Standardized and highly efficient expansion of Epstein-Barr virus-specific CD4+ T cells by using virus-like particles

Epstein-Barr virus (EBV)-specific T-cell lines generated by repeated stimulation with EBV-immortalized lymphoblastoid B-cell lines (LCL) have been successfully used to treat EBV-associated posttransplant lymphoproliferative disease (PTLD) in hematopoietic stem cell transplant recipients. However, PTLD in solid-organ transplant recipients and other EBV-associated malignancies respond less efficiently to this adoptive T-cell therapy. LCL-stimulated T-cell preparations are polyclonal and contain CD4(+) and CD8(+) T cells, but the composition varies greatly between lines. Because T-cell lines with higher CD4(+) T-cell proportions show improved clinical efficacy, we assessed which factors might compromise the expansion of this T-cell population. Here we show that spontaneous virus production by LCL and, hence, the presentation of viral antigens varies intra- and interindividually and is further impaired by acyclovir treatment of LCL. Moreover, the stimulation of T cells with LCL grown in medium supplemented with fetal calf serum (FCS) caused the expansion of FCS-reactive CD4(+) T cells, whereas human serum from EBV-seropositive donors diminished viral antigen presentation. To overcome these limitations, we used peripheral blood mononuclear cells pulsed with nontransforming virus-like particles as antigen-presenting cells. This strategy facilitated the specific and rapid expansion of EBV-specific CD4(+) T cells and, thus, might contribute to the development of standardized protocols for the generation of T-cell lines with improved clinical efficacy.     

A novel haplo-identical adoptive CTL therapy as a treatment for EBV-associated lymphoma after stem cell transplantation

Epstein–Barr virus (EBV)-related malignancies such as post-transplant lymphoproliferative disease (PTLD) are severe complications after allogeneic stem cell transplantation and solid-organ transplantation. In immunosuppressed transplant recipients, the activity of EBV-specific CTLs are often decreased or absent which leads to an increased risk of developing PTLD. If primary treatment modalities of PTLD fail, the most efficient way of treating the malignancy is adopting EBV-specific CTLs from the donor or, more recently, third-party donors. However, both are time consuming and expensive and often it is too late to administer cells to the patient. We have for the first time, using a rapid isolation protocol of EBV-specific T cells, treated and cured a patient suffering from PTLD with multiple-associated tissue lesions, using her haplo-identical mother as a donor. This treatment approach paves way for a new possibility to within-days treat patients with life-threatening EBV-associated malignancies.     

Dendritic cells transduced with an adenovirus vector encoding Epstein-Barr virus latent membrane protein 2B: a new modality for vaccination

Epstein-Barr virus (EBV) is a herpesvirus commonly associated with several malignancies, particularly in immunocompromised hosts. As a strategy for stimulating immunity against EBV for the treatment of EBV-associated tumors, we have genetically engineered dendritic cells (DC) to express EBV antigens, such as latent membrane protein 2B (LMP2B), using recombinant adenovirus vectors. CD8(+) T lymphocytes from HLA-A2.1(+), EBV-seropositive healthy donors were cultured with autologous DC infected with recombinant adenovirus vector AdEGFP, encoding an enhanced green fluorescent protein (EGFP), or AdLMP2B at a multiplicity of infection of 250. After 48 h, >95% of the DC were positive for EGFP expression as assessed by fluorescence-activated cell sorting analysis, indicating efficient gene transfer. AdLMP2-transduced DC were used to stimulate CD8(+) T cells. Responder CD8(+) T cells were tested for gamma interferon (IFN-gamma) release by enzyme-linked spot (ELISPOT) assay and cytotoxic activity. Prior to in vitro stimulation, the frequencies of T-cells directed against two HLA-A2-presented LMP2 peptides (LMP2 329-337 and LMP2 426-434) were very low as assessed by IFN-gamma spot formation (T-cell frequency, <0.003%). IFN-gamma ELISPOT assays performed at day 14 showed a significant (2-log) increase of the day 0 frequency of T cells reactive against the LMP2 329-337 peptide, from 0.003 to 0.3 (P < 0.001). Moreover, specific cytolytic activity was observed against the autologous EBV B-lymphoblastoid cell lines after 21 days of stimulation of T-cell responders with AdLMP2-transduced DC (P < 0.01). In summary, autologous mature DC genetically modified with an adenovirus encoding EBV antigens stimulate the generation of EBV-specific CD8(+) effector T cells in vitro, supporting the potential application of EBV-based adenovirus vector vaccination for the immunotherapy of the EBV-associated malignancies.     

