CLINICOPATHOLOGICAL,IMMUNOLOGICAL AND GENETIC STUDIES OF CD30+ ANAPLASTIC LARGE CELL LYMPHOMA OF B-CELL TYPE; ASSOCIATION WITH EPSTEIN–BARR VIRUS IN A JAPANESE POPULATION

The clinicopathological features, the immunophenotype, and the presence of Epstein–Barr virus (EBV)-associated genomes and gene products were examined in 17 cases of CD30⁺ anaplastic large cell lymphoma (ALCL) of B-cell type. Microscopically, the 17 cases were divided into ten cases of the monomorphic type and seven cases of the pleomorphic type. EBV was detected in 6 of 17 cases (38 per cent) by RNA in situ hybridization (ISH) with EBV-encoded RNA (EBER1). EBER1⁺ cases consisted of two cases (20 per cent) of the monomorphic type and four cases (57 per cent) of the pleomorphic type. The five EBER1⁺ cases showed clonality of the EBV genome by Southern blotting, consistent with the presence of EBV in a monoclonal proliferation. The EBV-encoded latent membrane protein 1 (LMP1) was found in all six EBER1⁺ cases and EBV-encoded nuclear antigen 2 (EBNA2) was present in two cases by immunohistochemistry. No expression of LMP1 or EBNA2 was observed in the EBER1⁻ cases. The EBER1⁺ cases had a tendency for a more favourable prognosis than the EBER1⁻ cases. It is concluded that EBV has an association with CD30⁺ ALCL of B-cell type in the Japanese population studied, and especially with the large pleomorphic type. EBV infection may play a pathoaetiological role and may influence clinical behaviour.     

Long-term carriers generate Epstein-Barr virus (EBV)-specific CD4(+) and CD8(+) polyfunctional T-cell responses which show immunodominance hierarchies of EBV proteins

T cells simultaneously producing multiple cytokines and possessing cytotoxic capacity termed polyfunctional cells (PFCs) are increasingly recognized as the immune correlate of protection against pathogenic viruses. We investigated co‐expression of four cytokines (interferon‐γ, macrophage inflammatory protein 1‐α, tumour necrosis factor‐α and interleukin‐2) and degranulation capacity (CD107a surface expression) of Epstein–Barr virus (EBV) ‐specific CD4⁺ and CD8⁺ T cells upon stimulation by overlapping peptides of EBV lytic (BZLF1) and latent (EBNA1, EBNA3 and LMP2) proteins, in 20 healthy Chinese long‐term carriers. Two patients with post‐transplant lymphoproliferative disorder (PTLD), who had impaired T‐cell immunity, were studied for comparison. Both EBV‐specific CD4⁺ and CD8⁺ PFCs were readily generated in long‐term carriers and showed immunodominance hierarchies of latent proteins (EBNA1 > EBNA3/LMP2 and EBNA3 > LMP2 > EBNA1 for CD4⁺ and CD8⁺ T cells, respectively), as evidenced by a higher proportion of PFCs generated by immunodominant EBV proteins than by subdominant viral proteins. In contrast, the proportion of EBV‐specific PFCs was markedly decreased in patients with PTLD. The EBV‐specific PFCs produced more cytokine per cell than single‐functional T cells and comprised different subsets. Five‐functional CD4⁺ and CD8⁺ T cells were detected and four‐functional CD4⁺ T cells were mainly CD107a negative and expressed all four cytokines whereas four‐functional CD8⁺ T cells were mainly CD107a positive and expressed three of the four cytokines (interleukin‐2‐negative). We conclude that EBV‐specific PFCs are generated in much higher proportions in the long‐term carriers than in the patients with PTLD and maintain the immunodominant characteristics of the virus.     

