Immunohistochemical detection of the Epstein-Barr virus-encoded latent membrane protein 2A in Hodgkin's disease and infectious mononucleosis

We describe two new monoclonal antibodies specific for the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) that are suitable for the immunohistochemical analysis of routinely processed paraffin sections. These antibodies were applied to the immunohistochemical detection of LMP2A in Hodgkin's disease (HD). LMP2A-specific membrane staining was seen in the Hodgkin and Reed-Sternberg (HRS) cells of 22 of 42 (52%) EBV-positive HD cases, but not in 39 EBV-negative HD cases. In lymphoid tissues from patients with acute infectious mononucleosis (IM), interfollicular immunoblasts were shown to express LMP2A. This is the first demonstration of LMP2A protein expression at the single-cell level in EBV-associated lymphoproliferations in vivo. The detection of LMP2A protein expression in HD and IM is of importance in view of the proposed role of this protein for maintaining latent EBV infection and its possible contribution for EBV-associated transformation. Because LMP2A provides target epitopes for EBV-specific cytotoxic T cells, the expression of this protein in HRS cells has implications for the immunotherapeutic approaches to the treatment of HD.     

Antigen presenting phenotype of Hodgkin Reed-Sternberg cells: analysis of the HLA class I processing pathway and the effects of interleukin-10 on epstein-barr virus-specific cytotoxic T-cell recognition

Approximately 40% of Hodgkin's disease (HD) cases in Western countries carry Epstein-Barr virus (EBV) in the malignant Hodgkin-Reed-Sternberg (H-RS) cells. HLA class I-restricted cytotoxic T lymphocytes (CTLs) with specificity for viral antigens expressed in H-RS cells therefore have therapeutic potential. However, a prerequisite for CTL therapy is that the tumor target be capable of processing and presenting endogenously expressed antigens via the transporter associated with antigen processing (TAP)-dependent HLA class I pathway. We have assessed the antigen-presenting phenotype of H-RS cells in two ways. First, immunohistochemical analysis of 38 HD biopsies showed that H-RS cells were uniformly TAP1/TAP2-positive and expressed HLA class I in the majority (18 of 24, 75%) of EBV-positive cases compared with only 4 of 14 (29%) of EBV-negative cases. Second, using a panel of 5 H-RS cell lines, we showed that 4 of 5 could process and present EBV proteins to HLA class I-restricted EBV-specific CTL clones. Others have reported that human interleukin-10 (IL-10), which is expressed by H-RS cells in the majority of EBV-positive HD cases, can abrogate CTL recognition in some circumstances. However, IL-10 pretreatment of the H-RS lines or of the EBV-specific CTLs had no such effect in this system. These results support the possibility that EBV-specific CTLs may be used to treat virus-positive HD.     

Explorers. Christopher Columbus and Leonardo da Vinci

Epstein-Barr virus (EBV) recently has been associated with Hodgkin's disease (HD) and the EBV genome was found in CD30-positive Reed-Sternberg cells. Therefore, tissue sections from 25 cases of HD, 35 cases of CD30-positive non-Hodgkin's lymphoma (NHL) (seven CD30-positive anaplastic large cell lymphomas [ALCLs] and 28 CD30-positive non-ALCLs), and 12 cases of CD30-negative NHL that previously had been screened for the presence of EBV by polymerase chain reaction and DNA in situ hybridization were studied by immunohistochemistry for the expression of the latent EBV proteins, latent membrane protein (LMP), and Epstein-Barr nuclear antigen-2 (EBNA-2). We also analyzed the expression of the B-cell activation molecule CD23 and the adhesion molecules LFA-1/CD11a and ICAM-1/CD54 because the upregulation of these molecules by LMP and/or EBNA-2 in vitro has been related to the EBV-induced lymphocyte growth. Latent membrane protein expression was found in Reed-Sternberg cells in nine of 25 cases (36%) of HD and in large, occasionally Reed-Sternberg-like tumor cells in six of 47 cases (12%) of NHL; these six tumors were CD30-positive, histologically high-grade NHL (one CD30-positive ALCL and five CD30-positive non-ALCLs). All the LMP-positive cases were also polymerase chain reaction EBV positive while LMP expression was not found in polymerase chain reaction EBV-negative HD and NHL. No staining for EBNA-2 was detected in our series. In view of the transforming potential of the LMP, these findings suggest that EBV may be associated with the development of some cases of HD and CD30-positive NHL. These findings also suggest a correlation between the expression of LMP and the detection of CD30 in tumor cells of HD and NHL. In contrast, no correlation was found between the expression of LMP and the detection of CD23, LFA-1/CD11a, and ICAM-1/CD54 in tumor cells of HD and NHL.     

