The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7644172219178472
Hydroa vacciniforme-like lymphoma (HVL) is a rare type of Epstein-Barr virus (EBV)-positive lymphoma of cytotoxic T-cell or natural killer cell origin that mainly affect children, characterized by a vesicopapular skin eruption that clinically resemble hydroa vacciniforme (HV). In current study, we report an adult patient with the tumor. The patient presented similar morphologic, immunophenotypic and genotypic changes of the disease with that occurred in children, whereas clinically, he showed a prolonged clinical course without hepatosplenomegaly or generalized lymphadenopathy. Whether there are some differences in biologic behavior between children and adults still remains unknown and it is necessary to collect more data to observe and to investigate in the future.
Hydroa vacciniforme-like lymphoma; Epstein-Barr virus; Adult
Hydroa vacciniforme (HV) is a photosensitivity disorder characterized by recurrent necrotic vesiculopapules on sun-exposed areas, which heal spontaneously during adolescence. Recently, an association has been reported between latent Epstein-Barr virus (EBV) infection and atypical HV-like eruption and malignant potential. However, latent EBV infection has also been reported in the setting of typical HV. An 11-year-old girl presented with recurrent, scattered, discrete vesicular eruptions with scarring on the face and the extensor surfaces of both forearms. In-situ hybridization was carried out to detect latent EBV infection. Based on the clinical and histopathological findings, typical EBV-associated HV was suspected.
Epstein-Barr virus; Hydroa vacciniforme
Reports of unusually severe lymphoproliferative disorders associated with extremely high antibody titers against Epstein-Barr virus (EBV) have recently increased. The syndrome, which we designated severe chronic active EBV infection syndrome, is characterized by persistent or intermittent fever, lymphadenopathy, and hepatosplenomegaly and primarily affects children and young adults. Polyclonal gammopathy and bone marrow suppression are generally observed, and some patients develop B-cell or T-cell lymphoproliferation or lymphoma. Frequently, EBV genomes are detectable in tissues infiltrated with lymphoid cells. Additionally, it is difficult to establish spontaneous or B95-8 EBV-induced cell lines despite the expression of an activated EBV infection. We review and report here the published medical literature and our own experience regarding patients with severe chronic active EBV infection syndrome in an attempt to understand this enigmatic syndrome and the possible pathogenetic mechanism(s) responsible for this disorder.
This study is to identify the spectrum of Epstein-Barr virus (EBV)-positive lymphoproliferative diseases (LPD) and relationships between these diseases in Korea. The EBV status and clinicopathology of 764 patients, including acute EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV (CAEBV) infections, B-LPD arising in chronic latent EBV infection, T & natural killer (NK) cell non-Hodgkin's lymphomas (NHL), B-NHLs, and Hodgkin's lymphomas (HD), were analyzed. T or NK cell NHLs were the most common forms of EBV-positive NHLs (107/167, 64%); among these, nasal-type NK/T cell lymphomas were the most common (89/107, 83%). According to the age, Burkitt's lymphoma was the most common in early childhood; in teenagers, chronic (active) EBV infection-associated LPD was the most common type. The incidence of NK/T cell lymphoma began to increase from the twenties and formed the major type of EBV-associated tumor throughout life. Diffuse large B cell lymphoma formed the major type in the sixties and seventies. In conclusion, primary infections in early childhood are complicated by the development of CAEBV infections that are main predisposing factors for EBV-associated T or NK cell malignancies in young adults. In old patients, decreased immunity associated with old age and environmental cofactors may provoke the development of peripheral T cell lymphoma, unspecified, and diffuse large B cell lymphoma.
Lymphoma; Epstein-Barr Virus; Lymphohistiocytosis, Hemophagocytic
Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH), EBV-positive systemic T-cell lymphoproliferative disease (STLPD) of childhood, and chronic active EBV (CAEBV) infection may develop after primary EBV infection. This study reviewed the clinicopathological spectrum of EBV-associated T- and natural killer (NK)-cell LPD, including STLPD and CAEBV infection, with an analysis of T-cell clonality.
Clinicopathological features of seven patients with EBV-associated HLH or STLPD and 12 patients with CAEBV infection were reviewed. Immunohistochemical staining and a T-cell receptor (TCR) gene rearrangement study were performed.