Epstein-Barr virus infection and its gene expression in gastric lymphoma of mucosa-associated lymphoid tissue

The role of Epstein-Barr virus (EBV) in the pathogenesis of gastric lymphoma of mucosa-associated lymphoid tissue (MALT) has not been well understood. The aim of the study was to investigate EBV infection and its gene expression in this tumor in order to understand its role in the pathogenesis. EBV infection was screened by in situ hybridization for EBV-encoded nonpolyadenylated RNA (EBER ISH) in 79 cases of gastric MALT lymphoma of nonimmunocompromised patients. The expression of EBV proteins [LMP1 (latent membrane protein 1), EBNA2 (EBV nuclear antigen 2), ZEBRA (switch protein encoded by BZLF1 gene)] was studied by immunohistochemistry in EBER-positive cases. EBV was detected with EBER ISH in 15 (19%) of the 79 cases. EBV was found in virtually all tumor cells in 2 cases of high-grade MALT lymphoma (2.5%) (EBV-associated), and was found only in occasional large or small lymphoid cells in 13 cases (16.5%). False positive EBER signal was detected in the mucinous glandular epithelial cells of gastric antrum with FITC-labeled oligonucleotide probe but not with digoxigenin or ³⁵S-labeled riboprobes. Type II latency (EBER+LMP1+ EBNA2-) was detected in both EBV-associated cases. Type III latency (EBER+LMP1+EBNA2+) was also identified in one EBV-associated case besides latency II. Double labeling showed coexpression of LMP1 and EBNA2 in a small number of tumor cells, indicating the presence of type III latency in single cell level. In cases with only occasional EBER-positive large or small lymphoid cells, LMP1 and EBNA2 were not detected. ZEBRA was negative in all the cases. These findings suggest that EBV may contribute to the pathogenesis of a small proportion of high-grade MALT lymphoma, where virtually all tumor cells harbored EBV and the oncogenic viral protein LMP1 was expressed. Moreover, latency III of EBV infection may exist in nonimmunocompromised patient. J. Med. Virol. 56:342–350, 1998 . © 1998 Wiley-Liss, Inc.     

Detection of Epstein–Barr virus‐specific memory CD4+ T cells using a peptide‐based cultured enzyme‐linked immunospot assay

Approaches to evaluate T‐cell responses to Epstein–Barr virus (EBV) include enzyme‐linked immunospot (ELISPOT), which quantifies cells capable of immediate interferon‐γ secretion upon antigen stimulation. However, evaluation of expandable EBV‐specific memory T cells in an ELISPOT format has not been described previously. We quantified EBV‐specific T‐cell precursors with high proliferative capacity by using a peptide‐based cultured interferon‐γ ELISPOT assay. Standard and cultured ELISPOT responses to overlapping peptide pools (15‐mers overlapping by 11 amino acids) covering the lytic (BZLF1 and BMRF1) and latent (EBNA1, EBNA3a, EBNA3b, EBNA3c, LMP1 and LMP2) EBV proteins were evaluated in 20 healthy subjects with remote EBV infection and, for comparison, in four solid organ transplant recipients. Cultured ELISPOT responses to both lytic and latent EBV antigens were significantly higher than standard ELISPOT responses. The distribution of EBV‐specific T‐cell responses detected in healthy virus carriers showed more consistent cultured ELISPOT responses compared with standard ELISPOT responses. T‐cell responses quantified by cultured ELISPOT were mainly mediated by CD4⁺ T cells and a marked pattern of immunodominance to latent‐phase antigens (EBNA1 > EBNA3 family antigens > LMP2 > LMP1) was shown. Both the magnitude and distribution of EBV‐specific T‐cell responses were altered in solid organ transplant recipients; in particular, cultured ELISPOT responses were almost undetectable in a lung‐transplanted patient with EBV‐associated diseases. Analysis of T‐cell responses to EBV by ELISPOT assays might provide new insights into the pathogenesis of EBV‐related diseases and serve as new tools in the monitoring of EBV infection in immunocompromised patients.     

