Human Epstein-Barr Virus Infection Research Articles

Cyclosporine A induces Epstein–Barr virus reactivation in tree shrew (Tupaia belangeri chinensis) model

Epstein–Barr virus (EBV) usually exists as a latent infection in immunocompetent hosts but immunosuppressed individuals are at risk for developing EBV reactivation that leads to the uncontrolled proliferation of B lymphocytes. In this study, we have mimicked the immunosuppressed microenvironment in the tree shrew model of EBV infection by using cyclosporine A (CsA). The results showed that EBV-cocultured peripheral blood mononuclear cells (PBMCs) proliferated vigorously in response to CsA treatment in vitro. However, EBV susceptibility in vivo depended on the timing of CsA administration. Reactivation of EBV occurred in the latently EBV-infected tree shrews after treatment with 25 mg/kg/day CsA (EBV > CsA group), whereas tree shrews were no longer susceptible to infection if CsA was administered for five weeks before EBV injection (CsA > EBV group). RNA-seq analysis of both groups identified a further link between immunosuppression and EBV infection. KEGG pathway enrichment analysis revealed a significant enrichment of viral infection-related pathways in the EBV > CsA group, whereas tumor-related pathways were significantly enriched in the CsA > EBV group. A protein–protein interaction network was constructed using Cytoscape for the purpose of identifying hub genes that were then verified using qRT-PCR. In conclusion, the tree shrew model of EBV infection exhibits certain features of EBV infection in humans and serves as a valuable platform for exploring the underlying mechanisms of EBV infection.

Protective anti-gB neutralizing antibodies targeting two vulnerable sites for EBV-cell membrane fusion

Epstein-Barr virus (EBV) infects more than 90% of the world's adult population and accounts for a significant cancer burden of epithelial and B cell origins. Glycoprotein B (gB) is the primary fusogen essential for EBV entry into host cells. Here, we isolated two EBV gB-specific neutralizing antibodies, 3A3 and 3A5; both effectively neutralized the dual-tropic EBV infection of B and epithelial cells. In humanized mice, both antibodies showed effective protection from EBV-induced lymphoproliferative disorders. Cryoelectron microscopy analyses identified that 3A3 and 3A5 bind to nonoverlapping sites on domains D-II and D-IV, respectively. Structure-based mutagenesis revealed that 3A3 and 3A5 inhibit membrane fusion through different mechanisms involving the interference with gB-cell interaction and gB activation. Importantly, the 3A3 and 3A5 epitopes are major targets of protective gB-specific neutralizing antibodies elicited by natural EBV infection in humans, providing potential targets for antiviral therapies and vaccines.

Tree Shrew Is a Suitable Animal Model for the Study of Epstein Barr Virus.

Epstein-Barr virus (EBV) is a human herpesvirus that latently infects approximately 95% of adults and is associated with a spectrum of human diseases including Infectious Mononucleosis and a variety of malignancies. However, understanding the pathogenesis, vaccines and antiviral drugs for EBV-associated disease has been hampered by the lack of suitable animal models. Tree shrew is a novel laboratory animal with a close phylogenetic relationship to primates, which is a critical advantage for many animal models for human disease, especially viral infections. Herein, we first identified the key residues in the CR2 receptor that bind the gp350 protein and facilitate viral entry. We found that tree shrew shares 100% sequence identity with humans in these residues, which is much higher than rabbits (50%) and rats (25%). In vitro analysis showed that B lymphocytes of tree shrews are susceptible to EBV infection and replication, as well as EBV-enhanced cell proliferation. Moreover, results of in vivo experiments show that EBV infection in tree shrews resembles EBV infection in humans. The infected animals exhibited transient fever and loss of weight accompanied by neutropenia and high viremia levels during the acute phase of the viral infection. Thereafter, tree shrews acted as asymptomatic carriers of the virus in most cases that EBV-related protein could be detected in blood and tissues. However, a resurgence of EBV infection occurred at 49 dpi. Nanopore transcriptomic sequencing of peripheral blood in EBV-infected animals revealed the dynamic changes in biological processes occurring during EBV primary infection. Importantly, we find that neutrophil function was impaired in tree shrew model as well as human Infectious Mononucleosis datasets (GSE85599 and GSE45918). In addition, retrospective case reviews suggested that neutropenia may play an important role in EBV escaping host innate immune response, leading to long-term latent infection. Our findings demonstrated that tree shrew is a suitable animal model to evaluate the mechanisms of EBV infection, and for developing vaccines and therapeutic drugs against EBV.

