T cell repertoire and Epstein-Barr virus-specific T cell response in chronic active Epstein-Barr virus infection: a case study

In a patient with chronic active Epstein-Barr virus infection associated with vasculitis and fulminant CD4+ T cell lymphoproliferative disorder, we probed the peripheral blood mononuclear cells (PBMC) for the presence of an EBV-specific T cell repertoire and tested the possible relationship between the lymphocytic infiltrate and the EBV-specific T cell response. Our results give credence to the presence of an apparently normal EBV-specific memory T cell response after in vitro reactivation of the patient's PBMC with autologous infected B lymphoblastoid cell lines. In keeping with the characterization of the vasculitis, certain T cell subsets were detected after expansion of skin lesion-infiltrating lymphocytes and were found to be infected with EBV. These particular T cell expansions were neither the effectors nor the targets of the in vitro reactivated EBV-specific T cells, thus excluding a simple relationship between EBV, the skin lesions, and the T cell expansions frequently observed in these patients.     

Association of virus infected-T cell in severe hepatitis caused by primary Epstein-Barr virus infection.

BACKGROUND: Infectious mononucleosis owing to primary Epstein-Barr virus (EBV) infection sometimes causes hepatitis, which is usually self-limiting with mildly elevated transaminases, but can rarely develop into severe hepatitis with jaundice. OBJECTIVE: To clarify the pathogenesis of severe hepatitis by primary EBV infection. METHODS: We experienced four cases of severe hepatitis with jaundice caused by primary EBV infection. These cases were analyzed virologically and histologically, and compared with infectious mononucleosis patients without jaundice. RESULTS AND DISCUSSION: Using real-time PCR, more EBV-DNA was detected in peripheral blood from patients with severe hepatitis, as compared to those without jaundice. Furthermore, CD3+, CD4+ or CD8+ cells contained more EBV DNA than did other cell populations, indicating that in severe hepatitis, T cells harbor most of the EBV. By contrast, mainly B cells were infected in infectious mononucleosis patients without jaundice. The liver was biopsied in three of the four cases. An in situ hybridization study showed that EBV infected lymphocytes, not hepatocytes. In addition, in one patient, it was confirmed that the infected lymphocytes were CD8+ T cells. These results suggest that a large EBV burden and T cell infection may play major roles in the mechanism of severe hepatitis caused by primary EB virus infection.     

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.     

Impaired cytotoxic T lymphocyte response to Epstein-Barr virus-infected NK cells in patients with severe chronic active EBV infection

Clinical evidence of a relationship between severe chronic active Epstein-Barr virus (EBV) infection and clonal expansion of EBV-infected T or NK cells has been accumulated. In order to clarify pathogenesis of EBV-infected cell proliferation in patients with severe chronic active EBV infection, cytotoxic T lymphocyte (CTL) responses of two patients against B-lymphoblastoid cell lines (B-LCL) and EBV-infected NK cells were examined in comparison with those of HLA-identical healthy siblings. Unexpectedly, patients' CTL activities induced by mixed culture with autologous B-LCLs were markedly reduced, although uncontrolled EBV-related B-cell proliferations have never been experienced. In contrast, limiting dilution analysis demonstrated that B-LCL-specific CTL precursor (CTLp) frequencies of patients were comparable to those of their healthy sisters. The existence of normal levels of B-LCL-specific T cell responses was confirmed by flow-cytometric analysis of IFN-gamma-producing T cells after stimulation with B-LCLs. Infected NK-cell-specific CTLp frequencies of the patients were at undetectable levels despite their expression of latent membrane protein (LMP) 1, suggesting mechanisms to escape immunologic surveillance. In the patients' HLA-identical healthy sisters, infected NK-cell-specific CTLps were detected, and infected NK-cell-specific CTL clones could be established. From these findings, two treatment options for severe chronic active EBV infection are offered for consideration: adoptive transfer of in vitro-cultured CTL, and bone marrow transplantation from HLA-identical donors.     

