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This article provides a retrospective on the life and career of Elliott Dan Kieff, a pioneer in the field of Epstein-Barr virus (EBV) cancer biology. Kieff's contributions to the understanding of EBV include characterizing the virus genome, identifying its latent and lytic cycle transcripts, and discovering key proteins involved in EBV-mediated B cell transformation. His work has shed light on the molecular mechanisms of virus-induced B cell transformation and the coevolution of EBV with its primate host. Additionally, Kieff was known for his dedication to scientific mentorship and supporting scientists from diverse backgrounds. His research has greatly advanced our understanding of EBV biology and its association with human tumors. [Extracted from the article]

Human herpesviruses are antigenically rich agents that induce strong CD8+T cell responses in primary infection yet persist for life, continually challenging T cell memory through recurrent lytic replication and potentially influencing the spectrum of antigen-specific responses. Here we describe the first lytic proteome-wide analysis of CD8+ T cell responses to a gamma1-herpesvirus, Epstein-Barr virus (EBV), and the first such proteome-wide analysis of primary versus memory CD8+ T cell responses to any human herpesvirus. Primary effector preparations were generated directly from activated CD8+ T cells in the blood of infectious mononucleosis (IM) patients by in vitro mitogenic expansion. For memory preparations, EBV-specific cells in the blood of long-term virus carriers were first re-stimulated in vitro by autologous dendritic cells loaded with a lysate of lytically-infected cells, then expanded as for IM cells. Preparations from 7 donors of each type were screened against each of 70 EBV lytic cycle proteins in combination with the donor’s individual HLA class I alleles. Multiple reactivities against immediate early (IE), early (E) and late (L) lytic cycle proteins, including many hitherto unrecognised targets, were detected in both contexts. Interestingly however, the two donor cohorts showed a different balance between IE, E and L reactivities. Primary responses targeted IE and a small group of E proteins preferentially, seemingly in line with their better presentation on the infected cell surface before later-expressed viral evasins take full hold. By contrast, target choice equilibrates in virus carriage with responses to key IE and E antigens still present but with responses to a select subset of L proteins now often prominent. We infer that, for EBV at least, long-term virus carriage with its low level virus replication and lytic antigen release is associated with a re-shaping of the virus-specific response. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV), originally discovered through its association with Burkitt lymphoma, is now aetiologically linked to a remarkably wide range of lymphoproliferative lesions and malignant lymphomas of B-, T- and NK-cell origin. Some occur as rare accidents of virus persistence in the B lymphoid system, while others arise as a result of viral entry into unnatural target cells. The early finding that EBV is a potent B-cell growth transforming agent hinted at a simple oncogenic mechanism by which this virus could promote lymphomagenesis. In reality, the pathogenesis of EBV-associated lymphomas involves a complex interplay between different patterns of viral gene expression and cellular genetic changes. Here we review recent developments in our understanding of EBV-associated lymphomagenesis in both the immunocompetent and immunocompromised host. [ABSTRACT FROM AUTHOR]

EBV persists in Ig class‐switched B cells, even when derived from T cell‐independent responses, and are transiently depleted in EBV‐infected children. The EBV is known to persist in memory B cells, but it remains unclear how this affects cell numbers and humoral immunity. We here studied EBV persistence in memory B cell subsets and consequences on B cell memory in young children. EBV genome loads were quantified in 6 memory B cell subsets in EBV+ adults. The effects of EBV infection on memory B cell numbers and vaccination responses were studied longitudinally in children within the Generation R population cohort between 14 mo and 6 yr of age. EBV genomes were more numerous in CD27+IgG+, CD27+IgA+, and CD27−IgA+ memory B cells than in IgM‐only, natural effector, and CD27−IgG+ B cells. The blood counts of IgM‐only, CD27+IgA+, CD27−IgG+, and CD27+IgG+ memory B cells were significantly lower in EBV+ children than in uninfected controls at 14 mo of age—the age when these cells peak in numbers. At 6 yr, all of these memory B cell counts had normalized, as had plasma IgG levels to previous primary measles and booster tetanus vaccinations. In conclusion, EBV persists predominantly in Ig class‐switched memory B cells, even when derived from T cell‐independent responses (CD27−IgA+), and EBV infection results in a transient depletion of these cells in young children. [ABSTRACT FROM AUTHOR]

