CD95 (Fas) ligand expression of Epstein-Barr virus (EBV)-infected lymphocytes: a possible mechanism of immune evasion in chronic active EBV infection

The Epstein-Barr virus (EBV) induces infectious mononucleosis (IM) and can be associated with chronic active EBV infection (CAEBV). Cytotoxic T lymphocytes (CTL) play an important role in excluding EBV-infected cells. Two cytotoxic mechanisms of CTL have been demonstrated: one perforin/granzyme-based and the other Fas (CD95)/Fas ligand (FasL)-based. To clarify these two pathways in CAEBV, we analyzed six patients with CAEBV and four patients with IM using immunohistochemical staining of the lymph nodes. In both CAEBV and IM, CD8⁺ T-cells increased in number, but CD56⁺ natural killer cells were rare. In four of six cases with CAEBV, approximately half the lymphocytes were positive for T cell-restricted intracellular antigens (TIA-1), which were recognized by the cytolytic granules of CTL. In IM, the number of TIA-1 positive cells was smaller than that in CAEBV. Fas-positive lymphocytes were frequently encountered in both CAEBV and IM. However, FasL-positive lymphocytes increased in three of six patients with CAEBV, but not in patients with IM. Except for one case with CAEBV, the number of perforin- and/or granzyme-positive cells was small in number in both CAEBV and IM cases. In double-staining FasL and EBV in situ hybridization, FasL-positive EBV-infected lymphocytes were detected in CAEBV but not in IM. In CAEBV, the Fas/FasL pathway and not perforin pathways appears to play an important role in the pathogenesis. The data suggest that EBV-infected lymphocytes may evade immune attack through the expression of FasL.     

Budd-Chiari syndrome and Epstein-Barr virus (EBV) associated plasmacytoma in a patient with chronic active EBV infection

A 42-year-old Japanese man with chronic active Epstein-Barr virus (EBV) infection initially responded to treatment with interleukin-2 (IL-2). Six months later he developed thrombosis in the hepatic veins, and Budd-Chiari syndrome associated with severe hepatic damage was diagnosed. He also developed a solitary EBV-positive plasmacytoma in the right femur. Since these rare complications occurred after long-term IL-2 therapy, the possibility that long-term IL-2 therapy might cause Budd-Chiari syndrome and liver damage as well as EBV-associated plasmacytoma is discussed.Abbreviations EBV Epstein-Barr virus - EBER1 EpsteinBarr virus-encoded RNA-1 - IL-2 interleukin-2 - NK natural killer - LMP latent membrane protein - LAK lymphokine activated killer     

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.     

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.     

Malignant lymphoma induction of rabbits with oral spray of Epstein-Barr virus-related herpesvirus from Si-IIA cells (HTLV-II-transformed Cynomolgus cell line): A possible animal model for Epstein-Barr virus infection and subsequent virus-related tumors in humans

Malignant lymphoma (ML) was Induced in eight of nine rabbb inoculated by oral spray of the cell-free pellets from SI-IIA culture (MLV-ll-transformed leukocyte cell line of the Cynomolgus-producing Epsteln-Barr virus (EBV)-related herpesvirus) after 64–141 days. None of the rabbits inoculated with EBV from B-95–8 cells or HTLV-II from MOT cells developed ML. Malignant lymphomas were usually of diffuse, large-cell or mixed type. HTLV-II infection was excluded by the polymerase chain reaction (PCR) and the particle agglutination test. EBV-encoded RNA-1 and EBV-related DNA were detected in the tumor tissues by In situ hybridization and PCR, respectively. Anti-viral capsid antigen of EBV antibody (anti-VCA) was observed 3 weeks after oral inoculation of Si-IIA cell-free pellets. Polymerase chain reaction revealed continuous detection of EBV-related virus DNA in the peripheral blood leukocytes from 3 days after oral inoculation. These results show that ML induced orally wtth SI-IIA cell-free pellets was caused by EBV-related herpesvirus harbored by Si-IIA cells. Oral spray of EBV from B-95–8 also induced EBV Infection in rabbits, which was confirmed both by the presence of anti-VCA and by PCR. These oral infection and mallgnant lymphoma induction systems of rabbit using EBV-related virus from Si-IIA or human EBV are useful animal models for the study of EBV infection and EBV-related lymphomas in humans.     