STLPD and EBV-positive HLH showed significantly overlapping clinicopathological findings. One patient with STLPD and one patient with EBV-positive HLH demonstrated moderate to severe atypia of the infiltrating lymphocytes, whereas the remaining patients lacked significant atypia. Twelve patients had CAEBV infection, four of whom suffered mosquito-bite hypersensitivity, five showed NK lymphocytosis, and one suffered hydroa vacciniforme. Infiltrating lymphocytes were predominantly small and devoid of atypia. Hemophagocytic histiocytosis was found in seven of 11 patients. Monoclonality was detected in three (50%) of the six patients with successful TCR gene analysis.
EBV-positive HLH and STLPD share similar clinicopathological findings and may constitute a continuous spectrum of acute EBV-associated T- or NK-cell proliferative disorders. The distinction of EBV-positive T-cell LPD from EBV-positive HLH may be difficult during routine diagnoses because of the technical limitations of clonality assessment.
Epstein-Barr virus infections; Lymphoma, T-cell; Killer cells, natural; Lymphoproliferative disorders; Clonality
Fulminant Epstein-Barr virus (EBV)-driven clonal T-cell lymphoproliferative disorder (T-LPD) is rare and most patients are of Asian origin. The disease usually develops shortly after primary acute EBV infection and the mechanism remains poorly understood. Here we report such a rare case in a 28-year-old Caucasian female with systemic lupus erythematosus (SLE). Immunophenotypic and molecular studies revealed that the proliferating lymphoid cells displayed a CD8+ T-cell phenotype with clonal rearrangement of the T-cell receptor gamma gene. Epstein-Barr virus-encoded RNA was also observed in the clonal lymphoid cells by in situ hybridization. The patient subsequently developed fatal virus-associated hemophagocytic syndrome one month after the primary acute EBV infection. The case represents the first report of fulminant EBV-driven CD8+ T-LPD occurring in an immunocompromised Caucasian SLE patient. This study, along with studies of similar Asian cases reported in the literature, suggests that dysregulated immunity due to either acquired or genetically determined susceptibility may result in an abnormal response to primary EBV infection and contribute to the pathogenesis of EBV-mediated fatal T-LPD.
Fatal infectious mononuleosis; Epstein-Barr virus; T-cell lymphoproliferative disorder; virus-associated hemophagocytic syndrome; hemophagocytosis; systemic lupus erythematosus
Post-transplantation lymphoproliferative disorders (PTLD) arise in the immunosuppressed and are frequently Epstein-Barr virus (EBV) associated. The most common PTLD histological sub-type is diffuse large B-cell lymphoma (EBV+DLBCL-PTLD). Restoration of EBV-specific T-cell immunity can induce EBV+DLBCL-PTLD regression. The most frequent B-cell lymphoma in the immunocompetent is also DLBCL. ‘EBV-positive DLBCL of the elderly’ (EBV+DLBCL) is a rare but well-recognized DLBCL entity that occurs in the overtly immunocompetent, that has an adverse outcome relative to EBV-negative DLBCL. Unlike PTLD (which is classified as viral latency III), literature suggests EBV+DLBCL is typically latency II, i.e. expression is limited to the immuno-subdominant EBNA1, LMP1 and LMP2 EBV-proteins. If correct, this would be a major impediment for T-cell immunotherapeutic strategies. Unexpectedly we observed EBV+DLBCL-PTLD and EBV+DLBCL both shared features consistent with type III EBV-latency, including expression of the immuno-dominant EBNA3A protein. Extensive analysis showed frequent polymorphisms in EB-NA1 and LMP1 functionally defined CD8+ T-cell epitope encoding regions, whereas EBNA3A polymorphisms were very rare making this an attractive immunotherapy target. As with EBV+DLBCL-PTLD, the antigen presenting machinery within lymphomatous nodes was intact. EBV+DLBCL express EBNA3A suggesting it is amenable to immunotherapeutic strategies.
