A cellular hierarchy in melanoma uncouples growth and metastasis.

Although melanoma is notorious for its high degree of heterogeneity and plasticity1,2, the origin and magnitude of cell-state diversity remains poorly understood. Equally, it is unclear whether growth and metastatic dissemination are supported by overlapping or distinct melanoma subpopulations. Here, by combining mouse genetics, single-cell and spatial transcriptomics, lineage tracing and quantitative modelling, we provide evidence of a hierarchical model of tumour growth that mirrors the cellular and molecular logic underlying the cell-fate specification and differentiation of the embryonic neural crest. We show that tumorigenic competence is associated with a spatially localized perivascular niche, a phenotype acquired through an intercellular communication pathway established by endothelial cells. Consistent with a model in which only a fraction of cells are fated to fuel growth, temporal single-cell tracing of a population of melanoma cells with a mesenchymal-like state revealed that these cells do not contribute to primary tumour growth but, instead, constitute a pool of metastatic initiating cells that switch cell identity while disseminating to secondary organs. Our data provide a spatially and temporally resolved map of the diversity and trajectories of melanoma cell states and suggest that the ability to support growth and metastasis are limited to distinct pools of cells. The observation that these phenotypic competencies can be dynamically acquired after exposure to specific niche signals warrant the development of therapeutic strategies that interfere with the cancer cell reprogramming activity of such microenvironmental cues.

How tech-savvy employees make the difference in core facilities: Recognizing core facility expertise with dedicated career tracks: Recognizing core facility expertise with dedicated career tracks.

Core facilities have a different mission than academic research labs. Accordingly, they require different career paths and structures.

Aspecific binding of anti-NK1.1 antibodies on myeloid cells in an experimental model for malaria-associated acute respiratory distress syndrome.

BACKGROUND: Conventional natural killer (cNK) cells play an important role in the innate immune response by directly killing infected and malignant cells and by producing pro- and anti-inflammatory cytokines. Studies on their role in malaria and its complications have resulted in conflicting results. METHODS: Using the commonly used anti-NK1.1 depletion antibodies (PK136) in an in-house optimized experimental model for malaria-associated acute respiratory distress syndrome (MA-ARDS), the role of cNK cells was investigated. Moreover, flow cytometry was performed to characterize different NK cell populations. RESULTS: While cNK cells were found to be dispensable in the development of MA-ARDS, the appearance of a NK1.1+ cell population was observed in the lungs upon infection despite depletion with anti-NK1.1. Detailed characterization of the unknown population revealed that this population consisted of a mixture of monocytes and macrophages that bind the anti-NK1.1 antibody in an aspecific way. This aspecific binding may occur via Fcγ receptors, such as FcγR4. In contrast, in vivo depletion using anti-NK1.1 antibodies was proved to be specific for cNK cells. CONCLUSION: cNK cells are dispensable in the development of experimental MA-ARDS. Moreover, careful flow cytometric analysis, with a critical mindset in relation to potential aspecific binding despite the use of commercially available Fc blocking reagents, is critical to avoid misinterpretation of the results.

The Attenuated Pseudorabies Virus Vaccine Strain Bartha Hyperactivates Plasmacytoid Dendritic Cells by Generating Large Amounts of Cell-Free Virus in Infected Epithelial Cells.