Brief report: killer cell defect and persistent immunological abnormalities in two patients with chronic active Epstein-Barr virus infection

Two members of a family have manifested a syndrome of chronic active Epstein-Barr virus (EBV) infection. A father and his daughter suffered prolonged or recurrent mononucleosis, with splenomegaly, anemia, and intermittent fever; persistent immunological abnormalities included defective natural killer (NK) cytotoxicity, inverted CD4/CD8 ratios, hyper IgG1, high EBV viral capsid antigen (VCA) and early antigen (EA) antibodies, and low or undetectable EBV nuclear antigen (EBNA) antibody titers. The EBV seronegative member of the family was free of these abnormalities. However, NK activity in the seronegative individual was low-normal and its EBV-specific antibody-dependent K-cell cytotoxicity (EBV-ADCC) was abnormally low, suggesting that this K-NK cell defect may be primary. The father, who suffered from the syndrome for more than 15 years, lacked (or lost) antibodies to EBV-envelope and infected cell membranes, such as antibody-dependent cellular cytotoxicity (ADCC), neutralizing (NT), and gp 350/220 antibodies. Slow improvement over a period of years was heralded by rising NK cytotoxicity.     

Epstein-Barr virus in nasal T-cell lymphomas (polymorphic reticulosis/midline malignant reticulosis) in Western China

Polymorphic reticulosis (PR) or midline malignant reticulosis (MMR) is considered to be malignant, or at least pre-malignant T-cell proliferations of the nose or midline area. Recent reports of small series of nasal T-cell lymphomas have shown a strong association with Epstein-Barr virus (EBV). Furthermore, a peculiar phenotype is described, with expression of CD56 and not of CD3, suggesting a possible origin from natural killer (NK) cells. We have analysed a series of 38 cases of PR/MMR for the presence of EBV by in situ hybridization (ISH) of the EBV-encoded RNAs 1 and 2 (EBER). Twenty cases were tested for expression of EBV-encoded latent membrane protein 1 (LMP-1). Special attention was also paid to the expression of CD3 and the NK cell-related marker CD56. Thirty-two cases (84 per cent) showed positive EBER ISH. In 5 of 20 cases, LMP-1 expression was detected. In three cases, a few scattered cells were positive, and in two cases, LMP-1 was detected in clusters of atypical cell. Most of the neoplasms showed expression of CD3 (89 per cent) and in 27 cases (71 per cent), CD56 was detected. These results are consistent with an aetiopathogenetic role for EBV in most, but not all, cases of PR/MMR. Our findings are less supportive of a major role for LMP-1 in tumour genesis. CD3 expression in most of the cases of PR/MMR underlines the T-cell origin of these neoplasms, often with aberrant expression of CD56.     

Immunopathology associated with Epstein-Barr virus (EBV) infection: Evidence for interactions with T-lymphocyte EBV receptor CD21

Epstein-Barr virus (EBV), also termed Human Herpes Virus 4, is the causative agent of infectious mononucleosis and may be a cofactor in some human cancers. The virus has also been suggested to play a role in human autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. X-linked lymphoproliferative syndrome caused by the deficiency of the Sarc homology 2 domain protein 1A, also termed signaling lymphocyte activation marker-associated protein, can result in immune dysfunction and death after EBV infection. The EBV-related immunopathology in X-linked lymphoproliferative syndrome and prototypical autoimmune syndromes is summarized in this review. A novel model of viral interaction with complement receptor CD21, which is also the receptor for EBV, is proposed to account for both the immunological abnormalities of X-linked lymphoproliferative syndrome and autoimmune diseases associated with EBV infection. The pathogenesis of both X-linked lymphoproliferative syndrome and EBV-associated immune diseases is proposed to result from increased direct infection of T cells by EBV through the T-cell complement receptor CD21 expressed on T cells. A prediction of this model is that therapy designed to decrease CD21-mediated EBV infection of T lymphocytes could also be beneficial in the treatment of some autoimmune diseases.     