Determination of HLA-A*02 antigen status in Hodgkin's disease and analysis of an HLA-A*02-restricted epitope of the Epstein-Barr virus LMP-2 protein

There is good evidence for an association between Epstein-Barr virus (EBV) and Hodgkin's disease (HD). In approximately one-third of cases, the EBV genome is detectable in Reed-Sternberg (RS) cells and there is expression of the viral nuclear antigen EBNA-1 and the latent membrane protein LMP-1. Expression of LMP-2 has been demonstrated at the mRNA level, and it is presumed that the protein is expressed alongside LMP-1. The LMP-2 protein is known to contain an epitope presented to cytotoxic T-cells which is restricted through the HLA class I antigen A*0201 in healthy seropositive individuals. Since most HLA-A*02-positive Caucasians are HLA-A*0201-positive, it was hypothesized that HLA-A*02-positive individuals would be under-represented among Caucasians with EBV-associated HD. HLA-A*02 status was determined, using flow cytometry and/or the polymerase chain reaction, for 276 individuals including 176 cases of HD. There was no significant difference between the frequency of HLA-A*02 positivity in HD cases and controls, and between EBV-associated and non-associated cases of HD. The A*02 alleles of 14 cases of EBV-associated HD were further subtyped using nested PCR; all except one case were found to be A*0201-positive. We therefore investigated whether there was any evidence for mutation of the epitope representing amino acids 426-434 of LMP-2a which is restricted through HLA-A*0201. In 10/11 cases the nucleotide sequence encoding this epitope was identical to the published sequence; in the remaining case there was a mutation which would not be expected to alter the conformation of the epitope. Overall, our data suggest that other mechanisms of immune escape must be operative in EBV-associated HD.     

The anti-MUC1 monoclonal antibody BCP8 can be used to isolate and identify putative major histocompatibility complex class I associated amino acid sequences

MHC class I molecules were isolated from the MUC1-positive human breast adenocarcinoma cell line MCF-7 by immunoaffinity using the panreactive anti-class I monoclonal antibodies (MAb) W6/32. Acid-eluted peptides from the class I molecules were separated twice by high-performance liquid chromatography and tested for reactivity with the MAb BCP8, which reacts with the minimal MUC1 core peptide sequence PDTRPA. A peak with strong and specific BCP8 reactivity was found in fractions eluting at 16.5-17.5 min. The protocol used for the MUC1+ pancreatic adenocarcinoma cell line CAPAN-1 (HLA.A2) was to perform sequential affinity purifications of class I molecules using MAb W6/32, followed by affinity purification of HLA.A2 molecules by the HLA.A2.1-specific MAb, MA2.1, and high-performance liquid chromatography fractionation of the acid-eluted material. A single peak with MAb BCP8 reactivity was noted at 18-19 min. The protocol for the MUC1+ breast adenocarcinoma cell line SKBr-3 (HLA.A11,B40), which used A11- and B40-specific MAbs, also resulted in the detection of BCP8-specific peaks at approximately 18-19 min. A preliminary mass spectral analysis of BCP8 affinity-purified class I associated material surprisingly revealed the presence of two 3-mer MUC1 amino acid sequences and one 6-mer sequence. A synthetic 9-mer MUC1 peptide, TSAPDTRPA, containing the isolated fragments was found to cause strong class I up-regulation in T2 cells as well as to serve as an epitope for CTL generated in a primary in vitro immune response. These studies suggest that MUC1-derived peptides are processed and presented in the context of MHC class I molecules on the surface of tumor cells and support the use of MAb BCP8 to further define MHC class I associated MUC1 motifs.     