Characterization of latent membrane protein 2 specificity in CTL lines from patients with EBV-positive nasopharyngeal carcinoma and lymphoma

Viral proteins expressed by EBV-associated tumors provide target Ags for immunotherapy. Adoptive T cell therapy has proven effective for posttransplant EBV-associated lymphoma in which all EBV latent Ags are expressed (type III latency). Application of immunotherapeutic strategies to tumors such as nasopharyngeal carcinoma and Hodgkin's lymphoma that have a restricted pattern of EBV Ag expression (type II latency) is under investigation. Potential EBV Ag targets for T cell therapy expressed by these tumors include latent membrane proteins (LMP) 1 and 2. A broad panel of epitopes must be identified from these target Ags to optimize vaccination strategies and facilitate monitoring of tumor-specific T cell populations after immunotherapeutic interventions. To date, LMP2 epitopes have been identified for only a limited number of HLA alleles. Using a peptide library spanning the entire LMP2 sequence, 25 CTL lines from patients with EBV-positive malignancies expressing type II latency were screened for the presence of LMP2-specific T cell populations. In 21 of 25 lines, T cell responses against one to five LMP2 epitopes were identified. These included responses to previously described epitopes as well as to newly identified HLA-A*0206-, A*0204/17-, A29-, A68-, B*1402-, B27-, B*3501-, B53-, and HLA-DR-restricted epitopes. Seven of the nine newly identified epitopes were antigenically conserved among virus isolates from nasopharyngeal carcinoma tumors. These new LMP2 epitopes broaden the diversity of HLA alleles with available epitopes, and, in particular, those epitopes conserved between EBV strains provide valuable tools for immunotherapy and immune monitoring.     

Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs

An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8⁺ and CD4⁺ T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8⁺ and CD4⁺ T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4⁺ T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4⁺ T cells was significantly correlated with that of CD8⁺ T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4⁺ T cell responses showed more significant changes than CD8⁺ T cell responses. CD8⁺ and CD4⁺ T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4⁺ T cells secreted significantly higher cytokine levels than did CD8⁺ T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.     

CD4+ and CD8+ T-Cell Responses to Latent Antigen EBNA-1 and Lytic Antigen BZLF-1 during Persistent Lymphocryptovirus Infection of Rhesus Macaques

Epstein-Barr virus (EBV) infection leads to lifelong viral persistence through its latency in B cells. EBV-specific T cells control reactivations and prevent the development of EBV-associated malignancies in most healthy carriers, but infection can sometimes cause chronic disease and malignant transformation. Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies and is a promising target for a therapeutic vaccine. Here, we studied the EBNA-1-specific immune response using the EBV-homologous rhesus lymphocryptovirus (rhLCV) infection in rhesus macaques. We assessed the frequency, phenotype, and cytokine production profiles of rhLCV EBNA-1 (rhEBNA-1)-specific T cells in 15 rhesus macaques and compared them to the lytic antigen of rhLCV BZLF-1 (rhBZLF-1). We were able to detect rhEBNA-1-specific CD4⁺ and/or CD8⁺ T cells in 14 of the 15 animals screened. In comparison, all 15 animals had detectable rhBZLF-1 responses. Most peptide-specific CD4⁺ T cells exhibited a resting phenotype of central memory (TCM), while peptide-specific CD8⁺ T cells showed a more activated phenotype, belonging mainly to the effector cell subset. By comparing our results to the human EBV immune response, we demonstrate that the rhLCV model is a valid system for studying chronic EBV infection and for the preclinical development of therapeutic vaccines.     

