Korneel Grauwet

Modulation of CD112 by the alphaherpesvirus gD protein suppresses DNAM-1–dependent NK cell-mediated lysis of infected cells

Significance Herpesviruses have developed fascinating mechanisms to evade elimination by key elements of the host immune system, allowing these pathogens to cause lifelong infections with periods of recurrent virus spread. Natural killer (NK) cells are central in the innate antiviral response. Here, we report that the gD glycoprotein of the alphaherpesviruses, pseudorabies virus and herpes simplex virus-2, displays previously uncharacterized immune evasion properties toward NK cells. Expression of the gD protein leads to degradation of CD112/nectin-2, a ligand for the NK-activating receptor DNAX accessory molecule 1 (DNAM-1). This impairs binding of DNAM-1 to the cell surface, thereby suppressing NK-mediated killing of virus-infected (or gD-transfected) cells. Identification of this previously unidentified immune evasion mechanism may contribute to the design of improved herpesvirus vaccines and herpesvirus-based therapeutic vectors. Natural killer (NK) cells are key players in the innate response to viruses, including herpesviruses. In particular, the variety of viral strategies to modulate the recognition of certain herpesviruses witnesses the importance of NK cells in the control of this group of viruses. Still, NK evasion strategies have remained largely elusive for the largest herpesvirus subfamily, the alphaherpesviruses. Here, we report that the gD glycoprotein of the alphaherpesviruses pseudorabies virus (PRV) and herpes simplex virus 2 (HSV-2) displays previously uncharacterized immune evasion properties toward NK cells. Expression of gD during infection or transfection led to degradation and consequent down-regulation of CD112, a ligand for the activating NK receptor DNAX accessory molecule 1 (DNAM-1). CD112 downregulation resulted in a reduced ability of DNAM-1 to bind to the surface of both virus-infected and gD-transfected cells. Consequently, expression of gD suppressed NK cell degranulation and NK cell-mediated lysis of PRV- or HSV-2–infected cells. These data identify an alphaherpesvirus evasion strategy from NK cells and point out that interactions between viral envelope proteins and host cell receptors can have biological consequences that stretch beyond virus entry.

Suppression of NK cell-mediated cytotoxicity against PRRSV-infected porcine alveolar macrophages in vitro

The adaptive immunity against PRRSV has already been studied in depth, but only limited data are available on the innate immune responses against this pathogen. In the present study, we analyzed the interaction between porcine natural killer (NK) cells and PRRSV-infected primary porcine alveolar macrophages (PAMs), since NK cells are one of the most important components of innate immunity and PAMs are primary target cells of PRRSV infection. NK cytotoxicity assays were performed using enriched NK cells as effector cells and virus-infected or mock-inoculated PAMs as target cells. The NK cytotoxicity against PRRSV-infected PAMs was decreased starting from 6h post inoculation (hpi) till the end of the experiment (12 hpi) and was significantly lower than that against pseudorabies virus (PrV)-infected PAMs. UV-inactivated PRRSV also suppressed NK activity, but much less than infectious PRRSV. Furthermore, co-incubation with PRRSV-infected PAMs inhibited degranulation of NK cells. Finally, using the supernatant of PRRSV-infected PAMs collected at 12 hpi showed that the suppressive effect of PRRSV on NK cytotoxicity was not mediated by soluble factors. In conclusion, PRRSV-infected PAMs showed a reduced susceptibility toward NK cytotoxicity, which may represent one of the multiple evasion strategies of PRRSV.

Role of NK cells in immunotherapy and virotherapy of solid tumors.

Although natural killer (NK) cells are endowed with powerful cytolytic activity against cancer cells, their role in different therapies against solid tumors has not yet been fully elucidated. Their interactions with various elements of the tumor microenvironment as well as their possible effects in contributing to and/or limiting oncolytic virotherapy render this potential immunotherapeutic tool still difficult to exploit at the bedside. Here, we will review the current literature with the aim of providing new hints to manage this powerful cell type in future innovative therapies, such as the use of NK cells in combination with new cytokines, specific mAbs (inducing ADCC), Tyr-Kinase inhibitors, immunomodulatory drugs and/or the design of oncolytic viruses aimed at optimizing the effect of NK cells in virotherapy.

Pseudorabies virus US3 protein kinase protects infected cells from NK cell-mediated lysis via increased binding of the inhibitory NK cell receptor CD300a

ABSTRACT Several reports have indicated that natural killer (NK) cells are of particular importance in the innate response against herpesvirus infections. As a consequence, herpesviruses have developed diverse mechanisms for evading NK cells, although few such mechanisms have been identified for the largest herpesvirus subfamily, the alphaherpesviruses. The antiviral activity of NK cells is regulated by a complex array of interactions between activating/inhibitory receptors on the NK cell surface and the corresponding ligands on the surfaces of virus-infected cells. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) displays previously uncharacterized immune evasion properties: it triggers the binding of the inhibitory NK cell receptor CD300a to the surface of the infected cell, thereby providing increased CD300a-mediated protection of infected cells against NK cell-mediated lysis. US3-mediated CD300a binding was found to depend on aminophospholipid ligands of CD300a and on group I p21-activated kinases. These data identify a novel alphaherpesvirus strategy for evading NK cells and demonstrate, for the first time, a role for CD300a in regulating NK cell activity upon contact with virus-infected target cells. IMPORTANCE Herpesviruses have developed fascinating mechanisms to evade elimination by key elements of the host immune system, contributing to their ability to cause lifelong infections with recurrent reactivation events. Natural killer (NK) cells are central in the innate antiviral response. Here we report that the US3 protein kinase of the alphaherpesvirus pseudorabies virus displays a previously uncharacterized capacity for evasion of NK cells. Expression of US3 protects infected cells from NK cell-mediated lysis via increased binding of the inhibitory NK cell receptor CD300a. We show that this US3-mediated increase in CD300a binding depends on aminophospholipids and on cellular p21-activated kinases (PAKs). The identification of this novel NK cell evasion strategy may contribute to the design of improved herpesvirus vaccines and may also have significance for other PAK- and CD300a-modulating viruses and cancer cells.