The impact of HLA class I and EBV latency‐II antigen‐specific CD8+ T cells on the pathogenesis of EBV+ Hodgkin lymphoma

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein–Barr virus (EBV) latency‐II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV⁺cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA‐A*02 is protective in EBV⁺cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA‐A*02^– versus HLA‐A*02⁺ EBV⁺cHL patients, suggesting that LMP2A‐specific CD8⁺ T cell anti‐tumoral immunity may be relatively ineffective in HLA‐A*02^– EBV⁺cHL. To ascertain the impact of HLA class I on EBV latency antigen‐specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV⁺cHL, the magnitude of ex‐vivo LMP1/2A‐specific CD8⁺ T cell responses was elevated in HLA‐A*02⁺ patients. Furthermore, in a controlled in‐vitro assay, LMP2A‐specific CD8⁺ T cells from healthy HLA‐A*02 heterozygotes expanded to a greater extent with HLA‐A*02‐restricted compared to non‐HLA‐A*02‐restricted cell lines. In an extensive analysis of HLA class I‐restricted immunity, immunodominant EBNA3A/3B/3C‐specific CD8⁺ T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A‐specific responses were confined largely to HLA‐A*02. Our results demonstrate that HLA‐A*02 mediates a modest, but none the less stronger, EBV‐specific CD8⁺ T cell response than non‐HLA‐A*02 alleles, an effect confined to EBV latency‐II antigens. Thus, the protective effect of HLA‐A*02 against EBV⁺cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency‐II antigen‐specific CD8⁺ T cell hierarchies.     

Association of Epstein-Barr virus infection with p53 protein accumulation but not bcl-2 protein in nasopharyngeal carcinoma

AIMS: The aim of this study was to investigate the association of Epstein-Barr virus (EBV) infection with status of p53 protein expression in nasopharyngeal carcinoma (NPC). The expression of EBV gene and gene product, p53 protein and bcl-2 protein in NPC was histopathologically studied. METHODS AND RESULTS: In-situ hybridization using oligonucleotide probe to EBV-encoded small RNAs (EBERs) and immunohistochemistry using monoclonal antibodies against EBV latent membrane protein 1 (LMP1), p53 protein and bcl-2 proteins were performed in 56 primary NPCs. EBERs were detected in 46 (82%) cases and LMP1 in 17 (30%) cases. While 30 of 32 (94%) cases in differentiated nonkeratinizing carcinoma (NKC, WHO type 2) and 16 of 17 (94%) cases in undifferentiated carcinoma (UC, WHO type 3) showed EBERs expression, neither five cases of keratinizing squamous cell carcinoma (KSCC, WHO type 1) nor two cases of adenocarcinoma showed EBERs. bcl-2 protein was detected in 50 (89%) cases, but its expression did not depend on expression of LMP1. p53 protein was detected in 31 (55%) cases, and there was a correlation between expression of EBERs and p53 protein (P < 0.05) but not between LMP1 and p53 protein. CONCLUSION: In this study, close association of NKC and UC but not KSCC with the latent infection with EBV was demonstrated. The induction of bcl-2 protein by LMP1, as shown in vitro, was not demonstrated. The association between overexpression of p53 protein and the presence of EBV suggests that some EBV-encoded protein, which may be different from LMP1, may play a role for nuclear accumulation of p53 protein.     

Epstein-Barr virus latent and replicative gene expression in oral hairy leukoplakia

Oral hairy leukoplakia is an epithelial lesion of the tongue associated with productive infection by Epstein-Barr virus (EBV). However, no data concerning the pattern of EBV latent gene expression have been reported, and it remains unresolved whether true latent infection occurs in basal cell layers of oral hairy leukoplakia. We have studied six cases of oral hairy leukoplakia using monoclonal antibody immunohistology for EBV latent--EB nuclear antigen (EBNA) 1, EBNA 2 and latent membrane protein 1 (LMP 1); immediate-early (BZLF1); and replicative (EA, VCA, MA) proteins, and for the EBV-receptor (CD21 antigen). EBV DNA was demonstrated by nucleic acid in situ hybridization. Mid- to upper-zone keratinocytes contained EBV DNA and co-expressed EBNA 1, EBNA 2 (5 of 6 cases), LMP 1, BZLF1 protein, EA, VCA and MA. No EBV genome or gene expression could be demonstrated in basal or parabasal cells. Spinous keratinocytes were labelled by anti-CD21 antibodies HB5 and B2, but did not express the EBV-receptor as defined by reactivity with OKB7. The co-expression of latent and replicative infection-associated antigens is striking, indicating possible functional roles for latent proteins during the productive cycle. Our results suggest that oral hairy leukoplakia is caused by repeated direct infection of upper epithelial cells with virus from saliva or adjacent replicatively infected cells, rather than by a latent EBV infection of basal epithelial cells with a differentiation-dependent switch to productive infection as previously proposed.     

