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.].

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.

Infectious diseases in humanized mice.

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.

Interleukins 12 and 15 induce cytotoxicity and early NK-cell differentiation in type 3 innate lymphoid cells.

Type 3 innate lymphoid cells (ILC3s) fulfill protective functions at mucosal surfaces via cytokine production. Although their plasticity to become ILC1s, the innate counterparts of type 1 helper T cells, has been described previously, we report that they can differentiate into cytotoxic lymphocytes with many characteristics of early differentiated natural killer (NK) cells. This transition is promoted by the proinflammatory cytokines interleukin 12 (IL-12) and IL-15, and correlates with expression of the master transcription factor of cytotoxicity, eomesodermin (Eomes). As revealed by transcriptome analysis and flow cytometric profiling, differentiated ILC3s express CD94, NKG2A, NKG2C, CD56, and CD16 among other NK-cell receptors, and possess all components of the cytotoxic machinery. These characteristics allow them to recognize and kill leukemic cells with perforin and granzymes. Therefore, ILC3s can be harnessed for cytotoxic responses via differentiation under the influence of proinflammatory cytokines.

Persistent KSHV Infection Increases EBV-Associated Tumor Formation In Vivo via Enhanced EBV Lytic Gene Expression.

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.

NK Cell Influence on the Outcome of Primary Epstein-Barr Virus Infection.

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.

Cognate HLA absence in trans diminishes human NK cell education.

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.

Animal models of Epstein Barr virus infection.

Epstein Barr virus (EBV) was the first human tumor virus to be described. Despite its discovery now more than fifty years ago, immune control of this virus is still not very well understood and no vaccine is available. This knowledge gap is due in part to the lack of a preclinical small animal model which can faithfully recapitulate EBV infection and immune control, and would allow testing of EBV specific vaccine candidates. With the advent of mice with reconstituted human immune system compartments (HIS mice) during the past decade this is changing. We will discuss which aspects of EBV infection and its immune control can already be modeled in HIS mice, and which shortcomings still need to be overcome in order to recapitulate the immunobiology of oncogenic EBV infection.

Human natural killer cells prevent infectious mononucleosis features by targeting lytic Epstein-Barr virus infection.

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 that predisposes for the development of distinct EBV-associated lymphomas. Why some individuals experience this symptomatic primary EBV infection, whereas the majority acquires the virus asymptomatically, remains unclear. Using a mouse model with reconstituted human immune system components, we show that depletion of human natural killer (NK) cells enhances IM symptoms and promotes EBV-associated tumorigenesis mainly because of a 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.