Regulatory network analysis of Epstein-Barr virus identifies functional modules and hub genes involved in infectious mononucleosis.

Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis (IM) and establishes lifetime infection associated with a variety of cancers and autoimmune diseases. The aim of this study was to develop an integrative gene regulatory network (GRN) approach and overlying gene expression data to identify the representative subnetworks for IM and EBV latent infection (LI). After identifying differentially expressed genes (DEGs) in both IM and LI gene expression profiles, functional annotations were applied using gene ontology (GO) and BiNGO tools, and construction of GRNs, topological analysis and identification of modules were carried out using several plugins of Cytoscape. In parallel, a human-EBV GRN was generated using the Hu-Vir database for further analyses. Our analysis revealed that the majority of DEGs in both IM and LI were involved in cell-cycle and DNA repair processes. However, these genes showed a significant negative correlation in the IM and LI states. Furthermore, cyclin-dependent kinase 2 (CDK2) - a hub gene with the highest centralityscore - appeared to be the key player in cell cycle regulation in IM disease. The most significant functional modules in the IM and LI states were involved in the regulation of the cell cycle and apoptosis, respectively. Human-EBV network analysis revealed several direct targets of EBV proteins during IM disease. Our study provides an important first report on the response to IM/LI EBV infection in humans. An important aspect of our data was the upregulation of genes associated with cell cycle progression and proliferation.

Mouse model for acute Epstein–Barr virus infection

Epstein-Barr Virus (EBV) infects human B cells and drives them into continuous proliferation. Two key viral factors in this process are the latent membrane proteins LMP1 and LMP2A, which mimic constitutively activated CD40 receptor and B-cell receptor signaling, respectively. EBV-infected B cells elicit a powerful T-cell response that clears the infected B cells and leads to life-long immunity. Insufficient immune surveillance of EBV-infected B cells causes life-threatening lymphoproliferative disorders, including mostly germinal center (GC)-derived B-cell lymphomas. We have modeled acute EBV infection of naive and GC B cells in mice through timed expression of LMP1 and LMP2A. Although lethal when induced in all B cells, induction of LMP1 and LMP2A in just a small fraction of naive B cells initiated a phase of rapid B-cell expansion followed by a proliferative T-cell response, clearing the LMP-expressing B cells. Interfering with T-cell activity prevented clearance of LMP-expressing B cells. This was also true for perforin deficiency, which in the human causes a life-threatening EBV-related immunoproliferative syndrome. LMP expression in GC B cells impeded the GC reaction but, upon loss of T-cell surveillance, led to fatal B-cell expansion. Thus, timed expression of LMP1 together with LMP2A in subsets of mouse B cells allows one to study major clinically relevant features of human EBV infection in vivo, opening the way to new therapeutic approaches.

The Incubation Period of Primary Epstein-Barr Virus Infection: Viral Dynamics and Immunologic Events.

Epstein-Barr virus (EBV) is a human herpesvirus that causes acute infectious mononucleosis and is associated with cancer and autoimmune disease. While many studies have been performed examining acute disease in adults following primary infection, little is known about the virological and immunological events during EBV’s lengthy 6 week incubation period owing to the challenge of collecting samples from this stage of infection. We conducted a prospective study in college students with special emphasis on frequent screening to capture blood and oral wash samples during the incubation period. Here we describe the viral dissemination and immune response in the 6 weeks prior to onset of acute infectious mononucleosis symptoms. While virus is presumed to be present in the oral cavity from time of transmission, we did not detect viral genomes in the oral wash until one week before symptom onset, at which time viral genomes were present in high copy numbers, suggesting loss of initial viral replication control. In contrast, using a sensitive nested PCR method, we detected viral genomes at low levels in blood about 3 weeks before symptoms. However, high levels of EBV in the blood were only observed close to symptom onset–coincident with or just after increased viral detection in the oral cavity. These data imply that B cells are the major reservoir of virus in the oral cavity prior to infectious mononucleosis. The early presence of viral genomes in the blood, even at low levels, correlated with a striking decrease in the number of circulating plasmacytoid dendritic cells well before symptom onset, which remained depressed throughout convalescence. On the other hand, natural killer cells expanded only after symptom onset. Likewise, CD4+ Foxp3+ regulatory T cells decreased two fold, but only after symptom onset. We observed no substantial virus specific CD8 T cell expansion during the incubation period, although polyclonal CD8 activation was detected in concert with viral genomes increasing in the blood and oral cavity, possibly due to a systemic type I interferon response. This study provides the first description of events during the incubation period of natural EBV infection in humans and definitive data upon which to formulate theories of viral control and disease pathogenesis.

