NKG2Cpos NK Cells Regulate the Expansion of Cytomegalovirus-Specific CD8 T Cells.

Infection with the human CMV associates with phenotypic alterations in lymphocyte subsets. A highly reproducible finding in CMV-seropositive individuals is an expansion of NKG2Cpos NK cells. In this study, we analyzed if the altered NK cell compartment in CMV-seropositive human donors may affect CMV-specific CD8 T cells. Resting CMV-specific CD8 T cells were terminally differentiated and expressed high levels of the NKG2C ligand HLA-E. Activation of CMV-specific CD8 T cells with the cognate Ag further increased HLA-E expression. In line with a negative regulatory effect of NKG2Cpos NK cells on HLA-Ehigh CD8 T cells, depletion of NKG2Cpos NK cells enhanced Ag-specific expansion of CMV-specific CD8 T cells in vitro. In turn, the activation of NK cells in coculture with CMV-specific CD8 T cells promoted a selective loss of HLA-Ehigh CD8 T cells. To test if NKG2Cpos NK cells can target HLA-Ehigh CD8 T cells, Jurkat T cells with and without stabilized HLA-E on the surface were used. NKG2Cpos NK cells stimulated with HLA-Ehigh Jurkat cells released higher levels of Granzyme B compared with NKG2Cneg NK cells and NKG2Cpos NK cells stimulated with HLA-Elow Jurkat cells. Moreover, intracellular levels of caspase 3/7 were increased in HLA-Ehigh Jurkat cells compared with HLA-Elow Jurkat cells, consistent with higher rates of apoptosis in HLA-Ehigh T cells in the presence of NKG2Cpos NK cells. Our data show that NKG2Cpos NK cells interact with HLA-Ehigh CD8 T cells, which may negatively regulate the expansion of CMV-specific CD8 T cells upon activation.

HCMV Infection in a Mesenchymal Stem Cell Niche: Differential Impact on the Development of NK Cells versus ILC3.

Human cytomegalovirus (HCMV) is highly prevalent in most populations worldwide and has a major influence on shaping the human immune system. Natural killer (NK) cells are important antiviral effectors that adapt to HCMV infection by expansion of virus-specific effector/memory cells. The impact of HCMV infection on the development of NK cells and innate lymphoid cells (ILC) in general is less well understood. In this context, we have recently established a novel in vitro platform to study human NK cell development in a stem cell niche based on human bone marrow-derived mesenchymal stem cells (MSC). Here, the system was modified by infecting MSC with HCMV to study the influence of virus infection on NK/ILC development. We show that cord blood-derived hematopoietic progenitor cells are successfully differentiated into mature CD56+CD94+NKG2A+ NK cells on HCMV-infected MSC with significant higher anti-viral cytokine production compared to NK cells developing on non-infected MSC. Furthermore, the generation of ILC3, characterized by expression of the signature transcription factor RAR-related orphan receptor gamma (RORγt) and the production of IL-22, was strongly impaired by HCMV infection. These observations are clinically relevant, given that ILC3 are associated with protection from graft-versus-host disease (GvHD) following stem cell transplantation and HCMV reactivation in turn is associated with increased incidence of GvHD.

hCMV-Mediated Immune Escape Mechanisms Favor Pathogen Growth and Disturb the Immune Privilege of the Eye.

Human retinal pigment epithelial (hRPE) cells are important for the establishment and maintenance of the immune privilege of the eye. They function as target cells for human cytomegalovirus (hCMV), but are able to restrict viral replication. hCMV causes opportunistic posterior uveitis such as retinitis and chorioretinitis. Both mainly occur in severely immunocompromised patients and rarely manifest in immunocompetent individuals. In this study, hRPE cells were infected with hCMV in vitro and activated with proinflammatory cytokines. The enzymatic activities of indoleamine 2,3-dioxygenase-1 (IDO1) and inducible nitric oxide synthase (iNOS) were determined. The antimicrobial capacity of both molecules was analyzed in co-infection experiments using Staphylococcus aureus (S. aureus) and Toxoplasma gondii (T. gondii), causing uveitis in patients. We show that an hCMV infection of hRPE cells blocks IDO1 and iNOS mediated antimicrobial defense mechanisms necessary for the control of S. aureus and T. gondii. hCMV also inhibits immune suppressive effector mechanisms in hRPE. The interferon gamma-induced IDO1 dependent immune regulation was severely blocked, as detected by the loss of T cell inhibition. We conclude that an active hCMV infection in the eye might favor the replication of pathogens causing co-infections in immunosuppressed individuals. An hCMV caused blockade of IDO1 might weaken the eye's immune privilege and favor the development of post-infectious autoimmune uveitis.