Rapid single-cell identification of Epstein–Barr virus-specific T-cell receptors for cellular therapy

Background and aimsEpstein–Barr virus (EBV) is associated with solid and hematopoietic malignancies. After allogeneic stem cell transplantation, EBV infection or reactivation represents a potentially life-threatening condition with no specific treatment available in clinical routine. In vitro expansion of naturally occurring EBV-specific T cells for adoptive transfer is time-consuming and influenced by the donor's T-cell receptor (TCR) repertoire and requires a specific memory compartment that is non-existent in seronegative individuals.The authors present highly efficient identification of EBV-specific TCRs that can be expressed on human T cells and recognize EBV-infected cells.Methods and ResultsMononuclear cells from six stem cell grafts were expanded in vitro with three HLA-B*35:01- or four HLA-A*02:01-presented peptides derived from six EBV proteins expressed during latent and lytic infection. Epitope-specific T cells expanded on average 42-fold and were single-cell-sorted and TCRαβ-sequenced. To confirm specificity, 11 HLA-B*35:01- and six HLA-A*02:01-restricted dominant TCRs were expressed on reporter cell lines, and 16 of 17 TCRs recognized their presumed target peptides. To confirm recognition of virus-infected cells and assess their value for adoptive therapy, three selected HLA-B*35:01- and four HLA-A*02:01-restricted TCRs were expressed on human peripheral blood lymphocytes. All TCR-transduced cells recognized EBV-infected lymphoblastoid cell lines.ConclusionsThe authors’ approach provides sets of EBV epitope-specific TCRs in two different HLA contexts. Resulting cellular products do not require EBV-seropositive donors, can be adjusted to cell subsets of choice with exactly defined proportions of target-specific T cells, can be tracked in vivo and will help to overcome unmet clinical needs in the treatment and prophylaxis of EBV reactivation and associated malignancies.     

The T cell repertoire selected in vitro against EBV: diversity, specificity, and improved purification through early IL-2 receptor alpha-chain (CD25)-positive selection

Polyclonal T cell lines specific for EBV proteins have proved efficient in preventing EBV-related immunoblastic lymphoma after allogeneic bone marrow transplantation. To gain insight into the composition of the EBV-specific T cell repertoire that ensured patient protection, we performed for the first time an extensive characterization of eight cytotoxic T cell lines selected in vitro against EBV-transformed autologous lymphoblastoid cell lines (BLCL). These T cell lines consist of 50-100 distinct T cell clones, of which 32-96% are specific for autologous BLCL. Moreover, we demonstrate that reactivities against only five EBV proteins (BZLF1, BMLF1, EBNA-3A, EBNA-3C, and LMP2) cover 86% (32/37) of the specificities detected. In addition, we describe an improved method of T cell harvesting using a CD25 selection procedure which reduces the time required to obtain specific T cells and improves the purity of EBV-specific T cells, thus showing promise for use in adoptive transfer protocols.     

Composite response of naive T cells to stimulation with the autologous lymphoblastoid cell line is mediated by CD4 cytotoxic T cell clones and includes an Epstein-Barr virus-specific component.

