A metabolic dependency of EBV can be targeted to hinder B cell transformation.

Following infection of B cells, Epstein Barr virus (EBV) engages host pathways that mediate cell proliferation and transformation, contributing to the propensity of the virus to drive immune dysregulation and lymphomagenesis. We found that the EBV protein EBNA2 initiates NAD de novo biosynthesis by driving expression of the metabolic enzyme IDO1 in infected B cells. Virus-enforced NAD production sustained mitochondrial complex I activity, to match ATP-production with bioenergetic requirements of proliferation and transformation. In transplant patients, IDO1 expression in EBV-infected B cells, and a serum signature of increased IDO1 activity, preceded development of lymphoma. In humanized mice infected with EBV, IDO1 inhibition reduced both viremia and lymphomagenesis. Virus-orchestrated NAD biosynthesis is, thus, a druggable metabolic vulnerability of EBV-driven B cell transformation-opening therapeutic possibilities for EBV-related diseases.

Reduced frequency of cytotoxic CD56dim CD16+ NK cells leads to impaired antibody-dependent degranulation in EBV-positive classical Hodgkin lymphoma.

Around 30-50% of classical Hodgkin lymphoma (cHL) cases in immunocompetent individuals from industrialized countries are associated with the B-lymphotropic Epstein-Barr virus (EBV). Although natural killer (NK) cells exhibit anti-viral and anti-tumoral functions, virtually nothing is known about quantitative and qualitative differences in NK cells in patients with EBV+ cHL vs. EBV- cHL. Here, we prospectively investigated 36 cHL patients without known immune suppression or overt immunodeficiency at diagnosis. All 10 EBV+ cHL patients and 25 out 26 EBV- cHL were seropositive for EBV antibodies, and EBV+ cHL patients presented with higher plasma EBV DNA levels compared to EBV- cHL patients. We show that the CD56dim CD16+ NK cell subset was decreased in frequency in EBV+ cHL patients compared to EBV- cHL patients. This quantitative deficiency translates into an impaired CD56dim NK cell mediated degranulation toward rituximab-coated HLA class 1 negative lymphoblastoid cells in EBV+ compared to EBV- cHL patients. We finally observed a trend to a decrease in the rituximab-associated degranulation and ADCC of in vitro expanded NK cells of EBV+ cHL compared to healthy controls. Our findings may impact on the design of adjunctive treatment targeting antibody-dependent cellular cytotoxicity in EBV+ cHL.

CD4 + T cells are found within endemic Burkitt lymphoma and modulate Burkitt lymphoma precursor cell viability and expression of pathogenically relevant Epstein-Barr virus genes.

Endemic Burkitt lymphoma (eBL) is an aggressive B cell cancer characterized by an IgH/c-myc translocation and the harboring of Epstein-Barr virus (EBV). Evidence accumulates that CD4 + T cells might contribute to eBL pathogenesis. Here, we investigate the presence of CD4 + T cells in primary eBL tissue and their potential dichotomous impact on an EBV-infected pre-eBL cell model using ex vivo material and in vitro co-cultures. In addition, we establish a novel method to study the effect of IgH/c-myc translocation in primary B cells by employing a CRISPR/Cas9 knock-in approach to introduce and tag de novo translocation. We unprecedently document that CD4 + T cells are present in primary eBL tumor tissue. Furthermore, we demonstrate that CD4 + T cells on the one hand suppress eBL development by killing pre-eBL cells lacking IgH/c-myc translocation in vitro and on the other hand indirectly promote eBL development by inducing crucial EBV Latency III to Latency I switching in pre-eBL cells. Finally, we show that while the mere presence of an IgH/c-myc translocation does not suffice to escape CD4 + T-cell-mediated killing in vitro, the CD4 + T-cell-mediated suppression of EBV's Latency III program in vivo may allow cells harboring an IgH/c-myc translocation and additional mutations to evade immune control and proliferate by means of deregulated c-myc activity, resulting in neoplasia. Thus, our study highlights the dichotomous effects of CD4 + T cells and the mechanisms involved in eBL pathogenesis, suggests mechanisms of their impact on eBL progression, and provides a novel in vitro model for further investigation of IgH/c-myc translocation.

Correction: Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

[This corrects the article DOI: 10.1371/journal.ppat.1008477.].

Co-infection of Cytomegalovirus and Epstein-Barr Virus Diminishes the Frequency of CD56dimNKG2A+KIR- NK Cells and Contributes to Suboptimal Control of EBV in Immunosuppressed Children With Post-transplant Lymphoproliferative Disorder.

