SUMMARY Primary infection with the human oncogenic Epstein Barr virus (EBV) can result in infectious mononucleosis (IM), a self-limiting disease caused by massive lymphocyte expansion, which predisposes for the development of distinct EBV-associated lymphomas. It remains unclear why some individuals experience this symptomatic primary EBV infection, while the majority acquires the virus asymptomatically. Using a mouse model with reconstituted human immune system components, we show here that depletion of human natural killer (NK) cells enhances IM symptoms and promotes EBV-associated tumorigenesis, mainly due to loss of immune control over lytic EBV infection. These data suggest that failure of innate immune control by human NK cells augments symptomatic lytic EBV infection, which drives lymphocyte expansion and predisposes for EBV-associated malignancies.

The human tumor viruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) establish persistent infections in B cells. KSHV is linked to primary effusion lymphoma (PEL), and 90% of PELs also contain EBV. Studies on persistent KSHV infection in vivo and the role of EBV co-infection in PEL development have been hampered by the absence of small animal models. We developed mice reconstituted with human immune system components as a model for KSHV infection and find that EBV/KSHV dual infection enhanced KSHV persistence and tumorigenesis. Dual-infected cells displayed a plasma cell-like gene expression pattern similar to PELs. KSHV persisted in EBV-transformed B cells and was associated with lytic EBV gene expression, resulting in increased tumor formation. Evidence of elevated lytic EBV replication was also found in EBV/KSHV dually infected lymphoproliferative disorders in humans. Our data suggest that KSHV augments EBV-associated tumorigenesis via stimulation of lytic EBV replication.

The herpesvirus Epstein–Barr virus (EBV) was discovered as the first human candidate tumor virus in Burkitt’s lymphoma more than 50 years ago. Despite its strong growth transforming capacity, more than 90% of the human adult population carries this virus asymptomatically under near perfect immune control. The mode of primary EBV infection is in part responsible for EBV-associated diseases, including Hodgkin’s lymphoma. It is, therefore, important to understand which circumstances lead to symptomatic primary EBV infection, called infectious mononucleosis (IM). Innate immune control of lytic viral replication by early-differentiated natural killer (NK) cells was found to attenuate IM symptoms and continuous loss of the respective NK cell subset during the first decade of life might predispose for IM during adolescence. In this review, we discuss the evidence that NK cells are involved in the immune control of EBV, mechanisms by which they might detect and control lytic EBV replication, and compare NK cell subpopulations that expand during different human herpesvirus infections.

NK cells are innate lymphocytes with protective functions against viral infections and tumor formation. Human NK cells carry inhibitory killer cell Ig-like receptors (KIRs), which recognize distinct HLAs. NK cells with KIRs for self-HLA molecules acquire superior cytotoxicity against HLA- tumor cells during education for improved missing-self recognition. Here, we reconstituted mice with human hematopoietic cells from donors with homozygous KIR ligands or with a mix of hematopoietic cells from these homozygous donors, allowing assessment of the resulting KIR repertoire and NK cell education. We found that co-reconstitution with 2 KIR ligand-mismatched compartments did not alter the frequency of KIR-expressing NK cells. However, NK cell education was diminished in mice reconstituted with parallel HLA compartments due to a lack of cognate HLA molecules on leukocytes for the corresponding KIRs. This change in NK cell education in mixed human donor-reconstituted mice improved NK cell-mediated immune control of EBV infection, indicating that mixed hematopoietic cell populations could be exploited to improve NK cell reactivity against leukotropic pathogens. Taken together, these findings indicate that leukocytes lacking cognate HLA ligands can disarm KIR+ NK cells in a manner that may decrease HLA- tumor cell recognition but allows for improved NK cell-mediated immune control of a human γ-herpesvirus

Key Points Human type 3 ILCs acquire features of early differentiated NK cells upon cytokine stimulation. IL-12 and IL-15–differentiated human ILC3s acquire cytotoxicity and kill leukemic targets.

