Evaluation of the antibody response to the EBV proteome in EBV-associated classical Hodgkin lymphoma.

The humoral immune response to Epstein-Barr virus (EBV) in classical Hodgkin lymphoma (cHL) stratified by EBV tumor status is unclear. We examined IgG and IgA antibody responses against 202 protein sequences representing 86 EBV proteins using a microarray and sera from 139 EBV-positive cHL cases, 70 EBV-negative cHL cases and 141 population-based controls frequency matched to EBV-positive cHL cases on sex and age by area (UK, Denmark and Sweden). We leveraged existing data on the proportion of circulating B-cells infected by EBV and levels of serum CCL17, a chemokine secreted by cHL tumor cells, from a subset of the cHL cases in the UK. Total IgG but not IgA response level was significantly different between EBV-positive cHL cases and controls. The distinct serological response included significant elevations in 16 IgG antibodies and 2 IgA antibodies, with odds ratioshighest vs. lowest tertile > 3 observed for the following EBV proteins: LMP1 (oncogene), BcLF1 (VCAp160, two variants) and BBLF1 (two variants). Our cHL IgG signature correlated with the proportion of circulating EBV-infected B-cells, but not serum CCL17 levels. We observed no differences in the anti-EBV antibody profile between EBV-negative cHL cases and controls. BdRF1(VCAp40)-IgG and BZLF1(Zta)-IgG were identified as the serological markers best able to distinguish EBV-positive from EBV-negative cHL tumors. Our results support the hypothesis that differences in the EBV antibody profile are specific to patients with EBV-positive cHL and are not universally observed as part of a systematically dysregulated immune response present in all cHL cases.

Early virological and immunological events in asymptomatic Epstein-Barr virus infection in African children.

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.

Modeling HLA associations with EBV-positive and -negative Hodgkin lymphoma suggests distinct mechanisms in disease pathogenesis.

HLA genotyping and genome wide association studies provide strong evidence for associations between Human Leukocyte Antigen (HLA) alleles and classical Hodgkin lymphoma (cHL). Analysis of these associations is complicated by the extensive linkage disequilibrium within the major histocompatibility region and recent data suggesting that associations with EBV-positive and EBV-negative cHL are largely distinct. To distinguish independent and therefore potentially causal associations from associations confounded by linkage disequilibrium, we applied a variable selection regression modeling procedure to directly typed HLA class I and II genes and selected SNPs from EBV-stratified patient subgroups. In final models, HLA-A*01:01 and B*37:01 were associated with an increased risk of EBV-positive cHL whereas DRB1*15:01 and DPB1*01:01 were associated with decreased risk. Effects were independent of a prior history of infectious mononucleosis. For EBV-negative cHL the class II SNP rs6903608 remained the strongest predictor of disease risk after adjusting for the effects of common HLA alleles. Associations with "all cHL" and differences by case EBV status reflected the subgroup analysis. In conclusion, this study extends previous findings by identifying novel HLA associations with EBV-stratified subgroups of cHL, highlighting those alleles likely to be biologically relevant and strengthening evidence implicating genetic variation associated with the SNP rs6903608.

Gammaherpesviruses and canine lymphoma: no evidence for direct involvement in commonly occurring lymphomas.

Lymphoma is the most common haematopoietic malignancy in dogs, but little is known about the aetiology of this heterogeneous group of cancers. In humans, the Epstein-Barr virus (EBV) is associated with several lymphoma subtypes. Recently, it was suggested that EBV or an EBV-like virus is circulating in dogs. We therefore investigated whether EBV, or a novel herpesvirus, is associated with canine lymphoma using both serological and molecular techniques. In an assay designed to detect antibodies to EBV viral capsid antigens, 41 % of dogs were positive. Dogs with cancers, including lymphoma, were more frequently positive than controls, but no particular association with B-cell lymphoma was noted. EBV-specific RNA and DNA sequences were not detected in lymphoma tissue by in situ hybridization or PCR, and herpesvirus genomes were not detected using multiple degenerate PCR assays with the ability to detect novel herpesviruses. We therefore found no evidence that herpesviruses are directly involved in common types of canine lymphoma although cannot exclude the presence of an EBV-like virus in the canine population.

Soluble CD30: a serum marker for Epstein-Barr virus-associated lymphoproliferative diseases.

The soluble form of CD30 (sCD30), a member of tumor necrosis factor receptor superfamily, has been used as a marker of disease activity in various lymphomas. Epstein-Barr virus (EBV) is a potent stimulator of CD30 expression. The study aims to evaluate whether sCD30 can be used as a diagnostic marker for EBV-associated infectious mononucleosis (IM) and post-transplant lymphoproliferative disease (PTLD). Plasma from EBV seropositive healthy controls (N = 90), acute IM patients (n = 90), non-PTLD heart/lung transplant recipients (N = 30) and EBV-positive PTLD patients (N = 23) was tested for sCD30 using a commercially available ELISA kit. EBV DNA was tested by real time quantitative polymerase chain reaction assay. Significantly higher sCD30 levels were observed in acute IM patients (median 242.9 ng/ml) compared to EBV seropositive controls (median 15.7 ng/ml; P < 0.0001). These levels were highest in IM patients within 14 days of onset of illness. PTLD patients had significantly higher sCD30 levels (median 94 ng/ml) than healthy controls (P < 0.0001) and transplant patients (median 27 ng/ml; P = 0.0007). EBV DNA was detected mostly in acute IM and PTLD patients. In both cases there was a significant correlation between sCD30 and EBV DNA levels in plasma (P < 0.0001). This study demonstrates that sCD30 and EBV DNA levels can be used as potential markers for diagnosis of IM and PTLD.