Post-transplant lymphoproliferative disorder (PTLD) is a rare but potentially life-threatening complication, frequently associated with Epstein-Barr virus (EBV), which develops after solid organ or stem cell transplantation. Immunosuppression received by transplant recipients has a significant impact on the development of PTLD by suppressing the function of T cells. The preferential proliferation of NKG2A-positive natural killer (NK) cells during primary symptomatic EBV infection known as infectious mononucleosis (IM) and their reactivity toward EBV-infected B cells point to a role of NK cell in the immune control of EBV. However, NK cell-mediated immune response to EBV in immunosuppressed transplant recipients who develop PTLD remains unclear. In this study, we longitudinally analyzed the phenotype and function of different NK cell subsets in a cohort of pediatric liver transplant patients who develop PTLD and compared them to those of children with IM. We found persistently elevated plasma EBV DNA levels in the PTLD patients indicating suboptimal anti-viral immune control. PTLD patients had markedly decreased frequency of CD56dimNKG2A+Killer Immunoglobulin-like receptor (KIR)- NK cells from the time of diagnosis through remission compared to those of IM patients. Whilst the proliferation of CD56dimNKG2A+KIR- NK cells was diminished in PTLD patients, this NK cell subset maintained its ability to potently degranulate against EBV-infected B cells. Compared to cytomegalovirus (CMV)-seropositive and -negative IM patients, PTLD patients co-infected with CMV and EBV had significantly higher levels of a CMV-associated CD56dimNKG2ChiCD57+NKG2A-KIR+ NK cell subset accumulating at the expense of NKG2A+KIR- NK cells. Taken together, our data indicate that co-infection of CMV and EBV diminishes the frequency of CD56dimNKG2A+KIR- NK cells and contributes to suboptimal control of EBV in immunosuppressed children with PTLD.

Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies.

CD8+ T cells retain protective functions despite sustained inhibitory receptor expression during Epstein-Barr virus infection in vivo.

Epstein Barr virus (EBV) is one of the most ubiquitous human pathogens in the world, persistently infecting more than 90% of the adult human population. It drives some of the strongest human CD8+ T cell responses, which can be observed during symptomatic primary infection known as infectious mononucleosis (IM). Despite high viral loads and prolonged CD8+ T cell stimulation during IM, EBV enters latency and is under lifelong immune control in most individuals that experience this disease. We investigated whether changes in T cell function, as frequently characterized by PD-1 up-regulation, occur during IM due to the prolonged exposure to high antigen levels. We readily detected the expansion of PD-1 positive CD8+ T cells together with high frequencies of Tim-3, 2B4, and KLRG1 expression during IM and in mice with reconstituted human immune system components (huNSG mice) that had been infected with a high dose of EBV. These PD-1 positive CD8+ T cells, however, retained proliferation, cytokine production, and cytotoxic abilities. Multiple subsets of CD8+ T cells expanded during EBV infection, including PD-1+Tim-3+KLRG1+ cells that express CXCR5 and TCF-1 germinal center homing and memory markers, and may also contain BATF3. Moreover, blocking the PD-1 axis compromised EBV specific immune control and resulted in virus-associated lymphomagenesis. Finally, PD-1+, Tim-3+, and KLRG1+ CD8+ T cell expansion coincided with declining viral loads during low dose EBV infection. These findings suggest that EBV infection primes PD-1 positive CD8+ T cell populations that rely on this receptor axis for the efficient immune control of this ubiquitous human tumor virus.

Two alternate strategies for innate immunity to Epstein-Barr virus: One using NK cells and the other NK cells and γδ T cells.

Most humans become infected with Epstein-Barr virus (EBV), which then persists for life. Infrequently, EBV infection causes infectious mononucleosis (IM) or Burkitt lymphoma (BL). Type I EBV infection, particularly type I BL, stimulates strong responses of innate immune cells. Humans respond to EBV in two alternative ways. Of 24 individuals studied, 13 made strong NK and γδ T cell responses, whereas 11 made feeble γδ T cell responses but stronger NK cell responses. The difference does not correlate with sex, HLA type, or previous exposure to EBV or cytomegalovirus. Cohorts of EBV+ children and pediatric IM patients include both group 1 individuals, with high numbers of γδ T cells, and group 2 individuals, with low numbers. The even balance of groups 1 and 2 in the human population points to both forms of innate immune response to EBV having benefit for human survival. Correlating these distinctive responses with the progress of EBV infection might facilitate the management of EBV-mediated disease.

Infectious Mononucleosis Triggers Generation of IgG Auto-Antibodies against Native Myelin Oligodendrocyte Glycoprotein.

A history of infectious mononucleosis (IM), symptomatic primary infection with the Epstein Barr virus, is associated with the development of autoimmune diseases and increases the risk to develop multiple sclerosis. Here, we hypothesized that immune activation during IM triggers autoreactive immune responses. Antibody responses towards cellular antigens using a HEp-2 based indirect immunofluorescence assay and native myelin oligodendrocyte glycoprotein (MOG) using a flow cytometry-based assay were determined in 35 patients with IM and in 23 control subjects. We detected frequent immunoglobulin M (IgM) reactivity to vimentin, a major constituent of the intermediate filament family of proteins, in IM patients (27/35; 77%) but rarely in control subjects (2/23; 9%). IgG autoantibodies binding to HEp-2 cells were absent in both groups. In contrast, IgG responses to native MOG, present in up to 40% of children with inflammatory demyelinating diseases of the central nervous system (CNS), were detectable in 7/35 (20%) patients with IM but not in control subjects. Normalization of anti-vimentin IgM levels to increased total IgM concentrations during IM resulted in loss of significant differences for anti-vimentin IgM titers. Anti-MOG specific IgG responses were still detectable in a subset of three out of 35 patients with IM (9%), even after normalization to increased total IgG levels. Vimentin-specific IgM and MOG-specific IgG responses decreased following clinical resolution of acute IM symptoms. We conclude from our data that MOG-specific memory B cells are activated in subset of patients with IM.