Intact antigen presentation for Epstein-Barr virus (EBV)-specific CTL by a lymphoblastoid cell line established from a patient with severe chronic active EBV infection

Severe chronic active Epstein-Barr virus (EBV) infection is a lymphoproliferative disease characterized by extremely high antibody titers to EBV, fever, lymphadenopathy, hepatosplenomegaly, and pancytopenia, without any prior immunological abnormality. A spontaneous lymphoblastoid cell line was established from a 4-year-old boy with severe chronic active EBV infection. Immunofluorescence and Western blotting analyses showed that the cell line was of B cell origin and expressed Epstein-Barr nuclear antigens 1, 2 3a, 3b and 3c, and latent membrane protein 1, which are reported to be targets for EBV-specific cytotoxic T lymphocytes (CTL). The cytotoxicity of peripheral blood mononuclear cells derived from the patient and his HLA-identical sister was assayed against the cell line. The cell line was recognized and killed by anti-EBV CTL derived from the HLA-identical sister, but the patient's peripheral blood mononuclear cells had no cytotoxicity. We conclude that antigen presentation in the EBV-infected cells from the patient is intact and sufficient for generation of an EBV-specific CTL response. These observations suggest that severe chronic active EBV infection may not be caused by impaired EBV-antigen presentation of the infected cells but by impaired cellular immune responses to the virus. Our results also suggest the therapeutic possibility that this disease may be treated by adoptive transfer of EBV-specific CTL or bone marrow transplantation from an HLA-matched donor whose immune response to EBV is intact.     

The effect of leucyl-leucine methyl ester on proliferation and Ig secretion of EBV-transformed human B lymphocytes.

The selective cytotoxicity of the lysosomotropic methyl esters of leucine or its lysosomal condensation product leucyl-leucine has been used to investigate the effect of cytolytic cells on the clonal outgrowth, cellular proliferation and antibody secretion of Epstein-Barr virus (EBV)-transformed human B cells. Large granular lymphocytes (LGL), monocytes, and a subset of T cells (CD8/CD11+) were permanently eliminated by the ester treatment. These lysosome-rich cells severely inhibit the clonal outgrowth of EBV-infected B cells, as determined by Poisson distribution calculations. Furthermore, leucyl-leucine methyl ester-treated and EBV-infected lymphocytes showed a significant increase in proliferative capability as well as immunoglobulin (Ig) production (three to 11 times) compared to non-treated but similarly infected lymphocytes. Since the effect of leucyl-leucine methyl ester treatment was also detectable in low-density (100 B cells/well) cultures, the suppression was unlikely to be exerted by EBV-specific T-cell clones, but pointed rather to the natural killer (NK) cells as effectors.     

Clonality analysis by sequence variation of the latent membrane protein 1 gene in patients with chronic active Epstein-Barr virus infection

Chronic active Epstein-Barr virus (EBV) infection is a severe systemic disease associated with high rates of mortality and morbidity. Recent studies suggest that the clonal expansion of EBV-infected T or natural killer cells plays a crucial role in the pathogenesis of chronic active EBV infection. However, it is not clear whether chronic active EBV infection is truly a monoclonal disorder that originates from one cell. The clonality of EBV was investigated by sequence variation of the latent membrane protein 1 (LMP1) gene, which has a high degree of sequence heterogeneity. Peripheral blood mononuclear cells were obtained from nine Japanese patients with chronic active EBV infection and four with infectious mononucleosis. A carboxyl-terminal region of the LMP1 gene was analyzed by polymerase chain reaction (PCR). The amplified PCR products were subcloned, and 18 clones from each sample were sequenced. Patients with chronic active EBV infection each had two to five different LMP1 nucleotide sequences, whereas patients with infectious mononucleosis each had one to seven different sequences. Patients with chronic active EBV infection and infectious mononucleosis both had one dominant sequence. Longitudinal analysis was performed in four patients with chronic active EBV infection, in whom the dominant strains were found to have remained unchanged for several years. The results suggest that EBV in patients with chronic active EBV infection was polyclonal, although clonal expansion may occur. Collectively, these findings are critical to clarify further the pathogenesis of chronic active EBV infection and aid in the development of effective treatment strategies.     

Epstein-Barr virus (EBV) infection visualized by EGFP expression demonstrates dependence on known mediators of EBV entry

Summary.  Studies of Epstein-Barr virus (EBV) infection and pathogenesis are limited by the lack of recombinant EBV bearing a reporter gene. Currently such studies typically utilize cellular transformation and immortalization as indicators of virus infection which takes several weeks. To investigate the dependence on known cellular receptors for EBV infection, a novel enhanced green fluorescent protein (EGFP) expressing virus, designated EBfaV-GFP, was used to infect a variety of different cell types. EBfaV-GFP infects B lymphoma-derived cell lines, lymphoblastoid cell lines (LCLs) and primary B cells, as judged by expression of EGFP. Ability of clonal cells to be infected with EBfaV-GFP correlates with expression of the known EBV entry mediators CD21 and HLA-DR. EGFP-expressing LCLs were derived by infection of primary B cells with EBfaV-GFP. Cells of the Jurkat line, derived from T lymphocytes, could not be infected, and infected primary lymphocytes did not include appreciable numbers of CD2-positive cells, showing that EBV rarely infects T cells. Expression of EGFP by EBV provides the opportunity to rapidly visualize infection in living cells and better delineate populations of infected cells. Received October 22, 1998. Accepted January 25, 1999     