We have approached the challenge of generating a primary T cell response to Epstein-Barr virus (EBV) in vitro by stimulating naive T cells with the autologous EBV-transformed lymphoblastoid cell line (LCL), a rich source of EBV-associated cytotoxic T lymphocyte (CTL) epitopes. Responsive T cells from three EBV-seronegative donors were cloned in agarose, phenotyped for T cell markers by flow cytometry, and their cytotoxic properties analyzed in the 51Cr release assay. Most clones (greater than 95%) expressed the CD4 phenotype and 59% of these clones showed cytotoxic properties. The dominant CTL response was specific for FCS-associated epitopes presented by FCS-grown autologous LCL target cells and was restricted by class II HLA antigens. Other clonal components included: (i) an EBV-specific response by HLA-restricted CD4 CTL clones that did not discriminate between A- and B-type EBV transformants; (ii) an EBV-specific response by an HLA-restricted CD4 CTL clone that discriminated between A- and B-type transformants, and (iii) a nonspecific cytotoxic response by CD3+,4+,8-, CD3+,4-,8-, and CD3-,4-,8- clones that were broadly allotypic or restricted to the lysis of K562 target cells. The EBV-specific CTL clones did not lyse the autologous EBV-negative B or T cell blasts and their specificity patterns of lysis were supported by the cold target competition data. These studies highlight the role of CD4 CTL in the establishment in vitro of a primary immune response to a human virus.     

Defective control of latent Epstein-Barr virus infection in systemic lupus erythematosus

EBV infection is more common in patients with systemic lupus erythematosus (SLE) than in control subjects, suggesting that this virus plays an etiologic role in disease and/or that patients with lupus have impaired EBV-specific immune responses. In the current report we assessed immune responsiveness to EBV in patients with SLE and healthy controls, determining virus-specific T cell responses and EBV viral loads using whole blood recall assays, HLA-A2 tetramers, and real-time quantitative PCR. Patients with SLE had an approximately 40-fold increase in EBV viral loads compared with controls, a finding not explained by disease activity or immunosuppressive medications. The frequency of EBV-specific CD69+ CD4+ T cells producing IFN-gamma was higher in patients with SLE than in controls. By contrast, the frequency of EBV-specific CD69+ CD8+ T cells producing IFN-gamma in patients with SLE appeared lower than that in healthy controls, although this difference was not statistically significant. These findings suggest a role for CD4+ T cells in controlling, and a possible defect in CD8+ T cells in regulating, increased viral loads in lupus. These ideas were supported by correlations between viral loads and EBV-specific T cell responses in lupus patients. EBV viral loads were inversely correlated with the frequency of EBV-specific CD69+ CD4+ T cells producing IFN-gamma and were positively correlated with the frequencies of CD69+ CD8+ T cells producing IFN-gamma and with EBV-specific, HLA-A2 tetramer-positive CD8+ T cells. These results demonstrate that patients with SLE have defective control of latent EBV infection that probably stems from altered T cell responses against EBV.     

Reconstitution of EBV-directed T cell immunity by adoptive transfer of peptide-stimulated T cells in a patient after allogeneic stem cell transplantation for AITL

Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRβ) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.     

Nasopharyngeal carcinoma-associated Epstein-Barr virus-encoded oncogene latent membrane protein 1 potentiates regulatory T-cell function

Sequence variation in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) oncogene structure may affect antigen-presenting cell (APC) function of infected B cells and immune escape by EBV-specific T cells and thus contribute to the development of malignancy. Normal B cell-associated LMP1 (B-LMP1) upregulates B cell APC function through activation of the necrosis factor (NF)-kappaB subunit, RelB. We examined the ability of B-LMP1 and a nasopharyngeal carcinoma-associated LMP1 (NPC-LMP1) to modulate B cell APC function and T-cell responses. B lymphoma cells transfected with NPC-LMP1 stimulated resting T cells in mixed lymphocyte reaction less efficiently than B-LMP1 transfectants. Unexpectedly, antigen presentation to CD4(+) T helper cells was reduced owing to potentiation of regulatory T-cell function by NPC-LMP1 transfectants, which produce increased levels of interleukin-10, rendering CD4(+) T cells hyporesponsive. Thus, after primary EBV infection, T cells may escape activation by NPC-LMP1. These observations have important implications for the establishment of EBV-associated malignancy in the context of infection with tumour-associated EBV LMP1 variants.     

