Mechanisms Of Innate Immune Evasion Research Articles

Abstract 3914: IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target

Abstract Background: Loss of MHC-I antigen presentation is often associated with T-cell excluded tumors, and represents a common mechanism of both primary and acquired resistance to immune checkpoint blockade (ICB) treatment. MHC-I normally acts as a marker of “self” and cells that lack MHC-I are recognized and killed by innate immunity. In this study, we investigated the mechanism of innate immune evasion in tumors with MHC-I antigen presentation defects. Methods: Integrating functional genomics, big data, and artificial intelligence, we discovered that IGSF8 expressed on cancer cells is an evolutionarily conserved innate immune checkpoint. IGSF8 is normally expressed in neuronal tissues and is not essential in vitro or in vivo. However, knockout of IGSF8 in B16-F10 melanoma cell line decreased tumor growth in vivo. For clinical relevance of IGSF8 in tumor immunity, we analyzed genomics, transcriptomics, and clinical data from The Cancer Genome Atlas (TCGA). We developed an antibody (GV20-0251) against IGSF8 and tested its function to induce immune cell killing of cancer cells in vitro and inhibit tumor growth in vivo. Results: IGSF8 has receptors on both natural killer (NK) cells and dendritic cells (DC) to strongly suppress NK cytotoxicity and antigen presentation. In the TCGA tumor profiles, IGSF8 has highest mRNA expression in melanoma and is significantly overexpressed in many cancer types. IGSF8 also shows frequent copy number amplifications. In addition, IGSF8 expression is associated with low antigen presentation, low immune infiltration, and worse overall survival in patients with low MHC class I expression. Immunohistochemistry staining of various cancers showed that IGSF8 expression was primarily observed in malignant cells with low MHC-I. We developed a cross-species reactive antibody against IGSF8 (GV20-0251) which blocks IGSF8 interaction with NK and DC. Flow cytometry of the tumor infiltrating CD45+ cells revealed that IGSF8 inhibition significantly increased NK and dendritic cell infiltration. GV20-0251 enhances NK killing of cancer cells in vitro and increases antigen presentation, NK-mediated cytotoxicity, and T cell signaling in vivo. In multiple syngeneic tumor models (B16-F10, CT26, LLC, 4T1 and EMT6), anti-IGSF8 shows single-agent efficacy and is synergistic with anti-PD1 in controlling tumor growth. Conclusions: IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target. A phase 1 study is ongoing to explore the IGSF8 inhibitor GV20-0251 in patients with advanced or metastatic solid tumors (NCT05669430). Citation Format: X. Shirley Liu, Yulong Li, Xiangyang Wu, Hailing Liu, Huizhu Liu, Caibin Sheng, Qingyang Ding, Yixuan Tang, Chao Liu, Bin Xie, Xi Xiao, Quan Yu, Rongbin Zheng, Xihao Hu, Tengfei Xiao. IGSF8 is a novel innate immune checkpoint and cancer immunotherapy target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3914.

The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localizes to mitochondria, where it inhibits innate immunity by restricting IFN-β production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterized mechanism of innate immune evasion by this important human pathogen.

Toll-like Receptor Response to Human Immunodeficiency Virus Type 1 or Co-Infection with Hepatitis B or C Virus: An Overview.

Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that play important roles in the early detection of pathogen-associated molecular patterns and shaping innate and adaptive immune responses, which may influence the consequences of infection. Similarly to other viral infections, human immunodeficiency virus type 1 (HIV-1) also modulates the host TLR response; therefore, a proper understanding of the response induced by human HIV-1 or co-infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), due to the common mode of transmission of these viruses, is essential for understanding HIV-1 pathogenesis during mono- or co-infection with HBV or HCV, as well as for HIV-1 cure strategies. In this review, we discuss the host TLR response during HIV-1 infection and the innate immune evasion mechanisms adopted by HIV-1 for infection establishment. We also examine changes in the host TLR response during HIV-1 co-infection with HBV or HCV; however, this type of study is extremely scarce. Moreover, we discuss studies investigating TLR agonists as latency-reverting agents and immune stimulators towards new strategies for curing HIV. This understanding will help develop a new strategy for curing HIV-1 mono-infection or co-infection with HBV or HCV.

Uncovering Novel Viral Innate Immune Evasion Strategies: What Has SARS-CoV-2 Taught Us?