Disruption of T cell memory during severe immune suppression results in reactivation of chronic viral infections, such as Epstein Barr virus (EBV) and Cytomegalovirus (CMV). How different subsets of memory T cells contribute to the protective immunity against these viruses remains poorly defined. In this study we examined the compartmentalization of virus-specific, tissue resident memory CD8+ T cells in human lymphoid organs. This revealed two distinct populations of memory CD8+ T cells, that were CD69+CD103+ and CD69+CD103—, and were retained within the spleen and tonsils in the absence of recent T cell stimulation. These two types of memory cells were distinct not only in their phenotype and transcriptional profile, but also in their anatomical localization within tonsils and spleen. The EBV-specific, but not CMV-specific, CD8+ memory T cells preferentially accumulated in the tonsils and acquired a phenotype that ensured their retention at the epithelial sites where EBV replicates. In vitro studies revealed that the cytokine IL-15 can potentiate the retention of circulating effector memory CD8+ T cells by down-regulating the expression of sphingosine-1-phosphate receptor, required for T cell exit from tissues, and its transcriptional activator, Kruppel-like factor 2 (KLF2). Within the tonsils the expression of IL-15 was detected in regions where CD8+ T cells localized, further supporting a role for this cytokine in T cell retention. Together this study provides evidence for the compartmentalization of distinct types of resident memory T cells that could contribute to the long-term protection against persisting viral infections. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three “first wave” proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501), as well as subdominant responses through common class I alleles (e.g. B7 and C*0304). Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that “first wave” antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design. [ABSTRACT FROM AUTHOR]

Human primary immunodeficiency (PID) states, where mutations in single immune system genes predispose individuals to certain infectious agents and not others, are experiments of nature that hold important lessons for the immunologist. The number of genetically defined PIDs is rising rapidly, as is the opportunity to learn from them. Epstein-Barr virus (EBV), a human herpesvirus, has long been of interest because of its complex interaction with the immune system. Thus, it causes both infectious mononucleosis (IM), an immunopathologic disease associated with exaggerated host responses, and at least one malignancy, EBV-positive lymphoproliferative disease, when those responses are impaired. Here, we describe the full range of PIDs currently linked with an increased risk of EBVassociated disease. These provide examples where IM-like immunopathology is fatally exaggerated, and others where responses impaired at the stage of induction, expansion, or effector function predispose to malignancy. Current evidence from this rapidly moving field supports the view that lesions in both natural killer cell and T cell function can lead to EBV pathology. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) is usually acquired silently early in life and carried thereafter as an asymptomatic infection of the B lymphoid system. However, many circumstances disturb the delicate EBV-host balance and cause the virus to display its pathogenic potential. Thus, primary infection in adolescence can manifest as infectious mononucleosis (IM), as a fatal illness that magnifies the immunopathology of IM in boys with the X-linked lymphoproliferative disease trait, and as a chronic active disease leading to life-threatening hemophagocytosis in rare cases of T or natural killer (NK) cell infection. Patients with primary immunodeficiencies affecting the NK and/or T cell systems, as well as immunosuppressed transplant recipients, handle EBV infections poorly, and many are at increased risk of virus-driven B-lymphoproliferative disease. By contrast, a range of other EBV-positive malignancies of lymphoid or epithelial origin arise in individuals with seemingly intact immune systems through mechanisms that remain to be understood. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) infection often occurs in early childhood and is asymptomatic. However, if delayed until adolescence, primary infection may manifest as acute infectious mononucleosis (AIM), a febrile illness characterised by global CD8+ T-cell lymphocytosis, much of it reflecting a huge expansion of activated EBV-specific CD8+ T-cells. While the events of AIM have been intensely studied, little is known about how these relate to asymptomatic primary infection. Here Gambian children (14–18 months old, an age at which many acquire the virus) were followed for the ensuing six months, monitoring circulating EBV loads, antibody status against virus capsid antigen (VCA) and both total and virus-specific CD8+ T-cell numbers. Many children were IgG anti-VCA-positive and, though no longer IgM-positive, still retained high virus loads comparable to AIM patients and had detectable EBV-specific T-cells, some still expressing activation markers. Virus loads and the frequency/activation status of specific T-cells decreased over time, consistent with resolution of a relatively recent primary infection. Six children with similarly high EBV loads were IgM anti-VCA-positive, indicating very recent infection. In three of these donors with HLA types allowing MHC-tetramer analysis, highly activated EBV-specific T-cells were detectable in the blood with one individual epitope response reaching 15% of all CD8+ T-cells. That response was culled and the cells lost activation markers over time, just as seen in AIM. However, unlike AIM, these events occurred without marked expansion of total CD8+ numbers. Thus asymptomatic EBV infection in children elicits a virus-specific CD8+ T-cell response that can control the infection without over-expansion; conversely, in AIM it appears the CD8 over-expansion, rather than virus load per se, is the cause of disease symptoms. [ABSTRACT FROM AUTHOR]

