Redirector of Vaccine-induced Effector Responses (RoVER) for specific killing of cellular targets.

BACKGROUND: In individuals with malignancy or HIV-1 infection, antigen-specific cytotoxic T lymphocytes (CTLs) often display an exhausted phenotype with impaired capacity to eliminate the disease. Existing cell-based immunotherapy strategies are often limited by the requirement for adoptive transfer of CTLs. We have developed an immunotherapy technology in which potent CTL responses are generated in vivo by vaccination and redirected to eliminate target cells using a bispecific Redirector of Vaccine-induced Effector Responses (RoVER). METHODS: Following Yellow fever (YF) 17D vaccination of 51 healthy volunteers (NCT04083430), single-epitope YF-specific CTL responses were quantified by tetramer staining and multi-parameter flow cytometry. RoVER-mediated redirection of YF-specific CTLs to kill antigen-expressing Raji-Env cells, autologous CD19+ B cells or CD4+ T cells infected in vitro with a full-length HIV-1-eGFP was assessed in cell killing assays. Moreover, secreted IFN-γ, granzyme B, and TNF-α were analyzed by mesoscale multiplex assays. FINDINGS: YF-17D vaccination induced strong epitope-specific CTL responses in the study participants. In cell killing assays, RoVER-mediated redirection of YF-specific CTLs to autologous CD19+ B cells or HIV-1-infected CD4+ cells resulted in 58% and 53% killing at effector to target ratio 1:1, respectively. INTERPRETATION: We have developed an immunotherapy technology in which epitope-specific CTLs induced by vaccination can be redirected to kill antigen-expressing target cells by RoVER linking. The RoVER technology is highly specific and can be adapted to recognize various cell surface antigens. Importantly, this technology obviates the need for adoptive transfer of CTLs. FUNDING: This work was funded by the Novo Nordisk Foundation (Hallas Møller NNF10OC0054577).

PD-L1 and PD-L2 immune checkpoint protein induction by type III interferon in non-small cell lung cancer cells.

INTRODUCTION: Despite the clinical success of PD-1/PD-1-ligand immunotherapy in non-small cell lung cancer (NSCLC), the appearance of primary and acquired therapy resistance is a major challenge reflecting that the mechanisms regulating the expression of the PD-1-ligands PD-L1 and PD-L2 are not fully explored. Type I and II interferons (IFNs) induce PD-L1 and PD-L2 expression. Here, we examined if PD-L1 and PD-L2 expression also can be induced by type III IFN, IFN-λ, which is peculiarly important for airway epithelial surfaces. METHODS: In silico mRNA expression analysis of PD-L1 (CD274), PD-L2 (PDCD1LG2), and IFN- λ signaling signature genes in NSCLC tumors and cell lines was performed using RNA sequencing expression data from TCGA, OncoSG, and DepMap portals. IFN-λ-mediated induction of PD-L1 and PD-L2 expression in NSCLC cell lines was examined by real-time quantitative polymerase chain reaction and flow cytometry. RESULTS: IFNL genes encoding IFN- λ variants are expressed in the majority of NSCLC tumors and cell lines along with the IFNLR1 and IL10R2 genes encoding the IFN-λ receptor subunits. The expression of PD-L1 and PD-L2 mRNA is higher in NSCLC tumors with IFNL mRNA expression compared to tumors without IFNL expression. In the NSCLC cell line HCC827, stimulation with IFN-λ induced both an increase in PD-L1 and PD-L2 mRNA expression and cell surface abundance of the corresponding proteins. In the NSCLC cell line A427, displaying a low basal expression of PD-L1 and PD-L2 mRNA and corresponding proteins, stimulation with IFN-λ resulted in an induction of the former. CONCLUSION: The type III IFN, IFN- λ, is capable of inducing PD-L1 and PD-L2 expression, at least in some NSCLC cells, and this regulation will need acknowledgment in the development of new diagnostic procedures, such as gene expression signature profiles, to improve PD-1/PD-1-ligand immunotherapy in NSCLC.

Two cGAS-like receptors induce antiviral immunity in Drosophila.

In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes1 and eukaryotes2-5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections6-8. Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3'2'-cGAMP, whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2'-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2'3'-cGAMP.

Selective Janus kinase inhibition preserves interferon-λ-mediated antiviral responses.