Generation of EBV-specific CD4+ cytotoxic T cells from virus naive individuals.

Adoptive immunotherapy with EBV-specific CTL (EBV-CTL) effectively prevents and treats EBV-driven lymphoproliferation in immunocompromised hosts. EBV-seronegative solid organ transplant recipients are at high risk of EBV-driven lymphoproliferation because they lack EBV-specific memory T cells. For the same reason, standard techniques for generating EBV-CTL in vitro from EBV-naive individuals are unsuccessful. To overcome this problem, we compared several methods of expanding EBV-CTL from seronegative adults and children. First, the standard protocol, using EBV-transformed lymphoblastoid B cell lines (LCL) as the source of APC, was compared with protocols using EBV-Ag-loaded dendritic cells as APC. Surprisingly, the standard protocol effectively generated CTL from all seronegative adults. The additional finding of EBV-DNA in the peripheral blood of three of these four adults suggested that some individuals may develop cellular, but not humoral, immune responses to EBV. By contrast, LCL failed to reactivate EBV-CTL from any of the six EBV-seronegative children. EBV-Ag-loaded dendritic cells could expand EBV-CTL, but only in a minority of children. However, the selective expansion of CD25-expressing T cells, 9-11 days after activation with LCL alone, proved to be a simple and reliable method for generating EBV-CTL from all seronegative children. The majority of these CTL were CD4(+) (71 +/- 26%) and demonstrated HLA class II-restricted, EBV-specific killing. Our results suggest that a negative EBV serology does not accurately identify EBV-negative individuals. In addition, our method for selecting EBV-specific CTL from naive individuals by precursor cell enrichment may be applicable to the immunotherapy of cancer patients with a low frequency of tumor- or virus-specific CTL.     

Different patterns of Epstein-Barr virus gene expression and of cytotoxic T-cell recognition in B-cell lines infected with transforming (B95.8) or nontransforming (P3HR1) virus strains

Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell lines have been converted to EBV genome positivity by in vitro infection with the transforming EBV strain B95.8 and with the nontransforming mutant strain P3HR1, which has a deletion in the gene encoding the nuclear antigen EBNA2. These B95.8- and P3HR1-converted lines have been compared for their patterns of expression of EBV latent genes (i.e., those viral genes constitutively expressed in all EBV-transformed lines of normal B-cell origin) and for their recognition by EBV-specific cytotoxic T lymphocytes (CTLs), in an effort to identify which latent gene products provide target antigens for the T-cell response. B95.8-converted lines on several different EBV-negative BL-cell backgrounds all showed detectable expression of the nuclear antigens EBNA1, EBNA2, and EBNA3 and of the latent membrane protein (LMP); such converts were also clearly recognized by EBV-specific CTL preparations with restriction through selected human leukocyte antigen (HLA) class I antigens on the target cell surface. The corresponding P3HR1-converted lines (lacking an EBNA2 gene) expressed EBNA1 and EBNA3 but, surprisingly, showed no detectable LMP; furthermore, these converts were not recognized by EBV-specific CTLs. Such differences in T-cell recognition were not due to any differences in expression of the relevant HLA-restricting determinants between the two types of convert, as shown by binding of specific monoclonal antibodies and by the susceptibility of both B95.8 and P3HR1 converts to allospecific CTLs directed against these same HLA molecules. The results suggest that in the normal infectious cycle, EBNA2 may be required for subsequent expression of LMP and that both EBNA2 and LMP (but not EBNA1 or EBNA3) may provide target antigens for the EBV-specific T-cell response.     