Epstein-Barr virus; diffuse large B-cell lymphoma; EBNA3A; T-cell; epitope; immunotherapy; posttransplantation lymphoproliferative disorder
Hydroa vacciniforme (HV) is a rare and chronic pediatric disorder that is characterized by photosensitivity and recurrent vesicles that heal with vacciniforme scarring. The pathogenesis of HV is unknown; no chromosome abnormality has been identified. HV patients have no abnormal laboratory results, so the diagnosis of HV is based on identifying the associated histological findings in a biopsy specimen and using repetitive ultraviolet phototesting to reproduce the characteristic vesicles on a patient's skin. Herein, we present a case of HV in a 7-year-old female who was diagnosed with HV according to histopathology and ultraviolet phototesting.
Hydroa vacciniforme; Photodermatosis; Phototest
Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a peculiar T cell lymphoma, as expanding B cell clones are often present besides the malignant T cell clones. In addition, large numbers of Epstein-Barr virus (EBV)-infected B cells are frequently observed. To analyze the differentiation status and clonal composition of EBV-harboring B cells in AILD, single EBV-infected cells were micromanipulated from lymph nodes of six patients with frequent EBV+ cells and their rearranged immunoglobulin (Ig) genes analyzed. Most EBV-infected B cells carried mutated Ig genes, indicating that in AILD, EBV preferentially resides in memory and/or germinal center B cells. EBV+ B cell clones observed in all six cases ranged from small polyclonal to large monoclonal expansions and often showed ongoing somatic hypermutation while EBV− B cells showed little tendency for clonal expansion. Surprisingly, many members of expanding B cell clones had acquired destructive mutations in originally functional V gene rearrangements and showed an unfavorable high load of replacement mutations in the framework regions, indicating that they accumulated mutations over repeated rounds of mutation and division while not being selected through their antigen receptor. This sustained selection-free accumulation of somatic mutations is unique to AILD. Moreover, the survival and clonal expansion of “forbidden” (i.e., Ig-deficient) B cells has not been observed before in vivo and thus represents a novel type of viral latency in the B cell compartment. It is likely the interplay between the microenvironment in AILD lymph nodes and the viral transformation that leads to the survival and clonal expansion of Ig-less B cells.
B lymphocytes; somatic hypermutation; EBV; immunoglobulin genes; single cell PCR
Epstein-Barr virus (EBV) is associated with non-Hodgkin’s lymphoma (NHL), occurring in immunocompetent individuals as well as those with immunodeficiency. In patients with immunodeficiency, the nature of EBV infection in the malignant cell determines the pattern of antigen expression and the associated presence of targets for cellular immunotherapy. EBV-expressing lymphoma cells in the setting of immunodeficiency express type III latency, characterized by expression of all nine latent-cycle EBV antigens, and strategies to restore EBV-specific immune responses have resulted in effective anti-tumour activity. In contrast, EBV-associated NHL in immunocompetent individuals is characterized by type II latency, where a more restricted array of EBV-associated antigens is expressed. In this setting, T-cell therapies are limited by inadequate persistence of transferred T cells and by tumour-evasion strategies. A number of strategies to genetically modify the infused T cells and modulate the host environment are under evaluation.
EBV; post-transplant lymphoproliferative disease; cytotoxic T lymphocytes; chimeric antigen receptors
Since the discovery of Epstein-Barr virus (EBV) from a cultured Burkitt's lymphoma cell line in 1964, the virus has been associated with Burkitt's lymphoma, nasopharyngeal carcinoma, and infectious mononucleosis. During the recent decade, EBV has been etiologically implicated in a broad spectrum of human diseases. The precise role of this virus in these diseases is not well understood, but clearly, defective immunosurveillance against the virus may permit an uncontrolled proliferation of EBV-infected cells. As a result, a growing number of cases of EBV-associated B-cell proliferative diseases or lymphoma have been noted in patients with primary and acquired immunodeficiencies. These lymphoproliferative diseases and others, such as chronic mononucleosis syndrome, are leading to new areas of investigation which are providing information regarding the pathogenetic mechanisms of EBV-induced diseases. The early accurate diagnosis of EBV infection can be achieved by performing EBV-specific serology, detecting for EBV-determined nuclear antigen in tissues, establishing spontaneous lymphoid cell lines, and using molecular hybridization techniques for demonstrating the presence of viral genome in affected lesions.
