Natural Killer Cells: From Innate to Adaptive Features.

Natural killer (NK) cells are innate lymphocytes that provide critical host defense against pathogens and cancer. Originally heralded for their early and rapid effector activity, NK cells have been recognized over the last decade for their ability to undergo adaptive immune processes, including antigen-driven clonal expansion and generation of long-lived memory. This review presents an overview of how NK cells lithely partake in both innate and adaptive responses and how this versatility is manifest in human NK cell-mediated immunity.

In Vitro Profiling of the Antiviral Peptide TAT-I24.

The synthetic peptide TAT-I24 (GRKKRRQRRRPPQCLAFYACFC) exerts antiviral activity against several double-stranded (ds) DNA viruses, including herpes simplex viruses, cytomegalovirus, some adenoviruses, vaccinia virus and SV40 polyomavirus. In the present study, in vitro profiling of this peptide was performed with the aim of characterizing and improving its properties for further development. As TAT-I24 contains three free cysteine residues, a potential disadvantageous feature, peptide variants with replacements or deletions of specific residues were generated and tested in various cell systems and by biochemical analyses. Some cysteine replacements had no impact on the antiviral activity, such as the deletion of cysteine 14, which also showed improved biochemical properties, while the cyclization of cysteines 14 and 20 had the most detrimental effect on antiviral activity. At concentrations below 20 µM, TAT-I24 and selected variants did not induce hemolysis in red blood cells (RBCs) nor modulated lipopolysaccharide (LPS)-induced release of cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), in human peripheral blood mononuclear cells (PBMCs). These data indicate that TAT-I24 or its peptide variants are not expected to cause unwanted effects on blood cells.

CD4+ T Cells Control Murine Cytomegalovirus Infection Indirectly.

CD4+ T cells are key to controlling cytomegalovirus infections. Salivary gland infection by murine cytomegalovirus (MCMV) provides a way to identify mechanisms. CD11c+ dendritic cells (DC) disseminate MCMV to the salivary glands, where they transfer infection to acinar cells. Antiviral CD4+ T cells are often considered to be directly cytotoxic for cells expressing major histocompatibility complex class II (MHCII). However, persistently infected salivary gland acinar cells are MHCII- and are presumably inaccessible to direct CD4 T cell recognition. Here, we show that CD4+ T cell depletion amplified infection of MHCII- acinar cells but not MHCII+ cells. MCMV-infected mice with disrupted MHCII on CD11c+ cells showed increased MHCII- acinar infection; antiviral CD4+ T cells were still primed, but their recruitment to the salivary glands was reduced, suggesting that engagement with local MHCII+ DC is important for antiviral protection. As MCMV downregulates MHCII on infected DC, the DC participating in CD4 protection may thus be uninfected. NK cells and gamma interferon (IFN-γ) may also contribute to CD4+ T cell-dependent virus control: CD4 T cell depletion reduced NK cell recruitment to the salivary glands, and both NK cell and IFN-γ depletion equalized infection between MHCII-disrupted and control mice. Taken together, these results suggest that CD4+ T cells protect indirectly against infected acinar cells in the salivary gland via DC engagement, requiring the recruitment of NK cells and the action of IFN-γ. Congruence of these results with an established CD4+ T cell/NK cell axis of gammaherpesvirus infection control suggests a common mode of defense against evasive viruses. IMPORTANCE Cytomegalovirus infections commonly cause problems in immunocompromised patients and in pregnancy. We lack effective vaccines. CD4+ T cells play an important role in normal infection control, yet how they act has been unknown. Using murine cytomegalovirus as an accessible model, we show that CD4+ T cells are unlikely to recognize infected cells directly. We propose that CD4+ T cells interact with uninfected cells that present viral antigens and recruit other immune cells to attack infected targets. These data present a new outlook on understanding how CD4+ T cell-directed control protects against persistent cytomegalovirus infection.

The Mouse Cytomegalovirus G Protein-Coupled Receptor Homolog, M33, Coordinates Key Features of In Vivo Infection via Distinct Components of Its Signaling Repertoire.