Mutations in the X-linked inhibitor of apoptosis ( XIAP) gene have been associated with XLP-like disease, including recurrent Epstein-Barr virus ( EBV)-related haemophagocytic lymphohystiocytosis (HLH), but the immunopathogenic bases of EBV-related disease in XIAP deficiency is unknown. We present the first analysis of EBV-specific T cell responses in functional XIAP deficiency. In a family of patients with a novel mutation in XIAP ( G466 X) leading to a late-truncated protein and varying clinical features, we identified gradual hypogammaglobulinaemia and large expansions of T cell subsets, including a prominent CD4+ CD8+ population. Extensive ex-vivo analyses showed that the expanded T cell subsets were dominated by EBV-specific cells with conserved cytotoxic, proliferative and interferon ( IFN)-γ secretion capacity. The EBV load in blood fluctuated and was occasionally very high, indicating that the XIAPG466X mutation could impact upon EBV latency. XIAP deficiency may unravel a new immunopathogenic mechanism in EBV-associated disease. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growthtransforming infections yet establishes persistence only in IgD+ CD27+ non-switched memory (NSM) and IgD- CD27+ switched memory (SM) B cells, not in IgD+ CD27- naïve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBVdriven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectivelyharvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naïve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCL's evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes. [ABSTRACT FROM AUTHOR]

Immunotherapies targeting peptides presented by allogeneic MHC molecules offer the prospect of circumventing tolerance to key tumor-associated self-antigens. However, the degree of antigen specificity mediated by alloreactive T cells, and their ability to discriminate normal tissues from transformed cells presenting elevated antigen levels, is poorly understood. We examined allorecognition of an HLA-A2—restricted Hodgkin's lymphoma-associated antigen and were able to isolate functionally antigen-specific allo-HLA-A2—restricted T cells from multiple donors. Binding and structural studies, focused on a prototypic allo-HLA-A2—restricted T-cell receptor (TCR) termed NB20 derived from an HLA-A3 homozygote, suggested highly peptide-specific allorecognition that was energetically focused on antigen, involving direct recognition of a distinct allopeptide presented within a conserved MHC recognition surface. Although NB20/HLA-A2 affinity was unremarkable, TCR/MHC complexes were very short-lived, consistent with suboptimal TCR triggering and tolerance to low antigen levels. These data provide strong molecular evidence that within the functionally heterogeneous alloreactive repertoire, there is the potential for highly antigen-specific "allo-MHC-restricted" recognition and suggest a kinetic mechanism whereby allo-MHC—restricted T cells may discriminate normal from transformed tissue, thereby outlining a suitable basis for broad-based therapeutic targeting of tolerizing tumor antigens. [ABSTRACT FROM AUTHOR]