Clinicopathological study of severe chronic active Epstein-Barr virus infection that developed in association with lymphoproliferative disorder and/or hemophagocytic syndrome

Chronlc active Epstein-Barr virus (CAEBV) infection has been prevlously reported to be sometimes associated with an aggresslve clinical course. However, the role of EBV in the CAEBV Is not well clarifled. A retrospective study was performed on nine adult and five child patients (eight males and six females). Histologlcally, at first admission, the presence of neoplastic lesions could not be confirmed. The lymph nodes In half of all cases revealed paracortical hyperplasla with transformed lymphocytes (hyperplastic type). Half of the cases showed nonsuppuratlve necrosis and an Increased number of histiocytes with phagocytosis (histiocytic type). Activated histiocytes with lymphokine positivity were frequently detected in the histiocytic type. In the phenotypical study, 10 of the examined 11 cases showed Increased numbers of natural killer (NK) cells and/or CD8 positive T lymphocytes. In situ hybridization (ISH) showed EBV-Infected lymphoid cells, but the number of EBV-infected cells varied. Double-labeling irnmunochemistryASH demonstrated EBV-infected T cells, including NK cells, but not B cells. In addition, three cases showed a monoclonal dissemination of EBV terminal repetitive sequence (TR), and two cases showed oligoclonal dissemination. From those findings, monoclonal, oligoclonal and polyclonal populations of EBV-infected T or NK cells were considered to be present in CAEBV states. During the clinical course, 12 of the 14 cases died within 5 years. Six cases died from EBV-associated hematopoietic tumors (histiocytlc tumor, T cell lymphoma, B cell lymphoma, plasmacytoma, and NK cell leukemia); one from non-EBV-associated acute myeloge-nous leukemia, and five due to hemophagocytic syndrome. The examined EBV-associated hematopoietic tumors showed monoclonal EBV terminal repetitive sequences. There is a possibility that the monoclonal dissemination of EBV-infected cells develops from oligoclonal or polyclonal EBV-infected cells. And active histlocytes with lymphokine positivity were frequently detected In the cases with histologically histiocytic type. These findings seem to be related with the causes of death due to hemophagocytic syndrome.     

Dendritic cells expand Epstein Barr virus specific CD8+ T cell responses more efficiently than EBV transformed B cells

Adoptive transfer of Epstein Barr virus (EBV) specific cytotoxic T lymphocytes (CTLs) has been successfully applied in the treatment of EBV associated post-transplant lymphoproliferative disease (PTLD). In most studies EBV transformed B cells (LCLs) have been used for the induction of EBV specific T cell lines. Application of this approach to other EBV associated tumors is difficult, because LCLs focus T cell expansion toward immunodominant EBV antigens that are not expressed in EBV associated Hodgkin's lymphoma and nasopharyngeal carcinoma. Therefore, we compared dendritic cells (DCs) with LCLs for CD8+ T cell stimulation against dominant and subdominant EBV antigens. DCs expanded tenfold more EBNA3A and LMP2 specific CD8+ T cells than LCL and also stimulated EBV specific CTL from PTLD patients. Both, DCs and LCLs stimulations led to the expansion of high affinity T cells, capable to target EBV transformed B cells. While LCLs and DCs expressed MHC class I and II products at similar levels, DCs showed a higher expression of costimulatory and adhesion molecules. This resulted in more efficient T cell conjugate formation with DCs than with LCLs. We propose the use of DCs for stimulation of EBV specific T cells in active or passive immunotherapy of EBV associated malignancies.     

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.     

Anti-Epstein-Barr virus memory T-cell response in Chediak-Higashi syndrome patients

Memory cytotoxic T-cell responses to Epstein-Barr virus transformed B cells were studied in two EBV seropositive Chediak-Higashi syndrome patients who were manifesting the terminal lymphoproliferative stage of the syndrome. The number of cells required for 50% regression of EBV-infected autologous lymphoblastoid cell lines was similar to that in the parents and healthy subjects. This indicates adequate memory cytotoxic T-cell control of EBV-infected B lymphocytes. Defective mechanisms other than those mediated by circulating cytotoxic cells, might be responsible for the chronic active EBV infection observed in these patients.     

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.     

Pathogenesis of chronic active Epstein‐Barr virus infection: Is this an infectious disease,lymphoproliferative disorder,or immunodeficiency?