Common to all cytomegalovirus (CMV) genomes analyzed to date is the presence of G protein-coupled receptors (GPCR). Animal models of CMV provide insights into their role in viral fitness. The mouse cytomegalovirus (MCMV) GPCR, M33, facilitates dendritic cell (DC)-dependent viremia, the extravasation of blood-borne infected DCs to the salivary gland, and the frequency of reactivation events from latently infected tissue explants. Constitutive G protein-coupled M33 signaling is required for these phenotypes, although the contribution of distinct biochemical pathways activated by M33 is unknown. M33 engages Gq/11 to constitutively activate phospholipase C ß (PLCß) and downstream cyclic AMP response-element binding protein (CREB) in vitro. Identification of a MCMV M33 mutant (M33ΔC38) for which CREB signaling was disabled but PLCß activation was preserved provided the opportunity to investigate their relevance in vivo. Following intranasal infection with MCMV M33ΔC38, the absence of M33 CREB Gq/11-dependent signaling correlated with reduced mobilization of lytically-infected DCs to the draining lymph node high endothelial venules (HEVs) and reduced viremia compared with wild type MCMV. In contrast, M33ΔC38-infected DCs within the vascular compartment extravasated to the salivary glands via a pertussis toxin-sensitive, Gi/o-dependent, and CREB-independent mechanism. In the context of MCMV latency, spleen explants from M33ΔC38-infected mice were markedly attenuated for reactivation. Taken together, these data demonstrate that key features of the MCMV life cycle are coordinated in diverse tissues by distinct pathways of the M33 signaling repertoire. IMPORTANCE G protein-coupled receptors (GPCRs) act as cell surface molecular "switches" that regulate the cellular response to environmental stimuli. All cytomegalovirus (CMV) genomes analyzed to date possess GPCR homologs with phylogenetic evidence for independent gene capture events, signifying important in vivo roles. The mouse CMV (MCMV) GPCR homolog, designated M33, is important for cell-associated virus spread and the establishment and/or reactivation of latent MCMV infection. The signaling repertoire of M33 is distinct from cellular GPCRs and little is known of the relevance of component signaling pathways for in vivo M33 function. In this report, we showed that temporal and tissue-specific M33 signaling was required to facilitate in vivo infection. Understanding the relevance of the viral GPCR signaling profiles for in vivo function will provide opportunities for future targeted interventions.

Characterization of M116.1p, a Murine Cytomegalovirus Protein Required for Efficient Infection of Mononuclear Phagocytes.

Broad tissue tropism of cytomegaloviruses (CMVs) is facilitated by different glycoprotein entry complexes, which are conserved between human CMV (HCMV) and murine CMV (MCMV). Among the wide array of cell types susceptible to the infection, mononuclear phagocytes (MNPs) play a unique role in the pathogenesis of the infection as they contribute both to the virus spread and immune control. CMVs have dedicated numerous genes for the efficient infection and evasion of macrophages and dendritic cells. In this study, we have characterized the properties and function of M116, a previously poorly described but highly transcribed MCMV gene region that encodes M116.1p, a novel protein necessary for the efficient infection of MNPs and viral spread in vivo. Our study further revealed that M116.1p shares similarities with its positional homologs in HCMV and RCMV, UL116 and R116, respectively, such as late kinetics of expression, N-glycosylation, localization to the virion assembly compartment, and interaction with gH-a member of the CMVs fusion complex. This study, therefore, expands our knowledge about virally encoded glycoproteins that play important roles in viral infectivity and tropism. IMPORTANCE Human cytomegalovirus (HCMV) is a species-specific herpesvirus that causes severe disease in immunocompromised individuals and immunologically immature neonates. Murine cytomegalovirus (MCMV) is biologically similar to HCMV, and it serves as a widely used model for studying the infection, pathogenesis, and immune responses to HCMV. In our previous work, we have identified the M116 ORF as one of the most extensively transcribed regions of the MCMV genome without an assigned function. This study shows that the M116 locus codes for a novel protein, M116.1p, which shares similarities with UL116 and R116 in HCMV and RCMV, respectively, and is required for the efficient infection of mononuclear phagocytes and virus spread in vivo. Furthermore, this study establishes the α-M116 monoclonal antibody and MCMV mutants lacking M116, generated in this work, as valuable tools for studying the role of macrophages and dendritic cells in limiting CMV infection following different MCMV administration routes.

Molecular dissection of plasmacytoid dendritic cell activation in vivo during a viral infection.

