Association of Epstein-Barr virus with human immunodeficiency virus-negative peripheral T-cell lymphomas in Japan

The association of Epstein-Barr virus (EBV) with human immunodeficiency virus-negative T-cell lymphoma was examined in 68 patients using the polymerase chain reaction (PCR) with DNA obtained from formalin-fixed paraffin-embedded tissues and an in situ hybridization technique. EBV-encoded RNA (EBER) was detected in 43 of 68 cases (63%) of peripheral T-cell lymphoma: in 100% (11 of 11 cases) of NK/T-cell lymphomas, 70% (14 of 20 cases) of angioimmunoblastic T-cell lymphomas (AILT) and 49% (18 of 37 cases) of other types of peripheral T-cell lymphoma. A positive band was also detected at high incidence (36 of 65 cases; 55%) in a PCR analysis using primers to detect the Bam HI-W fragment of EBV. In the immunohistochemical analysis using a monoclonal antibody to latent membrane protein 1 (LMP-1) of EBV, one of the EBV-encoded latent gene products, LMP-1, was found to be expressed in 13 of 64 cases (20%), but EBNA-2 was not expressed in all the cases examined (0 of 59 cases; 0%). The 5-yr survival rate was 28% for peripheral T-cell lymphomas overall, 0% for NK/T-cell lymphomas, 38% for AILTs and 28% for other types of peripheral T-cell lymphoma. The difference in the overall survival rate between NK/T-cell lymphoma and non-NK/T-cell lymphoma was significant (P = 0.0498 by Log-rank test). Among peripheral T-cell lymphoma patients overall, the group severely infected with EBV (EBER-ISH ++) had a lower 5-yr survival rate (8%) than the group slightly (EBER-ISH +) or not infected (38%; P = 0.0013).     

Frequent occurrence of B-cell lymphomas in angioimmunoblastic T-cell lymphoma and proliferation of Epstein-Barr virus-infected cells in early cases

Secondary lymphomas occurring in the setting of angioimmunoblastic T-cell lymphoma (AILT) are considered to be rare. Their occurrence has been attributed to Epstein-Barr virus (EBV)-associated lymphoproliferations. A previous study detected a dysregulated hypermutation process in B-cells of AILT. The present study aimed at estimating the frequency of B-cell lymphomas in AILT. By studying the expression of EBV and activation-induced cytidine deaminase (AID) as an indicator of hypermutating cells, we assessed whether B-cell lymphoproliferations in AILT were strictly associated with EBV and whether hypermutation might contribute to lymphomagenesis. Among 161 cases of AILT, diagnosed between 1996 and 2005 at the lymph node registry, Frankfurt, Germany, 19 cases were detected that also had B-cell non-Hodgkin lymphoma (NHL) and two cases had classical Hodgkin lymphoma (HL). EBV was detected in tumour cells of 7/18 NHL and both HL, suggesting that factors other than EBV contribute to lymphomagenesis. AID was expressed in AILT in large cells disseminated in the tissue, implying that the process of somatic hypermutation is ongoing in AILT, although the GC architecture is disrupted. This might be relevant in the development of secondary lymphomas.     

Epstein-Barr virus infection and associated products (LMP,EBNA2, vIL-10) in nodal non-Hodgkin's lymphoma of human immunodeficiency virus-negative Japanese

Sixty cases of B-cell nodal non-Hodgkin's malignant lymphoma (B-ML), and 46 cases of T-cell nodal lymphoma (T-ML) were surveyed for Epstein-Barr virus (EBV) genomes, RNA, and associated proteins. We used a Southern blot analysis, polymerase chain reaction (PCR), and EBV-encoded small RNA-1 (EBER-1) in situ hybridization to investigate the presence of EBV. We performed an immunohistochemical study on EBV-related oncoproteins, such as EBV-determined nuclear antigen-2 (EBNA-2), latent membrane protein (LMP), and viral interleukin-10 (vIL-10). In addition, we also analyzed the terminal repetitive sequence of EBV (EBV-TR) to investigate the EBV-infected cell clonality. Non-Hodgkin's lymphomas were grouped into three types by number of EBV-infected cells: I) almost all lymphoma cells showed an EBV presence; II) some scattered lymphoma cells showed an EBV presence; and III) only a few cells showed such a presence, which was probably due to a latent EBV infection. In 25 of 60 B-MLs, EBV-infected cells were found; 7 were type I, 1 was type II, and 17 were type III. In 27 of 46 T-MLs, EBV-infected cells were found; no cases were type I, 5 cases were type II, and 22 cases were type III. Seven B-MLs and 3 T cell lymphomas showed clonal TR bands. Expression of EBNA-2 was found in only three B-MLs, whereas LMP was seen in four B-MLs and six T-MLs. All EBNA-2/LMP-positive cases showed an EBV presence. In B-MLs, expression of EBNA-2 and LMP was detected in almost all lymphoma cells; In T-MLs, however, LMP was found in only a small portion of the lymphoma cells. Expression of IL-10 was closely associated with LMP. In summary, it was thus speculated that EBV infection was associated with the various states of lymphomagenesis. © 1996 Wiley-Liss, Inc.     