Inflammatory diseases are frequently treated with Janus kinase (JAK) inhibitors to diminish cytokine signaling. These treatments can lead to inadvertent immune suppression and may increase the risk of viral infection. Tyrosine kinase 2 (TYK2) is a JAK family member required for efficient type I interferon (IFN-α/ß) signaling. We report here that selective TYK2 inhibition preferentially blocked potentially detrimental type I IFN signaling, whereas IFN-λ-mediated responses were largely preserved. In contrast, the clinically used JAK1/2 inhibitor baricitinib was equally potent in blocking IFN-α/ß- or IFN-λ-driven responses. Mechanistically, we showed that epithelial cells did not require TYK2 for IFN-λ-mediated signaling or antiviral protection. TYK2 deficiency diminished IFN-α-induced protection against lethal influenza virus infection in mice but did not impair IFN-λ-mediated antiviral protection. Our findings suggest that selective TYK2 inhibitors used in place of broadly acting JAK1/2 inhibitors may represent a superior treatment option for type I interferonopathies to counteract inflammatory responses while preserving antiviral protection mediated by IFN-λ.

Disparate temperature-dependent virus-host dynamics for SARS-CoV-2 and SARS-CoV in the human respiratory epithelium.

Since its emergence in December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human upper and lower respiratory tract (33°C and 37°C, respectively) have been shown to affect the replication kinetics of several respiratory viruses, as well as host innate immune response dynamics, we investigated the impact of temperature on SARS-CoV-2 and SARS-CoV infection using the primary human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated to higher titers when infections were performed at 33°C rather than 37°C. Although both viruses were highly sensitive to type I and type III interferon pretreatment, a detailed time-resolved transcriptome analysis revealed temperature-dependent interferon and pro-inflammatory responses induced by SARS-CoV-2 that were inversely proportional to its replication efficiency at 33°C or 37°C. These data provide crucial insight on pivotal virus-host interaction dynamics and are in line with characteristic clinical features of SARS-CoV-2 and SARS-CoV, as well as their respective transmission efficiencies.

2'3'-cGAMP triggers a STING- and NF-κB-dependent broad antiviral response in Drosophila.

We previously reported that an ortholog of STING regulates infection by picorna-like viruses in Drosophila In mammals, STING is activated by the cyclic dinucleotide 2'3'-cGAMP produced by cGAS, which acts as a receptor for cytosolic DNA. Here, we showed that injection of flies with 2'3'-cGAMP induced the expression of dSTING-regulated genes. Coinjection of 2'3'-cGAMP with a panel of RNA or DNA viruses resulted in substantially reduced viral replication. This 2'3'-cGAMP-mediated protection was still observed in flies with mutations in Atg7 and AGO2, genes that encode key components of the autophagy and small interfering RNA pathways, respectively. By contrast, this protection was abrogated in flies with mutations in the gene encoding the NF-κB transcription factor Relish. Transcriptomic analysis of 2'3'-cGAMP-injected flies revealed a complex response pattern in which genes were rapidly induced, induced after a delay, or induced in a sustained manner. Our results reveal that dSTING regulates an NF-κB-dependent antiviral program that predates the emergence of interferons in vertebrates.

SARS-CoV-2 evades immune detection in alveolar macrophages.

Respiratory infections, like the current COVID-19 pandemic, target epithelial cells in the respiratory tract. Alveolar macrophages (AMs) are tissue-resident macrophages located within the lung. They play a key role in the early phases of an immune response to respiratory viruses. AMs are likely the first immune cells to encounter SARS-CoV-2 during an infection, and their reaction to the virus will have a profound impact on the outcome of the infection. Interferons (IFNs) are antiviral cytokines and among the first cytokines produced upon viral infection. In this study, AMs from non-infectious donors are challenged with SARS-CoV-2. We demonstrate that challenged AMs are incapable of sensing SARS-CoV-2 and of producing an IFN response in contrast to other respiratory viruses, like influenza A virus and Sendai virus, which trigger a robust IFN response. The absence of IFN production in AMs upon challenge with SARS-CoV-2 could explain the initial asymptotic phase observed during COVID-19 and argues against AMs being the sources of pro-inflammatory cytokines later during infection.

STEEP mediates STING ER exit and activation of signaling.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

Type I and III interferons disrupt lung epithelial repair during recovery from viral infection.