Plasmacytoid dendritic cells (pDC) are the major source of type I interferons (IFN-I) during viral infections, in response to triggering of endosomal Toll-like receptors (TLRs) 7 or 9 by viral single-stranded RNA or unmethylated CpG DNA, respectively. Synthetic ligands have been used to disentangle the underlying signaling pathways. The adaptor protein AP3 is necessary to transport molecular complexes of TLRs, synthetic CpG DNA, and MyD88 into endosomal compartments allowing interferon regulatory factor 7 (IRF7) recruitment whose phosphorylation then initiates IFN-I production. High basal expression of IRF7 by pDC and its further enhancement by positive IFN-I feedback signaling appear to be necessary for robust cytokine production. In contrast, we show here that in vivo during mouse cytomegalovirus (MCMV) infection pDC produce high amounts of IFN-I downstream of the TLR9-to-MyD88-to-IRF7 signaling pathway without requiring IFN-I positive feedback, high IRF7 expression, or AP3-driven endosomal routing of TLRs. Hence, the current model of the molecular requirements for professional IFN-I production by pDC, established by using synthetic TLR ligands, does not strictly apply to a physiological viral infection.

Murine Cytomegalovirus MCK-2 Facilitates In Vivo Infection Transfer from Dendritic Cells to Salivary Gland Acinar Cells.

The cytomegaloviruses (CMVs) spread systemically via myeloid cells and demonstrate broad tissue tropism. Human CMV (HCMV) UL128 encodes a component of the virion pentameric complex (PC) that is important for entry into epithelial cells and cell-cell spread in vitro. It possesses N-terminal amino acid sequences similar to those of CC chemokines. While the species specificity of HCMV precludes confirmation of UL128 function in vivo, UL128-like counterparts in experimental animals have demonstrated a role in salivary gland infection. How they achieve this has not been defined, although effects on monocyte tropism and immune evasion have been proposed. By tracking infected cells following lung infection, we show that although the UL128-like protein in mouse CMV (MCMV) (designated MCK-2) facilitated entry into lung macrophages, it was dispensable for subsequent viremia mediated by CD11c+ dendritic cells (DCs) and extravasation to the salivary glands. Notably, MCK-2 was important for the transfer of MCMV infection from DCs to salivary gland acinar epithelial cells. Acinar cell infection of MCMVs deleted of MCK-2 was not rescued by T-cell depletion, arguing against an immune evasion mechanism for MCK-2 in the salivary glands. In contrast to lung infection, peritoneal MCMV inoculation yields mixed monocyte/DC viremia. In this setting, MCK-2 again promoted DC-dependent infection of salivary gland acinar cells, but it was not required for monocyte-dependent spread to the lung. Thus, the action of MCK-2 in MCMV spread was specific to DC-acinar cell interactions. IMPORTANCE Cytomegaloviruses (CMVs) establish myeloid cell-associated viremias and persistent shedding from the salivary glands. In vitro studies with human CMV (HCMV) have implicated HCMV UL128 in epithelial tropism, but its role in vivo is unknown. Here, we analyzed how a murine CMV (MCMV) protein with similar physical properties, designated MCK-2, contributes to host colonization. We demonstrate that MCK-2 is dispensable for initial systemic spread from primary infection sites but within the salivary gland facilitates the transfer of infection from dendritic cells (DCs) to epithelial acinar cells. Virus transfer from extravasated monocytes to the lungs did not require MCK-2, indicating a tissue-specific effect. These results provide new information about how persistent viral tropism determinants operate in vivo.

Murine Cytomegalovirus Spread Depends on the Infected Myeloid Cell Type.

Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells. Murine CMV (MCMV) spreads from the lungs via infected CD11c+ cells, consistent with an important role for dendritic cells (DC). We show here that MCMV entering via the olfactory epithelium, a natural transmission portal, also spreads via infected DC. They reached lymph nodes, entered the blood via high endothelial venules, and then entered the salivary glands, driven by constitutive signaling of the viral M33 G protein-coupled receptor (GPCR). Intraperitoneal infection also delivered MCMV to the salivary glands via DC. However, it also seeded F4/80+ infected macrophages to the blood; they did not enter the salivary glands or require M33 for extravasation. Instead, they seeded infection to a range of other sites, including brown adipose tissue (BAT). Peritoneal cells infected ex vivo then adoptively transferred showed similar cell type-dependent differences in distribution, with abundant F4/80+ cells in BAT and CD11c+ cells in the salivary glands. BAT colonization by CMV-infected cells was insensitive to pertussis toxin inhibition of the GPCR signaling through Gi/o substrate, whereas salivary gland colonization was sensitive. Since salivary gland infection required both M33 and Gi/o-coupled signaling, whereas BAT infection required neither, these migrations were mechanistically distinct. MCMV spread from the lungs or nose depended on DC, controlled by M33. Infecting other monocyte populations resulted in unpredictable new infections.IMPORTANCE Cytomegaloviruses (CMVs) spread through the blood by infecting monocytes, and this can lead to disease. With murine CMV (MCMV) we can track infected myeloid cells and so understand how CMVs spread. Previous experiments have injected MCMV into the peritoneal cavity. MCMV normally enters mice via the olfactory epithelium. We show that olfactory infection spreads via dendritic cells, which MCMV directs to the salivary glands. Peritoneal infection similarly reached the salivary glands via dendritic cells. However, it also infected other monocyte types, and they spread infection to other tissues. Thus, infecting the "wrong" monocytes altered virus spread, with potential to cause disease. These results provide a basis for understanding how the monocyte types infected by human CMV might promote different infection outcomes.

Mouse Cytomegalovirus m153 Protein Stabilizes Expression of the Inhibitory NKR-P1B Ligand Clr-b.

Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germ line-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand Clr-b (encoded by Clec2d) on infected cells. Here we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using m153-deficient MCMV mutants (Δm144-m158 and Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during Δm153 mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of Clec2d transcription, imaging experiments revealed that the m153 and Clr-b proteins only minimally colocalize within the same subcellular compartments, and tagged versions of the proteins were refractory to coimmunoprecipitation under mild-detergent conditions. Surprisingly, the Δm153 mutant possesses enhanced virulence in vivo, independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlight a unique mechanism by which MCMV modulates NK ligand expression.IMPORTANCE Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells ("missing-self"). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.

Inhibition of Cytomegalovirus Replication with Extended-Half-Life Synthetic Ozonides.

Artesunate (AS), a semisynthetic artemisinin approved for malaria therapy, inhibits human cytomegalovirus (HCMV) replication in vitro, but therapeutic success in humans has been variable. We hypothesized that the short in vivo half-life of AS may contribute to the different treatment outcomes. We tested novel synthetic ozonides with longer half-lives against HCMV in vitro and mouse cytomegalovirus (MCMV) in vivo Screening of the activities of four ozonides against a pp28-luciferase-expressing HCMV Towne recombinant identified OZ418 to have the best selectivity; its effective concentration inhibiting viral growth by 50% (EC50) was 9.8 ± 0.2 µM, and cytotoxicity in noninfected human fibroblasts (the concentration inhibiting cell growth by 50% [CC50]) was 128.1 ± 8.0 µM. In plaque reduction assays, OZ418 inhibited HCMV TB40 in a concentration-dependent manner as well as a ganciclovir (GCV)-resistant HCMV isolate. The combination of OZ418 and GCV was synergistic in HCMV inhibition in vitro Virus inhibition by OZ418 occurred at an early stage and was dependent on the cell density at the time of infection. OZ418 treatment reversed HCMV-mediated cell cycle progression and correlated with the reduction of HCMV-induced expression of pRb, E2F1, and cyclin-dependent kinases 1, 2, 4, and 6. In an MCMV model, once-daily oral administration of OZ418 had significantly improved efficacy against MCMV compared to that of twice-daily oral AS. A parallel pharmacokinetic study with a single oral dose of OZ418 or AS showed a prolonged plasma half-life and higher unbound concentrations of OZ418 than unbound concentrations of AS. In summary, ozonides are proposed to be potential therapeutics, alone or in combination with GCV, for HCMV infection in humans.

Brain-resident memory CD8+ T cells induced by congenital CMV infection prevent brain pathology and virus reactivation.