The ongoing SARS-CoV-2 pandemic has tested the capabilities of public health and scientific community. Since the dawn of the twenty-first century, viruses have caused several outbreaks, with coronaviruses being responsible for 2: SARS-CoV in 2007 and MERS-CoV in 2013. As the border between wildlife and the urban population continue to shrink, it is highly likely that zoonotic viruses may emerge more frequently. Furthermore, it has been shown repeatedly that these viruses are able to efficiently evade the innate immune system through various strategies. The strong and abundant antiviral innate immunity evasion strategies shown by SARS-CoV-2 has laid out shortcomings in our approach to quickly identify and modulate these mechanisms. It is thus imperative that there be a systematic framework for the study of the immune evasion strategies of these viruses, to guide development of therapeutics and curtail transmission. In this review, we first provide a brief overview of general viral evasion strategies against the innate immune system. Then, we utilize SARS-CoV-2 as a case study to highlight the methods used to identify the mechanisms of innate immune evasion, and pinpoint the shortcomings in the current paradigm with its focus on overexpression and protein-protein interactions. Finally, we provide a recommendation for future work to unravel viral innate immune evasion strategies and suitable methods to aid in the study of virus-host interactions. The insights provided from this review may then be applied to other viruses with outbreak potential to remain ahead in the arms race against viral diseases.

TAMI-65. FASN-MEDIATED LIPID SYNTHESIS HAMPERS ANTI-CANCER IMMUNITY OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most aggressive type of primary brain tumor in adults. Radiation therapy (RT) is an essential modality for GBM treatment and is recognized to stimulate anti-cancer immunity by at least generating type I interferon (IFN-I) responses. However, RT also exacerbates potent immune inhibitory mechanisms that facilitate immune evasion. Notably, increased lipid synthesis by the fatty acid synthase (FASN) is an emerging mechanism that can account for the deceiving treatment efficacy and immune escape of GBM. Therefore, we hypothesize that FASN-mediated lipid synthesis represents an innate immune evasion mechanism of irradiated GBM. Supporting this hypothesis, we observed that 10 gray (Gy) irradiation of murine GBM cell lines, GL261 and CT2A, upregulates FASN protein expression and increase cellular lipid content determined by BODIPY staining and electronic microscopy. Interestingly, this effect was abrogated when GBM cells were incubated with an inhibitor of FASN. Next, to ask whether FASN was impairing RT-induced IFN-I, GL261 and CT2A cells were engineered to express an inducible shRNA silencing FASN (GBMshFASN) or its non-silencing control (GBMshNS). Irradiation of GBMshNS cells enhanced the secretion of IFN-beta and CXCL10, but this effect was more pronounced when FASN was blocked. Finally, GBMshNS and GBMshFASN cells were orthotopically implanted in mice on day 0. On day 10, selective irradiation (10Gy) was performed to the tumor. Tumor growth and immune contexture were evaluated on day 17. Magnetic resonance imaging revealed that FASN knockdown reduces tumor growth independently from RT. However, in situ immunofluorescence of GBMshFASN tumors showed increased infiltration of CD8+ T cells and CD11c+ cells only in irradiated mice bearing GBMshFASN tumors. Altogether, our data suggest that RT rewires the energy supply of GBM by promoting FASN-mediated lipid synthesis to foster immune evasion. Targeting FASN is a promising strategy to promote anti-cancer immunity and sensitize irradiated GBM to immunotherapies.

Herpes Simplex Virus 1 Infection of Neuronal and Non-Neuronal Cells Elicits Specific Innate Immune Responses and Immune Evasion Mechanisms.

Alphaherpesviruses (α-HV) are a large family of double-stranded DNA viruses which cause many human and animal diseases. There are three human α-HVs: Herpes Simplex Viruses (HSV-1 and HSV-2) and Varicella Zoster Virus (VZV). All α-HV have evolved multiple strategies to suppress or exploit host cell innate immune signaling pathways to aid in their infections. All α-HVs initially infect epithelial cells (primary site of infection), and later spread to infect innervating sensory neurons. As with all herpesviruses, α-HVs have both a lytic (productive) and latent (dormant) stage of infection. During the lytic stage, the virus rapidly replicates in epithelial cells before it is cleared by the immune system. In contrast, latent infection in host neurons is a life-long infection. Upon infection of mucosal epithelial cells, herpesviruses immediately employ a variety of cellular mechanisms to evade host detection during active replication. Next, infectious viral progeny bud from infected cells and fuse to neuronal axonal terminals. Here, the nucleocapsid is transported via sensory neuron axons to the ganglion cell body, where latency is established until viral reactivation. This review will primarily focus on how HSV-1 induces various innate immune responses, including host cell recognition of viral constituents by pattern-recognition receptors (PRRs), induction of IFN-mediated immune responses involving toll-like receptor (TLR) signaling pathways, and cyclic GMP‐AMP synthase stimulator of interferon genes (cGAS-STING). This review focuses on these pathways along with other mechanisms including autophagy and the complement system. We will summarize and discuss recent evidence which has revealed how HSV-1 is able to manipulate and evade host antiviral innate immune responses both in neuronal (sensory neurons of the trigeminal ganglia) and non-neuronal (epithelial) cells. Understanding the innate immune response mechanisms triggered by HSV-1 infection, and the mechanisms of innate immune evasion, will impact the development of future therapeutic treatments.