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency caused by mutations in SH2D1A which encodes SAP. SAP functions in signalling pathways elicited by the SLAM family of leukocyte receptors. A defining feature of XLP is exquisite sensitivity to infection with EBV, a B-lymphotropic virus, but not other viruses. Although previous studies have identified defects in lymphocytes from XLP patients, the unique role of SAP in controlling EBV infection remains unresolved. We describe a novel approach to this question using female XLP carriers who, due to random X-inactivation, contain both SAP+ and SAP2 cells. This represents the human equivalent of a mixed bone marrow chimera in mice. While memory CD8+ T cells specific for CMV and influenza were distributed across SAP+ and SAP2 populations, EBV-specific cells were exclusively SAP+. The preferential recruitment of SAP+ cells by EBV reflected the tropism of EBV for B cells, and the requirement for SAP expression in CD8+ T cells for them to respond to Ag-presentation by B cells, but not other cell types. The inability of SAP2 clones to respond to Ag-presenting B cells was overcome by blocking the SLAM receptors NTB-A and 2B4, while ectopic expression of NTB-A on fibroblasts inhibited cytotoxicity of SAP2 CD8+ T cells, thereby demonstrating that SLAM receptors acquire inhibitory function in the absence of SAP. The innovative XLP carrier model allowed us to unravel the mechanisms underlying the unique susceptibility of XLP patients to EBV infection in the absence of a relevant animal model. We found that this reflected the nature of the Ag-presenting cell, rather than EBV itself. Our data also identified a pathological signalling pathway that could be targeted to treat patients with severe EBV infection. This system may allow the study of other human diseases where heterozygous gene expression from random X-chromosome inactivation can be exploited. [ABSTRACT FROM AUTHOR]

Interactions between MHC class II (MHC II)-positive APCs and CD4+ T cells are central to adaptive immune responses. Using an Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line (LCL) as MHC II-positive APCs and CD4+ T-cell clones specific for two endogenously expressed EBV antigens, we found that shRNA knockdown of the tetraspanin protein CD63 in LCL cells consistently led to increased CD4+ T-cell recognition. This effect was not due to enhanced antigen processing nor to changes in MHC II expression since CD63 knockdown did not influence the amount or dimerization of MHC II in LCL cells. We therefore investigated the possible involvement of exosomes, small MHC II- and tetraspanin-abundant vesicles which are secreted by LCL cells and which we found could themselves activate the CD4+ T-cell clones in an MHC II-dependent manner. While equal loadings of exosomes purified from the control and CD63low LCLs stimulated T cells to a comparable degree, we found that exosome production significantly increased following CD63-knockdown, suggesting that this may underlie the greater T-cell stimulatory capacity of the CD63low LCLs. Taken together, our data reveal a new insight into the mechanisms by which tetraspanins are involved in the regulation of MHC II-dependent T-cell stimulation. [ABSTRACT FROM AUTHOR]

Whereas exogenously acquired proteins are the major source of antigens feeding the MHC class II pathway in antigen-presenting cells, some endogenously expressed antigens also access that pathway but the rules governing such access are poorly understood. Here we address this using Epstein-Barr virus (EBV)-coded nuclear antigen EBNA1, a protein naturally expressed in EBVinfected B lymphoblastoid cell lines (LCLs) and a source of multiple CD4+ T cell epitopes. Using CD4+ T cell clones against three indicator epitopes, we find thattwo epitopes are weakly displayed on the LCL surface whereas the third is undetectable, a pattern of limited epitope presentation that is maintained even when nuclear expression of EBNA1 is induced to high supraphysiological levels. Inhibitor and siRNA studies show that, of the two epitopes weakly presented under these conditions, one involves macroautophagy, and the second involves antigen delivery to the MHC II pathway by another endogenous route. In contrast, when EBNA1 is expressed as a cytoplasmic protein, all three CD4 epitopes are processed and presented much more efficiently, and all involve macroautophagy. We conclude that EBNA1's nuclear location limits its accessibility to the macroautophagy pathway and, in consequence, limits the level and range of EBNA1 CD4 epitopes naturally displayed on the infected cell surface. [ABSTRACT FROM AUTHOR]