Congenital HCMV infection is a leading infectious cause of long-term neurodevelopmental sequelae. Infection of newborn mice with mouse cytomegalovirus (MCMV) intraperitoneally is a well-established model of congenital human cytomegalovirus infection, which best recapitulates the hematogenous route of virus spread to brain and subsequent pathology. Here, we used this model to investigate the role, dynamics, and phenotype of CD8+ T cells in the brain following infection of newborn mice. We show that CD8+ T cells infiltrate the brain and form a pool of tissue-resident memory T cells (TRM cells) that persist for lifetime. Adoptively transferred virus-specific CD8+ T cells provide protection against primary MCMV infection in newborn mice, reduce brain pathology, and remain in the brain as TRM cells. Brain CD8+ TRM cells were long-lived, slowly proliferating cells able to respond to local challenge infection. Importantly, brain CD8+ TRM cells controlled latent MCMV and their depletion resulted in virus reactivation and enhanced inflammation in brain.

CD4 T cells are required for maintenance of CD8 TRM cells and virus control in the brain of MCMV-infected newborn mice.

Cytomegalovirus (CMV) infection is a significant public health problem. Congenital CMV infection is a leading infectious cause of long-term neurodevelopmental sequelae, including mental retardation and sensorineural hearing loss. Immune protection against mouse cytomegalovirus (MCMV) is primarily mediated by NK cells and CD8+ T cells, while CD4+ T cells are not needed for control of MCMV in majority of organs in immunocompetent adult mice. Here, we set out to determine the role of CD4+ T cells upon MCMV infection of newborn mice. We provide evidence that CD4+ T cells are essential for clearance of MCMV infection in brain of neonatal mice and for prevention of recurrence of latent MCMV. In addition, we provide evidence that CD4+ T cells are required for induction and maintenance of tissue-resident memory CD8+ T cells in the brain of mice perinatally infected with MCMV.

Lymphocyte-independent pathways underlie the pathogenesis of murine cytomegalovirus-associated secondary haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) constitutes a spectrum of immunological disorders characterized by uncontrolled immune activation and key symptoms such as fever, splenomegaly, pancytopenia, haemophagocytosis, hyperferritinaemia and hepatitis. In genetic or primary HLH, hyperactivated CD8+ T cells are the main drivers of pathology. However, in acquired secondary HLH, the role of lymphocytes remains vague. In the present study the involvement of lymphocytes in the pathogenesis of a cytomegalovirus-induced model of secondary HLH was explored. We have previously reported CD8+ T cells to be redundant in this model, and therefore focused on CD4+ helper and regulatory T cells. CD4+ T cells were activated markedly and skewed towards a proinflammatory T helper type 1 transcription profile in mice displaying a severe and complete HLH phenotype. Counter to expectations, regulatory T cells were not reduced in numbers and were, in fact, more activated. Therapeutic strategies targeting CD25high hyperactivated T cells were ineffective to alleviate disease, indicating that T cell hyperactivation is not a pathogenic factor in cytomegalovirus-induced murine HLH. Moreover, even though T cells were essential in controlling viral proliferation, CD4+ T cells, in addition to CD8+ T cells, were dispensable in the development of the HLH-like syndrome. In fact, no T or B cells were required for induction and propagation of HLH disease, as evidenced by the occurrence of cytomegalovirus-associated HLH in severe combined immunodeficient (SCID) mice. These data suggest that lymphocyte-independent mechanisms can underlie virus-associated secondary HLH, accentuating a clear distinction with primary HLH.

The Effects of IL10 and NK Cells on the Susceptibility to Mouse Cytomegalovirus in BALB/c Mice despite the Compensation of IFNγ.

OBJECTIVE: The aim of this study was to determine which factors lead to the susceptibility to mouse cytomegalovirus (MCMV) in the spleens of BALB/c mice. METHODS: BALB/c and C57BL/6 mice were randomly divided into a control group and an infection group and sacrificed on day 0, 1, 3, 7, 14, and 28 postinfection. The cytotoxicity of NK cells was determined by evaluating lactate dehydrogenase contents. Flow cytometry was used to analyze activated NK cells, IFNγ+ NK cells, and total NK cells in the spleen. The pathological changes of spleens in each group were analyzed by HE staining. The expression of IL10, IL18, IFNγ, Thpok, and IFNß of spleens was determined by quantitative reverse transcriptase PCR. The viral loads of MCMV in spleens and salivary glands were also detected. RESULTS: We found that spleen NK cells and IL10 in C57BL/6 mice possessed more powerful immunity to MCMV than BALB/c mice. In BALB/c mice, combined effects of the cytotoxicity of NK cells and IFNγ in spleens still ended up with deficient control of infection. CONCLUSION: The functional shortage of NK cells and inappropriate expression of IL10 result in the susceptibility to MCMV in BALB/c mice.

