Epstein-Barr virus (EBV) latent membrane protein-1-specific cytotoxic T lymphocytes targeting EBV-carrying natural killer cell malignancies

Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP) 1 is a potential target for immunotherapy of some proportion of Hodgkin's disease cases, nasopharyngeal carcinomas, EBV-associated natural killer (NK)/T lymphomas, and chronic active EBV infection (CAEBV). Since it is unknown whether EBV-infected NK/T cells are susceptible to lysis by LMP1-specific cytotoxic T lymphohcytes (CTL), we here tested the ability of mRNA-transduced antigen-presenting cells (APC) to stimulate rare LMP1-specific CTL. A 43-amino acid N-terminal deletion mutant LMP1 (DeltaLMP1) could be efficiently expressed in dendritic cells and CD40-activated B cells upon mRNA electroporation. DeltaLMP1-expressing APC were found to stimulate LMP1-specific CTL from a healthy donor and a CTL clone recognized a peptide, IIIILIIFI, presented by HLA-A*0206 molecules. Processing and presentation of the antigenic peptide proved dependent on expression of an immunoproteasome subunit, low-molecular-weight protein-7, as confirmed by RNA interference gene silencing. Furthermore, an EBV-infected NK cell line derived from a patient with CAEBV, and another from an NK lymphoma with enforced HLA-A*0206 expression, were specifically lysed by the CTL. Overall, these data suggest that immunotherapy targeting LMP1 in EBV-associated NK lymphomas and CAEBV might serve as an alternative treatment modality.     

Role of Epstein-Barr virus encoded latent membrane protein 1 in the carcinogenesis of nasopharyngeal carcinoma

Epstein-Barr virus （EBV）-encoded latent membrane protein 1 （LMPI） has been known to have oncogenic properties during latent infection in nasopharyngeal carcinoma （NPC）. Our studies focused on the role of LMP1 in NPC, and showed that LMP1 triggers the NF-κB, AP-1 and STAT signaling pathways. Strikingly, LMP1 was found to mediate the formation of a new heterodimer between c-Jun and JunB. Also, we have identified JAK/STAT and PI-PLC-PKC activation triggered by LMP1 through upregulating the expression of JAK3 and enhancing the phosphorylation of STATo The constitutive activation of these signaling cascades explains LMP1＇s ability to induce such a diverse array of morphological and phenotypic effects in cells and provides insight into how LMP1 may induce cell transformation, in which multihit targeted genes in the downstream play an essential role. All signaling cascades triggered by LMP1 ultimately lead to the disruption of the cell cycle： the acceleration of G1/S phase and the arrest of G2/M phase. We also found that LMP1 induced the expression of hTERT and promoted cell immortalization. Importantly, by intervening physical intracellular signal transduction pathways and disturbing the progression of the cell cycle, LMP1, an important oncoprotein encoded by EBV, is thought to be a key modulator in the pathogenesis of NPC. Interfering LMP1 signaling could be a promising strategy to target the malignant phenotype of NPC. Cellular ＆ Molecular Immunology.     

Deletion of Epstein-Barr virus latent membrane protein 1 gene in united states and Brazilian Hodgkin's disease and reactive lymphoid tissue: High frequency of a 30-bp deletion