Pediatric human immunodeficiency virus infection and circulating IgD+ memory B cells

Levels of circulating naive and memory B cells were measured in human immunodeficiency virus (HIV)-infected children and control subjects to determine whether the irreversible depletion of memory B cells described in HIV-infected adults occurs in children with HIV infection. Depletion of circulating IgD+ memory B cells was seen in HIV-infected children despite control of the HIV load with highly active antiretroviral therapy (HAART) (P =. 04). IgD+ memory B cell percentages did not correlate with CD4+ cell percentages (P =. 027) or disease duration (P =. 026). Naive/transitional and IgD- memory B cell numbers were not affected. Pediatric HIV infection is associated with selective depletion of circulating IgD+ memory B cells despite control of the HIV load with HAART.     

Detection of Epstein-Barr virus in Korean peripheral T-cell lymphoma

One hundred thirty-seven patients with peripheral T-cell lymphomas (PTL) were examined for the presence of Epstein-Barr virus (EBV) using in situ hybridization for EBV-encoded RNA (EBER) and Bam H-fragment, lower strand frame (BHLF) and immunohistochemical stain for latent membrane protein (LMP). EBER was detected in tumor cells in 79 cases (58%); 26/66 PTL, unspecified (39%), 3/4 AILD (75%), 47/51 angiocentric lymphomas (AL) (92%), and 3/13 anaplastic large cell lymphoma (ALCL) (23%) by Revised European-American Lymphoma (REAL) classification. EBER was detected in 17/36 nodal (47%) vs. 62/101 extranodal PTLs (61%); 21/24 nasal, 12/32 Waldeyer's ring, 9/13 gastrointestinal, and 20/32 skin and soft tissue PTL. AL was consistently associated with the highest frequency of EBER among the extranodal PTL: nose (19/20), GI tracts (3/3), skin (14/15), and Waldeyer's ring (11/14). In extranodal lymphomas, coagulative-type zonal necrosis was seen almost exclusively in AL and showed correlation with EBER-positivity (P < 0.01). LMP was detected in 24 among 107 cases tested (22%). No signal for BHLF was detected in 76 cases tested, implying absent or negligible incidence of lytic infection. In conclusion, high incidence of EBV was observed in PTL among Koreans, with predilection for angiocentric lymphomas and extranodal presentation, especially involving nose, skin, and gastrointestinal tract.     

Heterogeneous Epstein-Barr virus infection patterns in peripheral T-cell lymphoma of angioimmunoblastic lymphadenopathy type.

In this study, 32 cases of T-cell lymphoma of angioimmunoblastic lymphadenopathy type (AILD-TCL) were investigated for their association with Epstein-Barr virus (EBV). For this purpose, three different approaches were applied: polymerase chain reaction (PCR) for the presence of EBV-DNA, in situ hybridization (ISH) for EBV-encoded small nuclear RNAs (EBER), and immunohistology for EBV-encoded latent membrane protein (LMP). PCR and EBER-ISH produced almost identical results, showing that all but one case of AILD-TCL contained EBV genomes. Three distinctive patterns of EBV infection were observed after immunophenotypical characterization of EBER-positive cells: (1) in 26% of the cases, B and T cells were infected, the majority of which were B cells of immunoblastic morphology located in the remnants of lymphoid follicles; (2) in 42% of the cases, the vast majority of infected cells were neoplastic T cells diffusely distributed in the lymph nodes, but infected B cells were also present; and (3) in 32% of the cases, there were only a few infected small lymphoid cells. Detectable LMP was frequent in cases exhibiting patterns 1 and 2. These findings suggest that in AILD-TCL patients, B cells and especially T cells are highly susceptible to a persistent EBV infection, which often leads to a growth advantage of the infected cells. Thus EBV, in conjunction with genetic abnormalities and selective defects of the immune system, might be involved in the pathogenesis of AILD-TCL.     