CD21非依赖性EB病毒对人胃印戒细胞癌细胞系的感染

目的　探讨CD2 1非依赖性EB病毒 (EBV)对人胃印戒细胞癌细胞系 (HSC 39)的感染作用。方法　用Akata和P3HR 1EBV毒株感染HSC 39,有限稀释法对感染细胞进行克隆。结果　两种EBV毒株感染细胞中均可检测到EBV编码的小RNA(EBER)的表达 ,两种EBV毒株感染的亲代细胞及大多数细胞克隆表达EBV核抗原 (EBNA1) ,但不表达EBNA2、潜伏期膜蛋白 (LMP1)和LMP2A。表现为潜伏Ⅰ型感染。未感染的HSC 39细胞及P3HR 1感染的细胞克隆CD2 1表达阴性 ,而AkataEBV感染的部分细胞克隆CD2 1mRNA阳性。结论　EBV可能通过不依赖CD2 1受体的途径感染HSC 39,印戒细胞癌细胞系可用作EBV感染的靶细胞。     

鼻咽癌组织中EB病毒潜伏相关基因的表达

目的： 通过检测鼻咽癌组织中EB病毒的潜伏膜蛋白LMP1的序列以及LMP1、EBNA1、EBNA2的mRNA表达来探讨EB病毒的感染状态及其表达产物与鼻咽癌的关系。方法: 应用PCR法检测鼻咽癌组织中LMP1 DNA的存在，并对鼻咽癌来源的LMP1和EB病毒永生化狨猴B淋巴细胞系B95-8来源的LMP1进行测序，比较序列的差异。利用巢式RT-PCR检测鼻咽癌组织中LMP1、EBNA1、EBNA2的mRNA表达。结果: 47例鼻咽癌组织均含有LMP1 DNA，所有鼻咽癌来源的LMP1 DNA与B95-8来源的LMP1 DNA序列比较均存在着多个单核苷酸变异，最明显的是XhoⅠ酶切位点的丢失。测序后显示鼻咽癌来源的LMP1 DNA有30个核苷酸的丢失。巢式RT-PCR显示LMP1、EBNA1、EBNA2在鼻咽癌中的mRNA表达率分别为76.6％、80.0％和74.5％。其中EBNA1的表达是由Qp启动的，而B95-8细胞中EBNA1的表达是由Cp启动的。结论: 鼻咽癌中EB病毒的作用途径比较复杂，LMP1、EBNA1、EBNA2等潜伏期基因还有早期裂解基因BARF1均可能参与鼻咽癌的发生发展过程。     

2005-2010年北京地区儿童原发性EBV感染流行株EBNA1与LMP1基因特征分析

目的 分析2005-2010年北京地区儿童原发性EBV感染流行株EBNA1与LMP1基因特征,为研究EBV变异株与疾病临床表型间是否存在相关性提供背景资料.方法 应用PCR方法扩增EBV的EBNA3C、EBNA1和LMP1基因片段,测序后应用BioEdit 7.0.9和Mega 4.0.2软件进行序列分析.结果 62例进行了EBV分型,以EBV-Ⅰ型为主,检出率为98％.62例EBNA1基因扩增阳性,其中V-val亚型(均是Vvv1变异株)为98％.50例LMP1基因羧基段扩增阳性,以China 1为主,其检出率为90％.Vvv1变异株和China 1变异株在EBV-IM与EBV-HLH中的检出率差异均无统计学意义(P=1.00).40例进行了多基因连锁分析,其中EBV-Ⅰ型、EBNA1-Vvv1变异株和LMP1-China 1变异株高度连锁,其连锁检出率为90％.35例患儿LMP1基因全长扩增阳性,CG1-CG4的检出率分别为85％、6％、6％和3％.结论 北京地区儿童原发性EBV感染疾病中,EBV亚型以EBV-Ⅰ型为主,Vvv1变异株和China 1变异株分别是EBNA1和LMP1变异株中的优势变异株,且二者高度连锁.儿童原发性EBV感染流行株可以分为CG1-4组,其中以CG1为主.     