Modeling EBV infection and pathogenesis in new-generation humanized mice.

The development of highly immunodeficient mouse strains has allowed the reconstitution of functional human immune system components in mice. New-generation humanized mice generated in this manner have been extensively used for modeling viral infections that are exclusively human tropic. Epstein–Barr virus (EBV)-infected humanized mice reproduce cardinal features of EBV-associated B-cell lymphoproliferative disease and EBV-associated hemophagocytic lymphohistiocytosis (HLH). Erosive arthritis morphologically resembling rheumatoid arthritis (RA) has also been recapitulated in these mice. Low-dose EBV infection of humanized mice results in asymptomatic, persistent infection. Innate immune responses involving natural killer cells, EBV-specific adaptive T-cell responses restricted by human major histocompatibility and EBV-specific antibody responses are also elicited in humanized mice. EBV-associated T-/natural killer cell lymphoproliferative disease, by contrast, can be reproduced in a distinct mouse xenograft model. In this review, recent findings on the recapitulation of human EBV infection and pathogenesis in these mouse models, as well as their application to preclinical studies of experimental anti-EBV therapies, are described.

Natural EBV infection in humans induces an expression profile similar to cytokine storm illnesses that is unique from other acute viral infections (P6326)

Abstract Herpesvirus infections are common in humans, and while generally not lethal, lead to lifelong infection associated with cancer and autoimmune disease. To better understand immunity to persistent viral infection, we performed a prospective study in otherwise healthy college students of naturally acquired infection by the gamma herpesvirus, Epstein Barr virus (EBV). Transcriptome analysis defined a striking and reproducible profile during acute infection regardless of disease severity and included genes related to cell cycle, interferon response, and immune function. Even with the highly sensitive approach taken, however, no lasting gene changes were apparent during latent infection. Comparing the EBV response profile to multiple other acute viral infections revealed similarity only to Dengue virus. The signature shared by EBV and Dengue was also present in inflammatory syndrome patients. Interestingly, while EBV induced a type I interferon response, a subset of interferon induced genes, including MX1 and OAS1, were not upregulated, despite these genes being conventional contributors in a type I interferon response. Furthermore, IL1B was downregulated, together suggesting potential mechanisms of viral antagonism. These data provide an important first description of the response to natural herpesvirus infection in humans during multiple stages of infection.

Humanized mouse models of epstein-barr virus infection and associated diseases.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus infecting more than 90% of the adult population of the world. EBV is associated with a variety of diseases including infectious mononucleosis, lymphoproliferative diseases, malignancies such as Burkitt lymphoma and nasopharyngeal carcinoma, and autoimmune diseases including rheumatoid arthritis (RA). EBV in nature infects only humans, but in an experimental setting, a limited species of new-world monkeys can be infected with the virus. Small animal models, suitable for evaluation of novel therapeutics and vaccines, have not been available. Humanized mice, defined here as mice harboring functioning human immune system components, are easily infected with EBV that targets cells of the hematoimmune system. Furthermore, humanized mice can mount both cellular and humoral immune responses to EBV. Thus, many aspects of human EBV infection, including associated diseases (e.g., lymphoproliferative disease, hemophagocytic lymphohistiocytosis and erosive arthritis resembling RA), latent infection, and T-cell-mediated and humoral immune responses have been successfully reproduced in humanized mice. Here we summarize recent achievements in the field of humanized mouse models of EBV infection and show how they have been utilized to analyze EBV pathogenesis and normal and aberrant human immune responses to the virus.

Human Solid Tumor Xenografts in Immunodeficient Mice Are Vulnerable to Lymphomagenesis Associated with Epstein-Barr Virus