Despite many theoretical incompatibilities between mouse and human cells, mice with reconstituted human immune system components contain nearly all human leukocyte populations. Accordingly, several human-tropic pathogens have been investigated in these in vivo models of the human immune system, including viruses such as human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV), as well as bacteria such as Mycobacterium tuberculosis and Salmonella enterica Typhi. While these studies initially aimed to establish similarities in the pathogenesis of infections between these models and the pathobiology in patients, recent investigations have provided new and interesting functional insights into the protective value of certain immune compartments and altered pathology upon mutant pathogen infections. As more tools and methodologies are developed to make these models more versatile to study human immune responses in vivo, such improvements build toward small animal models with human immune components, which could predict immune responses to therapies and vaccination in human patients. Keywords:Dengue virus r EBV r HIV r Mycobacterium tuberculosis r Salmonella enterica typhi IntroductionThe complexity of infections and the corresponding elicited immune responses are best investigated in animal models that allow the manipulation of the timing and dose of infection, as well as of the responding immune compartments. Small animal models, such as the mouse, are preferred for these types of investigations due to low costs and ease of handling. However, divergent evolution between these small mammals and humans in the past 65 million years has rendered the immune system the third most different organ system between the two species, after olfaction and reproduction [1]. Many of these differences are found in the innate immune system, which ensures the initial survival of the infected host and also recognizes pathogens by their molecular patterns [2]. This divergence probably results from the different Correspondence: Prof. Christian Münz e-mail: christian.muenz@uzh.ch infectious disease challenges associated with the respective ecological niches that these two species inhabit. Unfortunately, these differences between the mouse and human immune systems also result in dissimilar inflammatory responses to burns, trauma, and endotoxemia at the gene expression level, such as integrin, ICOS-ICOSL, CD28, and PKC signaling [3]. Therefore, alternatives to classical mouse models, which more closely model human immune system behavior during infection in vivo, would be of significant benefit for the development of immunomodulatory treatments.The category of new models, which comes closest to achieving this goal, is mice with reconstituted human immune system components. These mice are mainly generated by neonatal injection of human hematopoietic progenitor cells in mice that lack murine innate and adaptive lymphocytes, namely NOD-scid γ c −/− (NSG), (Fig. 1). For some studies, a fetal organoid of liver and thymic tissue is implanted ...

Epstein Barr virus (EBV) was the first human tumor virus to be identified. Despite 50years of research on this oncogenic virus, no therapeutic or prophylactic vaccine is available against this pathogen. In part, the development of such a vaccine is hampered by the lack of in vivo models for EBV infection and immune control. However, with the advent of mice with reconstituted human immune system components (HIS mice), certain aspects of EBV associated diseases and immune responses can be modeled in vivo. In this review, we will discuss the insights that can be gained from these experiments, and how immune system components can be manipulated to interrogate their function during EBV infection. Finally, we will compare EBV immunobiology in HIS mice to infection by EBV-related viruses in monkeys, and we will outline the strengths and weaknesses of these two in vivo models of EBV infection. Both of these models show great promise as a platform for preclinical EBV vaccine testing. AbstractEpstein Barr virus (EBV) was the first human tumor virus to be identified. Despite 50 years of research on this oncogenic virus, no therapeutic or prophylactic vaccine is available against this pathogen. In part, the development of such a vaccine is hampered by the lack of in vivo models for EBV infection and immune control. However, with the advent of mice with reconstituted human immune system components (HIS mice), certain aspects of EBV associated diseases and immune responses can be modeled in vivo. In this review, we will discuss the insights that can be gained from these experiments, and how immune system components can be manipulated to interrogate their function during EBV infection. Finally, we will compare EBV immunobiology in HIS mice to infection by EBV-related viruses in monkeys, and we will outline the strengths and weaknesses of these two in vivo models of EBV infection. Both of these models show great promise as a platform for preclinical EBV vaccine testing. Chatterjee et al. 3

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 lymphoproliferationassociated 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.