A 30-basepair (bp) deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene has been reported in nasopharyngeal carcinoma and EBV-associated malignant lymphomas. Prior studies have found the deletion in about 10% to 28% of cases of Hodgkin's disease (HD), particularly in cases with aggressive histology. We studied the prevalence of 30-bp LMP1 gene deletion in EBV-positive HD in the United States (US) (12 cases) and Brazil (26 cases) with comparison to reactive lymphoid tissues (21 cases) and HD without EBV-positive Reed-Sternberg cells (15 cases). We studied the status of the LMP1 gene by Southern blot hybridization of polymerase chain reaction (PCR) products obtained after amplification with primers spanning the site of the deletion. We also performed EBV typing, EBER1 in situ hybridization, and LMP1 protein immunohistochemistry. EBV was detected in (46%) cases of HD from the US and (96%) cases of Brazilian HD. The 30-bp LMP1 gene deletion was observed in (33%) cases of EBV-positive HD from US, and (46%) cases of Brazilian EBV-positive HD, including 3 cases of type B EBV, as compared with (57%) reactive lymphoid tissues and (60%) cases of EBV-negative HD. US and Brazilian HD showed a higher prevalence of the 30-bp LMP1 gene deletion, compared with studies of others. The unexpected finding of high incidence of 30-bp deletion in LMP1 gene in reactive lymphoid tissue and HD without EBV-positive Reed-Sternberg cells suggests that this deletion may not be relevant to HD pathogenesis in most cases.     

Frequent expression of Epstein-Barr virus latent membrane protein-1 in tumour cells of Hodgkin's disease in HIV-positive patients.

Epstein-Barr virus (EBV) is believed to be implicated in the aetiology of non-Hodgkin's lymphomas developing in immunodeficient individuals including AIDS patients. EBV has also been associated with Hodgkin's disease (HD), where the genomes have been demonstrated in the Hodgkin and Reed-Sternberg cells in some of the cases. Recent evidence has shown that EBV genomes are transcribed in these cells, because the EBV-encoded latent membrane protein-1 (LMP-1) can be demonstrated in the tumour cells in about half of the HD cases in HIV-negative patients using immunohistochemistry. LMP-1 is of special interest as a possible oncogenic agent because of its strong transforming capacity in vitro. In this study we have examined the expression of LMP-1 in HD of HIV-positive patients compared with HD in HIV-negative patients. We investigated 18 lymph nodes from 16 HIV-positive patients with HD (eight mixed cellularity, nine nodular sclerosis, one unclassified) using the CS.1-4 anti-LMP-1 monoclonal antibodies, which can usually be applied successfully to archival biopsy material. In each case, 50-90 per cent of the tumour cells were labelled. Staining was excellent for both fixatives used (4 per cent buffered formalin, Bouin's fluid). It is concluded that EBV-encoded LMP-1 is firmly associated with HD of HIV-positive patients. This is most conspicuous in the nodular sclerosing subtype HD in HIV-positive patients, in which 100 per cent were LMP-1 positive as compared with 32 per cent of nodular sclerosis HD in HIV-negative cases in a previously published series. This difference is statistically significant (P < 0.001). The possible biological and clinical significance of this difference should therefore be studied in larger series.     

Epstein-Barr virus latent membrane protein expression by Hodgkin and Reed-Sternberg-like cells in acute infectious mononucleosis.

In the light of reports of latent membrane protein (LMP) expression by Hodgkin and Reed-Sternberg (HRS) cells, paraffin sections of tonsil (two cases), lymph nodes (eight cases; three cervical, one axillary, and four inguinal) and spleen (four cases) from 14 patients with acute infectious mononucleosis (IM) have been examined for the presence of HRS-like cells and immunostained with an antibody to LMP. Sections of the tonsils and one lymph node were also stained with a panel of antibodies which characterize HRS cells of Hodgkin's disease. The tonsils contained abundant HRS-like cells, mainly adjacent to the crypts, which were highlighted by strong LMP expression. The immunophenotype of these cells closely, but not completely, resembled that of HRS cells of Hodgkin's disease. The lymph nodes and spleens showed the typical changes of acute IM but only few LMP-positive HRS-like cells were present in the cervical lymph nodes and hardly any were present in the inguinal nodes and spleen. These findings suggest that tonsillar crypt squamous epithelium may play a role in the formation of LMP-positive HRS-like cells; these cells could be progenitors of Hodgkin's disease HRS cells and, if so, this might explain the restricted sites of presentation of Hodgkin's disease.     