Xenografting primary human solid tumor tissue into immunodeficient mice is a widely used tool in studies of human cancer biology; however, care must be taken to prove that the tumors obtained recapitulate parent tissue. We xenografted primary human hepatocellular carcinoma (HCC) tumor fragments or bulk tumor cell suspensions into immunodeficient mice. We unexpectedly observed that 11 of 21 xenografts generated from 16 independent patient samples resembled lymphoid neoplasms rather than HCC. Immunohistochemistry and flow cytometry analyses revealed that the lymphoid neoplasms were comprised of cells expressing human CD45 and CD19/20, consistent with human B lymphocytes. In situ hybridization was strongly positive for Epstein-Barr virus (EBV) encoded RNA. Genomic analysis revealed unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements in each B-cell neoplasm. These data demonstrate that the lymphoid neoplasms were EBV-associated human B-cell lymphomas. Analogous to EBV-associated lymphoproliferative disorders in immunocompromised humans, the human lymphomas in these HCC xenografts likely developed from reactivation of latent EBV in intratumoral passenger B lymphocytes following their xenotransplantation into immunodeficient recipient mice. Given the high prevalence of latent EBV infection in humans and the universal presence of B lymphocytes in solid tumors, this potentially confounding process represents an important pitfall of human solid tumor xenografting. This phenomenon can be recognized and avoided by routine phenotyping of primary tumors and xenografts with human leukocyte markers, and provides a compelling biological rationale for exclusion of these cells from human solid tumor xenotransplantation assays.

Primary Epstein-Barr virus infection does not erode preexisting CD8+ T cell memory in humans

Acute Epstein-Barr virus (EBV) infection results in an unusually robust CD8(+) T cell response in young adults. Based on mouse studies, such a response would be predicted to result in attrition of preexisting memory to heterologous infections like influenza A (Flu) and cytomegalovirus (CMV). Furthermore, many studies have attempted to define the lymphocytosis that occurs during acute EBV infection in humans, but it is unclear whether bystander T cells contribute to it. To address these issues, we performed a longitudinal prospective study of primary EBV infection in humans. During acute EBV infection, both preexisting CMV- and Flu-specific memory CD8(+) T cells showed signs of bystander activation, including up-regulation of granzyme B. However, they generally did not expand, suggesting that the profound CD8(+) lymphocytosis associated with acute EBV infection is composed largely of EBV-specific T cells. Importantly, the numbers of CMV- and Flu-specific T cells were comparable before and after acute EBV infection. The data support the concept that, in humans, a robust CD8(+) T cell response creates a new memory CD8(+) T cell niche without substantially depleting preexisting memory for heterologous infections.

Antibodies to Lytic Infection Proteins in Lymphocryptovirus-Infected Rhesus Macaques: a Model for Humoral Immune Responses to Epstein-Barr Virus Infection

Humoral immune responses to rhesus lymphocryptovirus (rhLCV) lytic infection proteins were evaluated in the rhesus macaque animal model for Epstein-Barr virus (EBV) infection. We found a hierarchy of humoral responses to 14 rhLCV lytic infection proteins in naturally infected rhesus macaques, with (i) widespread and robust responses to four glycoproteins expressed as late proteins, (ii) frequent but less robust responses to a subset of early proteins, and (iii) low-level responses to immediate-early proteins. This hierarchy of humoral responses was similar to that reported for EBV-infected humans, with the notable exception of the response to rhBARF1. Serum antibodies to rhBARF1 were frequently detected in healthy rhLCV-infected macaques, but in humans, anti-BARF1 antibodies have been reported primarily in patients with EBV-positive nasopharyngeal carcinoma (NPC). The macaque data accurately predicted that serum antibodies against BARF1 are a normal response to EBV infection when human serum samples are analyzed. The rhesus macaque animal provides a unique perspective on humoral responses to EBV infection in humans and can be a valuable model for EBV vaccine development.

The immune response of foals to natural infection with equid herpesvirus-2 and its association with febrile illness

Equid herpesvirus-2 (EHV-2) infection is ubiquitous in horses. Although EHV-2 infection has been associated with several disease syndromes, its true pathogenic significance in horses remains uncertain. Epstein-Barr virus (EBV), another gammaherpesvirus, has been shown to cause febrile illness in humans related to its immunopathologic effects. Thus, the purpose of this study was to describe the ontogeny of the immune response of a cohort of 9 foals to natural infection with EHV-2 by evaluating serial complete blood counts, lymphocyte morphology, cytokine gene expression in peripheral blood mononuclear cells (PBMC), viral load in nasal swabs and blood, and antigen-specific cellular immune responses of PBMC, in conjunction with clinical evaluation of the foals. The occurrence of fever in foals was not related to lymphocytosis or specific changes in lymphocyte morphology, cytokine gene expression, or viral load, but tended to be associated ( P ≤ 0.10) with increased EHV-2-specific responsiveness of PBMC. These results support the conclusion that the cellular immune response to EHV-2 may lead to an immunologically mediated disease of foals that is analogous to infectious mononucleosis caused by EBV infection in humans.