Viruses that naturally infect cells expressing both MHC I and MHC II molecules render themselves potentially visible to both CD8+ and CD4+ T cells through the de novo expression of viral antigens. Here we use one such pathogen, the Blymphotropic Epstein-Barr virus (EBV), to examine the kinetics of these processes in the virally-infected cell, comparing newly synthesised polypeptides versus the mature protein pool as viral antigen sources for MHC I- and MHC II-restricted presentation. EBV-transformed B cell lines were established in which the expression of two cognate EBV antigens, EBNA1 and EBNA3B, could be induced and then completely suppressed by doxycycline-regulation. These cells were used as targets for CD8+ and CD4+ T cell clones to a range of EBNA1 and EBNA3B epitopes. For both antigens, when synthesis was induced, CD8 epitope display rose quickly to near maximum within 24 h, well before steady state levels of mature protein had been reached, whereas CD4 epitope presentation was delayed by 36-48 h and rose only slowly thereafter. When antigen expression was suppressed, despite the persistence of mature protein, CD8 epitope display fell rapidly at rates similar to that seen for the MHC I/epitope half-life in peptide pulse-chase experiments. By contrast, CD4 epitope display persisted for many days and, following peptide stripping, recovered well on cells in the absence of new antigen synthesis. We infer that, in virally-infected MHC I/II-positive cells, newly-synthesised polypeptides are the dominant source of antigen feeding the MHC I pathway, whereas the MHC II pathway is fed by the mature protein pool. Hence, newly-infected cells are rapidly visible only to the CD8 response; by contrast, latent infections, in which viral gene expression has been extinguished yet viral proteins persist, will remain visible to CD4+ T cells. [ABSTRACT FROM AUTHOR]

The gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8+ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8+ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (ΔBNLF2a) and compared the ability of ΔBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8+ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by ΔBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8+ T cells remained equally poor when presented by both wild-type and ΔBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet ΔBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle. [ABSTRACT FROM AUTHOR]

Two factors contribute to Burkitt lymphoma (BL) pathogenesis, a chromosomal translocation leading to c-myc oncogene deregulation and infection with Epstein-Barr virus (EBV). Although the virus has B cell growth-transforming ability, this may not relate to its role in BL since many of the transforming proteins are not expressed in the tumor. Mounting evidence supports an alternative role, whereby EBV counteracts the high apoptotic sensitivity inherent to the c-myc-driven growth program. In that regard, a subset of BLs carry virus mutants in a novel form of latent infection that provides unusually strong resistance to apoptosis. Uniquely, these virus mutants use Wp (a viral promoter normally activated early in B cell transformation) and express a broader-than-usual range of latent antigens. Here, using an inducible system to express the candidate antigens, we show that this marked apoptosis resistance is mediated not by one of the extended range of EBNAs seen in Wp-restricted latency but by Wp-driven expression of the viral bcl2 homologue, BHRF1, a protein usually associated with the virus lytic cycle. Interestingly, this Wp/BHRF1 connection is not confined to Wp-restricted BLs but appears integral to normal B cell transformation by EBV. We find that the BHRF1 gene expression recently reported in newly infected B cells is temporally linked to Wp activation and the presence of W/BHRF1-spliced transcripts. Furthermore, just as Wp activity is never completely eclipsed in in vitro-transformed lines, low-level BHRF1 transcripts remain detectable in these cells longterm. Most importantly, recognition by BHRF1-specific T cells confirms that such lines continue to express the protein independently of any lytic cycle entry. This work therefore provides the first evidence that BHRF1, the EBV bcl2 homologue, is constitutively expressed as a latent protein in growth-transformed cells in vitro and, in the context of Wp-restricted BL, may contribute to virus-associated lymphomagenesis in vivo. [ABSTRACT FROM AUTHOR]

Background. To investigate how intense Plasmodium falciparum infection predisposes to Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL), we analyzed the effect of acute malaria on existing EBV-host balance. Methods. EBV genome loads in peripheral blood mononuclear cells were assayed by quantitative polymerase chain reaction, and EBV-specific CD8+ T cell responses were assayed by interferon-γ enzyme-linked immunospot assay. Results. Gambian children, from whom samples were obtained during an acute malaria attack and again up to 6 weeks later, had extremely high viral loads, reaching levels that in the United Kingdom are seen only in patients with infectious mononucleosis. Gambian control subjects (children and adults with no recent history of malaria) had lower median viral loads, although they were still >10-fold above the median for healthy UK adults. Limited experiments with EBV epitope peptides (restricted through the HLA-B*3501 and HLA-B*5301 alleles) also suggested an impairment of virus-specific CD8+ T cell function in children with malaria, but only during acute disease. Conclusions. Acute malaria is associated with sustained increase in EBV load and, possibly, a transient decrease in EBV-specific T cell surveillance. We infer that the unusually high set point of virus carriage in P. falciparum-challenged populations, allied with the parasite's capacity to act as a chronic B cell stimulus, probably contributes to the pathogenesis of endemic BL. [ABSTRACT FROM AUTHOR]