Lytic viral replication and immunopathology in a cytomegalovirus-induced mouse model of secondary hemophagocytic lymphohistiocytosis.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a rare immunological disorder caused by unbridled activation of T cells and macrophages, culminating in a life-threatening cytokine storm. A genetic and acquired subtype are distinguished, termed primary and secondary HLH, respectively. Clinical manifestations of both forms are frequently preceded by a viral infection, predominantly with herpesviruses. The exact role of the viral infection in the development of the hemophagocytic syndrome remains to be further elucidated. METHODS: We utilized a recently developed murine model of cytomegalovirus-associated secondary HLH and dissected the respective contributions of lytic viral replication and immunopathology in its pathogenesis. RESULTS: HLH-like disease only developed in cytomegalovirus-susceptible mouse strains unable to clear the virus, but the severity of symptoms was not correlated to the infectious viral titer. Lytic viral replication and sustained viremia played an essential part in the pathogenesis since abortive viral infection was insufficient to induce a full-blown HLH-like syndrome. Nonetheless, a limited set of symptoms, in particular anemia, thrombocytopenia and elevated levels of soluble CD25, appeared less dependent of the viral replication but rather mediated by the host's immune response, as corroborated by immunosuppressive treatment of infected mice with dexamethasone. CONCLUSION: Both virus-mediated pathology and immunopathology cooperate in the pathogenesis of full-blown virus-associated secondary HLH and are closely entangled. A certain level of viremia appears necessary to elicit the characteristic HLH-like symptoms in the model.

Natural killer cell biology: an update and future directions.

Natural killer (NK) cells constitute a minor subset of normal lymphocytes that initiate innate immune responses toward tumor and virus-infected cells. They can mediate spontaneous cytotoxicity toward these abnormal cells and rapidly secrete numerous cytokines and chemokines to promote subsequent adaptive immune responses. Significant progress has been made in the past 2 decades to improve our understanding of NK cell biology. Here we review recent discoveries, including a better comprehension of the "education" of NK cells to achieve functional competence during their maturation and the discovery of "memory" responses by NK cells, suggesting that they might also contribute to adaptive immunity. The improved understanding of NK cell biology has forged greater awareness that these cells play integral early roles in immune responses. In addition, several promising clinical therapies have been used to exploit NK cell functions in treating patients with cancer. As our molecular understanding improves, these and future immunotherapies should continue to provide promising strategies to exploit the unique functions of NK cells to treat cancer, infections, and other pathologic conditions.

Combined Host-Pathogen Fate Mapping to Investigate Lung Macrophages in Viral Infection.

Macrophage identity, as defined by epigenetic, transcriptional, proteomic, and functional programs, is greatly impacted by cues originating from the microenvironment. As a consequence, immunophenotyping based on surface marker expression is established and reliable in homeostatic conditions, whereas environmental challenges, in particular infections, severely hamper the determination of identity states. This has become more evident with recent discoveries that macrophage-inherent plasticity may go beyond limits of lineage-defining immunophenotypes. Therefore, transgenic fate mapping tools, such as the phage-derived loxP-cre-system, are essential for the analysis of macrophage adaptation in the tissue under extreme environmental conditions, for example, upon encounter with pathogens. In this chapter, we describe an advanced application of the loxP-cre-system during infection. Here, the host encodes a cell type-specific cre-recombinase, while the pathogen harbors a STOP-floxed fluorescent reporter gene. As an instructive example for the versatility of the system, we demonstrate that alveolar macrophages are predominantly targeted after respiratory tract infection with mouse cytomegalovirus (MCMV). Combined host-pathogen fate mapping not only enables to distinguish between infected and non-infected (bystander) macrophages but also spurs exploration of phenotypic adaptation and tracing of cellular localization in the context of MCMV infection. Moreover, we provide a gating strategy for resolving the diversity of pulmonary immune cell populations.

Epidemiological and phylogenetic analysis of institutional mouse parvoviruses.