RNA Sensors as a Mechanism of Innate Immune Evasion among SARSCoV2, HIV and Nipah Viruses.

Innate immunity is the first line of defence elicited by the host immune system to fight against invading pathogens such as viruses and bacteria. From this elementary immune response, the more complex antigen-specific adaptive responses are recruited to provide a long-lasting memory against the pathogens. Innate immunity gets activated when the host cell utilizes a diverse set of receptors known as pattern recognition receptors (PRR) to recognize the viruses that have penetrated the host and responds with cellular processes like complement system, phagocytosis, cytokine release and inflammation and destruction of NK cells. Viral RNA or DNA or viral intermediate products are recognized by receptors like toll-like receptors(TLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) thereby, inducing type I interferon response (IFN) and other proinflammatory cytokines in infected cells or other immune cells. But certain viruses can evade the host innate immune response to replicate efficiently, triggering the spread of the viral infection. The present review describes the similarity in the mechanism chosen by viruses from different families -HIV, SARSCoV- 2 and Nipah viruses to evade the innate immune response and how efficiently they establish the infection in the host. The review also addresses the stages of developments of various vaccines against these viral diseases and the challenges encountered by the researchers during vaccine development.

A Review of the Innate Immune Evasion Mechanisms and Status of Vaccine Development of Klebsiella pneumonia

Klebsiella pneumoniae (KP) is a human pathogen causing a broad spectrum of diseases such as urinary tract infections (UTI), pneumonia, pyogenic liver abscess, bloodstream infections, and sepsis. Neonate, geriatric and immunocompromised individuals are the most vulnerable to KP infections. The success of KP as an infectious agent is due to the evolution of various mechanisms to evade the host's immune system. These diverse mechanisms have led to the dominance of KP infections in community settings where hypervirulent strains predominate and in hospital-acquired infections where multidrug-resistant strains predominate. KP infections in the past decades have been increasingly associated with high morbidity and mortality due to the emergence of multidrug-resistant and hypervirulent strains capable of evading both the internal immune defense mechanisms and external antimicrobial agents. The pharmaceutical industries have very few and often expensive new antibiotics in the pipeline, offering little hope for antibiotic therapy. The development of new therapeutic strategies such as polyvalent, biconjugate vaccines that can provide protective immunity, especially against vulnerable populations, can mitigate the effects of KP infections. In this review, we discuss the virulence mechanisms of KP and how it evades the innate host immunity, and the interplay between the virulence and immune evasion strategies. The progress in the search for a vaccine to protect against KP infections will also be highlighted.

RNA Sensors as a Mechanism of Innate Immune Evasion among SARSCoV2, HIV and Nipah Viruses

RNA Sensors as a Mechanism of Innate Immune Evasion among SARSCoV2, HIV and Nipah Viruses

Viral Innate Immune Evasion and the Pathogenesis of Emerging RNA Virus Infections.

Positive-sense single-stranded RNA (+ssRNA) viruses comprise many (re-)emerging human pathogens that pose a public health problem. Our innate immune system and, in particular, the interferon response form the important first line of defence against these viruses. Given their genetic flexibility, these viruses have therefore developed multiple strategies to evade the innate immune response in order to optimize their replication capacity. Already many molecular mechanisms of innate immune evasion by +ssRNA viruses have been identified. However, research addressing the effect of host innate immune evasion on the pathology caused by viral infections is less prevalent in the literature, though very relevant and interesting. Since interferons have been implicated in inflammatory diseases and immunopathology in addition to their protective role in infection, antagonizing the immune response may have an ambiguous effect on the clinical outcome of the viral disease. Therefore, this review discusses what is currently known about the role of interferons and host immune evasion in the pathogenesis of emerging coronaviruses, alphaviruses and flaviviruses.