Epitope specificity and clonality of EBV-specific CTLs used to treat posttransplant lymphoproliferative disease.

In a recent phase II clinical trial using banked allogeneic CTL lines to treat EBV-associated posttransplant lymphoproliferative disease, a response rate of 52% was recorded 6 mo posttreatment. Tumor response was associated with an increase in both CTL/recipient HLA matches and CD4(+) T cells within the infused CTL lines. The present study was undertaken to correlate tumor response with CTL specificity. The majority of CTL lines infused recognized EBV-encoded nuclear Ag-3 proteins, but CTL protein specificity itself did not correlate with tumor response. Specificity in conjunction with donor/recipient functional HLA matching as opposed to HLA matching alone, however, was important for tumor response. CTL receptor TCR beta-chain variable gene subfamilies were polyclonal, with no preferential use of a particular family. However, tumor response was improved in those receiving CTL lines with polyclonal vs clonal distribution for subfamilies 2, 3, and 9. Interestingly, in five of six tumors (five Hodgkin's-like and one Burkitt's-like posttransplant lymphoproliferative disease) with restricted viral gene expression a complete response was recorded, although in some cases the tumor cells did not express the proteins recognized by the infused CTL. Thus CTL were advantageous when functionally HLA matched but for certain tumor types complete responses occurred in the absence of detectable specific CTL/tumor recognition. We suggest that either the allogenic CTL contained small, undetectable, EBV-specific, HLA-matched T cell populations or perhaps they stimulated nonspecific inflammatory responses in vivo, which were beneficial for tumor regression. These observations should be considered when designing and implementing CTL therapies.

Infectious mononucleosis in university students in the United kingdom: evaluation of the clinical features and consequences of the disease.

BACKGROUND: Infectious mononucleosis (IM) is common among university students. We undertook to analyze the clinical features and sequelae of the disease in a cohort of students at Edinburgh University. METHODS: Consecutive IM case patients were recruited from 2000 through 2002 at the University Health Service after diagnosis of IM. RESULTS: IM resulted in marked reductions in student study time, physical exercise, and non-exercise-related social activities, and sustained increases in reported number of hours of sleep. The disease profile differed between the sexes, with significantly more females reporting fatigue, which was more likely to be prolonged (P = .003) and to lead to loss of study time (P = .013). Female case patients were more likely to discontinue their studies following IM (16% vs 0%; P = .056). Within the typically elevated lymphocyte counts in IM, we identified an elevated gammadelta T cell component that may contribute to the disease pathogenesis. CONCLUSIONS: IM results in substantial morbidity among university students, reported as more profound in females, and affecting academic studies, physical exercise, and social activities. Immunization to prevent IM and strategies to reduce post-IM disability would be beneficial in this population.

HLA class I polymorphisms are associated with development of infectious mononucleosis upon primary EBV infection.

Infectious mononucleosis (IM) is an immunopathological disease caused by EBV that occurs in young adults and is a risk factor for Hodgkin lymphoma (HL). An association between EBV-positive HL and genetic markers in the HLA class I locus has been identified, indicating that genetic differences in the HLA class I locus may alter disease phenotypes associated with EBV infection. To further determine whether HLA class I alleles may affect development of EBV-associated diseases, we analyzed 2 microsatellite markers and 2 SNPs located near the HLA class I locus in patients with acute IM and in asymptomatic EBV-seropositive and -seronegative individuals. Alleles of both microsatellite markers were significantly associated with development of IM. Specific alleles of the 2 SNPs were also significantly more frequent in patients with IM than in EBV-seronegative individuals. IM patients possessing the associated microsatellite allele had fewer lymphocytes and increased neutrophils relative to IM patients lacking the allele. These patients also displayed higher EBV titers and milder IM symptoms. The results of this study indicate that HLA class I polymorphisms may predispose patients to development of IM upon primary EBV infection, suggesting that genetic variation in T cell responses can influence the nature of primary EBV infection and the level of viral persistence.

Cytolytic mechanisms and T-cell receptor Vbeta usage by ex vivo generated Epstein-Barr virus-specific cytotoxic T lymphocytes.