Pseudorabies virus (PRV) is a porcine alphaherpesvirus and the causative agent of Aujeszky's disease. Successful eradication campaigns against PRV have largely relied on the use of potent PRV vaccines. The live attenuated Bartha strain, which was produced by serial passaging in cell culture, represents one of the hallmark PRV vaccines. Despite the robust protection elicited by Bartha vaccination, very little is known about the immunogenicity of the Bartha strain. Previously, we showed that Bartha-infected epithelial cells trigger plasmacytoid dendritic cells (pDC) to produce much higher levels of type I interferons than cells infected with wild-type PRV. Here, we show that this Bartha-induced pDC hyperactivation extends to other important cytokines, including interleukin-12/23 (IL-12/23) and tumor necrosis factor alpha (TNF-α) but not IL-6. Moreover, Bartha-induced pDC hyperactivation was found to be due to the strongly increased production of extracellular infectious virus (heavy particles [H-particles]) early in infection of epithelial cells, which correlated with a reduced production of noninfectious light particles (L-particles). The Bartha genome is marked by a large deletion in the US region affecting the genes encoding US7 (gI), US8 (gE), US9, and US2. The deletion of the US2 and gE/gI genes was found to be responsible for the observed increase in extracellular virus production by infected epithelial cells and the resulting increased pDC activation. The deletion of gE/gI also suppressed L-particle production. In conclusion, the deletion of US2 and gE/gI in the genome of the PRV vaccine strain Bartha results in the enhanced production of extracellular infectious virus in infected epithelial cells and concomitantly leads to the hyperactivation of pDC. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha has been and still is critical in the eradication of PRV in numerous countries. However, little is known about how this vaccine strain interacts with host cells and the host immune system. Here, we report the surprising observation that Bartha-infected epithelial porcine cells rapidly produce increased amounts of extracellular infectious virus compared to wild-type PRV-infected cells, which in turn potently stimulate porcine plasmacytoid dendritic cells (pDC). We found that this phenotype depends on the deletion of the genes encoding US2 and gE/gI. We also found that Bartha-infected cells secrete fewer pDC-inhibiting light particles (L-particles), which appears to be caused mainly by the deletion of the genes encoding gE/gI. These data generate novel insights into the interaction of the successful Bartha vaccine with epithelial cells and pDC and may therefore contribute to the development of vaccines against other (alphaherpes)viruses.

The Pseudorabies Virus Glycoprotein gE/gI Complex Suppresses Type I Interferon Production by Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDC) play a central role in the antiviral immune response, both in the innate response and in shaping the adaptive response, mainly because of their ability to produce massive amounts of type I interferon (TI-IFN). Here, we report that cells infected with the live attenuated Bartha vaccine strain of porcine alphaherpesvirus pseudorabies virus (PRV) trigger a dramatically increased TI-IFN response by porcine primary pDC compared to cells infected with wild-type PRV strains (Becker and Kaplan). Since Bartha is one of the relatively few examples of a highly successful alphaherpesvirus vaccine, identification of factors that may contribute to its efficacy may provide insights for the rational design of other alphaherpesvirus vaccines. The Bartha vaccine genome displays several mutations compared to the genome of wild-type PRV strains, including a large deletion in the unique short (US) region, encompassing the glycoprotein E (gE), gI, US9, and US2 genes. Using recombinant PRV Becker strains harboring the entire Bartha US deletion or single mutations in the four affected US genes, we demonstrate that the absence of the viral gE/gI complex contributes to the observed increased IFN-α response. Furthermore, we show that the absence of gE leads to an enhanced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in pDC, which correlates with a higher TI-IFN production by pDC. In conclusion, the PRV Bartha vaccine strain triggers strongly increased TI-IFN production by porcine pDC. Our data further indicate that the gE/gI glycoprotein complex suppresses TI-IFN production by pDC, which represents the first alphaherpesvirus factor that suppresses pDC activity.IMPORTANCE Several alphaherpesviruses, including herpes simpex virus, still lack effective vaccines. However, the highly successful Bartha vaccine has contributed substantially to eradication of the porcine alphaherpesvirus pseudorabies virus (PRV) in several countries. The impact of Bartha on the immune response is still poorly understood. Type I interferon (TI-IFN)-producing plasmacytoid dendritic cells (pDC) may play an important role in vaccine development. Here, we show that Bartha elicits a dramatically increased type I interferon (TI-IFN) response in primary porcine pDC compared to wild-type strains. In addition, we found that the gE/gI complex, which is absent in Bartha, inhibits the pDC TI-IFN response. This is the first description of an immune cell type that is differentially affected by Bartha versus wild-type PRV and is the first report describing an alphaherpesvirus protein that inhibits the TI-IFN response by pDC. These data may therefore contribute to the rational design of other alphaherpesvirus vaccines.

Pseudorabies Virus US3-Induced Tunneling Nanotubes Contain Stabilized Microtubules, Interact with Neighboring Cells via Cadherins, and Allow Intercellular Molecular Communication.