Nasopharyngeal carcinoma (NPC), an Epstein–Barr virus (EBV)-associated tumour common in Southern Chinese populations, is a potentially important target for T cell-based immunotherapy. The tumour cells are HLA class I- and II-positive and express a limited subset of EBV latent proteins, namely the nuclear antigen EBNA1 and the latent membrane proteins LMP2 and (in some cases) LMP1. To ask whether the tumour develops in the presence of a potentially protective host response or in its absence, we set out to determine the prevailing levels of CD4+ and CD8+ T cell memory to these proteins in NPC patients at tumour diagnosis. We first screened healthy Chinese donors against Chinese strain EBNA1, LMP1 and LMP2 sequences in Elispot assays of interferon-γ release and identified the immunodominant CD4+ and CD8+ epitope peptides presented by common Chinese HLA alleles. Then, comparing 60 patients with >70 healthy controls on peptide epitope mini-panels, we found that T cell memory to CD4 epitopes in all three proteins was unimpaired in the blood of patients at diagnosis. In most cases NPC patients also showed detectable responses to CD8 epitopes relevant to their HLA type, the one consistent exception being the absence in patients of a B*4001-restricted response to LMP2. We infer that NPC arises in patients whose prevailing levels of T cell memory to tumour-associated EBV proteins is largely intact; the therapeutic goal must therefore be to re-direct the existing memory repertoire more effectively against antigen-expressing tumour cells. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) provides a useful model to study cellular immunity to a genetically stable, persistent human virus. Different sets of proteins expressed during EBV's lyric and cell transforming infections induce qualitatively different cellular immune responses. The factors governing immunodominance hierarchies and the biological effectiveness of these different responses are now being revealed. Analysis of infectious mononucleosis (IN[), a clinical syndrome that can arise during primary EBV infection, has allowed the evolution of the responses to be tracked over time, giving an understanding of the immune response kinetics and of those determinants affecting selection into memory. Furthermore, following IM, expression of the receptor for the homeostatic cytokine IL- 15 on NK and T cells is lost within these individuals. This experiment of nature provides a system to advance understanding of immunological homeostasis in humans, illustrating how data obtained from the study of EBV have wider significance to the immunological community. [ABSTRACT FROM AUTHOR]

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus-associated disease with high prevalence in Southern Chinese. Using multiparametric flow cytometry, we identified significant expansions of circulating naïve and memory CD4+CD25high T cells in 56 NPC patients compared with healthy age- and sex-matched controls. These were regulatory T cells (Treg), as they overexpressed Foxp3 and GITR, and demonstrated enhanced suppressive activities against autologous CD4+CD25− T-cell proliferation in functional studies on five patients. Abundant intraepithelial infiltrations of Treg with very high levels of Foxp3 expression and absence of CCR7 expression were also detected in five primary tumours. Our current study is the first to demonstrate an expansion of functional Treg in the circulation of NPC patients and the presence of infiltrating Treg in the tumour microenvironment. As Treg may play an important role in suppressing antitumour immunity, our findings provide critical insights for clinical management of NPC.British Journal of Cancer (2007) 96, 617–622. doi:10.1038/sj.bjc.6603580 www.bjcancer.com Published online 30 January 2007 [ABSTRACT FROM AUTHOR]

In humans, circulating CD8(+) memory T cells to a nonpersistent virus (influenza) lie within CCR7(+)CD45RA(-) central memory, whereas memory to Epstein-Barr virus (EBV) latent, EBV lytic, and cytomegalovirus (CMV) antigens are progressively larger in size and are more biased toward CCR7(-)CD45RA(-) effector memory and CCR7(-)CD45RA(+) terminally differentiated compartments. We found that these populations are also distinguished by progressively lower expression of the interleukin-7 receptor (IL-7R alpha) and by lower IL-7 responsiveness; indeed, percentage IL-7R alpha -positive values showed a tight inverse correlation with population size. However, these relationships among size, differentiation phenotype, and IL-7R alpha status in blood did not hold in tonsillar tissue. In tonsil tissue, although EBV reactivities outnumbered their CMV and influenza counterparts, the distinct CCR7/CD45 isoform signatures of the different virus-specific populations were retained. Moreover, all detectable reactivities showed high levels of IL-7R alpha expression. As a discriminator between different virus-specific populations, IL-7R alpha therefore appears to be more susceptible to tissue location than the classical CCR7/CD45 markers. [ABSTRACT FROM AUTHOR]