Nasal NK/T-cell lymphoma is an aggressive subtype of non-Hodgkin lymphoma (NHL) that is closely associated with Epstein–Barr virus (EBV). The clonal expansion of EBV-infected NK or T cells is also seen in patients with chronic active EBV (CAEBV) infection, suggesting that two diseases might share a partially similar mechanism by which EBV affects host cellular gene expression. To understand the pathogenesis of EBV-associated NK/T-cell lymphoproliferative disorders (LPD) and design new therapies, we employed a novel EBV DNA microarray to compare patterns of EBV expression in six cell lines established from EBV-associated NK/T-cell LPD. We found that expression of BZLF1, which encodes the immediate-early gene product Zta, was expressed in SNK/T cells and the expression levels were preferentially high in cell lines from CAEBV infection. We also analyzsd the gene expression patterns of host cellular genes using a human oligonucleotide DNA microarray. We identified a subset of pathogenically and clinically relevant host cellular genes, including TNFRSF10D, CDK2, HSPCA, IL12A as a common molecular biological properties of EBV-associated NK/T-cell LPD and a subset of genes, such as PDCD4 as a putative contributor for disease progression. This study describes a novel approach from the aspects of viral and host gene expression, which could identify novel therapeutic targets in EBV-associated NK/T-cell LPD.
Epstein Barr virus; nasal NK/T-cell lymphoma; EBV DNA microarray; CAEBV infection
The Epstein-Barr virus (EBV) has an important and multifaceted role in liver pathology. As a member of the herpes virus family, EBV establishes a persistent infection in more than 90% of adults. Besides acute hepatitis during primary infection, many clinical syndromes of interest for the hepatologist are associated with EBV infection. The role of EBV in the evolution of chronic hepatitis from hepatotropic viruses is considered. Chronic EBV-associated hepatitis is suspected in immunocompetent adults with compatible serology, suggestive histology and detection of the viral genome in the liver and/or increase of specific circulating cytotoxic T-lymphocytes. EBV is the main cause of post-transplant lymphoproliferative disorders which occur in up to 30% of cases. EBV-driven lymphoproliferative diseases are also recognized in non-immunocompromised patients and liver is involved in up to a third of the cases. Directly implicated in the pathogenesis of different tumors, EBV has a disputable role in hepatocellular carcinoma carcinogenesis. Further research is required in order to establish or reject the role of EBV in human liver cancer. This paper attempts to discuss the range of EBV-associated chronic liver diseases in immunocompetent patients, from mild, self-limiting mononuclear hepatitis to liver cancer.
Epstein-Barr virus; Chronic hepatitis; Liver disease; Chronic active Epstein-Barr virus; Post-transplant lymphoproliferative disorder; Infectious mononucleosis
Up to 40% of Hodgkin lymphoma (HL) cases are associated with the Epstein-Barr virus (EBV). Clonal viral genomes can be found in the HL tumor cells, the Hodgkin Reed-Sternberg cells (HRS). The latent infection results in expression of the viral oncogenes LMP1 and LMP2A which contribute to generate the particular phenotype of the HRS cells. EBV does not only undergo epigenetic changes of its genome during latency, but also induces epigenetic changes in the host genome. The presence of EBV may alter the composition and activity of the immune cells surrounding the HRS cells. EBV favours a Th1 reaction, but this attempt at a cell mediated immune response appears to be ineffective. The presence of EBV in HL is associated with several clinicopathological characteristics: It is more frequent in cases with mixed cellular histology, in males, in children and older adults, and in developing countries, while the young-adult onset HL of nodular sclerosis type in industrialized countries is typically EBV-negative. Countries in the Mediterranean area often show an intermediate epidemiological pattern. Recent studies suggest a genetic predisposition to develop EBV-associated HL. Circulating EBV-DNA may serve as a biomarker to monitor response to therapy, and eventually, EBV will become a target for therapeutic intervention also in HL.
