive lymphophagocytosis. Electron microscopic and cytologic examinations showed the presence of dense cytoplasmic granules. Immunophenotyping by flow cytometry and by immunohistochemistry yielded surface markers consistent with a natural killer cell lymphoma. The Epstein-Barr virus genome and monoclonality were detected by in situ hybridization and Southern blot analysis. Polymerase chain reaction confirmed the presence of type A Epstein-Barr virus. T-cell receptor gene rearrangement could not be identified by Southern blot analysis or polymerase chain reaction. To the best of our knowledge, this is the first reported case of designated natural killer cell lymphoma from the mediastinum, as well as the first reported case of natural killer cell lymphoma in a patient with acquired immunodeficiency syndrome. This tumor disseminated early and pursued a highly aggressive course. Epstein-Barr virus may play a role in the pathogenesis of this disease.     

Epstein-Barr virus in B-cell lymphomas associated with chronic suppurative inflamation

Chronic antigenic stimulation is considered to play an important role in neoplastic lymphoid transformation. This paper describes three cases of non-Hodgkin lymphomas (NHLs) associated with long-standing chronic suppuration. Two were primary bone lymphomas associated with long-standing chronic osteomyelitis and one was a primary skin lymphoma associated with chronic venous ulcers with a latent period of 13 years. All were diffuse large B-cell lymphomas, with plasmacytoid differentiation in two cases. Epstein–Barr virus (EBV)-encoded RNAs were demonstrated in virtually all tumour cells in all cases by in situ hybridization. Immunohistochemistry revealed EBV-encoded latent membrane protein (LMP)-1 expression in one case and BZLF1 protein expression in all three cases, whilst the EBV-encoded nuclear antigen (EBNA)-2 was not detected. Kaposi's sarcoma-associated herpesvirus (KSHV) sequences were absent in two cases studied. Our cases show similarities with pyothorax-associated pleural lymphomas reported mainly from Japan and recently from France, which are invariably associated with EBV. These findings suggest a causal effect of EBV in the development of this type of lymphoma complicating long-standing chronic suppuration. Localized immunodepression induced by chronic inflammation or immunosuppressive cytokines may favour the clonal proliferation of EBV-infected B cells. © 1997 John Wiley & Sons, Ltd.     

Heterogeneity of Epstein-Barr virus infection in angioimmunoblastic lymphadenopathy type T-cell lymphoma

To investigate the relationship of Epstein-Barr virus (EBV) and angioimmunoblastic lymphadenopathy with dysproteinemia, we performed DNA analysis using the polymerase chain reaction (PCR), Southern blot, in situ hybridization, and immunohistochemical analysis of lymph nodes in five patients who were followed up and biopsied more than once. In the course of the disease, nodal architecture diminished, cellular atypia worsened, and clear cells increased in number. In the DNA analysis of the receptor genes, the clonal population increased in number. EBV nucleic acid sequences were found by either PCR or in situ hybridization in all examined nodes. The number of EBV-positive cells varied widely among the cases and throughout the course of the disease in the same patients. The analysis of EBV terminal repeats or lymphocyte-determined membrane antigen genes showed polyclonal populations of EB-infected cells. EBV-positive cells possessed intermediate- to large-sized nuclei, and the cells with large nuclei, especially, expressed latent membrane protein of EBV. These large cells varied in number among the cases. Double-labelling immunohistochemistry/in situ hybridization studies demonstrated that most of the EBV-positive cells expressed B-cell antigen (CD20). The presence of EBV seems to be associated with the selective defects of the immune system, rather than with the direct pathogenesis of angioimmunoblastic lymphadenopathy.     

Human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) antigens and genome in lymph nodes of HIV-positive patients affected by persistent generalized lymphadenopathy (PGL)

The presence of human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) antigens and genome has been investigated in 50 lymph nodes involved by persistent generalized lymphadenopathy (PGL). All the patients were HIV infected and most of them (42 of 50) also had anti-EBV serum antibodies. At lymph node level, HIV and EBV antigens were studied by immunohistochemistry using monoclonal antibodies directed against viral core proteins. The HIV p24 protein was detected in 43 of 50 lymph nodes within the B-cell germinal centers with a reticular pattern. Few cells with positive results for EBV antigens were found in only 2 of 50 lymph nodes. These rare EBV-positive centrocyte-like cells were mainly located in the germinal centers. The presence of HIV and EBV genome was also studied in lymph nodes involved by PGL, with the use of in situ and Southern blot hybridization. A positive reaction for HIV genome was detected in only 1 of 14 lymph nodes with the Southern blot hybridization, and the presence of EBV genome was never demonstrated in these lymph nodes with the use of both in situ and Southern blot hybridization. The expression of EBV antigens and genome was also investigated in the peripheral blood of 15 patients with PGL in which cells with positive results for EBV antigens were detected in a single case with a frequency of 1 X 10(-4). No evidence of EBV genome was found with the use of the in situ hybridization. These results suggest that EBV is not present in lymph nodes during the PGL phase and that its possible implication in the pathogenesis of acquired immune deficiency syndrome (AIDS)-associated lymphoma might be a late event.     