EB病毒对人胃癌细胞系HSC-39感染的研究

目的 探讨EB病毒（EBV）对胃癌细胞系的感染作用。方法 用Akata和P3HR－1 EBV毒株感染人胃癌印戒细胞系（HSC－39），有限稀释法对感染细胞进行克隆。结果 EBV感染细胞中可检测到EBV编码的核抗原（EBNA)，2种EBV毒株感染的细胞克隆表现有不同的形态学特征及生长方式。EBV感染的亲代细胞及大部分克隆表达EBNA1，但不表达EBNA2、潜伏期膜蛋白（LMP）1和LMP2A；亲代细胞及所有细胞克隆未观察到裂解感染，EBNA启动子Qp表达阳性，而启动子Cp和Wp未见表达。结论 HSC－39对2种EBV毒株均易感，EBV感染可改变HSC－39的细胞表型，且不同EBV毒株对其影响不同，提示印戒细胞癌细胞系可用作EBV感染的靶细胞。     

Lack of expression of the Epstein-Barr Virus (EBV) gene products,EBERs, EBNA1, LMP1, and LMP2A,in breast cancer cells

Epstein-Barr virus (EBV), a gamma herpesvirus, has been associated with a variety of human malignancies such as Burkitt's lymphoma, Hodgkin's lymphoma, NPC, and gastric cancer. A controversy regarding the association of EBV with breast cancers has recently been reported in the literature. These reports have mainly used the DNA detection techniques of polymerase chain reaction and Southern blot hybridization, with the inherent lacunae associated with these techniques for signal localization. Our group has studied EBV association with breast cancer by using in situ hybridization for detecting nonpolyadenylated EBV RNA (EBERs), along with using protein localization technique of immunohistochemistry, studying the EBV nuclear antigen 1 (EBNA1) and the latent membrane proteins (LMP1 and LMP2A). This is the first article analyzing the expression of LMP2A in breast cancer cells. In all of our 43 female breast cancer cases under study, we failed to detect expression of any of the EBV viral gene products tested.     

A naturally derived gastric cancer cell line shows latency I Epstein-Barr virus infection closely resembling EBV-associated gastric cancer

In a process seeking out a good model cell line for Epstein-Barr virus (EBV)-associated gastric cancer, we found that one previously established gastric adenocarcinoma cell line is infected with type 1 EBV. This SNU-719 cell line from a Korean patient expressed cytokeratin without CD19 or CD21 expression. In SNU-719, EBNA1 and LMP2A were expressed, while LMP1 and EBNA2 were not. None of the tested lytic EBV proteins were detected in this cell line unless stimulated with phorbol ester. EBV infection was also shown in the original carcinoma tissue of SNU-719 cell line. Our results support the possibility of a CD21-independent EBV infection of gastric epithelial cells in vivo. As the latent EBV gene expression pattern of SNU-719 closely resembles that of the EBV-associated gastric cancer, this naturally derived cell line may serve as a valuable model system to clarify the precise role of EBV in gastric carcinogenesis.     

EPSTEIN–BARR VIRUS IN HODGKIN'S DISEASE: MORE THAN JUST AN INNOCENT BYSTANDER

Several lines of evidence suggest that the Epstein-Barr virus (EBV) is an important factor in the pathogenesis of Hodgkin's disease (HD). This Editorial focuses on two pathogenic mechanisms probably influenced by the presence of EBV in the Hodgkin and Reed-Sternberg (H-RS) cells: resistance of the H-RS cells to apoptosis; and escape of H-RS cells from a cytotoxic T lymphocyte (CTL) mediated immune response. In addition, data are summarized implicating the latent membrane protein 1 (LMP1) as the most likely EBV-encoded protein responsible for this putative EBV-mediated pathogenic effect. It is known that, using conventional therapy regimens, the presence of EBV bears little influence on clinical presentation and treatment outcome of HD. However, the differences in regulation of both apoptosis and immune escape mechanisms between EBV⁺ and EBV⁻ cases may be important determinants of the success of immunotherapy to treat Hodgkin's disease. Thus, clarification of these mechanisms will be essential to the development of successful immunotherapeutic strategies in Hodgkin's disease. © 1997 by John Wiley & Sons, Ltd.     