Molecular analysis of presentation by HLA-A2.1 of a promiscuously binding V3 loop peptide from the HIV-envelope protein to human cytotoxic T lymphocytes

P18(IIIB) is a highly immunogenic peptide from the V3 loop of the HIV-1 gp160 envelope protein that is presented promiscuously by multiple class I MHC molecules. Understanding the molecular basis for promiscuous presentation may have many practical applications. As the highly prevalent HLA-A2.1 class I molecule is known to present P18(IIIB) for recognition by cytotoxic T lymphocytes (CTL) found in peripheral blood mononuclear cells of HIV+ donors, a P18(IIIB)-specific CTL line was generated from and HLA-A2(+), HIV- donor in order to define the molecular basis for, and ultimately improve upon the binding of, this peptide to HLA-A2.1. The minimal epitope recognized by the line was a decamer, I10, with the sequence RGPGRAFVTI. Interestingly, this decamer is identical to the minimal epitope from P18(IIIB) seen by murine CTL restricted by H-2Dd. A panel of Ala-substituted peptides was employed in MHC-binding and T cell response studies to identify MHC- and TCR-binding residues. Notably, many of the agretopic and epitopic residues identified were identical to those involved in the corresponding interactions of I10 with the H-2Dd MHC molecule and murine I10-specific CTL. The I10 peptide does not contain the described HLA-A2.1 binding motif. Instead a Pro at P3, a Phe at P7 and an Ile at P10 are utilized for MHC binding. Agretopic residue similarities with the hepatitis B nucleocapsid decamer suggest that these residues may comprise an alternative motif of anchors utilized by decamers for binding to HLA-A2.1.     

Unusually high frequency of Epstein-Barr virus genetic variants in Papua New Guinea that can escape cytotoxic T-cell recognition: implications for virus evolution

Cytotoxic T lymphocytes (CTLs) which recognize viral antigens in association with human leukocyte antigens (HLAs) play an important role in controlling persistent virus infections. These viruses use several mechanisms to evade the immune response, including mutations that affect either T-cell receptor recognition or binding of viral epitopes to the HLA. It has recently been proposed that the distribution of HLA frequencies and the specific CTL response may influence the long-term evolution of Epstein-Barr virus (EBV) by selecting variants which lack immunodominant CTL epitopes. To test this hypothesis, we have studied EBV isolates from two genetically distinct Papua New Guinea (PNG) populations, residing in coastal and highland regions, for polymorphism within seven viral CTL epitope sequences restricted through several class I HLAs. Surprisingly, all EBV isolates analyzed displayed identical amino acid substitutions within HLA A11-, B35- and B8-restricted CTL epitope sequences which completely abrogated CTL recognition and binding of synthetic peptides to HLA molecules. Furthermore, these substitutions revealed no correlation with the contemporary distribution of HLAs in the different PNG populations, which argues for a minimal influence of immune pressure. The sequence homology between EBV isolates from coastal and highland PNG suggests that the virus may have had a single origin and, more importantly, that these isolates are genetically distinct from those present in a Caucasian population.     

Deficient major histocompatibility complex class II antigen presentation in a subset of Hodgkin's disease tumor cells

Hodgkin's disease is a common malignancy of the lymphoid system. Although the scarce Hodgkin and Reed-Sternberg (HRS) tumor cells in involved tissue synthesize major histocompatibility complex (MHC) class II and costimulatory molecules such as CD40 or CD86, it is unclear whether these tumor cells are operational antigen-presenting cells (APC). We developed an immunofluorescence-based assay to determine the number of MHC class II molecules present on the surface of single living HRS cells. We found that in fresh Hodgkin's disease lymph node biopsies, a subset of HRS cells express a substantial number of surface MHC class II molecules that are occupied by MHC class II-associated invariant chain peptides (CLIP), indicating deficient loading of MHC class II molecules with antigenic peptides. Cultured Hodgkin's disease-derived (HD) cell lines, however, were found to express few MHC class II molecules carrying CLIP peptides on the cell surface and were shown to generate sodium dodecyl sulphate (SDS)-stable MHC class II alphabeta dimers. In addition to showing deficient MHC class II antigen presentation in a subset of HRS cells, our results show that the widely used HD-cell lines are not ideal in vitro models for the disease. The disruption of MHC class II-restricted antigen presentation in HRS cells could represent a key mechanism by which these tumor cells escape immune surveillance.     