Frequent presence of latent Epstein-Barr virus infection in lymphoepithelioid cell lymphoma (Lennert's lymphoma)

This study deals with the investigation of the biological significance of an Epstein–Barr virus (EBV) infection in lymphoepithelioid cell lymphoma. A selection of EBV-detection techniques was applied to 15 cases, including polymerase chain reaction (PCR) for the detection of EBV-DNA, in situ hybridization (ISH) for the cellular localization of EBV-encoded small nuclear (EBER 1 and EBER 2) and immediate-early (BHLF) RNAs, and immunohistology for the detection of EBV-encoded latent membrane protein (LMP) expression. PCR and EBER-ISH produced congruent results in those cases with amplifiable DNA, leading to an EBV presence in 11/15 lymphoepithelioid cell lymphoma cases (73%). EBER-ISH combined with immunohistology localized the virus predominantly in several B immunoblasts and small B lymphocytes in eight of the EBV-positive cases, five of which also contained single infected lymphocytes expressing T-cell characteristic antigens. LMP was detected using immunohistology in only a proportion of immunoblasts in four of these cases. The remaining three EBV-positive lymphoepithelioid cell lymphoma cases contained only single EBER-positive small B lymphocytes without LMP expression. No case contained BHLF-RNA expressing cells. These data imply that, although latently EBV-infected cells are frequently present in lymphoepithelioid cell lymphoma cases, the virus is probably not directly involved in the pathogenesis of this entity.     

Epstein-Barr virus (EBV) latent membrane protein-1-specific cytotoxic T lymphocytes targeting EBV-carrying natural killer cell malignancies

Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP) 1 is a potential target for immunotherapy of some proportion of Hodgkin's disease cases, nasopharyngeal carcinomas, EBV-associated natural killer (NK)/T lymphomas, and chronic active EBV infection (CAEBV). Since it is unknown whether EBV-infected NK/T cells are susceptible to lysis by LMP1-specific cytotoxic T lymphohcytes (CTL), we here tested the ability of mRNA-transduced antigen-presenting cells (APC) to stimulate rare LMP1-specific CTL. A 43-amino acid N-terminal deletion mutant LMP1 (DeltaLMP1) could be efficiently expressed in dendritic cells and CD40-activated B cells upon mRNA electroporation. DeltaLMP1-expressing APC were found to stimulate LMP1-specific CTL from a healthy donor and a CTL clone recognized a peptide, IIIILIIFI, presented by HLA-A*0206 molecules. Processing and presentation of the antigenic peptide proved dependent on expression of an immunoproteasome subunit, low-molecular-weight protein-7, as confirmed by RNA interference gene silencing. Furthermore, an EBV-infected NK cell line derived from a patient with CAEBV, and another from an NK lymphoma with enforced HLA-A*0206 expression, were specifically lysed by the CTL. Overall, these data suggest that immunotherapy targeting LMP1 in EBV-associated NK lymphomas and CAEBV might serve as an alternative treatment modality.     

Cutaneous EBV-related lymphoproliferative disorders

This article will focus on the cutaneous lymphoproliferative disorders associated with EBV, with an emphasis on the upcoming changes in the revised 4th Edition of the WHO classification of tumors of the hematopoietic system, many of which deal with cutaneous disorders derived from NK-cells or T-cells. Extranodal NK/T-cell lymphoma usually presents in the upper aerodigestive tract, but can involve the skin secondarily. EBV-associated T- and NK-cell lymphoproliferative disorders (LPD) in the pediatric age group include the systemic diseases, chronic active EBV infection (CAEBV) and systemic EBV+ T-cell lymphoma of childhood. Hydroa vacciniforme (HV)-like LPD is a primarily cutaneous form of CAEBV and encompasses the lesions previously referred to as HV and HV-like lymphoma (HVLL). All the T/NK-cell-EBV-associated diseases occur with higher frequency in Asians, and indigenous populations from Central and South America and Mexico. Among the B-cell EBV-associated LPD two major changes have been introduced in the WHO. The previously designated EBV-positive diffuse large B-cell lymphoma (EBV-DLBCL) of the elderly, has been changed to EBV-DLBCL with ‘not otherwise specified’ as a modifier (NOS). A new addition to the WHO system is the more recently identified EBV+ mucocutaneous ulcer, which involves skin and mucosal-associated sites.     

Activation of the lytic program of the Epstein-Barr virus in Burkitt's lymphoma cells leads to a two steps downregulation of expression of the proapoptotic protein BimEL, one of which is EBV-late-gene expression dependent

The Epstein-Barr virus (EBV) generally latently infects its target cells with expression of genes conferring resistance to apoptosis. However, the modulation of apoptotic signals during lytic cycle remains poorly understood. We show here that resulting from viral reactivation in the EBV-positive Mutu-I and Akata Burkitt's lymphoma cell lines, a two steps proteasome-dependent downregulation of expression of the proapoptotic protein BimEL occurs. The first drop might be EBV-independent, is ERK 1/2 dependent, and BimEL is phosphorylated on Ser69. A second dramatic drop of the BimEL level observed during the lytic cycle is dependent of EBV-late-gene expression, ERK 1/2 independent, and no further phosphorylation of BimEL on Ser69 occurred. These results demonstrate for the first time, that the lytic cycle contributes to downregulation of BimEL and then could add to protection against apoptosis.