An animal model for Epstein-Barr virus (EBV)-associated lymphomagenesis in the human: malignant lymphoma induction of rabbits by EBV-related herpesvirus from cynomolgus

It is very important to develop and analyze animal models of Epstein-Barr virus (EBV)-associated tumors in the human. However, only a few reports on the animal models of EBV infection have been reported. Here we review those previous models and describe the details on our newly developed rabbit model of malignant lymphoma induced by EBV-related virus from cynomolgus. In brief, Si-IIA-EBV or Cyno-EBV induced T-cell lymphomas in rabbits inoculated intravenously (77-90%), orally (82-89%), subcutaneously (3/3) and intraperitoneally (2/3) about 2-5 months later. EBV-DNA was detected in peripheral blood by polymerase chain reaction 2 days after oral inoculation of Cyno-EBV while antiviral capsid antigen immunoglobulin G (IgG) was raised 3 weeks after the inoculation. Rabbit lymphomas and their cell lines contained EBV-DNA and expressed EBV-encoded small RNA-1 and EBV-associated nuclear antigen. Rabbit lymphoma cell lines, some of which have specific chromosomal abnormality, showed tumorigenicity in nude mice. The significance and further research subjects of this animal model will be discussed. We believe that the present rabbit model of lymphoma with specific chromosomal abnormalities is very useful for clarifying the role of EBV in human EBV-associated lymphoma and provides a means for studying prophylactic and therapeutic regimens.     

Biclonal expansion of T cells infected with monoclonal Epstein-Barr virus (EBV) in a patient with chronic,active EBV infection

Recent studies have suggested that a high percentage of Epstein-Barr virus (EBV)-infected lymphocytes in peripheral blood of patients with chronic, active EBV infection (CAEBV) is of T cell origin. Although T cells are expanded oligoclonally in CAEBV, it is not clear whether the restricted diversity of T cells arise from immune reaction against EBV-related antigens or from proliferation of EBV-infected cells. We experienced a patient with CAEBV who had biclonal expansion of peripheral blood T cells. We identified clonotypes of these two T cell clones in detail and purified the T cell clones. EBV infected mainly the two T cell clones, whereas the viral loads in peripheral blood cells other than these T cell clones were low or undetectable. The EBV strains infecting the two T cells clones were indistinguishable from each other by a series of genotype analyses of the virus. These results suggest that the two T cell clones infected with the same monoclonal EBV proliferated in peripheral blood of the patient.     

Chronic active Epstein-Barr virus infection associated with low expression of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) on natural killer cells

Chronic active Epstein–Barr virus (CAEBV) infection is an uncommon and severe manifestation of EBV infection that frequently leads to the development of natural killer (NK) or T cell lymphomas. We report here that NK cells derived from a boy with CAEBV showed decreased cytotoxic function and impaired interferon- secretion against a variety of transformed target cell lines. In addition, NK cells from the patient lacked expression of the broadly expressed NK receptor, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). These data suggest that CAEBV is associated with low expression of LAIR-1 on NK cells.     

Clinical significance of variations in levels of Epstein-Barr Virus (EBV) antigen and adaptive immune response during chronic active EBV infection in children

Abstract

Pediatric patients were recruited to analyze differences in Epstein-Barr virus (EBV) copy numbers and adaptive immune reactions in children with chronic active vs acute EBV infection (CAEBVI vs AEBVI), as well as to examine the relationship between these parameters and the pathogenesis of CAEBVI. Fluorescent qPCR was used to assess EBV-DNA levels, while ELISA, antibody affinity, flow cytometry, and heterophil agglutination (HA) assays were used to evaluate patient EBV-adaptive humoral and cellular immunity. Lastly, ELISPOT was employed to assess interferon (IFN)-γ secretory functions of EBV-specific cytotoxic T-lymphocytes (CTL) as a marker of subject EBV-specific adaptive cellular immunity. The results indicated that, compared with AEBVI patients or normal children, there was a dramatic elevation in viral copy levels, viral capsid antigen (VCA)-IgA, early antigen (EA)-IgA, and EA-IgG, but a lack of EBV nuclear antigen (EBNA)-IgG and a negative HA in CAEBVI patients (p?<?0.01). These subjects also had decreased CD4⁺, CD8⁺ (naïve), CD8⁺CD38⁺, and effective memory T-lymphocyte levels compared with AEBVI patients (p?<?0.01), and decreased EBV-specific CTL function compared with normal children (p?<?0.01). These results suggest that there is a disturbance in EBV antigen availability and in both the adaptive humoral and cellular immune responses in patients with CAEBVI, and that these outcomes may be associated with the chronic active re-infection process itself associated with CAEBVI.     