The FDA-Approved Oral Drug Nitazoxanide Amplifies Host Antiviral Responses and Inhibits Ebola Virus.

SummaryHere, we show that the US Food and Drug Administration-approved oral drug nitazoxanide (NTZ) broadly amplifies the host innate immune response to viruses and inhibits Ebola virus (EBOV) replication. We find that NTZ enhances retinoic-acid-inducible protein I (RIG-I)-like-receptor, mitochondrial antiviral signaling protein, interferon regulatory factor 3, and interferon activities and induces transcription of the antiviral phosphatase GADD34. NTZ significantly inhibits EBOV replication in human cells through its effects on RIG-I and protein kinase R (PKR), suggesting that it counteracts EBOV VP35 protein's ability to block RIG-I and PKR sensing of EBOV. NTZ also inhibits a second negative-strand RNA virus, vesicular stomatitis virus (VSV), through RIG-I and GADD34, but not PKR, consistent with VSV's distinct host innate immune evasion mechanisms. Thus, NTZ counteracts varied virus-specific immune evasion strategies by generally enhancing the RNA sensing and interferon axis that is triggered by foreign cytoplasmic RNA exposure, and holds promise as an oral therapy against EBOV.

Arterivirus nsp4 Antagonizes Interferon Beta Production by Proteolytically Cleaving NEMO at Multiple Sites.

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose major threats for the horse- and swine-breeding industries worldwide. A previous study suggested that PRRSV nsp4, a 3C-like protease, antagonizes interferon beta (IFN-β) production by cleaving the NF-κB essential modulator (NEMO) at a single site, glutamate 349 (E349). Here, we demonstrated that EAV nsp4 also inhibited virus-induced IFN-β production by targeting NEMO for proteolytic cleavage and that the scission occurred at four sites: E166, E171, glutamine 205 (Q205), and E349. Additionally, we found that, besides the previously reported cleavage site E349 in NEMO, scission by PRRSV nsp4 took place at two additional sites, E166 and E171. These results imply that while cleaving NEMO is a common strategy utilized by EAV and PRRSV nsp4 to antagonize IFN induction, EAV nsp4 adopts a more complex substrate recognition mechanism to target NEMO. By analyzing the abilities of the eight different NEMO fragments resulting from EAV or PRRSV nsp4 scission to induce IFN-β production, we serendipitously found that a NEMO fragment (residues 1 to 349) could activate IFN-β transcription more robustly than full-length NEMO, whereas all other NEMO cleavage products were abrogated for the IFN-β-inducing capacity. Thus, NEMO cleavage at E349 alone may not be sufficient to completely inactivate the IFN response via this signaling adaptor. Altogether, our findings suggest that EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is critical for disarming the innate immune response for viral survival.IMPORTANCE The arterivirus nsp4-encoded 3C-like protease (3CLpro) plays an important role in virus replication and immune evasion, making it an attractive target for antiviral therapeutics. Previous work suggested that PRRSV nsp4 suppresses type I IFN production by cleaving NEMO at a single site. In contrast, the present study demonstrates that both EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is essential for disruption of type I IFN production. Moreover, we reveal that EAV nsp4 also cleaves NEMO at glutamine 205 (Q205), which is not targeted by PRRSV nsp4. Notably, targeting a glutamine in NEMO for cleavage has been observed only with picornavirus 3C proteases (3Cpro) and coronavirus 3CLpro In aggregate, our work expands knowledge of the innate immune evasion mechanisms associated with NEMO cleavage by arterivirus nsp4 and describes a novel substrate recognition characteristic of EAV nsp4.

Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

SummaryVaccinia virus (VACV) has numerous immune evasion strategies, including multiple mechanisms of inhibition of interferon regulatory factor 3 (IRF-3), nuclear factor κB (NF-κB), and type I interferon (IFN) signaling. Here, we use highly multiplexed proteomics to quantify ∼9,000 cellular proteins and ∼80% of viral proteins at seven time points throughout VACV infection. A total of 265 cellular proteins are downregulated >2-fold by VACV, including putative natural killer cell ligands and IFN-stimulated genes. Two-thirds of these viral targets, including class II histone deacetylase 5 (HDAC5), are degraded proteolytically during infection. In follow-up analysis, we demonstrate that HDAC5 restricts replication of both VACV and herpes simplex virus type 1. By generating a protein-based temporal classification of VACV gene expression, we identify protein C6, a multifunctional IFN antagonist, as being necessary and sufficient for proteasomal degradation of HDAC5. Our approach thus identifies both a host antiviral factor and a viral mechanism of innate immune evasion.