Tunneling nanotubes (TNTs) are long bridge-like structures that connect eukaryotic cells and mediate intercellular communication. We found earlier that the conserved alphaherpesvirus US3 protein kinase induces long cell projections that contact distant cells and promote intercellular virus spread. In this report, we show that the US3-induced cell projections constitute TNTs. In addition, we report that US3-induced TNTs mediate intercellular transport of information (e.g., green fluorescent protein [GFP]) in the absence of other viral proteins. US3-induced TNTs are remarkably stable compared to most TNTs described in the literature. In line with this, US3-induced TNTs were found to contain stabilized (acetylated and detyrosinated) microtubules. Transmission electron microscopy showed that virus particles are individually transported in membrane-bound vesicles in US3-induced TNTs and are released along the TNT and at the contact area between a TNT and the adjacent cell. Contact between US3-induced TNTs and acceptor cells is very stable, which correlated with a marked enrichment in adherens junction components beta-catenin and E-cadherin at the contact area. These data provide new structural insights into US3-induced TNTs and how they may contribute to intercellular communication and alphaherpesvirus spread.IMPORTANCE Tunneling nanotubes (TNT) represent an important and yet still poorly understood mode of long-distance intercellular communication. We and others reported earlier that the conserved alphaherpesvirus US3 protein kinase induces long cellular protrusions in infected and transfected cells. Here, we show that US3-induced cell projections constitute TNTs, based on structural properties and transport of biomolecules. In addition, we report on different particular characteristics of US3-induced TNTs that help to explain their remarkable stability compared to physiological TNTs. In addition, transmission electron microscopy assays indicate that, in infected cells, virions travel in the US3-induced TNTs in membranous transport vesicles and leave the TNT via exocytosis. These data generate new fundamental insights into the biology of (US3-induced) TNTs and into how they may contribute to intercellular virus spread and communication.

The US3 Protein of Pseudorabies Virus Drives Viral Passage across the Basement Membrane in Porcine Respiratory Mucosa Explants.

Passage of the basement membrane (BM), which forms a barrier between the epithelium and the underlying lamina propria, represents an important step in the early pathogenesis of different alphaherpesviruses. Rho GTPase signaling plays an important role in transmigration of cells across the BM during physiological and pathological processes. We reported earlier that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) interferes with Rho GTPase signaling and causes a reorganization of the host cell cytoskeleton, which as a consequence, enhances viral cell-to-cell spread in epithelial cell cultures. Here, using an ex vivo system of porcine nasal respiratory mucosa explants that allows to study PRV invasion through the BM, we found that a PRV strain that lacks US3 expression (ΔUS3 PRV) showed a reduced spread in mucosal epithelium and was virtually unable to breach the BM, in contrast to isogenic wild-type (WT) or US3 rescue PRV strains. Interestingly, addition of IPA3, an inhibitor of p21-activated kinases that blocks the effects of US3 on the cytoskeleton, suppressed the ability of WT PRV to spread across the BM. In addition, artificial suppression of RhoA signaling using CPC3 (cell-permeable C3 transferase) to mimic the effects of US3 on Rho GTPase signaling, significantly increased passage of ΔUS3 PRV through the BM, whereas it did not significantly affect BM passage of WT or US3 rescue PRV. In conclusion, these data indicate that US3 plays an important role in PRV mucosal invasion across the BM, which involves its interference with Rho GTPase signaling. This is the first report describing an alphaherpesvirus protein that drives viral BM passage. IMPORTANCE: Many viruses, including alphaherpesviruses, primarily replicate in epithelial cells of surface mucosae, such as the respiratory mucosa. Some of these viruses breach the basement membrane underlying these epithelial cells to reach underlying connective tissue and blood vessels and invade the host. Hence, epithelial spread and basement membrane passage represent crucial but still poorly understood early steps in (alphaherpes)virus pathogenesis. Here, using ex vivo porcine respiratory mucosa explants, we show that the conserved US3 protein of the porcine alphaherpesvirus pseudorabies virus (PRV) is critical for passage of PRV across the basement membrane and contributes to efficient viral epithelial spread. In addition, we show that US3-mediated viral epithelial spread and passage across the basement membrane depend at least in part on the ability of this viral protein to modulate cellular Rho GTPase signaling. This is the first report that identifies an alphaherpesvirus protein that drives viral basement membrane passage.

Tumor endothelial cell autophagy is a key vascular-immune checkpoint in melanoma.

Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T-cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh /AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8+ T-cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.