Chronic active Epstein-Barr virus infection (CAEBV) is characterised by chronic or recurrent infectious mononucleosis-like symptoms, such as fever, hepatosplenomegaly, persistent hepatitis and extensive lymphadenopathy. Patients with CAEBV have high viral loads in their peripheral blood and/or an unusual pattern of EBV-related antibodies. This disease is rare but severe with high morbidity and mortality. Nearly three decades have passed since this disease was first identified, and recent advances in technology have increased our understanding of CAEBV pathophysiology. There is accumulating evidence that the clonal expansion of EBV-infected T or natural killer (NK) cells plays a central role in the pathogenesis of CAEBV. However, it remains unclear whether CAEBV is truly a monoclonal lymphoproliferative disorder. EBV-infected T or NK cells are able to evade the host cellular immune system due to the limited expression of viral proteins of reduced antigenicity. Recent studies suggest that infection of T or NK cells is a common event during primary EBV infection. A defect or single nucleotide polymorphism in host immune-modulating genes may allow for the expansion of virus infected cells giving rise to CAEBV. In this review, I summarise our current understanding of the pathogenesis of CAEBV and propose a model of CAEBV pathogenicity.     

Differential cellular susceptibility to Epstein-Barr virus infection in a patient with X-linked lymphoproliferative disease

Epstein-Barr virus (EBV) is often associated with lethal lymphoproliferative diseases in immunologically compromised individuals. Recently, we have studied a 20-month-old boy with X-linked lymphoproliferative disease (XLP) who had succumbed to infectious mononucleosis (IM) complicated by fulminant hepatitis and virus-associated hemophagocytic syndrome following EBV infection. EBV genomes were detected in peripheral blood lymphocytes (PBL), cervical and mesenteric lymph nodes, liver, spleen, thymus, and bone marrow. According to restriction endonuclease analyses, the EBV-DNA pattern was similar in all samples except for the EBV-DNA from the bone marrow. Additionally, circular EBV-DNA (suggesting a latent infection) predominated in spontaneously established lymphoblastoid cell lines (LCLs) derived from both the lymph node and cord lymphocytes co-cultured with PBL. In contrast, both circular and linear EBV-DNA (suggesting a lytic infection) were noted in spontaneously established LCLs derived from his PBL. Furthermore, LCLs derived from both the lymph node and cord lymphocytes co-cultured with PBL expressed fewer reactive cells for early antigen (EA) and viral capsid antigen (VCA) than spontaneous LCLs from his PBL, thus providing evidence for different B cellular susceptibility to EBV infection in this patient with XLP. Finally, defective EBV-specific cytotoxic T cell activity was observed in this patient. Latent EBV infected cells may easily escape immunosurveillance by the host. These findings may explain the fatal course of EBV infection in this patient.     

Transient immune deficiency in patients with acute Epstein-Barr virus infection

To study the effect of primary Epstein-Barr virus (EBV) infection on antigen-specific antibody production, we immunized 17 college students who had developed acute infectious mononucleosis with the T-cell dependent neoantigen bacteriophage phi X174. During the early phase of infectious mononucleosis, the proportion of peripheral blood lymphocytes displaying Ia and T8 (CD8) phenotypes was increased and the T helper/suppressor (T4/T8) ratio was decreased (less than 1). These abnormalities disappeared during the convalescent phase. Correlating with EBV-induced changes in T lymphocytes, we demonstrated depressed humoral immune responses to bacteriophage phi X174 both in vivo and in vitro. In vitro coculture experiments indicated that the Ia+ suppressor T cells could inhibit antibody production and isotype switch. Removal of T8+ lymphocytes from patient T cells normalized in vitro antibody synthesis. In addition, impaired B-cell function was shown to be in part responsible for deficient antibody production. These studies demonstrate that infection with EBV affects both B and T lymphocytes and causes a broad-based transient immune deficiency in patients with uncomplicated infectious mononucleosis.     