Productive infection of T cells in lymphoid tissues during primary and early human immunodeficiency virus infection

Current models suggest that during human immunodeficiency virus type 1 (HIV-1) transmission virions are selected that use the CCR5 chemokine receptor on macrophages and/or dendritic cells. A gradual evolution to CXCR4 chemokine receptor use causes a shift in the proportion of productively infected cells to the CD4 cell population. Productively infected cells during acute and early infection in lymphoid tissue were assessed, as well as the impact of productive infection on the T cell population in 21 persons who had biopsies performed on days 2-280 after symptoms of acute HIV-1 seroconversion. Even in the earliest stages of infection, most productively infected cells were T lymphocytes. There were sufficient infected cells in lymphoid tissue (LT) to account for virus production and virus load in plasma. Despite the relatively high frequency of productively infected cells in LT, the impact on the size of the T cell population in LT at this stage was minor.     

High expression of latent membrane protein 1 of Epstein-Barr virus and BCL-2 oncoprotein in acquired immunodeficiency syndrome-related primary brain lymphomas

Nearly all primary brain lymphomas in acquired immunodeficiency syndrome (AIDS) patients are associated withEpstein-Barr virus (EBV). The role of EBV in lymphomagenesis is not totally elucidated. One possible mechanism is the overexpression of the BCL-2 oncoprotein, because the latent membrane protein 1 (LMP1) has been reported to transactivate the bcl-2 gene in vitro. To study the interrelationship beetween LMP1 and BCL-2 in vivo, we have analyzed and compared their expression in 11 AIDS-related primary brain lymphomas and 57 AIDS- related systemic lymphomas by immunoperoxidase technique on frozen sections. In AIDS-related primary brain lymphoma, LMP1 and BCL-2 were expressed in all cases but 1. All positive cases exhibited morphologic immunoblastic features. In contrast, the only negative case was histologically close to Burkitt's lymphoma. In systemic lymphomas, LMP1 was expressed in 21 cases, whereas BCL-2 was positive in only 3 cases, all of which were extranodal. These results indicate that, in addition to the histologic type, the role of EBV genes and BCL-2 expression in lymphomatous cells differ as a function of their localization. In AIDS- related primary brain lymphomas, this correlation between LMP1 and BCL- 2 overexpression may have a major implication in lymphomagenesis.     

Simultaneous multiorgan presence of human herpesvirus 8 and restricted lymphotropism of Epstein-Barr virus DNA sequences in a human immunodeficiency virus-negative immunodeficient infant

Because a profound dysregulation of the immune system occurs in primary immunodeficiencies, viral infections are not uncommon. Human herpesvirus (HHV)-8 DNA was detected by polymerase chain reaction (PCR) analysis, Southern blotting, and in situ hybridization (ISH) in peripheral blood mononuclear cells and lymphoid organs (bone marrow, spleen, and lymph nodes) and endothelial and epithelial cells and macrophages from several organs (skin, lung, esophagus, intestine, choroid plexus [but not in brain or cerebellum], heart, striated muscle, liver, and kidney) of a human immunodeficiency virus-negative infant with DiGeorge anomaly who died of disseminated infection. Epstein-Barr virus DNA sequences were detected in the spleen and lymph nodes (by PCR and ISH) and in bone marrow (only by ISH) but not in blood or nonlymphoid organs. This report is believed to be the first of multiorgan dissemination of HHV-8 in a primary immunodeficiency.     

Influence of Epstein-Barr virus infection in adult T-cell leukemia

The involvement of adult T-cell leukemia (ATL) cells in organs such as the skin and lymph nodes is observed in about 50% of cases of ATL. Epstein-Barr virus (EBV) infection has often been observed in the clinical course of ATL. In this study, we established two B-cell lines from peripheral blood of patients with ATL. EBV DNA, proviral DNA for HTLV-1 and Tax mRNA were detected in both lines. As part of the characterization of these cells, an enhanced expression of intercellular adhesion molecule-1 (ICAM-1) (CD54) or ICAM-3 (ICAM-3) (CD50), lymphocyte function-1 (LFA-1) (CD11a/CD18), and Mac-1 (CD11b/CD18) was observed. To investigate the role of the interaction of these viruses, we transfected EBV and/or HTLV-1 into a healthy donor's lymphocytes, an EBV-infected B cell line, Raji, and a HTLV-1 negative T-cell line, Jurkat. Enhanced expression of adhesion molecules was also observed in double transfectants (EBV and HTLV-1). In the clinical course of ATL, LMP-1, EBNA-2, CD50 and CD54 were detected in lymph nodes and skin specimens by immunohistochemical staining. Furthermore, high levels of interleukin-4 (IL-4) were detected in these cell lines and transfectants. The results indicated that coinfection with HTLV-1 and EBV may induce aggressive organ involvement through the enhanced expression of adhesion molecules via IL-4 signaling. A new mechanism of ATL involvement is discussed.     