Epstein–Barr virus can infect rabbits by the intranasal or peroral route: An animal model for natural primary EBV infection in humans

Epstein-Barr virus (EBV) is spread universally in humans, and it causes infectious mononucleosis and sometimes induces serious EBV-associated disease. The detailed mechanism of primary infection in humans has remained unclear, because it is difficult to examine the dynamics of EBV in vivo. In this study, a natural EBV-infection rabbit model by intranasal or peroral inoculation is described. Ten male rabbits were examined for EBV-DNA or mRNA expression and anti-EBV antibodies in blood. Four of 10 rabbits showed the evidence of EBV infection; detection of EBV-DNA or EBV-related genes mRNA in peripheral blood mononuclear cells, increased EBV antibodies in the plasma, and the presence of lymphocytes expressing EBER1 and EBV-related gene proteins in the lymphoid tissues of a rabbit. Three of four infected rabbits were detected transiently EBV-DNA and/or mRNA of EBV-related genes such as EBNA1, EBNA2, BZLF1, and EA in blood, while in one of four, EBV-DNA and/or mRNA were detected for more than 200 days after viral inoculation. The level of EA-IgG increased and its level was maintained in all infected rabbits, whereas those of VCA-IgM and VCA-IgG increased transiently, and EBNA-IgG was not elevated. Pathological examination of a rabbit infected transiently revealed some scattered lymphocytes expressing EBER1, LMP1, and EBNA2 in the spleen and lymph nodes. EA expression was also observed in the spleen. These findings suggest that EBV can infect the rabbit by the intranasal or peroral route, and that this rabbit model is useful for examining the pathophysiology of natural primary EBV infection in humans.

CD34 + Cord Blood Cell-Transplanted Rag2 −/− γ c−/− Mice as a Model for Epstein-Barr Virus Infection

Recent studies suggest that Epstein-Barr virus (EBV) can infect naïve B cells, driving them to differentiate into resting memory B cells via the germinal center reaction. This hypothesis has been inferred from parallels with the biology of normal B cells but has never been proven experimentally. Rag2(-/-) gamma(c)(-/-) mice that were transplanted with human CD34(+) cord blood cells as newborns were recently shown to develop human B, T, and dendritic cells, constituting lymphoid organs in situ. Here we used this model to better define the strategy of EBV infection of human B cells in vivo and to compare this model system with different conditions of EBV infection in humans. Our results support the model of EBV persistence in vivo in cases that were characterized by follicular hyperplasia and a relatively normal CD4(+) and CD8(+) T-cell distribution. Intriguingly, in cases that were characterized by nodular and diffuse proliferation with a preponderance of CD8(+) T cells, similar to infectious mononucleosis, EBV still infects naïve B cells but also induces clonal expansion and ongoing somatic mutations without germinal center reactions. Our results reveal different strategies of EBV infection in B cells that possibly result from variations in the host immune response. Future experiments might allow understanding of the mechanisms responsible for persistent EBV infection and provide targets for more highly tailored therapeutic interventions.

A new animal model for primary and persistent Epstein–Barr virus infection: Human EBV‐infected rabbit characteristics determined using sequential imaging and pathological analysis

Most adults have persistent Epstein-Barr virus (EBV)-infection. Adolescents and young adults with primary EBV-infection frequently develop infectious mononucleosis. Latent EBV-infection is associated with various diseases, neoplasms, and hematological disorders. In vivo animal models of human EBV infection, such as non-human primates, have had limited success. A new rabbit model for primary human EBV-infection is described in this study. Seven male rabbits inoculated intravenously with EBV were sequentially imaged by ultrasonography and computed tomography, and examined for anti-EBV-VCA titer and EBV-DNA levels in blood. Six rabbits demonstrated transient splenomegaly, increased anti-EBV-VCA titers and/or EBV-DNA in blood. Transient infiltration of some EBER1-positive lymphocytes was observed in biopsied liver tissues. After splenomegaly, two rabbits tested continuously negative, two alternatively positive and negative, and one consistently positive EBV detection in blood for 470 days. One tested negative for both EBV DNA and splenomegaly. On the 14th day, mild to moderate numbers of EBER1-positive lymphocytes expressing LMP1, EBNA2, or ZEBRA infiltrated mainly in enlarged white pulps of two splenectomized materials. These cells included both B and T cells. EBV clonality analysis revealed an oligoclonal pattern. These indicate that EBV-inoculated rabbits exhibiting heterogenous host reactions are a good model for primary and persistent human EBV infection.

Epstein–Barr virus-associated tumours: an update for the attention of the working pathologist

Epstein–Barr virus (EBV) is a herpesvirus associated with approximately 1% of tumours worldwide. EBV is the epitome of B lymphotropic viruses, but the spectrum of tumours it is associated with...