Lung cancer: intermittent irradiation synchronized with respiratory motion--results of a pilot study

Pyothorax-associated lymphoma (PAL) is a newly-described entity developing several decades after artificial pneumothorax treatment for pulmonary or pleural tuberculosis. It is known to be associated with Epstein-Barr virus (EBV) with constant expression of the two latent membrane proteins: latent membrane protein (LMP)-1 and EBV-associated nuclear antigen (EBNA)-2. We are reporting three new cases of PAL. All of the tumours were of B-cell lineage and classified as large-cell diffuse lymphomas according to the International Working Formulation for the Classification of Lymphomas. The EBV genome was detected in two of the cases with LMP-1 and EBNA-2 expression. No EBV could be detected in the third case suggesting that different mechanisms may be involved in the pathogenesis of the disease. Body cavity-based high grade lymphomas (BCBL) represent a new disease, developing mainly in human immunodeficiency virus (HIV) infected patients: the tumoural cells often contain both human herpes virus (HHV)-8 (or Kaposi's sarcoma herpes virus) and EBV genomes, suggesting that these viruses might co-operate in the pathogenesis of the disease. The pleural location and the association of EBV have led to speculation that PAL could also be related to HHV-8 infection. However, no HHV-8 genome could be detected in any of the 14 tested cases already reported in the literature nor in the two cases we studied (one EBV-positive and one EBV-negative), suggesting that PAL and BCBL are two different entities.     

Cross-reactive memory T cells for Epstein-Barr virus augment the alloresponse to common human leukocyte antigens: degenerate recognition of major histocompatibility complex-bound peptide by T cells and its role in alloreactivity

In the present report, cytotoxic T lymphocyte (CTL) clones are described that display dual specificity for one of two common human leukocyte antigens (HLA B14 or B35) as alloantigens, and an immunodominant epitope (FLRGRAYGL) from Epstein-Barr virus (EBV) that binds to HLA B8. These T cell clonotypes were isolated from several unrelated HLA B8+, EBV-exposed individuals, and each distinct cross-reactivity pattern was associated with a common, public T cell receptor (TCR). In some individuals, CTL cross-reactive with these alloantigens completely dominated the memory response to this EBV epitope. Moreover, these memory T cells to EBV could be reactivated as a significant component of the repertoire of CTL responding to allogeneic stimulator cells expressing either HLA B14 or B35. These data illustrate how a history of infection with an immunogenic virus such as EBV can augment responsiveness to particular alloantigens; such influences may underlie the observed clinical association between herpesvirus infection and both allograft rejection and graft-versus-host disease. We have also explored the molecular basis for T cell cross-reactivity with alloantigens using the HLA B35 allo-reactive CTL clonotype. To elucidate the structural features of peptides that may be cross-recognized by these T cells, mono-substituted analogs of the viral epitope were screened for recognition, revealing broad specificity for major histocompatibility complex (MHC)-bound peptide. Based on the particular amino acid changes tolerated by the CTL at each peptide position, the human protein sequence database was searched for possible sequences that were recognized in association with HLA B35. Four peptides were identified (MPEATVYGL, IPIAPVYGM, KPSPPYFGL, and KPIVVLHGY) that were powerful activating ligands for the CTL when presented on HLA B35 but not B8. Thus, equivalent epitopes, capable of fully activating a single TCR, were formed by peptides with minimal obvious sequence homology bound to either HLA B8 or B35. These data indicate that degenerate peptide recognition by TCR may play an important role in the vigorous response of self-MHC-restricted T cells to alloantigens.     

Targeting Epstein-Barr virus nuclear antigen 1 (EBNA1) through the class II pathway restores immune recognition by EBNA1-specific cytotoxic T lymphocytes: evidence for HLA-DM-independent processing

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all malignancies associated with EBV and there is now convincing evidence to suggest that EBNA1 is not recognized by MHC class I-restricted cytotoxic T lymphocytes (CTL). The lack of recognition of EBNA1 has been attributed to a cis-acting inhibitory effect of glycine-alanine repetitive (G-Ar) sequences on the endogenous processing of this antigen through the class I pathway. In the present study we have explored the possibility of targeting EBNA1 through an alternative mechanism using the MHC class II pathway. Using purified EBNA1 protein, we demonstrate here that CD4+ CTL can efficiently recognize EBV-transformed B cells and Burkitt's lymphoma cells following exogenous sensitization with this antigen, and this immune recognition is not affected by the G-Ar domain within EBNA1. Analysis of the processing mechanism revealed that intracellular loading of class II molecules with an EBNA1 epitope occurs through an HLA-DM-independent pathway. These results highlight a novel mechanism for immune recognition of EBNA1 and also demonstrate that the G-Ar-mediated protection from processing can be overridden if this antigen is presented through the class II pathway.     