Reconstitution of EBV latent but not lytic antigen-specific CD4+ and CD8+ T cells after HIV treatment with highly active antiretroviral therapy

The incidence of (EBV-related) malignancies in HIV-infected subjects has declined since the introduction of highly active antiretroviral therapy (HAART). To investigate the effect of HAART on EBV infection, we performed a longitudinal analysis of the T cell response to both a latent and a lytic Ag and EBV viral load in 10 subjects from early in HIV infection up to 5 years after HAART. All individuals responded to HAART by a decline in HIV viral load, a restoration of total CD4+ T cell numbers, and a decline in T cell immune activation. Despite this, EBV load remained unaltered, even after 5 years of therapy, although a decline in both CD4+ and CD8+ T cells specific for the lytic EBV protein BZLF1 suggested a decreased EBV reactivation rate. In contrast, latent EBV Ag EBNA1-specific CD4+ and CD8+ T cell responses were restored after 5 years of treatment to levels comparable to healthy individuals. In two individuals who were treated by HAART late during HIV progression, a lymphoma developed shortly after initiation of HAART, despite restoration of EBV-specific CD4+ and CD8+ T cells. In conclusion, long-term HAART does not alter the EBV DNA load, but does lead to a restoration of EBNA1-specific T cell responses, which might allow better control of EBV-infected cells when applied early enough during HIV infection.     

Autoactivation of the Epstein-Barr virus oncogenic protein LMP1 during type II latency through opposite roles of the NF-kappaB and JNK signaling pathways

Epstein-Barr virus (EBV) is associated with several human malignancies where it expresses limited subsets of latent proteins. Of the latent proteins, latent membrane protein 1 (LMP1) is a potent transforming protein that constitutively induces multiple cell signaling pathways and contributes to EBV-associated oncogenesis. Regulation of LMP1 expression has been extensively described during the type III latency of EBV. Nevertheless, in the majority of EBV-associated tumors, the virus is commonly found to display a type II latency program in which it is still unknown which viral or cellular protein is really involved in maintaining LMP1 expression. Here, we demonstrate that LMP1 activates its own promoter pLMP1 through the JNK signaling pathway emerging from the TES2 domain. Our results also reveal that this activation is tightly controlled by LMP1, since pLMP1 is inhibited by LMP1-activated NF-kappaB signaling pathway. By using our physiological models of EBV-infected cells displaying type II latency as well as lymphoblastoid cell lines expressing a type III latency, we also demonstrate that this balanced autoregulation of LMP1 is shared by both latency programs. Finally, we show that this autoactivation is the most important mechanism to maintain LMP1 expression during the type II latency program of EBV.     

Functional reversion of antigen-specific CD8+ T cells from patients with Hodgkin lymphoma following in vitro stimulation with recombinant polyepitope

Recent studies on Hodgkin's lymphoma (HL) have indicated that patients with active disease display functional impairment of Ag-specific CD8+ T cells due to expansion of regulatory T cells at sites of disease and in the peripheral blood. Adoptive cellular immunotherapy based on EBV-specific CD8+ T cells has been explored with limited success to date. It has been proposed that improved targeting of these CD8+ T cells toward viral Ags that are expressed in HL may enhance future therapeutic vaccine strategies. In this study, we have developed a novel replication-deficient adenoviral Ag presentation system that is designed to encode glycine alanine repeat-deleted EBV nuclear Ag 1 covalently linked to multiple CD8+ T cell epitopes from latent membrane proteins 1 and 2. A single stimulation of CD8+ T cells from healthy virus carriers, and patients with HL with this adenoviral construct in combination with IL-2, was sufficient to reverse the functional T cell impairment and restored both IFN-gamma production and cytolytic function. More importantly, these activated CD8+ T cells responded to tumor cells expressing membrane proteins and recognized novel EBNA1 epitopes. Flow cytometric analysis revealed that a large proportion of T cells expanded from patients with HL were CD62L(high) and CD27(high), and CCR7(low), consistent with early to mid effector T cells. These findings provide an important platform for translation of Ag-specific adoptive immunotherapy for the treatment of EBV-associated malignancies such as HL and nasopharyngeal carcinoma.     