In humans, circulating CD8+ memory T cells to a nonpersistent virus (influenza) lie within CCR7+CD45RA- central memory, whereas memory to Epstein-Barr virus (EBV) latent, EBV lytic, and cytomegalovirus (CMV) antigens are progressively larger in size and are more biased toward CCR7-CD45RA- effector memory and CCR7-CD45RA+ terminally differentiated compartments. We found that these populations are also distinguished by progressively lower expression of the interleukin-7 receptor (IL-7Rα) and by lower IL-7 responsiveness; indeed, percentage IL-7Rα-positive values showed a tight inverse correlation with population size. However, these relationships among size, differentiation phenotype, and IL-7Ra status in blood did not hold in tonsillar tissue. In tonsil tissue, although EBV reactivities outnumbered their CMV and influenza counterparts, the distinct CCR7/CD45 isoform signatures of the different virus-specific populations were retained. Moreover, all detectable reactivities showed high levels of IL-7Rα expression. As a discriminator between different virus-specific populations, IL-7Rα therefore appears to be more susceptible to tissue location than the classical CCR7/CD45 markers. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV), a human herpesvirus, transforms B cell growth in vitro through expressing six virus-coded Epstein-Barr nuclear antigens (EBNA5) and two latent membrane proteins (LMPs). In many EBV-associated tumors, however, viral antigen expression is more restricted, and the aetiological role of the virus is unclear. For example, endemic Burkitt lymphoma (BL) classically presents as a monoclonal, c-myc-translocation-positive tumor in which every cell carries EBV as an EBNA1-only (Latency I) infection; such homogeneity among EBV-positive cells, and the lack of EBV-negative comparators, hampers attempts to understand EBV's role in BL pathogenesis. Here, we describe an endemic BL that was unusually heterogeneous at the single-cell level and, in early passage culture, yielded a range of cellular clones, all with the same c-myc translocation but differing in EBV status. Rare EBV-negative cells were isolated alongside EBV-positive cells displaying one of three forms of restricted latency: (,) conventional Latency I expressing EBNA1 only from a WI virus genome, (ii) Wp-restricted latency expressing EBNAs 1, 3A, 3B, 3C, and-IP only from an EBNA2-deleted genome, and (iii) a previously undescribed EBNA2+/LMP1- latency in which all six EBNA5 are expressed again in the absence of the LMP5. Intercional comparisons showed that each form of EBV infection was associated with a specific degree of protection from apoptosis. Our work suggests that EBV acts as an antiapoptotic rather than a growth-promoting agent in BL by selecting among three transcriptional programs, all of which, unlike the full virus growth-transforming program, remain compatible with high c-myc expression. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV), an orally transmitted herpesvirus, efficiently targets B lymphocytes through binding of the viral envelope glycoprotein gp350 to the complement receptor CD21. How the virus accesses epithelial cells is less well understood, because such cells are largely resistant to infection with cell-free virus in vitro. Here, we show that, after binding to primary B cells, most Epstein-Barr virions are not internalized but remain on the B cell surface and from there can transfer efficiently to CD21-negative epithelial cells, increasing epithelial infection by 10³- to 104-fold compared with cell-free virus. Transfer infection is associated with the formation of B cell-epithelial conjugates with gp350/CD21 complexes focused at the intercellular synapse; transfer involves the gp85 and gp110 viral glycoproteins but is independent of gp42, the HLA class II ligand that is essential for B cell entry. Therefore, through efficient binding to the B cell surface, EBV has developed a means of simultaneously accessing both lymphoid and epithelial compartments; in particular, infection of pharyngeal epithelium by orally transmitted virus becomes independent of initial virus replication in the B cell system. [ABSTRACT FROM AUTHOR]