MXB inhibits murine cytomegalovirus.

The MX dynamin GTPases inhibit diverse viruses at early post-entry phases. While MXA acts antiviral against influenza viruses, the anti HIV-1 activity of MXB was discovered recently. Here, we have studied the antiviral effect of MX proteins on murine cytomegalovirus (MCMV). Our data demonstrate that human MXB but not other human or murine MX proteins inhibit MCMV propagation. Evidently, the viral protein expression was delayed and the viral DNA amount in nucleus was diminished in MXB expressing cells indicating an obstruction of nuclear entry. Of note, MCMV did not deplete MX proteins. Considering the role of capsid on HIV-1 sensitivity to MXB, MXB binding to tested MCMV capsids was not detected. Moreover, MCMV restriction occurred only when MXB contained both the nuclear localization signal and a functional GTPase domain. Hence, we propose a new mode of inhibition of MCMV by MXB that is conspicuously different from that of HIV-1.

Anti-inflammatory consequences of bile acid accumulation in virus-infected bile duct ligated mice.

Cholestatic patients exhibiting high bile acid serum levels were reported to be more susceptible to bacterial and viral infections. Animal studies in bile duct ligated (BDL) mice suggest that cholestasis leads to an aggravation of hepatic bacterial infections. We have investigated the impact of cholestasis on mouse cytomegalovirus (MCMV)-induced immune responses and viral replication. While MCMV did not aggravate BDL-induced liver damage, BDL markedly reduced MCMV-triggered chemokine expression and immune cell recruitment to the liver. MCMV-infected BDL mice showed diminished trafficking of Ly6C+/F4/80+ myeloid cells and NK1.1+ NK cells to the liver compared to MCMV infected control mice. Moreover, virus-driven expression of CCL7, CCL12, CXCL9 and CXCL10 was clearly impaired in BDL- compared to sham-operated mice. Furthermore, production of the anti-inflammatory cytokine IL-10 was massively augmented in infected BDL mice. In contrast, intra- and extrahepatic virus replication was unaltered in BDL-MCMV mice when compared to sham-MCMV mice. Cholestasis in the BDL model severely impaired pathogen-induced chemokine expression in the liver affecting CCR2- and CXCR3-dependent cell trafficking. Cholestasis resulted in reduced recruitment of inflammatory monocytes and NK cells to the liver.

RAIDD Mediates TLR3 and IRF7 Driven Type I Interferon Production.

BACKGROUND/AIMS: Viral infections represent a global health problem with the need for new viral therapies and better understanding of the immune response during infection. The most immediate and potent anti-viral defense mechanism is the production of type I interferon (IFN-I) which are activated rapidly following recognition of viral infection by host pathogen recognition receptors (PRR). The mechanisms of innate cellular signaling downstream of PRR activation remain to be fully understood. In the present study, we demonstrate that CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD/RAIDD) is a critical component in type I IFN production. METHODS: The role of RAIDD during IFN-I production was investigated using western blot, shRNA mediated lentiviral knockdown, immunoprecipitation and IFN-I driven dual luciferase assay. RESULTS: Immunoprecipitation analysis revealed the molecular interaction of RAIDD with interferon regulatory factor 7 (IRF7) and its phosphorylating kinase IKKε. Using an IFN-4α driven dual luciferase analysis in RAIDD deficient cells, type I IFN activation by IKKε and IRF7 was dramatically reduced. Furthermore, deletion of either the caspase recruitment domain (CARD) or death domain (DD) of RAIDD inhibited IKKε and IRF7 mediated interferon-4α activation. CONCLUSION: We have identified that the adaptor molecule RAIDD coordinates IKKε and IRF7 interaction to ensure efficient expression of type I interferon.

Anti-apoptotic Bcl-XL but not Mcl-1 contributes to protection against virus-induced apoptosis.