Epstein-Barr virus (EBV) is associated to the etio-pathogenesis of an increasing number of tumors. Detection of EBV in pathology samples is relevant since its high prevalence in some cancers makes the virus a promising target of specific therapies. RNA in situ hybridization (RISH) is the standard diagnostic procedure, while polymerase chain reaction (PCR)-based methods are used for strain (EBV type-1 or 2) distinction. We performed a systematic comparison between RISH and PCR for EBV detection, in a group of childhood B-cell Non-Hodgkin lymphomas (NHL), aiming to validate PCR as a first, rapid method for the diagnosis of EBV-associated B-cell NHL.
EBV infection was investigated in formalin fixed paraffin-embedded tumor samples of 41 children with B-cell NHL, including 35 Burkitt's lymphoma (BL), from Rio de Janeiro, Brazil, by in situ hybridization of EBV-encoded small RNA (EBER-RISH) and PCR assays based on EBNA2 amplification.
EBV genomes were detected in 68% of all NHL. Type 1 and 2 accounted for 80% and 20% of EBV infection, respectively. PCR and RISH were highly concordant (95%), as well as single- and nested-PCR results, allowing the use of a single PCR round for diagnostic purposes. PCR assays showed a sensitivity and specificity of 96% and 100%, respectively, with a detection level of 1 EBV genome in 5,000–10,000 EBV-negative cells, excluding the possibility of detecting low-number EBV-bearing memory cells.
We describe adequate PCR conditions with similar sensitivity and reliability to RISH, to be used for EBV diagnostic screening in high grade B-NHL, in "at risk" geographic regions.
The latent membrane protein (LMP) of Epstein-Barr virus (EBV) forms patches associated with the vimentin intermediate filament system in EBV-transformed lymphoblastoid cell lines, EBV-infected Burkitt's lymphoma cells, and LMP-transfected, EBV-negative Burkitt's lymphoma cells. By gene transfer, LMP induces the expression of vimentin and B-cell activation antigens in EBV-negative Burkitt's lymphoma cells. We have now expressed LMP in an EBV-positive Burkitt's lymphoma cell line, Daudi, which does not express any LMP or vimentin. In these Daudi transfectants, LMP still formed plasma membrane patches in the absence of vimentin. LMP did not resist nonionic detergent extraction in Daudi cells as it does in vimentin-expressing cells. LMP still retained functional activity as judged by induction of B-cell activation antigens. These data indicate that LMP can form plasma membrane patches and induce B-lymphocyte activation independent of vimentin association.
The aged immune system is characterized by clonal expansions of CD8+ T cells of which a substantial portion are directed against Epstein-Barr virus (EBV) and cytomegalovirus (CMV). It is unknown if these expansions represent increased viral reactivation or simply reflect an accumulation over time. We investigated herpesvirus reactivation in young and old subjects co-infected with CMV and EBV. Using molecular and serological techniques, we found significant increases in both the frequency and magnitude of EBV and CMV reactivation in elderly subjects. CMV DNA was frequently detected in the urine of elderly subjects; EBV load in peripheral blood was also significantly increased. Notably, EBV DNA in plasma was detected in a majority of the elderly subjects which was supported by frequent transcription of late structural genes. Furthermore, CD8+ T cells specific for EBV structural antigens were detected in samples from the elderly. Samples from our younger control group were negative for EBV DNA in plasma, CMV DNA in urine, expression of structural transcripts, and lacked CD8+ T cells specific for EBV structural antigens. These findings indicate that the aged immune system is no longer able to control EBV and CMV reactivation that could now be characterized as chronic instead of latent.