Excessive cytokine signaling frequently exacerbates lung tissue damage during respiratory viral infection. Type I (IFN-α and IFN-ß) and III (IFN-λ) interferons are host-produced antiviral cytokines. Prolonged IFN-α and IFN-ß responses can lead to harmful proinflammatory effects, whereas IFN-λ mainly signals in epithelia, thereby inducing localized antiviral immunity. In this work, we show that IFN signaling interferes with lung repair during influenza recovery in mice, with IFN-λ driving these effects most potently. IFN-induced protein p53 directly reduces epithelial proliferation and differentiation, which increases disease severity and susceptibility to bacterial superinfections. Thus, excessive or prolonged IFN production aggravates viral infection by impairing lung epithelial regeneration. Timing and duration are therefore critical parameters of endogenous IFN action and should be considered carefully for IFN therapeutic strategies against viral infections such as influenza and coronavirus disease 2019 (COVID-19).

Systemic juvenile idiopathic arthritis and recurrent macrophage activation syndrome due to a CASP1 variant causing inflammasome hyperactivation.

OBJECTIVES: We investigated a patient with systemic juvenile idiopathic arthritis (sJIA) and recurrent macrophage activation syndrome (MAS) to discover genetic and immunological contributing factors. METHODS: Severe recurrent MAS motivated whole exome sequencing (WES) to identify genetic variants potentially involved in disease pathogenesis. In vitro peripheral blood mononuclear cell (PBMC) stimulations for cytokine expression and caspase-1 activity assays as well as NF-κB reporter luciferase assays were performed to functionally characterize variants. RESULTS: WES revealed an extremely rare heterozygous missense variant, c.482G>A, p.R161H in the CASP1 gene encoding pro-caspase-1. Lipopolysaccharide (LPS) stimulation of patient PBMCs induced high levels of IL-6 compared to controls, and activation of the NLRP3 inflammasome resulted in increased production of IL-1ß and IL-18 as well as significantly elevated caspase-1 activity. Constitutive and inducible levels of IL-18 and IFNγ in whole blood were markedly elevated. Expression of the CASP1 variant in an NF-κB reporter luciferase assay induced increased NF-κB activation in a RIP2-dependent manner. The disease course of the patient was complicated by severe recurrent MAS. However, dual IL-1 and IL-6 blockade caused disease remission. CONCLUSION: For the first time, we demonstrate the involvement of a CASP1 variant in sJIA and recurrent MAS. This variant is gain-of-function for both inflammasome and NF-κB activation leading to increased production of IL-6, IL-1ß and IL-18. Although dual IL-1 and IL-6 blockade may be beneficial in patients, in whom single treatment is not sufficient to control MAS, caution should be practiced, since interstitial lung disease may progress despite apparent clinical and biochemical remission.

The IFNL4 Gene Is a Noncanonical Interferon Gene with a Unique but Evolutionarily Conserved Regulation.

Interferon lambda 4 (IFN-λ4) is a recently identified enigmatic member of the interferon (IFN) lambda family. Genetic data suggest that the IFNL4 gene acts in a proviral and anti-inflammatory manner in patients. However, the protein is indistinguishable in vitro from the other members of the interferon lambda family. We have investigated the gene regulation of IFNL4 in detail and found that it differs radically from that of canonical antiviral interferons. Being induced by viral infection is a defining characteristic of interferons, but viral infection or overexpression of members of the interferon regulatory factor (IRF) family of transcription factors only leads to a minute induction of IFNL4 This behavior is evolutionarily conserved and can be reversed by inserting a functional IRF3 binding site into the IFNL4 promoter. Thus, the regulation of the IFNL4 gene is radically different and might explain some of the atypical phenotypes associated with the IFNL4 gene in humans.IMPORTANCE Recent genetic evidence has highlighted how the IFNL4 gene acts in a counterintuitive manner, as patients with a nonfunctional IFNL4 gene exhibit increased clearance of hepatitis C virus (HCV) but also increased liver inflammation. This suggests that the IFNL4 gene acts in a proviral and anti-inflammatory manner. These surprising but quite clear genetic data have prompted an extensive examination of the basic characteristics of the IFNL4 gene and its gene product, interferon lambda 4 (IFN-λ4). We have investigated the expression of the IFNL4 gene and found it to be poorly induced by viral infections. A thorough investigation of the IFNL4 promoter revealed a highly conserved and functional promoter, but also one that lacks the defining characteristic of interferons (IFNs), i.e., the ability to be effectively induced by viral infections. We suggest that the unique function of the IFNL4 gene is related to its noncanonical transcriptional regulation.