Infection of mammalian cells with viruses often induces apoptosis. How the recognition of viruses leads to apoptosis of the infected cell and which host cell factors regulate this cell death is incompletely understood. In this study, we focussed on two major anti-apoptotic proteins of the host cell, whose abundance and activity are important for cell survival, the Bcl-2-like proteins Mcl-1 and Bcl-XL. During infection of epithelial cells and fibroblasts with modified vaccinia virus Ankara (MVA), Mcl-1 protein levels dropped but the MVA Bcl-2-like protein F1L could replace Mcl-1 functionally; a similar activity was found in vaccinia virus (VACV)-infected cells. During infection with murine cytomegalovirus (MCMV), Mcl-1-levels were not reduced but a viral Mcl-1-like activity was also generated. Infection of mouse macrophages with any of these viruses, on the other hand, induced apoptosis. Virus-induced macrophage apoptosis was unaltered in the absence of Mcl-1. However, apoptosis was substantially increased in infected Bcl-XL-deficient macrophages or macrophages treated with the Bcl-2/Bcl-XL-inhibitor ABT-737. Genetic loss of Bcl-XL or treatment of macrophages with ABT-737 reduced the generation of infectious VACV. These data show that Mcl-1 is dispensable for the regulation of apoptosis during infection with different large DNA viruses, either because the viruses replace its function (in fibroblasts and epithelial cells) or because the pro-apoptotic activity generated by the infection appears not to be blocked by it (in macrophages). Bcl-XL, on the other hand, can be important to maintain survival of virus-infected cells, and its activity can determine outcome of the infection.

Bile Acids Act as Soluble Host Restriction Factors Limiting Cytomegalovirus Replication in Hepatocytes.

UNLABELLED: The liver constitutes a prime site of cytomegalovirus (CMV) replication and latency. Hepatocytes produce, secrete, and recycle a chemically diverse set of bile acids, with the result that interactions between bile acids and cytomegalovirus inevitably occur. Here we determined the impact of naturally occurring bile acids on mouse CMV (MCMV) replication. In primary mouse hepatocytes, physiological concentrations of taurochenodeoxycholic acid (TCDC), glycochenodeoxycholic acid, and to a lesser extent taurocholic acid significantly reduced MCMV-induced gene expression and diminished the generation of virus progeny, while several other bile acids did not exert antiviral effects. The anticytomegalovirus activity required active import of bile acids via the sodium-taurocholate-cotransporting polypeptide (NTCP) and was consistently observed in hepatocytes but not in fibroblasts. Under conditions in which alpha interferon (IFN-α) lacks antiviral activity, physiological TCDC concentrations were similarly effective as IFN-γ. A detailed investigation of distinct steps of the viral life cycle revealed that TCDC deregulates viral transcription and diminishes global translation in infected cells. IMPORTANCE: Cytomegaloviruses are members of the Betaherpesvirinae subfamily. Primary infection leads to latency, from which cytomegaloviruses can reactivate under immunocompromised conditions and cause severe disease manifestations, including hepatitis. The present study describes an unanticipated antiviral activity of conjugated bile acids on MCMV replication in hepatocytes. Bile acids negatively influence viral transcription and exhibit a global effect on translation. Our data identify bile acids as site-specific soluble host restriction factors against MCMV, which may allow rational design of anticytomegalovirus drugs using bile acids as lead compounds.

Highly individual patterns of virus-immune IgG effector responses in humans.

IgG responses are fundamental to adaptive immunity and document immunological memory of previous pathogen encounter. While specific antigen recognition is mediated by the variable F(ab')2 domain of IgG, various effector functions become activated via the constant Fcγ part bridging IgG-opsonized targets to FcγR-expressing immune effector cells. Traditionally, neutralizing IgG is considered the most appropriate correlate of protective humoral immunity to viruses. However, evidence is increasing that antiviral IgG mediates protection to viruses via activation of FcγRs. Using a test system allowing quantitative detection of virus-immune IgG able to activate FcγRs, sera of healthy individuals and vaccinees were assessed with regard to two prototypical human pathogenic viruses: measles and human cytomegalovirus. Marked differences in the capacity of individuals to generate FcγRI-, FcγRII- and FcγRIII-activating responses were noted. Comparison of FcγR-activating IgG with neutralizing and ELISA IgG concentrations did not correlate for HCMV and only very poorly for MV. Since neither neutralizing IgG nor overall IgG responses faithfully predict the activation of FcγRs, only the simultaneous quantification of IgGs activating defined FcγRs will aid to delineate individual "immunograms" of virus IgG immunity. Such new multiparametric assessment of antiviral IgG qualities could be instrumental in defining correlates of protection and disease progression.