Leptospira interrogans outer membrane protein LipL21 is a potent inhibitor of neutrophil myeloperoxidase

ABSTRACT Leptospirosis is a widespread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. After entrance in the host, pathogenic leptospires evade the host natural defense mechanisms in order to propagate and disseminate to multiple organs. Myeloperoxidase is an enzyme stored in neutrophils azurophilic granules, and is released upon neutrophil activation to produce mainly hypochlorous acid, a strong oxidant and potent antimicrobial agent. In the present investigation, we studied the modulation of myeloperoxidase activity by L. interrogans serovar Copenhageni. We show that leptospires and their culture supernatants are able to inhibit both peroxidase and chlorination activities of myeloperoxidase, without interfering with neutrophil degranulation. By leptospiral outer membrane protein extraction and fractionation, we identified the proteins LipL21 and LipL45 as myeloperoxidase inhibitors, constituting new Leptospira virulence factors. Accordingly, we propose a function for the protein LipL21, one of the most expressed leptospiral outer membrane proteins. Our results show a novel innate immune evasion mechanism by which leptospires interfere with the host response in order to cope with the host oxidative stress and efficiently achieve dissemination and colonization.

Topoisomerase II Inhibitors Induce DNA Damage-Dependent Interferon Responses Circumventing Ebola Virus Immune Evasion.

ABSTRACTEbola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication.

Zoonotic Potential of Emerging Paramyxoviruses: Knowns and Unknowns.

The risk of spillover of enzootic paramyxoviruses and the susceptibility of recipient human and domestic animal populations are defined by a broad collection of ecological and molecular factors that interact in ways that are not yet fully understood. Nipah and Hendra viruses were the first highly lethal zoonotic paramyxoviruses discovered in modern times, but other paramyxoviruses from multiple genera are present in bats and other reservoirs that have unknown potential to spillover into humans. We outline our current understanding of paramyxovirus reservoir hosts and the ecological factors that may drive spillover, and we explore the molecular barriers to spillover that emergent paramyxoviruses may encounter. By outlining what is known about enzootic paramyxovirus receptor usage, mechanisms of innate immune evasion, and other host-specific interactions, we highlight the breadth of unexplored avenues that may be important in understanding paramyxovirus emergence.

Trypanosoma brucei metabolite indolepyruvate decreases HIF-1α and glycolysis in macrophages as a mechanism of innate immune evasion

The parasite Trypanasoma brucei causes African trypanosomiasis, known as sleeping sickness in humans and nagana in domestic animals. These diseases are a major burden in the 36 sub-Saharan African countries where the tsetse fly vector is endemic. Untreated trypanosomiasis is fatal and the current treatments are stage-dependent and can be problematic during the meningoencephalitic stage, where no new therapies have been developed in recent years and the current drugs have a low therapeutic index. There is a need for more effective treatments and a better understanding of how these parasites evade the host immune response will help in this regard. The bloodstream form of T. brucei excretes significant amounts of aromatic ketoacids, including indolepyruvate, a transamination product of tryptophan. This study demonstrates that this process is essential in bloodstream forms, is mediated by a specialized isoform of cytoplasmic aminotransferase and, importantly, reveals an immunomodulatory role for indolepyruvate. Indolepyruvate prevents the LPS-induced glycolytic shift in macrophages. This effect is the result of an increase in the hydroxylation and degradation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). The reduction in HIF-1α levels by indolepyruvate, following LPS or trypanosome activation, results in a decrease in production of the proinflammatory cytokine IL-1β. These data demonstrate an important role for indolepyruvate in immune evasion by T. brucei.

Hepatitis C Virus Stimulates Murine CD8α-Like Dendritic Cells to Produce Type I Interferon in a TRIF-Dependent Manner.