Positive Epstein-Barr virus heterophile antibody tests in patients with primary human immunodeficiency virus infection

PURPOSE: To describe three cases of primary human immunodeficiency virus (HIV) infection in patients who had laboratory studies consistent with infectious mononucleosis. SUBJECTS: We describe 3 patients who presented with a viral syndrome, had a positive heterophile antibody test, and were diagnosed with primary HIV infection. RESULTS: The results of Epstein-Barr virus serology studies in each of these patients were consistent with chronic, but not acute, Epstein-Barr virus infection. HIV antibody tests were negative, and HIV RNA was >500,000 copies/mL in each patient. CONCLUSIONS: Clinicians should recognize that a positive heterophile antibody test in the setting of an acute viral illness does not exclude the diagnosis of primary HIV infection, although reactivation of latent Epstein-Barr virus infection cannot be ruled out. Patients presenting with nonspecific viral syndromes should be assessed for HIV risk behaviors and tested for primary HIV infection when appropriate.     

CD8 T cells specific for human immunodeficiency virus, Epstein-Barr virus, and cytomegalovirus lack molecules for homing to lymphoid sites of infection.

CD8 T cells are classified as na?ve, effector, or memory cells on the basis of CD45RA, CD62L, and CCR7 expression. Sequential engagement of cell-surface CD62L and CCR7 receptors is required for efficient trafficking to lymphoid tissue by means of high endothelial venules. Na?ve CD8 T cells are CCR7(+)CD62L(+) CD45RA(+), whereas long-term memory cells are CCR7(+)CD62L(+)CD45RA(-). Effector cytotoxic T cells are thought to be CCR7(-)CD45RA(+). The distribution of CD8 subsets and cytolytic protein expression in healthy donors and donors seropositive for human immunodeficiency virus (HIV) were compared. In HIV-infected subjects, CCR7(-) CD8 T cells expanded at the expense of na?ve and long-term memory cells. In both healthy donors and HIV-infected donors, CCR7(+) CD8 T cells were uniformly negative for perforin. In all subsets, perforin and granzyme A were not coordinately expressed, with perforin expression being more tightly regulated. The properties of CD8 T cells specific for cytomegalovirus, Epstein-Barr virus (EBV), and HIV were studied by staining with major histocompatibility complex peptide tetramers. Antigen-specific cells for chronic infections with these viruses were uniformly CCR7(-) and predominantly CD62L(-). In 2 HIV-seropositive donors, 3- to 4-fold fewer EBV-tetramer-positive cells were present in lymph nodes compared with blood. Antigen-specific CD8 T cells are therefore preferentially excluded from lymphoid sites, even when infection is primarily in lymphoid tissue. This may protect lymphoid tissues from immunopathological changes but compromise immune defense against viruses, such as HIV and EBV, that target lymphocytes. HIV-specific CD8 T cells do not express CD45RA, whereas EBV- and CMV-specific CD8 T cells are heterogeneous in CD45RA(+) expression. Lack of CD45RA expression may indicate incomplete differentiation of HIV-specific CD8 T cells to cytotoxic T cells.     

Filarial infections increase susceptibility to human immunodeficiency virus infection in peripheral blood mononuclear cells in vitro

Because helminth infections and human immunodeficiency virus (HIV) coexist in areas where the spread of AIDS is most dramatic, their in vitro interaction was explored. Cryopreserved peripheral blood mononuclear cells (PBMC) from patients with filarial infections (n=24) and from unexposed control subjects (n=12) were depleted of CD8 T cells and were infected with macrophage (M)- and T cell-tropic viruses. A trend toward increased HIV replication in PBMC from filaria-infected patients was observed. Furthermore, PBMC from 6 filaria-infected patients before antifilarial treatment were significantly more susceptible to replication of M-tropic virus than their posttreatment PBMC (P=.03). No intergroup differences were found in the surface expression of HLA-DR, CD25, CCR5, CXCR4, CCR3 on CD4 T cells, or monocytes before infection. PBMC from filaria-infected patients produced less RANTES (P=.02) but more intracellular interleukin-4 than those of control subjects. Thus, PBMC from persons with filarial infections appear to have enhanced susceptibility to HIV-1 infection mediated by an undetermined mechanism.     