Ex-vivo-generated Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) have been used for cellular adoptive immunotherapy of EBV-associated lymphomas. Here we investigated the phenotypes, cytolytic mechanisms, polyfunctionality and T-cell receptor (TCR) usage in growing and established CTL, generated by weekly stimulation with an EBV-transformed autologous lymphoblastoid cell line (LCL). Our results showed that phenotypically mature CTL developed within the first 4 weeks of culture, with an increase in CD45RO and CD69, and a decrease in CD45RA, CD62L, CD27 and CD28 expression. Spectratyping analysis of the variable beta-chain of the TCR revealed that TCR repertoire remained diverse during the course of culture. Cytotoxicity of CTL was significantly inhibited by concanamycin A (P < 0.0001) and ethylene glycol-bis tetraacetic acid (P < 0.0001), indicating that a calcium and perforin-mediated exocytosis pathway with the release of granzyme B was the principal cytotoxic mechanism. The CTL mainly produced interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN-gamma and TNF-alpha. Granzyme B, perforin and Fas ligand were detected in CD8(+) and CD4(+) cells in all CTL; however, a greater proportion of CD8(+) than CD4(+) T cells expressed granzyme B (P < 0.0001) and more granzyme B was detected in CD8(+) T cells than in CD4(+) T cells (P = 0.001). This difference was not observed with Fas ligand or perforin expression. Our results provide insight into the basic characteristics of ex-vivo-generated CTL.

EBV+ diffuse large B-cell lymphoma arising within atrial myxoma. An example of a distinct primary cardiac EBV+ DLBCL of immunocompetent patients.

Cardiac myxoma and diffuse large B-cell lymphoma are uncommon tumors, yet four composite tumors have been reported since 2009. We are reporting on the fifth case providing detailed immunohistochemical and FISH analyses. The lymphoma was present as superficially located nests of large cells with patchy necrosis in the background of a typical atrial myxoma. It displayed features of DLBCL with non-germinal center phenotype, expressed EBER, LMP1, EBNA2 and shared the following features with the previously reported cases: B-cell lineage, high-grade cytology, high proliferation rate, EBV infection in latency type 3 with one tested case and an excellent outcome. The lymphomas arising within myxoma may follow a pathogenic pathway driven by EBV, whose transformation potential is unleashed in the cytokine-rich milieu of a myxoma, presumably accentuating age-related decline of adaptive immunity known as immune senescence. DLBCL arising within atrial myxoma grouped together with EBV+ DLBCL associated with valve prosthesis and with an atrial thrombus differs in the immunocompetent patients from primary cardiac DLBCL, not otherwise specified, in clinical presentation, pathological features and a course of the disease. Distinction between these groups may have important therapeutic consequences.

Role of the HLA system in the association between multiple sclerosis and infectious mononucleosis.

OBJECTIVE: To determine whether multiple sclerosis (MS) and infectious mononucleosis (IM) share common HLA associations. DESIGN: A prospective cohort study was conducted from October 1, 1999, through September 30, 2003. SETTING: University of Edinburgh Richard Verney Health Centre, Edinburgh, Scotland. PATIENTS: Participants included 179 individuals who underwent asymptomatic Epstein-Barr virus seroconversion and 175 patients who developed IM. INTERVENTION: Genotyping for 5 classical HLA loci (HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1). MAIN OUTCOME MEASURE: Diagnosis of IM and allele frequency. RESULTS: Allelic analysis showed that HLA-DRB1*01:01 was significantly associated with the development of IM (odds ratio, 3.2; P < .001). Patients with IM and HLA-DRB1*01:01 had a lower Epstein-Barr virus viral load compared with those without the allele (median, 783 vs 7366 copies/10(6) peripheral blood mononuclear cells; P = .03). CONCLUSION: HLA-DRB1*01:01 is protective against developing MS; thus, a common genetic basis between IM and MS is not supported.

Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion.

BACKGROUND & AIMS: Studies have indicated that stem cells have unexpected plasticity and can differentiate down a multitude of nonhematopoietic cell lineages in rodents. Our aim was to identify whether human cord blood cells, which are a rich source of stem cells, would be able to differentiate into hepatocytes when infused into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. We also wanted to test whether such differentiated cells were the result of cellular fusion or true stem cell transdifferentiation. METHODS: Unsorted mononuclear cell preparations of human cord blood were infused into sublethally irradiated NOD-SCID mice. After death, immunohistologic analysis of murine livers was performed using human specific hepatocyte, biliary, and endothelial markers. Fluorescent in situ hybridization (FISH) for mouse and human DNA was also performed. RESULTS: We show that human cord blood cells have the ability to engraft into NOD-SCID liver and become mature hepatocytes. We were unable to identify any biliary or endothelial differentiation. Furthermore, we do not detect any evidence of cell fusion in any of the human cells found in the mouse liver, suggesting that human cord blood cells are capable of true transdifferentiation into hepatocytes in vivo. CONCLUSIONS: We conclude that hepatocytes can derive from human cord blood cells when infused into NOD-SCID mice in the absence of fusion. The demonstration that human stem cell differentiation can occur in this murine model permits comprehensive study of human stem cell plasticity in vivo.