The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation

The linear virion form of Epstein-Barr virus (EBV) DNA has variable numbers of direct tandem 500 bp repeats at each terminus. The terminal restriction endonuclease fragments and the fused terminal fragments in the intracellular episomal form are heterogeneous in size, and vary by increments of 500 bp. The structure of the termini of EBV in carcinomas of the nasopharynx and the parotid gland was compared with the EBV termini in monoclonal and polyclonal tissues or cell lines. A single band representing the EBV joined termini was detected in each of the carcinomas and in the monoclonal lymphoid proliferations. Polyclonal cell lines contained multiple forms of the joined termini. The detection of a homogeneous episomal population suggests that EBV-associated epithelial malignancies are clonal expansions of a single EBV-infected progenitor cell.     

CD4+ T cell-induced differentiation of EBV-transformed lymphoblastoid cells is associated with diminished recognition by EBV-specific CD8+ cytotoxic T cells

EBV transformation of human B cells in vitro results in establishment of immortalized cell lines (lymphoblastoid cell lines (LCL)) that express viral transformation-associated latent genes and exhibit a fixed, lymphoblastoid phenotype. In this report, we show that CD4(+) T cells can modify the differentiation state of EBV-transformed LCL. Coculture of LCL with EBV-specific CD4(+) T cells resulted in an altered phenotype, characterized by elevated CD38 expression and decreased proliferation rate. Relative to control LCL, the cocultured LCL were markedly less susceptible to lysis by EBV-specific CD8(+) CTL. In contrast, CD4(+) T cell-induced differentiation of LCL did not diminish sensitivity of LCL to lysis by CD8(+) CTL specific for an exogenously loaded peptide Ag or lysis by alloreactive CD8(+) CTL, suggesting that differentiation is not associated with intrinsic resistance to CD8(+) T cell cytotoxicity and that evasion of lysis is confined to EBV-specific CTL responses. CD4(+) T cell-induced differentiation of LCL and concomitant resistance of LCL to lysis by EBV-specific CD8(+) CTL were associated with reduced expression of viral latent genes. Finally, transwell cocultures, in which direct LCL-CD4(+) T cell contact was prevented, indicated a major role for CD4(+) T cell cytokines in the differentiation of LCL.     

The signaling pathways of Epstein-Barr virus-encoded latent membrane protein 2A (LMP2A) in latency and cancer

Epstein-Barr virus (EBV) is a ubiquitous virus with infections commonly resulting in a latency carrier state. Although the exact role of EBV in cancer pathogenesis remains not entirely clear, it is highly probable that it causes several lymphoid and epithelial malignancies, such as Hodgkin’s lymphoma, NK-T cell lymphoma, Burkitt’s lymphoma, and nasopharyngeal carcinoma. EBV-associated malignancies are associated with a latent form of infection, and several of these EBV-encoded latent proteins are known to mediate cellular transformation. These include six nuclear antigens and three latent membrane proteins. Studies have shown that EBV displays distinct patterns of viral latent gene expression in these lymphoid and epithelial tumors. The constant expression of latent membrane protein 2A (LMP2A) at the RNA level in both primary and metastatic tumors suggests that this protein might be a driving factor in the tumorigenesis of EBV-associated malignancies. LMP2A may cooperate with the aberrant host genome, and thereby contribute to malignant transformation by intervening in signaling pathways at multiple points, especially in the cell cycle and apoptotic pathway. This review summarizes the role of EBV-encoded LMP2A in EBV-associated viral latency and cancers. We will focus our discussions on the molecular interactions of each of the conserved motifs in LMP2A, and their involvement in various signaling pathways, namely the B-cell receptor blockade mechanism, the ubiquitin-mediated (Notch and Wnt) pathways, and the MAPK, PI3-K/Akt, NK-κB and STAT pathways, which can provide us with important insights into the roles of LMP2A in the EBV-associated latency state and various malignancies.