Neural crest requires Impdh2 for development of the enteric nervous system, great vessels, and craniofacial skeleton.

Mutations that impair the proliferation of enteric neural crest-derived cells (ENCDC) cause Hirschsprung disease, a potentially lethal birth defect where the enteric nervous system (ENS) is absent from distal bowel. Inosine 5' monophosphate dehydrogenase (IMPDH) activity is essential for de novo GMP synthesis, and chemical inhibition of IMPDH induces Hirschsprung disease-like pathology in mouse models by reducing ENCDC proliferation. Two IMPDH isoforms are ubiquitously expressed in the embryo, but only IMPDH2 is required for life. To further understand the role of IMPDH2 in ENS and neural crest development, we characterized a conditional Impdh2 mutant mouse. Deletion of Impdh2 in the early neural crest using the Wnt1-Cre transgene produced defects in multiple neural crest derivatives including highly penetrant intestinal aganglionosis, agenesis of the craniofacial skeleton, and cardiac outflow tract and great vessel malformations. Analysis using a Rosa26 reporter mouse suggested that some or all of the remaining ENS in Impdh2 conditional-knockout animals was derived from cells that escaped Wnt1-Cre mediated DNA recombination. These data suggest that IMPDH2 mediated guanine nucleotide synthesis is essential for normal development of the ENS and other neural crest derivatives.

MAPKAP kinase 2 regulates IL-10 expression and prevents formation of intrahepatic myeloid cell aggregates during cytomegalovirus infections.

BACKGROUND & AIMS: The kinase p38(MAPK) and its downstream target MAPKAP kinase (MK) 2 are critical regulators of inflammatory responses towards pathogens. To date, the relevance of MK2 for regulating IL-10 expression and other cytokine responses towards cytomegalovirus (CMV) infection and the impact of this pathway on viral replication in vitro and in vivo is unknown and the subject of this study. METHODS: The effect of MK2, interferon-α receptor (IFNAR)1, tristetraprolin (TTP) and IL-10 on mouse (M)CMV virus titres, cytokine expression, signal transduction, transcript stability, liver enzymes release, immune cell recruitment and aggregation in response to MCMV infection were studied ex vivo in hepatocytes and macrophages, as well as in vivo. RESULTS: MK2 is critical for MCMV-induced production of IL-10, IFN-α2 and 4, IFN-ß, IL-6, and TNF-α but not for IFN-γ. The MCMV-induced IL-10 production requires activation of IFNAR1 and is further regulated by MK2 and TTP-dependent stabilization of IL-10 transcripts. MK2(-/-) mice are able to control acute MCMV replication, despite deregulated cytokine production. This may be related to the observation that MCMV-infected MK2(-/-) mice show enhanced formation of focal intrahepatic lymphocyte infiltrates resembling intrahepatic myeloid cell aggregates of T cell expansion (iMATEs), which were also observed in MCMV-infected IL-10(-/-) mice but are almost absent in MCMV-infected wild-type controls. CONCLUSIONS: The data suggest that MK2 is critical for regulating cytokine responses towards acute MCMV infection, including that of IL-10 via IFNARI-mediated circuits. MCMV stimulates expression of MK2-dependent cytokines, in particular IL-10 and thereby prevents enhanced formation of intrahepatic iMATE-like cellular aggregates.

Regulatory T cells and T-cell-derived IL-10 interfere with effective anti-cytomegalovirus immune response.