Cooperative interactions among p53, bcl-2 and Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma cells

Interactions among p53, bcl-2 and Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma (NPC) cells were evaluated by gene cotransfections. The data showed that bcl-2 expression was not only able to prevent the growth suppression induced by wild-type p53 but was also paradoxically able to inhibit the growth enhancement induced by mutant p53. Latent membrane protein 1 was shown to be capable of overcoming the growth inhibition induced by wild-type p53 and the synergistic cooperation with bcl-2 to enhance cellular growth. Latent membrane protein 1 could also cooperate with mutant p53 to provide a growth advantage for NPC cells. Most NPC revealed detectable overexpression of p53, and the majority of those were a wild type possibly responding to EBV infection. The coexpression of bcl-2 and LMP1 was thought to inhibit the growth suppression induced by wild-type p53 in NPC. But there was no associated expression between LMP1 and bcl-2 because we demonstrated that transfected LMP1 failed to induce bcl-2 expression in NPC cells in contrast to the findings in B cells. It is theorized that the cooperative expression of bcl-2 and LMP1 exists in the majority of NPC, while a minority of NPC have cooperative expression of LMP1 and mutant p53. Each cooperative interaction could play an important role in the development and progression of NPC.     

The c-Jun N-terminal kinase pathway is critical for cell transformation by the latent membrane protein 1 of Epstein-Barr virus

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) transforms cells activating signal transduction pathways such as NF-kappaB, PI3-kinase, or c-Jun N-terminal kinase (JNK). Here, we investigated the functional role of the LMP1-induced JNK pathway in cell transformation. Expression of a novel dominant-negative JNK1 allele caused a block of proliferation in LMP1-transformed Rat1 fibroblasts. The JNK-specific inhibitor SP600125 reproduced this effect in Rat1-LMP1 cells and efficiently interfered with proliferation of EBV-transformed lymphoblastoid cells (LCLs). Inhibition of the LMP1-induced JNK pathway in LCLs caused the downregulation of c-Jun and Cdc2, the essential G2/M cell cycle kinase, which was accompanied by a cell cycle arrest of LCLs at G2/M phase transition. Moreover, SP600125 retarded tumor growth of LCLs in a xenograft model in SCID mice. Our data support a critical role of the LMP1-induced JNK pathway for proliferation of LMP1-transformed cells and characterize JNK as a potential target for intervention against EBV-induced malignancies.     

LMP1对鼻咽癌细胞系CNE1癌基因微小RNA表达谱的影响

目的:比较鼻咽癌细胞系CNE1与其EB病毒潜伏膜蛋白1(LMP1)稳定转染细胞系CNE1-LMP1的癌基因微小RNA(oncomiRs)表达谱的差异,探讨LMP1对鼻咽癌细胞系CNE1 oncomiRs表达的影响。方法:采用包含132个oncomiRs分子的microRNA芯片分析CNE1与CNE1-LMP1的表达差异,并采用荧光定量PCR验证差异表达明显的oncomiRs分子。结果:二者共检出30个miRNA分子,CNE1中检出miRNA分子19个;其中11个为CNE1-LMP1特异性表达。在共同表达的19个miRNA分子中,在CNE1-LMP1中表达升高2倍以上的miRNA分子有6个:hsa-miR-19b、hsa-miR-17-3p、hsa-miR-22、hsa-miR-149、hsa-miR-150和hsa-miR-188。没有表达降低2倍以上的分子。在CNE1-LMP1特异性表达的11个miRNA中,hsa-miR-122a呈高水平表达,其表达水平超过内对照。通过荧光定量PCR验证6个差异分子的表达,发现改变倍数与芯片结果一致(P0.01)。结论:LMP1能影响oncomiRs表达谱,可能是LMP1作为病毒癌基因发挥作用的另一重要途径。     

Deletions and single-base mutations within the carboxy-terminal region of the latent membrane protein 1 oncogene in Epstein-Barr virus-related gastric cancers of Southern Japan