aging; herpesvirus; Epstein-Barr Virus; Cytomegalovirus
Epstein-Barr virus (EBV) causes various diseases, such as infectious mononucleosis (IM), fatal IM, EBV-associated hemophagocytic syndrome (EBVAHS), and chronic active EBV infection (CAEBV). In the present study, cell-free EBV DNA was detected in the plasma of patients with EBV-associated diseases by PCR assay. The patients included 20 patients with IM, 2 patients with fatal IM, 4 patients with EBVAHS, 4 patients with CAEBV, and 38 healthy children (20 EBV seropositive and 18 EBV seronegative). In patients with IM, plasma samples were positive for EBV DNA in all patients (100%) in the acute phase and in 44% of the patients in the convalescent phase, but plasma samples from the 38 healthy control children were negative (0%) for EBV DNA. Quantitative PCR assay revealed that plasma from patients with IM contained the highest amount of virus DNA within 7 days following the onset of disease (mean, 6 x 10(4) copies per ml). The EBV DNA concentration decreased thereafter as the patients recovered. Plasma from patients with fatal IM contained more than 100 times more copies of EBV DNA (3 x 10(7) copies per ml) than plasma from patients with IM. Plasma from patients with the acute phase of EBVAHS contained 10 times more copies of EBV DNA (5 x 10(5) copies per ml) than plasma from IM, and then patients with the number of copies decreased similarly in both groups of patients in the convalescent phase (2 x 10(4) copies per ml). The amount of virus DNA in patients with CAEBV (6 x 10(4) copies per ml) was similar to that noted in patients with IM; however, it became higher (1 x 10(6) copies per ml) when the patients' clinical status deteriorated.(ABSTRACT TRUNCATED AT 250 WORDS)
Background: Recently novel Epstein–Barr virus (EBV) lymphoproliferative diseases (LPDs) have been identified in non-immunocompromised hosts, both in Asia and Western countries. These include aggressive T-cell and NK-cell LPDs often subsumed under the heading of chronic active Epstein–Barr virus (CAEBV) infection and EBV-driven B-cell LPDs mainly affecting the elderly.
Design: To better define the pathogenesis, classification, and treatment of these disorders, participants from Asia, The Americas, Europe, and Australia presented clinical and experimental data at an international meeting.
Results: The term systemic EBV-positive T-cell LPD, as adopted by the WHO classification, is preferred as a pathological classification over CAEBV (the favored clinical term) for those cases that are clonal. The disease has an aggressive clinical course, but may arise in the background of CAEBV. Hydroa vacciniforme (HV) and HV-like lymphoma represent a spectrum of clonal EBV-positive T-cell LPDs, which have a more protracted clinical course; spontaneous regression may occur in adult life. Severe mosquito bite allergy is a related syndrome usually of NK cell origin. Immune senescence in the elderly is associated with both reactive and neoplastic EBV-driven LPDs, including EBV-positive diffuse large B-cell lymphomas.
Conclusion: The participants proposed an international consortium to facilitate further clinical and biological studies of novel EBV-driven LPDs.
chronic active EBV infection; diffuse large B-cell lymphoma; hemophagocytic syndrome; hydroa vacciniforme; immune senescence; senile EBV-positive lymphoproliferative disease; systemic EBV-positive lymphoproliferative disease
The nasal type, extranodal natural killer or T(NK/T)-cell lymphoma is usually associated with latent Epstein–Barr virus (EBV) infection. In order to elucidate the EBV gene expression patterns in vivo, we examined eight patients with cutaneous EBV-related NK/T-cell lymphomas, including six patients with a NK-cell phenotype and two patients with a T-cell phenotype. The implication of EBV in the skin lesions was determined by the presence of EBV-DNA, EBV-encoded nuclear RNA (EBER) and a clonality of EBV-DNA fragments containing the terminal repeats. Transcripts of EBV-encoded genes were screened by reverse transcription- polymerase chain reaction (RT-PCR), and confirmed by Southern blot hybridization. The expression of EBV-related antigens was examined by immunostaining using paraffin-embedded tissue sections and cell pellets of EBV-positive cell lines. Our study demonstrated that all samples from the patients contained EBV nuclear antigen (EBNA)-1 mRNA which was transcribed using the Q promoter, whereas both the Q promoter and another upstream promoter (Cp/Wp) were used in EBV-positive cell lines, B95.8, Raji and Jiyoye. Latent membrane protein-1 (LMP-1) mRNA was detected in seven of eight patients and all cell lines, whereas EBNA-2 transcripts were found only in the cell lines. Immunostaining showed no LMP-1, EBNA-2 or ZEBRA antigens in the paraffin-embedded tissue sections, although they were positive in the cell line cells. Latent BHRF1 transcripts encoding bcl-2 homologue and BCRF1 transcripts encoding viral interleukin (vIL)-10 were detected in one and two of eight patients, respectively. A patient with NK-cell lymphoma expressing both transcripts died of rapid progression of the illness. Our results indicate that the restricted expression of the latency-associated EBV genes and the production of vIL-10 and bcl-2 homologue may favour tumour growth, evading the host immune surveillance. © 2001 Cancer Research Campaign http://www.bjcancer.com
Epstein–Barr virus; NK/T-cell lymphomas; latency; viral IL-10; bcl-2 homologue; LMP; EBNA
Certain classes of human T-cell lymphomas have been shown to be persistently infected with Epstein-Barr virus (EBV). To achieve an experimental system of persistent EBV infection in T cells, we used EBV recombinants with a positive selection marker. Infection of the human T-cell line MT-2 with EBV recombinants that had a hygromycin resistance gene and subsequent selection with this drug permitted isolation and long-term maintenance of EBV-infected MT-2 clones. For each clone, essentially 100% of cells were positive for EBV nuclear antigen. These MT-2 clones harbor monoclonal episomes of EBV DNA and stably express two EBV latent proteins, EBV nuclear antigen 1 and latent membrane protein 1. The growth of these EBV-infected MT-2 clones was slower than that of uninfected clones, suggesting that EBV affects regulation of T-cell proliferation. EBV recombinants with a positive selection marker will be a useful tool in establishing experimental systems of persistent EBV infection in certain non-B-cell lineages.