Hepatitis C virus (HCV) induces interferon (IFN) stimulated genes in the liver despite of distinct innate immune evasion mechanisms, suggesting that beyond HCV infected cells other cell types contribute to innate immune activation. Upon coculture with HCV replicating cells, human CD141+ myeloid dendritic cells (DC) produce type III IFN, whereas plasmacytoid dendritic cells (pDC) mount type I IFN responses. Due to limitations in the genetic manipulation of primary human DCs, we explored HCV mediated stimulation of murine DC subsets. Coculture of HCV RNA transfected human or murine hepatoma cells with murine bone marrow-derived DC cultures revealed that only Flt3-L DC cultures, but not GM-CSF DC cultures responded with IFN production. Cells transfected with full length or subgenomic viral RNA stimulated IFN release indicating that infectious virus particle formation is not essential in this process. Use of differentiated DC from mice with genetic lesions in innate immune signalling showed that IFN secretion by HCV-stimulated murine DC was independent of MyD88 and CARDIF, but dependent on TRIF and IFNAR signalling. Separating Flt3-L DC cultures into pDC and conventional CD11b-like and CD8α-like DC revealed that the CD8α-like DC, homologous to the human CD141+ DC, release interferon upon stimulation by HCV replicating cells. In contrast, the other cell types and in particular the pDC did not. Injection of human HCV subgenomic replicon cells into IFN-β reporter mice confirmed the interferon induction upon HCV replication in vivo. These results indicate that HCV-replicating cells stimulate IFN secretion from murine CD8α-like DC independent of infectious virus production. Thus, this work defines basic principles of viral recognition by murine DC populations. Moreover, this model should be useful to explore the interaction between dendritic cells during HCV replication and to define how viral signatures are delivered to and recognized by immune cells to trigger IFN release.

MOV10 Provides Antiviral Activity against RNA Viruses by Enhancing RIG-I-MAVS-Independent IFN Induction.

Moloney leukemia virus 10, homolog (MOV10) is an IFN-inducible RNA helicase, associated with small RNA-induced silencing. In this article, we report that MOV10 exhibits antiviral activity, independent of its helicase function, against a number of positive- and negative-strand RNA viruses by enhancing type I IFN induction. Using a number of genome-edited knockout human cells, we show that IFN regulatory factor 3-mediated IFN induction and downstream IFN signaling through IFN receptor was necessary to inhibit virus replication by MOV10. MOV10 enhanced IFN regulatory factor 3-mediated transcription of IFN. However, this IFN induction by MOV10 was unique and independent of the known retinoic acid-inducible gene I/mitochondrial antiviral-signaling protein-mediated RNA-sensing pathway. Upon virus infection, MOV10 specifically required inhibitor of κB kinase ε, not TANK-binding kinase 1, for its antiviral activity. The important role of MOV10 in mediating antiviral signaling was further supported by the finding that viral proteases from picornavirus family specifically targeted MOV10 as a possible innate immune evasion mechanism. These results establish MOV10, an evolutionary conserved protein involved in RNA silencing, as an antiviral gene against RNA viruses that uses an retinoic acid-inducible gene I-like receptor-independent pathway to enhance IFN response.

Natural killer cells require monocytic Gr-1+/CD11b+ myeloid cells to eradicate orthotopically engrafted glioma cells

ABSTRACTMalignant gliomas are resistant to natural killer (NK) cell immune surveillance. However, the mechanisms used by these cancers to suppress antitumor NK cell activity remain poorly understood. We have recently reported on a novel mechanism of innate immune evasion characterized by the overexpression of the carbohydrate-binding protein galectin-1 by both mouse and rat malignant glioma. Here, we investigate the cytokine profile of galectin-1-deficient GL26 cells and describe the process by which these tumors are targeted by the early innate immune system in RAG1−/− and C57BL/6J mice. Our data reveal that galectin-1 knockdown in GL26 cells heightens their inflammatory status leading to the rapid recruitment of Gr-1+/CD11b+ myeloid cells and NK1.1+ NK cells into the brain tumor microenvironment, culminating in tumor clearance. We show that immunodepletion of Gr-1+ myeloid cells in RAG1−/− mice permits the growth of galectin-1-deficient glioma despite the presence of NK cells, thus demonstrating an essential role for myeloid cells in the clearance of galectin-1-deficient glioma. Further characterization of tumor-infiltrating Gr-1+/CD11b+ cells reveals that these cells also express CCR2 and Ly-6C, markers consistent with inflammatory monocytes. Our results demonstrate that Gr-1+/CD11b+ myeloid cells, often referred to as myeloid-derived suppressor cells (MDSCs), are required for antitumor NK cell activity against galectin-1-deficient GL26 glioma. We conclude that glioma-derived galectin-1 represents an important factor in dictating the phenotypic behavior of monocytic Gr-1+/CD11b+ myeloid cells. Galectin-1 suppression may be a valuable treatment approach for clinical glioma by promoting their innate immune-mediated recognition and clearance through the concerted effort of innate myeloid and lymphoid cell lineages.