The 30-base pair (bp) deletion of the cytoplasmic carboxy-terminal domain of the latent membrane protein 1 (LMP-1) gene was analyzed in 37 frozen tissues from patients with Epstein-Barr virus (EBV)-related gastric cancer and 18 throat washings from healthy adults in southern Japan. The 30-bp deletion was identified in 33 (91.7%) of 36 specimens of EBV-related gastric cancers and in 15 (83.3%) of 18 throat washings from healthy adults. In one case of gastric cancer, an additional 9-bp deletion was identified downstream of the 30-bp deletion. From the last transmembane domain to the end of the carboxy terminal of LMP-1, mutations were examined in 37 cases of gastric cancers and in three cases of throat washings. Twenty-eight nonsilent mutations were identified in this region of EBV-related gastric cancer and throat washings. Five nonsilent mutations at positions 168,755, 168,746, 168,687, 168,357, and 168,355 were identified in all 30-bp-deleted cases of EBV-related gastric cancers and throat washings. However, these nonsilent mutations were not identified in three patients without the 30-bp deletion. Although the deletion and single-base mutations of the LMP-1 gene in gastric cancers and throat washings were similar to those of nasopharyngeal carcinoma in Taiwan and China, more single-base mutations were found in southern Japan. These data indicate that high prevalence of the 30-bp deletion of the LMP-1 gene in gastric cancers may reflect the prevalence of the deletion variant in the normal population in southern Japan. J. Med. Virol. 57:152–158, 1999. © 1999 Wiley-Liss, Inc.     

Endogenous latent membrane protein 1 in Epstein-Barr virus-infected nasopharyngeal carcinoma cells attracts T lymphocytes through upregulation of multiple chemokines

Tumor-infiltrating T lymphocytes are considered to facilitate development of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), but how EBV in NPC tumor cells directs T cell infiltration remains unclear. Here we compare EBV-infected NPC cells with and without spontaneous expression of viral latent membrane protein 1 (LMP1) and find that culture supernatants of LMP1-positive NPC cells exert enhanced chemoattraction to primary T cells. Knockdown of endogenous LMP1 in the cells suppresses the chemotactic activity. Endogenous LMP1 in NPC cells upregulates multiple chemokines, among which MIP-1α, MIP-1β and IL-8 contribute to T cell chemotaxis. We further reveal that LMP1-induced production of MIP-1α and MIP-1β in NPC cells requires not only two carboxyl-terminal activation regions of LMP1 but also their downstream NF-κB and JNK pathways. This study corroborates that endogenous LMP1 in EBV-infected NPC cells induces multiple chemokines to promote T cell recruitment and perhaps other pathogenic events in NPC.     

Nuclear factor κB represses the expression of latent membrane protein 1 in Epstein-Barr virus transformed cells

AIM: To investigate the role of nuclear factor κB(NF-κB) in the regulation of Epstein-Barr virus(EBV) latent membrane protein 1(LMP1) in EBV transformed cells. METHODS: LMP1 expression was examined in EBV transformed human B lymphocytes with modulation of NF-κB activity. RESULTS: EBV infection is associated with several human cancers. EBV LMP1 is required for efficient transformation of adult primary B cells in vitro, and is expressed in several pathogenic stages of EBVassociated cancers. Regulation of EBV LMP1 involves both viral and cellular factors. LMP1 activates NF-κB signaling pathway that is a part of the EBV transformation program. However, the relation between NF-κB and LMP1 expression is not well established yet. In this report, we found that blocking the NF-κB activity by Inhibitor of κB stimulated LMP1 expression, while the overexpression of NF-κB repressed LMP1 expression in EBV-transformed IB4 cells. In addition, LMP1 repressed its own promoter activities in reporter assays, and the repression was associated with the activation of NF-κB. Moreover, NF-κB alone is sufficient to repress LMP1 promoter activities. CONCLUSION: Our data suggest LMP1 may repress its own expression through NF-κB in EBV transformed cells and shed a light on LMP1 regulation during EBV transformation.