Mouse parvoviruses (MPVs) are small, single-stranded, 5 kb DNA viruses that are subclinical and endemic in many laboratory mouse colonies. MPVs cause more distinctive deleterious effects in immune-compromised or genetically-engineered mice than immuno-competent mice. At the University of Louisville (U of L), there was an unexpected increase of MPV sero-positivity for MPV infections in mouse colonies between January 2006 and February 2007, resulting in strategic husbandry changes aimed at controlling MPV spread throughout the animal facility. To investigate these MPVs, VP2 genes of seven MPVs were cloned and sequenced from eight documented incidences by PCR technology. The mutations in these VP2 genes were compared to those found at the Genbank database (NCBI; http://www.ncbi.nlm.nih.gov) and an intra-institutional phylogenetic tree for MPV infections at U of L was constructed. We discovered that the seven MPV isolates were different from those in Genbank and were not identical to each other. These MPVs were designated MPV-UL1 to 7; none of them were minute virus of mice (MVMs). Four isolates could be classified as MPV1, one was classified as MPV2, and two were defined as novel types with less than 96% and 94% homology with existing MPV types. Considering that all seven isolates had mutations in their VP2 genes and no mutations were observed in VP2 genes of MPV during a four-month time period of incubation, we concluded that all seven MPVs isolated at U of L between 2006 and 2007 probably originated from different sources. Serological survey for MPV infections verified that each MPV outbreak was controlled without further contamination within the institution.

The Synthesis and Anti-Cytomegalovirus Activity of Piperidine-4-Carboxamides.

Treatment options for human cytomegalovirus (CMV) remain limited and are associated with significant adverse effects and the selection of resistant CMV strains in transplant recipients and congenitally infected infants. Although most approved drugs target and inhibit the CMV DNA polymerase, additional agents with distinct mechanisms of action are needed for the treatment and prevention of CMV. In a large high throughput screen using our CMV-luciferase reporter Towne, we identified several unique inhibitors of CMV replication. Here, we synthesize and test in vitro 13 analogs of the original NCGC2955 hit (1). Analogs with no activity against the CMV-luciferase at 10 µM and 30 µM (2-6, 10-14) were removed from further analysis. Three analogs (7-9) inhibited CMV replication in infected human foreskin fibroblasts. The EC50 of (1) was 1.7 ± 0.6 µM and 1.99 ± 0.15 µM, based on luciferase and plaque assay, respectively. Compounds 7, 8, and 9 showed similar activities: the EC50 values of 7 were 0.21 ± 0.06 µM (luciferase) and 0.55 ± 0.06 (plaque), of 8: 0.28 ± 0.06 µM and 0.42 ± 0.07, and of 9: 0.30 ± 0.05 µM (luciferase) and 0.35 ± 0.07 (plaque). The CC50 for 7, 8, and 9 in non-infected human foreskin fibroblasts was > 500µM, yielding a selectivity index of >1500. Compounds 1, 7, and 8 were also tested in CMV-infected primary human hepatocytes and showed a dose-response against CMV by luciferase activity and viral protein expression. None of the active compounds inhibited herpes simplex virus 1 or 2. Compounds 7 and 8 inhibited mouse CMV replication in vitro. Both inhibited CMV at late stages of replication; 7 reduced virus yield at all late time points, although not to the same degree as letermovir. Finally, the activity of analog 8 was additive with newly identified CMV inhibitors (MLS8969, NFU1827, MSL8554, and MSL8091) and with ganciclovir. Further structural activity development should provide promising anti-CMV agents for use in clinical studies.

Recent Advancements in Understanding Primary Cytomegalovirus Infection in a Mouse Model.

Animal models that mimic human infections provide insights in virus-host interplay; knowledge that in vitro approaches cannot readily predict, nor easily reproduce. Human cytomegalovirus (HCMV) infections are acquired asymptomatically, and primary infections are difficult to capture. The gap in our knowledge of the early events of HCMV colonization and spread limits rational design of HCMV antivirals and vaccines. Studies of natural infection with mouse cytomegalovirus (MCMV) have demonstrated the olfactory epithelium as the site of natural colonization. Systemic spread from the olfactory epithelium is facilitated by infected dendritic cells (DC); tracking dissemination uncovered previously unappreciated DC trafficking pathways. The olfactory epithelium also provides a unique niche that supports efficient MCMV superinfection and virus recombination. In this review, we summarize recent advances to our understanding of MCMV infection and spread and the tissue-specific mechanisms utilized by MCMV to modulate DC trafficking. As these mechanisms are likely conserved with HCMV, they may inform new approaches for preventing HCMV infections in humans.