Pseudorabies virus US3 leads to filamentous actin disassembly and contributes to viral genome delivery to the nucleus

The conserved alphaherpesvirus US3 tegument protein induces rearrangements of the actin cytoskeleton, consisting of protrusion formation and stress fiber breakdown. Although US3 does not affect levels of total actin protein, it remains unclear whether US3 modulates the total levels of filamentous (F) actin. In this report, we show that the pseudorabies virus (PRV) US3 protein, via its kinase activity, leads to disassembly of F-actin in porcine ST cells. F-actin disassembly has been reported before to contribute to host cell entry of HIV. In line with this, in the current study, we report that US3 has a previously uncharacterized role in viral genome delivery to the nucleus, since quantitative polymerase chain reaction (qPCR) assays on nuclear fractions demonstrated a reduced nuclear delivery of US3null PRV compared to wild type PRV genomes. Treatment of cells with the actin depolymerizing drug cytochalasin D enhanced virus genome delivery to the nucleus, particularly of US3null PRV, supporting a role for F-actin disassembly during certain aspects of viral entry. In conclusion, the US3 kinase of PRV leads to F-actin depolymerization, and US3 and F-actin disassembly contribute to viral genome delivery to the nucleus.

Role of Mast Cells in Regulation of T Cell Responses in Experimental and Clinical Settings

Mast cells secrete a wide spectrum of stored or newly synthesized pro-inflammatory, anti-inflammatory, and/or immunosuppressive mediators and express several costimulatory and inhibitory surface molecules. Mast cells finely tune activities of T cells, B cells, and regulatory cells and effectively contribute to the development of different T cell-associated responses by influencing their recruitment, activation, proliferation, and differentiation. The interaction between mast cells and T cells, with regard to cellular functionality and immune responses, can be assessed in both activating and inhibitory regulations. While Th2 cytokines, including IL-5 and IL-9, stimulate stem cell factor (SCF)-dependent proliferation of mast cells, Th1 cytokine IFN-γ suppresses SCF-mediated differentiation of mast cell progenitors. Mast cell mediators such as CCL5 have a role in the recruitment of CD8+ T cells to viral infection sites where their ability in clearance of viral reservoirs is needed. The capacity of mast cells in presenting antigens by classes I and II MHC molecules to CD4+ and CD8+ T cells respectively is considered one of the main antigen-dependent interactions of mast cells with T cells. Interestingly, Tregs recruit mast cells to different sites through secretion of IL-9, while the OX40L (expressed on mast cell)-OX40(expressed on T cell) interaction inhibits the extent of the mast cell degranulation. Recently, the capability of exosomes to carry regulatory receptors of the mast cell surface and their role in T cell activation has been investigated. Functional interplay between mast cells and T cell subsets has been suggested primarily by investigating their co-localization in inflamed tissues and involvement of mast cells in autoimmune diseases. In this review, the interactions of mast cells with T cells are reviewed in cell-to-cell, cytokine, and exosome categories.

Glioma and microglia, a double entendre

Microglia are important facilitators of glioma proliferation and invasion. An important component of this process seems to be glioma-mediated suppression of microglial activation via S-nitrosylation of microglial caspase-3.

Differences in uptake and killing of pathogenic and non-pathogenic bacteria by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone)

Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non-pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi-granulocytes) have the main function in phagocytosis of both pathogenic and non-pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non-virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post-inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non-virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.

Comparative immune responses against Psoroptes ovis in two cattle breeds with different susceptibility to mange

The sheep scab mite, Psoroptes ovis, is a major problem in the beef cattle industry, especially in Belgian Blue (BB) cattle. This breed is naturally more predisposed to psoroptic mange but reasons for this high susceptibility remain unknown. Different immune responses could be a potential cause; thus in this study, the cutaneous immune response and in vitro cellular immune response after antigen re-stimulation were examined in naturally infested BB. Cytokine production in the skin and in circulating re-stimulated peripheral blood mononuclear cells (PBMC) demonstrated a mixed pro-inflammatory Th2/Th17 profile, with transcription of IL-4, IL-13, IL-6 and IL-17. Strong IL-17 up-regulation in the skin of BB was associated with an influx of eosinophils and other immune cells, potentially leading towards more severe symptoms. Virtually no changes in cutaneous IFN-γ transcription were detected, while there was substantial IFN-γ up-regulation in re-stimulated PBMC from infested and uninfested animals, potentially indicating a role of this pro-inflammatory cytokine in the innate immune response. In Holstein–Friesian (HF) cattle, generally more resistant to P. ovis infection, a largely similar immunologic response was observed. Differences between HF and BB were the lack of cutaneous IL-17 response in infested HF and low transcription levels of IFN-γ and high IL-10 transcription in re-stimulated PBMC from both infested and uninfested animals. Further research is needed to identify potential cell sources and biological functions for these cytokines and to fully unravel the basis of this different breed susceptibility to P. ovis.