Epstein Barr virus (EBV) causes lymphomas in immune competent and, at increased frequencies, in immune compromised patients. In the presence of an intact immune system, EBV associated lymphomas express in most cases only three or fewer EBV antigens at the protein level, always including the nuclear antigen 1 of EBV (EBNA1). EBNA1 is a prominent target for EBV specific CD4+ T cell and humoral immune responses in healthy EBV carriers. Here we demonstrate that patients with EBV associated lymphomas, primarily Hodgkin's lymphoma, lack detectable EBNA1 specific CD4+ T cell responses and have slightly altered EBNA1 specific antibody titers at diagnosis. In contrast, the majority of EBV negative lymphoma patients had detectable IFNγ expression and proliferation by CD4+ T cells in response to EBNA1, and carry EBNA1 specific immunoglobulins at levels similar to healthy virus carriers. Other EBV antigens, which were not present in the tumors, were recognized in less EBV positive, than negative lymphoma patients, but detectable responses reached similar CD8+ T cell frequencies in both cohorts. Patients with EBV positive and negative lymphomas did not differ in T cell responses in influenza specific CD4+ T cell proliferation and in antibody titers against tetanus toxoid. These data suggest a selective loss of EBNA1 specific immune control in EBV associated lymphoma patients, which should be targeted for immunotherapy of these malignancies.
EBNA1; CD4+ T cells; Hodgkin's lymphoma
To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2 to over 107 copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mononuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in the PBMNC was 103.7 copies/μg of DNA in patients with EBV-related lymphoproliferative disorders, 104.1 copies/μg of DNA in patients with chronic active EBV infections, and 102.2 copies/μg of DNA in patients with infectious mononucleosis. These numbers were significantly larger than those in either posttransplant patients or immunocompetent control patients without EBV-related diseases. In a patient with infectious mononucleosis, the virus load decreased as the symptoms resolved. The copy number of EBV DNA in PBMNC from symptomatic EBV infections was correlated with the EBV-positive cell number determined by the in situ hybridization assay (r = 0.842; P < 0.0001). These results indicate that the real-time PCR assay is useful for diagnosing symptomatic EBV infection and for monitoring the virus load.
In the present study, we established an in vitro system representing the Burkitt’s lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.
X-linked lymphoproliferative disease (XLP) is an immunodeficiency caused by defects in the adaptor molecule SAP. The manifestations of XLP generally occur following Epstein-Barr virus (EBV) infection and include fulminant mononucleosis, hypogammaglobulinemia and lymphoma. In this report, we describe two unrelated patients with fatal T cell-mediated central nervous system vasculitis for whom repeated serologic and molecular testing for EBV was negative. In both patients, clonal T cell populations were observed, but neither demonstrated evidence of lymphoma. Thus, loss of SAP function can lead to dysregulated immune responses characterized by the uncontrolled expansion and activation of T cells independent of EBV infection.
X-linked lymphoproliferative disease; central nervous system; vasculitis