Cytomegalovirus (CMV) establishes lifelong chronic infection in its host with mostly asymptomatic or only mild disease, but under immunosuppressive conditions the virus can reactivate and infection can cause life-threatening disease. CMV has evolved several mechanisms to escape from host's immunity, allowing persistence of the virus. Until now, it remains elusive whether regulatory T cells (Tregs) have an impact on insufficient host immune response against the virus in this context. In the present study, we provide evidence that CD4(+)Foxp3(+) naturally occurring Tregs (nTregs) as well as CD4(+)Foxp3(-)IL-10(+)-induced Tregs (iTregs) interfere with an effective anti-mouse CMV (mCMV) immune response. Depletion of Foxp3(+) Tregs by using DEREG mice resulted in enhanced T-cell activation as measured by the expression of CD62L, granzyme B and interferon (IFN)-γ and was associated with reduced viral titers in salivary glands, the organ where mCMV mainly persists. Moreover, we identified CD4(+)Foxp3(-) T cells to produce elevated levels of the immunosuppressive cytokine IL-10 at early time points during mCMV infection. Analysis of T-cell activation and viral replication in mCMV-infected IL-10(flox/flox) × CD4-cre mice and IL-10(flox/flox) × FIC mice revealed that T-cell-specific inactivation of IL-10, but not Foxp3(+) Treg-specific IL-10 ablation alone, resulted in elevated IFN-γ production by T cells associated with a significant decrease in viral loads in salivary glands. Thus, our data illustrate a crucial role for CD4(+)Foxp3(+) nTregs as well as IL-10-producing CD4(+)Foxp3(-) iTregs in the regulation of appropriate T-cell responses and viral clearance during mCMV infection.

Human cytomegalovirus Fcγ binding proteins gp34 and gp68 antagonize Fcγ receptors I, II and III.

Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.

Cytomegalovirus infection impairs immunosuppressive and antimicrobial effector functions of human multipotent mesenchymal stromal cells.

Human mesenchymal stromal cells (MSC) possess immunosuppressive and antimicrobial effects that are partly mediated by the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). Therefore MSC represent a promising novel cellular immunosuppressant which has the potential to control steroid-refractory acute graft versus host disease (GvHD). In addition, MSC are capable of reducing the risk of infection in patients after haematopoietic stem cell transplantation (HST). Recent data indicate that signals from the microenvironment including those from microbes may modulate MSC effector functions. As Cytomegalovirus (CMV) represents a prominent pathogen in immunocompromised hosts, especially in patients following HST, we investigated the impact of CMV infection on MSC-mediated effects on the immune system. We demonstrate that CMV-infected MSC lose their cytokine-induced immunosuppressive capacity and are no longer able to restrict microbial growth. IDO expression is substantially impaired following CMV infection of MSC and this interaction critically depends on intact virus and the number of MSC as well as the viral load. Since overt CMV infection may undermine the clinical efficacy of MSC in the treatment of GvHD in transplant patients, we recommend that patients scheduled for MSC therapy should undergo thorough evaluation for an active CMV infection and receive CMV-directed antiviral therapy prior to the administration of MSC.

MicroRNA editing facilitates immune elimination of HCMV infected cells.

The human cytomegalovirus (HCMV) is extremely prevalent in the human population. Infection by HCMV is life threatening in immune compromised individuals and in immune competent individuals it can cause severe birth defects, developmental retardation and is even associated with tumor development. While numerous mechanisms were developed by HCMV to interfere with immune cell activity, much less is known about cellular mechanisms that operate in response to HCMV infection. Here we demonstrate that in response to HCMV infection, the expression of the short form of the RNA editing enzyme ADAR1 (ADAR1-p110) is induced. We identified the specific promoter region responsible for this induction and we show that ADAR1-p110 can edit miR-376a. Accordingly, we demonstrate that the levels of the edited-miR-376a (miR-376a(e)) increase during HCMV infection. Importantly, we show that miR-376a(e) downregulates the immune modulating molecule HLA-E and that this consequently renders HCMV infected cells susceptible to elimination by NK cells.

Checks and balances between human cytomegalovirus replication and indoleamine-2,3-dioxygenase.

Despite a rigorous blockade of interferon-γ (IFN-γ) signalling in infected fibroblasts as a mechanism of immune evasion by human cytomegalovirus (HCMV), IFN-γ induced indoleamine-2,3-dioxygenase (IDO) has been proposed to represent the major antiviral restriction factor limiting HCMV replication in epithelial cells. Here we show that HCMV efficiently blocks transcription of IFN-γ-induced IDO mRNA both in infected fibroblasts and epithelial cells even in the presence of a preexisting IFN-induced antiviral state. This interference results in severe suppression of IDO bioactivity in HCMV-infected cells and restoration of vigorous HCMV replication. Depletion of IDO expression nonetheless substantially alleviated the antiviral impact of IFN-γ treatment in both cell types. These findings highlight the effectiveness of this IFN-γ induced effector gene in restricting HCMV productivity, but also the impact of viral counter-measures.