Epstein-Barr virus induces Fas (CD95) in T cells and Fas ligand in B cells leading to T-cell apoptosis.

Epstein-Barr virus (EBV) acute infectious mononucleosis (AIM) is characterized by transient immunosuppression in vivo and increased T-cell apoptosis after ex vivo culture of AIM peripheral blood mononuclear cells. We undertook experiments to test whether EBV or purified virion envelope glycoprotein gp350 could contribute to Fas-mediated T-cell apoptosis. Our in vitro results indicate that EBV increased Fas expression in CD4(+) T cells and Fas ligand (FasL) expression in B cells and macrophages. Purified gp350 was also shown to significantly increase CD95 expression in CD4(+) T cells. When T-cell CD95 was cross-linked, EBV-stimulated T cells underwent apoptosis. The induction of T-cell CD95 by EBV followed by CD95 cross-linking with anti-CD95 monoclonal antibody resulted in a loss in the number of T cells responding to the T-cell mitogens, anti-CD3 antibody, and interleukin-2. These results indicate that, in addition to serving as a principal ligand for the attachment of virus to target cells, gp350 may also act as an immunomodulatory molecule that promotes T-cell apoptosis.     

肠道T细胞淋巴瘤中EB病毒感染的研究

目的 了解我国肠道T细胞淋巴瘤（ITCL）中EB病毒（EBV）潜伏感染的状态，其亚型的感染情况，基因产物表达与EBV阳性细胞的性质。方法采用PCR检测42例ITCL中的不同EBV亚型的核抗原基因（EBNA－3C），并对其扩增产的行DNA序列分析。运用EBER1／2－RNA原位杂交证实EBV潜伏感染，IHC／ISH双重染色技术判断EBV阳性细胞性质。免疫组化检测EBV基因产物（LMP－1，EBNA－2）的表达。结果 42例ITCL中EBV的阳性率为97．6％，以A型EBV感染居多（32／38例，84．2％），B型为2／38例（5．3％），混合型为4／38例（10．5％）。EBNA－3C基因的PCR产物DNA序列中存在个别碱基的缺失和插入.EBER1/2的检出率为85．7％。EBER1／2阳性细胞同时表达CD45RO和TIA－1，且表达CD4，CD8及CD56的肿瘤细胞呈EBER1／2阳性。16／42例ITCL（38．1％）表达LMP－1。ITCL中EBV的潜伏感染模式多为I型（24／42，66．7％），Ⅱ型次之（12／42，33．4％）。结论 在我国，ITCL中存在高水平的EBV潜伏感染，且多为A型EBV感染，感染模式为Ⅰ型和Ⅱ型。部分ITCL可能与鼻NK／T细胞淋巴瘤属于同一疾病谱系。     

Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection

To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred syngeneic lymphocytes after ex vivo expansion and transduction with a chimeric receptor gene (CD4/CD3-zeta) between identical twins discordant for HIV infection. Single and multiple infusions of 10(10) genetically modified CD8(+) T cells resulted in peak fractions in the circulation of approximately 10(4) to 10(5) modified cells/10(6) mononuclear cells at 24 to 48 hours, followed by 2- to 3-log declines by 8 weeks. In an effort to provide longer high-level persistence of the transferred cells and possibly enhance anti-HIV activity, we administered a second series of infusions in which both CD4(+ )and CD8(+) T cells were engineered to express the chimeric receptor and were costimulated ex vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions of approximately 10(3) to 10(4) modified cells/10(6) total CD4(+) or CD8(+) cells persisted for at least 1 year. Assessment of in vivo trafficking of the transferred cells by lymphoid tissue biopsies revealed the presence of modified cells in proportions equivalent to or below those in the circulation. The cell infusions were well tolerated and were not associated with substantive immunologic or virologic changes. Thus, adoptive transfer of genetically modified HIV-antigen-specific T cells was safe. Sustained survival in the circulation was achieved when modified CD4(+ )and CD8(+) T cells were infused together after ex vivo costimulation, indicating the important role played by antigen-specific CD4(+) T cells in providing "help" to cytotoxic effectors. (Blood. 2000;96:467-474)