Distribution of the Epstein-Barr virus in the normal stomach and gastric lesions in Thai population

The Epstein-Barr virus (EBV) is one of the infectious agents found in stomach tissue. Recently, EBV-associated gastric carcinoma (EBVaGC) was classified as a new subtype of gastric carcinoma. To date, there is a lack of knowledge about the distribution and prevalence of EBV infection in both the normal stomach and various gastric lesions, including EBVaGC, in the Thai population. In this study, we detected EBV in the normal stomach (NS; n = 19), chronic gastritis (CG; n = 36), intestinal metaplasia (IM; n = 40), gastric dysplasia (GD; n = 15), and gastric adenocarcinoma (GC; n = 33) by polymerase chain reaction (PCR) amplification of the latent membrane protein (LMP1) gene of EBV. EBV-PCR amplification was positive in 42.1%, 36.1%, 22.5%, 13.3%, and 33.3% of NS, CG, IM, GD, and GC, respectively. For further clarification in EBVaGC, we performed EBV-encoded small RNA in situ hybridization (EBER-ISH) in PCR-positive cases of GD and GC. Four GC cases were EBER-ISH positive (12.1%), while both GD cases were EBER-ISH negative. In addition, we determined the distribution of the EBV strain (type A or B) based on EBNA3C sequence and EBV variants based on LMP1 variation (wild-type and 30-bp deletion variants; wt-LMP1 or del-LMP1). The results showed that type A and wt-LMP1 were the most prevalent in all lesions. In conclusion, EBV is common in both the NS and gastric lesions, and the frequency of EBVaGC was 12.1% in Thai patients.     

The expression pattern of Epstein-Barr virus latent genes in vivo is dependent upon the differentiation stage of the infected B cell

Epstein-Barr virus-infected B cells in vivo demonstrate three distinct patterns of latent gene expression, depending on the differentiation stage of the cell. Tonsillar naive B cells express the EBNA2-dependent lymphoblastoid phenotype, characteristic of direct infection. Germinal center centroblasts and centrocytes as well as tonsillar memory B cells express a more restricted pattern of latent genes (EBNA1(Q-K)+, LMP1+, LMP2+, EBNA2-) that has only been seen previously in EBV-positive tumors. Peripheral memory cells express an even more restricted pattern where no latent genes are expressed, with the possible exception of LMP2. These results are consistent with a model where EBV uses the normal biology of B lymphocytes to gain access to and persist within the long-lived memory B cell compartment.     

Transporter (TAP)-independent processing of a multiple membrane-spanning protein,the Epstein-Barr virus latent membrane protein 2

Antigen presentation to CD8⁺ cytotoxic T lymphocytes (CTL) usually involves proteolytic cleavage of antigen in the cytosol and the delivery of epitope peptides onto major histocompatibility complex class I molecules in the endoplasmic reticulum (ER) via the heterodimeric peptide transporter TAP1/TAP2. In the few exceptional cases where TAP-independent presentation of an endogenously expressed protein has been observed, the epitope-containing domain of the protein either has naturally accessed or has been directed into the ER lumen where it is thought to become susceptible to ER proteases. Here, we describe a novel example of TAP-independent processing involving the Epstein-Barr virus (EBV) latent membrane protein LMP2, a multiple membrane-spanning protein with minimal projection into the ER. Expression of LMP2 in the TAP⁻ T2 cell line, whether from the resident EBV genome or from a recombinant vaccinia virus vector vacc-LMP2, rendered the cells sensitive to recognition by CTL clones specific for two HLA-A2.1-restricted peptide epitopes, LMP2 329–337 or 426–434. Vacc-LMP2-mediated sensitization to lysis required expression of the antigen de novo in T2 cells and was blocked by brefeldin A. In the same experiments, two other EBV-specific CTL epitopes, one derived from LMP2 but restricted through a different HLA allele (A11), the other restricted through A2.1 but derived from a different viral protein (BMLF1), did not display TAP-independent processing. The results are discussed in relation to the unusual topology of LMP2 in the membrane and the position of the epitope peptides within that structure.