Clinicopathological analysis of the age-related differences in patients with Epstein-Barr virus (EBV)-associated extranasal natural killer (NK)/T-cell lymphoma with reference to the relationship with aggressive NK cell leukaemia and chronic active EBV infection-associated lymphoproliferative disorders

Takahashi E, Ohshima K, Kimura H, Hara K, Suzuki R, Kawa K, Eimoto T & Nakamura S for the NK‐cell Tumor Study Group
(2011) Histopathology 59 , 660–671 Clinicopathological analysis of the age‐related differences in patients with Epstein–Barr virus (EBV)‐associated extranasal natural killer (NK)/T‐cell lymphoma with reference to the relationship with aggressive NK cell leukaemia and chronic active EBV infection‐associated lymphoproliferative disorders Aims: Extranodal natural killer (NK)/T‐cell lymphoma (NKTL), comprising nasal NKTL and extranasal NKTL (ENKTL), is associated with Epstein–Barr virus (EBV). A bimodal age distribution was noted in NKTL patients. We examined the clinicopathological differences between two age groups of ENKTL patients (n = 23) and compared the findings with those of aggressive NK cell leukaemia (ANKL; n = 10) and monoclonal chronic active EBV infection‐associated T/NK‐cell lymphoproliferative disorders [chronic active EBV infection/TNK‐lymphoproliferative disorders (CAEBV/TNK‐LPD)] of NK‐cell type (n = 45). Methods and results: Distinct differences existed between elderly (>50 years; n = 13) and younger (≤50 years; n = 10) ENKTL patients; the latter showed a higher disease stage (P = 0.0286), worse performance status (P = 0.0244), more frequent B symptoms (P = 0.0286) and more frequent liver, spleen and bone marrow involvement (P = 0.0222, 0.0005 and 0.0259, respectively). Few clinicopathological differences existed between younger ENKTL and ANKL patients. Patients with monoclonal CAEBV/TNK‐LPD of NK‐cell type (n = 45) showed features similar to those in younger ENKTL/ANKL patients, except a more juvenile onset of CAEBV‐related symptoms and better prognosis. However, the onset age of overt leukaemia/lymphoma in CAEBV/TNK‐LPD patients and overall survival thereafter were similar to those in younger ENKTL/ANKL patients. Conclusions: ENKTL (≤50 years) is distinct from that in elderly patients and may encompass ANKL and overlap in the clinicopathological profile with NK‐cell type CAEBV/TNK‐LPD.     

Prognostic significance of CD20 expression and Epstein‐Barr virus (EBV) association in classical Hodgkin lymphoma in Japan: A clinicopathologic study

To investigate the clinicopathological significance of CD20 expression and Epstein‐Barr virus (EBV) association in Hodgkin and Reed–Sterberg cells of classical Hodgkin lymphoma (CHL), CD20 expression and EBV positivity (by EBER in situ hybridization) were investigated in 389 CHL patients in Japan. They included 74 CD20‐positive cases (19%) and 315 CD20‐negative cases (81%). CD20‐positive cases showed significantly older age at onset (P = 0.018) and higher association with EBV (P = 0.002). Multivariate analysis identified EBV‐positivity (but not CD20‐positivity), presence of B symptoms, thrombocytopenia, elevated serum lactate dehydrogenase and performance status >1 as poor prognostic factors for overall survival (OS). We constructed a new prognostic model with these five factors classifying patients into three groups: low risk, 0–1 adverse factor; intermediate risk, 2–3 factors; high risk, 4–5 factors. This prognostic model could stratify the prognosis of CHL patients (P < 0.0001). For 144 patients (58%) classified into the low‐risk group, the 5‐year OS was 91%. For 92 patients (37%) in the intermediate group, the 5‐year OS was 66%; for 11 patients (5%) in the high‐risk group, the 5‐year OS was 36%. In conclusion, EBV is identified as an independent poor prognostic factor for CHL patients. Therefore, examination of EBV association in CHL is recommended as routine pathologic practice especially in countries where EBV infection prevails.     