Recent work using a heteroduplex tracking assay (HTA) to identify resident viral sequences has suggested that patients with infectious mononucleosis (IM) who are undergoing primary Epstein-Barr virus (EBV) infection frequently harbor different EBV strains. Here, we examine samples from patients with IM by use of a new Epstein-Barr nuclear antigen 2 HTA alongside the established latent membrane protein 1 HTA. Coresident allelic sequences were detected in ex vivo blood and throat wash samples from 13 of 14 patients with IM; most patients carried 2 or more type 1 strains, 1 patient carried 2 type 2 strains, and 1 patient carried both virus types. In contrast, coresident strains were detected in only 2 of 14 patients by in vitro B cell transformation, despite screening >20 isolates/patient. We infer that coacquisition of multiple strains is common in patients with IM, although only 1 strain tends to be rescued in vitro; whether nonrescued strains are present in low abundance or are transformation defective remains to be determined. [ABSTRACT FROM AUTHOR]

Leukocyte immunoglobulin-like receptor-1 (LIR-1) is an inhibitory receptor that negatively regulates T cell effector functions after interaction with host class I major histocompatibility complex molecules and, additionally, binds to UL18, a human cytomegalovirus (HCMV)-encoded class I homologue. Here, we demonstrate that virus-specific cytotoxic T lymphocytes (CTLs) differentially express LIR-1, with high frequencies of expression on HCMV-specific CD8+ T cells and intermediate and low frequencies of expression on influenza virus-specific and Epstein-Barr virus (EBV)-specific CTLs, respectively. Expression of LIR-1 was dependent on CTL-antigen specificity and was associated with a differentiated effector memory phenotype, as demonstrated by decreased expression of CD28 and increased expression of CD57. During primary HCMV and EBV infections, expression of LIR-1 on virus-specific CTLs was low and increased slowly. These results indicate that expression of LIR-1 increases during differentiation of virus-specific CD8+ effector T cells. Furthermore, they suggest that a potential immunoregulatory function of UL18 may be to preferentially target highly differentiated HCMV-specific effector memory T cells during persistent infection. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) was discovered 40 years ago from examining electron micrographs of cells cultured from Burkitt's lymphoma, a childhood tumour that is common in sub-Saharan Africa, where its unusual geographical distribution-which matches that of holoendemic malaria-indicated a viral aetiology. However, far from showing a restricted distribution, EBV-a?-herpesvirus-was found to be widespread in all human populations and to persist in the vast majority of individuals as a lifelong, asymptomatic infection of the B-lymphocyte pool. Despite such ubiquity, the link between EBV and 'endemic' Burkitt's lymphoma proved consistent and became the first of an unexpectedly wide range of associations discovered between this virus and tumours. [ABSTRACT FROM AUTHOR]

Examines the effects of MYC gene overexpression on Epstein-Barr virus-infected B cells. Generation of lymphoblastoid cell lines; Examination on the immunogenicity of cell lines; Features of the nonimmunogenic BL phenotype.

Summary Most tumours do not stimulate effective antitumour immune responses in vivo . In order to enhance the immunogenicity of human tumour cells, we fused a variety of tumour cell lines with an Epstein–Barr virus transformed B‐lymphoblastoid cell line (EBV B‐LCL) in vitro , to produce stable hybrid cells. Hybrid cell lines showed a marked increase in their ability to stimulate primary allogeneic T‐cell responses in vitro , as compared with the parent tumour cells. The hybrid cells induced proliferation of naive (CD45RA+ ) as well as memory (CD45RO+ ) T lymphocytes, and both CD4+ and CD8+ subpopulations of T cells were directly stimulated. The stimulatory hybrids expressed human leucocyte antigen (HLA) class I and II, and a wide range of surface accessory molecules, including the T‐cell co‐stimulatory ligand molecules CD40, CD80 (B7.1) and CD86 (B7.2), the expression of which was required for optimal stimulation of T‐cell responses. Fusion of the EBVB‐LCL with a melanoma cell line (518.A2) yielded hybrid cells that expressed the melanoma‐associated antigens MAGE‐1 and MAGE‐3, and presented these antigens to antigen‐specific, HLA class I‐restricted cytotoxic T‐lymphocyte clones with greater efficiency than the parent melanoma cell line. These findings suggest that the generation of human antigen‐presenting cell/tumour cell hybrids offers promise as an approach to cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Discusses the reversal of Epstein-Barr virus (EBV)-driven lymphoproliferations in bone marrow transplant patients following cytotoxic T lymphocyte (CTL) infusion. Memory and primary CTL responses; Immunobiology of EBV persistence; EBV control of EBV-associated tumors.