Frequent detection of Epstein–Barr virus-infected B cells in peripheral T-cell lymphomas

Although Epstein–Barr virus (EBV) positivity has been described in peripheral T-cell lymphomas (PTCLs) in Chinese patients, the cellular lineage of EBV-harbouring cells is unknown. Forty-four cases of PTCL were therefore studied by in situhybridization (ISH) for EBV-encoded small non-polyadenylated RNA 1 and 2 (EBER), and the lineage of the EBER+ cells was determined by double labelling. The findings were further correlated with the clonality of EBV and the genotype of these EBER+ tumours. The results for the detection of EBV by ISH show that 23 of the 44 cases were EBER+. In 5/23 of the EBER+ cases, EBER was found in around 50 per cent of atypical cells and in 18/23 cases, EBER was found in a subpopulation of atypical cells. Amongst the EBER+ cases, all 15 tested showed clonal T-cell receptor gene rearrangement by Southern blot hybridization. Double labelling was successfully done in 11 EBER+ cases, and by comparison, EBER+/CD20+ B cells outnumbered the EBER+/CD3+ T cells in all these cases. EBV clonality analysis revealed that EBV was monoclonal in six EBER+ cases and biclonal in three cases. With the predominance of EBV+ B cells over EBV+ neoplastic T cells being observed in most of these cases, it is possible that the EBV-infected clonal population may be of B-cell lineage. This was supported in some cases where a faint clonal band was seen over a background smear in the gene rearrangement study of immunoglobulin heavy chain gene by polymerase chain reaction (PCR), indicating a minor B-cell clone. It is concluded that in EBV+ PTCL, EBV is preferentially localized in B cells rather than neoplastic T cells. The neoplastic T cells may support the clonal proliferation of a subpopulation of EBV+ B cells in PTCLs. © 1998 John Wiley & Sons, Ltd.     

Epstein-Barr virus-related lymph node lesion resembling autoimmune disease-like clinicopathological findings in elderly patients. Report of three cases

Three cases of Epstein-Barr virus (EBV)-related lymphoproliferative disorders in elderly patients showing autoimmune disease-associated lymphadenopathy-like clinicopathological findings have been reported. Clinically, they were characterized by systemic lymphadenopathy, "B" symptoms, polyclonal hypergammaglobulinemia, elevated serum LDH and transient presence of various autoantibodies, and absence of atypical lymphocytosis in peripheral blood. One case was associated with idiopathic thrombocytopenic purpura. The clinical course was self-limiting. Histologically, they exhibited numerous lymphoid follicles with hyperplastic germinal centers and atypical interfollicular widening with prominent vascular proliferation. In the paracortical area, there was a mixed infiltrate comprising small to medium-sized lymphocytes and plasma cells, and variable numbers of eosinophils and T- and B-immunoblasts. In situ hybridization demonstrated a varying number of EBV-infected lymphocytes in the germinal center as well as in the interfollicular area. Polymerase chain reaction demonstrated that neither clonal rearrangement of T-cell receptor gamma-gene nor immunoglobulin heavy-chain rearrangement was detected in two of the cases examined. Although acute EBV infection rarely occurs in older adults, EBV related to reactive lymphoproliferative disorder should be added to the differential diagnosis of autoimmune disease-associated lymphadenopathy and node-based peripheral T-cell lymphoma in elderly patients.     

Plasmacytic hyperplasia in age-related Epstein-Barr virus-associated lymphoproliferative disorders: a report of two cases

Age-related Epstein-Barr virus (EBV)+B-cell lymphoproliferative disorder (LPD) is a recently recognized entity that occurs in patients over 40 years of age without any known immunodeficiency. However, this entity is thought to be related to the immunological deterioration that is part of the aging process. Histologically, age-related EBV+B-cell LPDs are classified into the polymorphous subtype and large B-cell lymphoma subtypes. However, plasmacytic hyperplasia, which is thought to be the earliest recognizable EBV+PT-LPD, has not been reported among EBV+B-cell LPDs. We report here two cases of age-related EBV-associated LPD and demonstrate the histological evolution. Pathologically, the initial lymph node biopsy specimens from both cases showed the classical Hodgkin lymphoma-like polymorphous subtype. Hodgkin (H) and Reed-Sternberg (RS) cells were CD3-, CD20+, CD15-. CD30+, CD45RB+, and latent membrane antigen-1+. In-situ hybridization (ISH) study demonstrated that numerous H and RS cells contained EBV-encoded small RNA (EBER)+. Repeated lymph node biopsy specimens from each case contained a mixture of lymphoid cells with prominent plasma cell differentiation, including immunoblasts without atypia. A portion of B-immunoblasts were CD30+ and EBER+. As assessed by polymerase chain reaction (PCR) assay, only the initial biopsy specimen in Case 1 displayed a solitary faint immunoglobulin heavy chain (IgH) gene rearrangement, consistent with the presence of a small clonal B-cell population. However, PCR analyses for EBV-genomes demonstrated the same single clonal infection of EBV in the initial and recurrent lymph node lesions in the present two cases. These two cases demonstrated the presence of plasmacytic hyperplasia in age-related EBV+B-cell LPDs, and plasmacytic hyperplasia also appears to be the earliest lesion of age-related EBV+B-cell LPDs.