An age-related increase in the number of CD8+ T cells carrying receptors for an immunodominant Epstein-Barr virus (EBV) epitope is counteracted by a decreased frequency of their antigen-specific responsiveness

The aim of this study was to provide a basis for investigating the effects of one very common environmental factor, Epstein-Barr virus (EBV), on age-related changes in the immune system. To this end, the frequency of CD8(+) T cells carrying receptors for an immunodominant EBV lytic epitope was assessed by direct staining with HLA-peptide tetrameric complexes in 19 very old (>87 years) and 12 young (20-40 years) EBV carriers. The frequency of EBV-tetramer-positive cells within the CD8(+) subset was significantly greater in the old compared to the young group (P=0.001). However, the frequency of EBV antigen-specific IFN-gamma producing T cells, as determined by ELISPOT, was significantly lower in the old (P=0.001). Therefore, the absolute number of functional EBV-specific T cells in the elderly and the young was probably similar. These data suggest CD8 clonal expansions in the elderly, resulting in an accumulation of dysfunctional EBV-specific cells which possibly fill the 'immunological space' and could lead to a shrinking of the T cell repertoire for other novel antigens. This may help to explain the increased incidence and case-fatality caused by viruses and intracellular pathogens in the elderly.     

Non‐neutralizing epitopes induce robust hepatitis C virus (HCV)‐specific antibody‐dependent CD56+ natural killer cell responses in chronic HCV‐infected patients

Natural killer (NK) cell‐mediated antibody‐dependent cellular cytotoxicity (NK‐ADCC) is of considerable interest in viral infection. However, little is known about NK‐ADCC responses in chronic hepatitis C virus (HCV) infection. In this study, impaired non‐specific antibody‐dependent CD56⁺ NK cell responses were observed in chronic HCV infection, as shown by decreased degranulation (extracellular CD107a expression) and interferon (IFN)‐γ production in response to antibody‐bound P815 cells. A peptide pool composed of epitopes recognized by anti‐HCV‐E1/E2 antibodies could induce pronounced HCV‐specific antibody‐dependent NK cell responses in sera from approximately half the chronic HCV carriers. Additionally, HCV‐specific epitopes with the capacity to induce robust NK‐ADCC activity were identified. Five linear NK‐ADCC epitopes (aa211‐aa217, aa384‐aa391, aa464‐aa475, aa544‐aa551 and aa648‐aa659 of the HCV envelope) were identified and do not overlap with putative linear neutralizing epitopes. This study revealed the dysfunctional characteristics of antibody‐dependent CD56⁺ NK cell responses in chronic HCV carriers. The key non‐neutralizing NK‐ADCC epitopes identified in this study may act as new targets for immunological intervention.     

Vascular lesion in a patient of chronic active Epstein-Barr virus infection with hypersensitivity to mosquito bites: vasculitis induced by mosquito bite with the infiltration of nonneoplastic Epstein-Barr virus-positive cells and subsequent development of natural killer/T-cell lymphoma with angiodestruction

This report describes a vasculitis and subsequently developing angiodestructive lymphoma in an 11-year-old Japanese-Filipino girl exhibiting mosquito allergy with the background of chronic active Epstein-Barr virus (EBV) infection. She developed necrotic skin ulcer at the site of mosquito bite, and histopathological examination revealed EBV-positive mononuclear cell infiltration throughout the wall of small-sized muscular artery. These EBV-positive lymphoid cells were oligoclonal in Southern blot analysis for EBV terminal repeats. Effectiveness of steroid therapy also supports the nonneoplastic nature. Approximately 1 year later, she developed progressive large skin ulcer without mosquito bites. Microscopically, the angiocentric or angiodestructive pattern of EBV-positive atypical cells supported the diagnosis of extranodal natural killer/T-cell lymphoma. Southern blot analysis revealed the monoclonal neoplastic nature of EBV-positive cells. In contrast to the primary mosquito bite lesion, natural killer/T-cell lymphoma cells exhibited the higher expression of EBV latent membrane protein 1 mRNA and the apparent protein expression detected by immunohistochemistry.