Epstein–Barr virus infection in humans: from harmless to life endangering virus–lymphocyte interactions

After the primary infection, that may or may not cause infectious mononucleosis, the ubiquitous Epstein-Barr virus (EBV) is carried for lifetime. The great majority of adult humans are virus carriers. EBV was discovered in a B-cell lymphoma (Burkitt lymphoma). EBV infection in humans is the example for the power of immune surveillance against virus transformed, potentially malignant cells. Although the virus can transform B lymphocytes in vitro into proliferating lines, it induces malignancy directly only in immunosuppressed hosts. EBV-induced growth transformation occurs only in B lymphocytes. It is the result of a complex interaction between virally encoded and cellular proteins. Different forms of the virus-cell and the cell-host interactions have evolved during a long period of coexistence between the virus and all Old World (but not New World) primates. The asymptomatic carrier state is based on a viral-strategy that downregulates the expression of the transforming proteins in the virus-carrying cell. In addition to the silent viral-gene carriers and the expressors of the nine virus-encoded genes that drive the growth program, virus carrying cells exist that show other patterns of gene expression, depending on the differentiated state of the host cell. Certain combinations contribute to malignant transformation, but only in conjunction with additional cellular changes. These are induced by direct or cytokine-mediated interactions with normal cells of the immune system.

Expression of Leukocyte Immunoglobulin-Like Receptors and Natural Killer Receptors on Virus-Specific CD8+T Cells during the Evolution of Epstein-Barr Virus-Specific Immune Responsesin Vivo

Antigen-primed cytotoxic T lymphocytes (CTL) may express leukocyte immunoglobulin-like receptors (LILRs) and natural killer receptors (NKRs). Published work suggests that expression of some of these receptors confers survival advantage, leading to the idea that cells expressing such receptors may accumulate as an antigen-specific response evolves. Here we tested this hypothesis by analyzing expression of CD85j (also known as LILRB1 or ILT2), KIRs, CD94, and CD161 by Epstein- Barr virus (EBV)-specific CTL during the primary and persistent phases of EBV infection in humans. During primary infection, few EBV-specific CTL expressed these receptors and this proportion was equally low in early persistent infection. Thus, expression of these molecules does not influence capacity to survive downregulation of the primary response. However, in donors persistently infected with EBV for many years, a significantly higher proportion of EBV-specific CTL expressed CD85j and NKRs, suggesting that cells expressing these receptors can accumulate with time. Using FACS analysis, we confirmed, at a single cell level, that expression of CD85j, defined by staining with the antibody VMP55, was associated with reduced capacity of EBV-specific CD8+ T cells to respond to antigen. Thus, in the later stages of persistent infection, protective immunity to EBV may be reduced due to the preferential accumulation of hyporesponsive EBV-specific CD8+ T cells.

NKT cells depend on SAP

An intracellular adaptor protein is indispensable for the development of natural killer T (NKT) cells, according to Pasquier et al. (page 695). The loss of these cells might contribute to the genesis of a lethal immunodeficiency syndrome. The adaptor protein in question—SAP (SLAM-associated protein)—is expressed in T, NK, and NKT cells and responds to SLAM family receptors by recruiting and activating the downstream tyrosine kinase Fyn. Fyn was known to be required for NKT cell development in the thymus, but the upstream cell surface signals remained unknown. Pasquier and colleagues now implicate the SLAM family of receptors in NKT cell development by showing that the loss of SAP results in a complete absence of NKT cells in both mice and humans. SAP-transmitted signaling events were uniquely required for the development of NKT cells, as conventional T cells and NK cells developed normally in the absence of SAP. The need for SAP-mediated signals may reflect the unique requirements for positive selection of NKT cells in the thymus. Whereas conventional T cells interact with thymic epithelial cells for selection, NKT cells must interact with CD1d-expressing thymocytes via their invariant T cell receptors. This unique interaction might also provide the required SLAM-mediated signals. In an intriguing twist, mutations in SAP cause a fatal disease called X-linked lymphoproliferative syndrome (XLP), which is characterized by uncontrolled Epstein-Barr virus (EBV) infections and B cell lymphomas. The loss of SAP impacts the function of multiple cell types including NK cells and T cells, but based on the current study, NKT cells may also contribute to XLP. Studying NKT cell activation during human EBV infection may give clues about why the lack of SAP, which results in so many immune cell defects, would preferentially increase susceptibility to only one type of virus.