Conventional murine gene targeting.

Murine gene knockout models engineered over the last two decades have continued to demonstrate their potential as invaluable tools in understanding the role of gene function in the context of normal human development and disease. The more recent elucidation of the human and mouse genomes through sequencing has opened up the capability to elucidate the function of every human gene. State-of-the-art mouse model generation allows, through a multitude of experimental steps requiring careful standardization, gene function to be reliably and predictably ablated in a live model system. The application of these standardized methodologies to directly target gene function through murine gene knockout has to date provided comprehensive and verifiable genetic models that have contributed tremendously to our understanding of the cellular and molecular pathways underlying normal and disease states in humans. The ensuing chapter provides an overview of the latest steps and procedures required to ablate gene function in a murine model.

Cytomegalovirus impairs the induction of indoleamine 2,3-dioxygenase mediated antimicrobial and immunoregulatory effects in human fibroblasts.

Human fibroblasts provide immunosuppressive functions that are partly mediated by the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). Moreover, upon stimulation with inflammatory cytokines human fibroblasts exhibit broad-spectrum antimicrobial effector functions directed against various clinically relevant pathogens and these effects are also IDO-dependent. Therefore human fibroblasts are suggested to be involved in the control of immune reactions during infectious diseases. As human cytomegalovirus (HCMV) represents a pathogen frequently found in immunocompromised hosts and IDO is involved in the control of HCMV growth, we here investigated the impact of HCMV infection on IDO-mediated antimicrobial and immunoregulatory effects. We show that infection with HCMV substantially impairs IFN-γ-induced IDO-activity in human fibroblasts in a dose and time dependent fashion. Consequently, these cells are no longer able to restrict bacterial and parasitic growth and, furthermore, loose their IDO-mediated immunosuppressive capacity. Our results may have significant implications for the course of HCMV infection during solid organ transplantation: we suggest that loss of IDO-mediated antimicrobial and immunoregulatory functions during a HCMV infection might at least in part explain the enhanced risk of organ rejection and infections observed in patients with HCMV reactivation after solid organ transplantation.

A novel assay for detecting virus-specific antibodies triggering activation of Fcγ receptors.

IgG responses are crucial in antiviral defence and instrumental for the serodiagnosis of infections. Fcγ receptors (FcγRs), which recognize the Fc-part of IgG, differ regarding their IgG binding affinity, IgG subclass preference, cellular expression profile and pathogen elimination mechanisms elicited upon activation. Assessing their activation in vitro is of fundamental importance, but technically difficult. Therefore, a novel assay for measuring antiviral IgG antibodies triggering activation of individual host Fcγ receptors was established. The assay comprises the co-cultivation of virus-infected target cells with immune IgG antibodies and mouse BW5147 hybridoma cells stably expressing chimeric FcγR-CD3ζ chain molecules consisting of the extracellular domain of human FcγRIIIA, FcγRIIA or FcγRI, respectively, fused to the transmembrane and intracellular domains of the mouse CD3ζ chain. Triggering of the chimeric FcγR receptors by immune complexes formed on the surface of IgG-opsonized virus-infected target cells resulted in FcγR activation leading to IL-2 secretion by BW5147 cells, which was quantified as a surrogate marker in an ELISA. Target cells infected with various human pathogenic viruses including herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), measles virus (MV), and respiratory syncytial virus (RSV) or displaying human immunodeficiency virus-1 (HIV-1) gp120 evoke dose-dependent IgG responses demonstrating the universal applicability of the assay. Taken together, a new reliable and simple tool for measuring antibodies triggering activation of Fcγ receptors was established. This assay will be instrumental for defining novel correlates of IgG immunity and the design of new therapeutic IgGs.

Complete genome sequence of the english isolate of rat cytomegalovirus (Murid herpesvirus 8).

The complete genome of the English isolate of rat cytomegalovirus (RCMV-E) was determined. RCMV-E has a 202,946-bp genome with noninverting repeats but without terminal repeats. Thus, it differs significantly in size and genomic arrangement from closely related rodent cytomegaloviruses (CMVs). To account for the differences between the rat CMV isolates of Maastricht and England, RCMV-E was classified as Murid herpesvirus 8 by the International Committee on Taxonomy of Viruses.