Epstein-Barr virus contributes to the malignant phenotype and to apoptosis resistance in Burkitt's lymphoma cell line Akata

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.     

中国人经典霍奇金淋巴瘤中人类白细胞共同抗原表达的研究

目的 既往的研究发现在高加索人中,经典霍奇金淋巴瘤肿瘤细胞人类白细胞共同抗原(HLA)的表达与EB病毒感染密切相关,研究在亚洲人中两者的相关性.方法 随机选取北京大学医学部病理学系常规外检及会诊病例中确诊为经典霍奇金淋巴瘤的145例,所有病例均有石蜡包埋组织蜡块.常规HE染色、形态学观察,根据WHO分类标准对所有病例进行重新分类.原位杂交方法检测EB病毒编码的小RNA(EBER)以提示肿瘤与EB病毒的相关性.HLA-Ⅰ类抗原的表达使用HC-10和β 2-微球蛋白抗体检测,而HLA-Ⅱ类抗原的表达使用CR3/43抗体检测.结果 145例中,40%(58例)的病例为EB病毒相关性.EB病毒阳性病例中,混合细胞型较结节硬化型更为常见(71%比16%,P＜0.001).HLA-Ⅰ类抗原在EB病毒阳性病例中的表达率明显高于EB病毒阴性的病例(79%比30%,P＜0.001).而HLA-Ⅱ类抗原的阳性率在EB病毒阳性和阴性病例中差异无统计学意义(52%比43%,P=0.277).结论 中国人经典霍奇金淋巴瘤HLA-Ⅰ类抗原的表达与EB病毒感染密切相关,与高加索人一样,但HLA-Ⅱ类抗原的表达与EB病毒感染无显著相关性.     

Intracellular delivery of a cytolytic T-lymphocyte epitope peptide by pertussis toxin to major histocompatibility complex class I without involvement of the cytosolic class I antigen processing pathway

A CD8(+) cytolytic T-lymphocyte (CTL) response to antigen-presenting cells generally requires intracellular delivery or synthesis of antigens in order to access the major histocompatibility complex (MHC) class I processing and presentation pathway. To test the ability of pertussis toxin (PT) to deliver peptides to the class I pathway for CTL recognition, we constructed fusions of CTL epitope peptides with a genetically detoxified derivative of PT (PT9K/129G). Two sites on the A (S1) subunit of PT9K/129G tolerated the insertion of peptides, allowing efficient assembly and secretion of the holotoxin fusion by Bordetella pertussis. Target cells incubated with these fusion proteins were specifically lysed by CTLs in vitro, and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A, suggesting a dependence on intracellular trafficking events, but was not inhibited by the proteasome inhibitors lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL). Furthermore, the activity was present in mutant antigen-presenting cells lacking the transporter associated with antigen processing, which transports peptides from the cytosol to the endoplasmic reticulum for association with MHC class I molecules. PT may therefore bypass the proteasome-dependent cytosolic pathway for antigen presentation and deliver epitopes to class I molecules via an alternative route.     

B7-CD28 costimulation unveils the hierarchy of tumor epitopes recognized by major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes

Immunization of mice with tumors genetically engineered to express the B7 costimulatory molecules amplifies the antitumor immune response mediated by CD8+ cytolytic T lymphocytes (CTL). In this report, we examined the effect of B7-CD28 costimulation on the hierarchy of tumor epitopes. Using a combination of affinity chromatography/reversed-phase high performance liquid chromatography and CTL cloning, we show that major histocompatibility complex (MHC) class I molecules from EL4 lymphoma cells can present at least six distinct CTL epitopes presented by MHC class I molecules. Nevertheless, mice immunized with wild-type B7-negative EL4 cells develop CTL only to one immunodominant epitope. In contrast, immunization with B7-transduced EL4 cells led to not only the amplification of the CTL response to this immunodominant epitope, but also to the recognition of five otherwise silent subdominant epitopes. The adoptive transfer of a CTL clone against such a subdominant epitope cured mice bearing EL4 lymphoma growing as an ascites tumor. The fact that CTL response can be spread to normally silent epitopes as a result of B7-CD28 costimulation suggests a novel approach to manipulate the hierarchy of CTL epitopes and offers an opportunity to explore novel targets for T cell-mediated cancer therapy.