Role for early-differentiated natural killer cells in infectious mononucleosis.

A growing body of evidence suggests that the human natural killer (NK)-cell compartment is phenotypically and functionally heterogeneous and is composed of several differentiation stages. Moreover, NK-cell subsets have been shown to exhibit adaptive immune features during herpes virus infection in experimental mice and to expand preferentially during viral infections in humans. However, both phenotype and role of NK cells during acute symptomatic Epstein-Barr virus (EBV) infection, termed infectious mononucleosis (IM), remain unclear. Here, we longitudinally assessed the kinetics, the differentiation, and the proliferation of subsets of NK cells in pediatric IM patients. Our results indicate that acute IM is characterized by the preferential proliferation of early-differentiated CD56(dim) NKG2A(+) immunoglobulin-like receptor(-) NK cells. Moreover, this NK-cell subset exhibits features of terminal differentiation and persists at higher frequency during at least the first 6 months after acute IM. Finally, we demonstrate that this NK-cell subset preferentially degranulates and proliferates on exposure to EBV-infected B cells expressing lytic antigens. Thus, early-differentiated NK cells might play a key role in the immune control of primary infection with this persistent tumor-associated virus.

Oropharyngeal group A streptococcal colonization disrupts latent Epstein-Barr virus infection.

Epstein-Barr virus (EBV) infects >90% of the human population within the first 2 decades of life and establishes reversible latent infection in B cells. The stimuli that lead to switching from latent to lytic EBV infection in vivo are still elusive. Group A streptococci (GAS) are a common cause of bacterial pharyngotonsillitis in children and adolescents and colonize the tonsils and pharynx of up to 20% of healthy children. Thus, concomitant presence of EBV and GAS in the same individual is frequent. Here, we show that EBV carriers who are colonized with GAS shed EBV particles in higher numbers in their saliva, compared with EBV carriers not colonized with GAS. Messenger RNA levels of the master lytic regulatory EBV gene BZLF1 were more frequently detected in tonsils from EBV carriers colonized with GAS than from EBV carriers not colonized. Heat-killed GAS, potentially mimicking GAS colonization, elicited lytic EBV in latently infected lymphoblastoid cell lines (LCLs) partially via Toll-like receptor 2 triggering, as did purified GAS peptidoglycan. Thus, colonization by GAS might benefit EBV by increasing the EBV load in saliva and thereby enhancing the likelihood of EBV spread to other hosts.

A distinct subpopulation of human NK cells restricts B cell transformation by EBV.

NK cells constitute the first line of defense against pathogens and transformed cells. They mature in secondary lymphoid organs, including tonsils, where common pathogens, such as EBV, enter the host and potentially imprint differentiating cells, which then patrol the body via the blood stream. Therefore, we set out to characterize a distinct human NK cell population in tonsils that produces high amounts of the immunomodulatory and antiviral cytokine IFN-γ. We found that the tonsilar IFN-γ(high) NK cell subset is CD56(bright)NKG2A(+)CD94(+)CD54(+)CD62L(-), is present in tonsils ex vivo and is more mature than other CD56(bright) NK cells in tonsils and less mature than other NK cells in blood, shows very low plasticity even after prolonged cytokine stimulation, accumulates in tonsils of EBV carriers, and is able to potently restrict EBV-induced transformation of B cells. Thus, we characterized a distinct and stable IFN-γ(high) NK cell subpopulation that can specifically restrict malignant transformation of EBV-infected B cells. This subset should be exploited for future development of cell-based therapeutic approaches in EBV-associated malignancies.

Innate immune responses against Epstein Barr virus infection.

EBV persists life-long in >95% of the human adult population. Whereas it is perfectly immune-controlled in most infected individuals, a minority develops EBV-associated diseases, primarily malignancies of B cell and epithelial cell origin. In recent years, it has become apparent that the course of primary infection determines part of the risk to develop EBV-associated diseases. Particularly, the primary symptomatic EBV infection or IM, which is caused by exaggerated T cell responses, resulting in EBV-induced lymphocytosis, predisposes for EBV-associated diseases. The role of innate immunity in the development of IM remains unknown. Therefore, it is important to understand how the innate immune response to this virus differs between symptomatic and asymptomatic primary EBV infection. Furthermore, the efficiency of innate immune compartments might determine the outcome of primary infection and could explain why some individuals are susceptible to IM. We will discuss these aspects in this review with a focus on intrinsic immunity in EBV-infected B cells, as well as innate immune responses by DCs and NK cells, which constitute promising immune compartments for the understanding of early immune control against EBV and potential targets for EBV-specific immunotherapies.