The Influence of the rs30461 Single Nucleotide Polymorphism on IFN-λ1 Activity and Secretion.

Genetic variation within the IFNL loci is associated with several diseases and evidence indicates that the IFNL genes have been subjects of strong selection during recent human evolution. The nonsynonymous rs30461 single nucleotide polymorphism (SNP), generating interferon (IFN)-λ1 D188N, shows a strong signature of positive selection in European and Asian populations. Nevertheless, genetic association studies have failed to show any coupling of rs30461 to diseases such as psoriasis and periodontitis. Based on these observations, we purified IFN-λ1 N188 and IFN-λ1 D188 to compare the biological activity of these 2 IFN-λ1 versions. Furthermore, we evaluated the secretion of the 2 different IFN-λ1 versions. We were unable to observe any differences between IFN-λ1 N188 and IFN-λ1 D188 based on biological activity or secretion that could account for the positive selection.

The role of IFN in the development of NAFLD and NASH.

Non-alcoholic fatty liver disease (NAFLD) and its progressive inflammatory form non-alcoholic steatohepatitis (NASH) are major health challenges due to a significant increase in their incidence and prevalence. While NAFLD is largely benign, the chronic liver inflammation in NASH patients may cause progression to liver cirrhosis and hepatocellular carcinoma. There is an urgent need for a better understanding of the factors, which drive the progression from NAFLD to NASH and how to use this information both to improve diagnostic and to develop new treatment strategies. Increasing evidence points to interferons (IFNs) as key players in NAFLD and particular in the progression to NASH. IFNs crucial role in disease development is supported by both genetic evidence and animal studies. In this review, we describe the involvement of both type I and type III IFNs in the development and progression of NAFLD and NASH.

The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi's sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein.

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few oncogenic human viruses known to date. Its large genome encodes more than 85 proteins and includes both unique viral proteins as well as proteins conserved amongst herpesviruses. KSHV ORF20 is a member of the herpesviral core UL24 family, but the function of ORF20 and its role in the viral life cycle is not well understood. ORF20 encodes three largely uncharacterized isoforms, which we found were localized predominantly in the nuclei and nucleoli. Quantitative affinity purification coupled to mass spectrometry (q-AP-MS) identified numerous specific interacting partners of ORF20, including ribosomal proteins and the interferon-stimulated gene product (ISG) oligoadenylate synthetase-like protein (OASL). Both endogenous and transiently transfected OASL co-immunoprecipitated with ORF20, and this interaction was conserved among all ORF20 isoforms and multiple ORF20 homologs of the UL24 family in other herpesviruses. Characterization of OASL interacting partners by q-AP-MS identified a very similar interactome to that of ORF20. Both ORF20 and OASL copurified with 40S and 60S ribosomal subunits, and when they were co-expressed, they associated with polysomes. Although ORF20 did not have a global effect on translation, ORF20 enhanced RIG-I induced expression of endogenous OASL in an IRF3-dependent but IFNAR-independent manner. OASL has been characterized as an ISG with antiviral activity against some viruses, but its role for gammaherpesviruses was unknown. We show that OASL and ORF20 mRNA expression were induced early after reactivation of latently infected HuARLT-rKSHV.219 cells. Intriguingly, we found that OASL enhanced infection of KSHV. During infection with a KSHV ORF20stop mutant, however, OASL-dependent enhancement of infectivity was lost. Our data have characterized the interaction of ORF20 with OASL and suggest ORF20 usurps the function of OASL to benefit KSHV infection.

IFNλ is a potent anti-influenza therapeutic without the inflammatory side effects of IFNα treatment.

Influenza A virus (IAV)-induced severe disease is characterized by infected lung epithelia, robust inflammatory responses and acute lung injury. Since type I interferon (IFNαß) and type III interferon (IFNλ) are potent antiviral cytokines with immunomodulatory potential, we assessed their efficacy as IAV treatments. IFNλ treatment of IAV-infected Mx1-positive mice lowered viral load and protected from disease. IFNα treatment also restricted IAV replication but exacerbated disease. IFNα treatment increased pulmonary proinflammatory cytokine secretion, innate cell recruitment and epithelial cell death, unlike IFNλ-treatment. IFNλ lacked the direct stimulatory activity of IFNα on immune cells. In epithelia, both IFNs induced antiviral genes but no inflammatory cytokines. Similarly, human airway epithelia responded to both IFNα and IFNλ by induction of antiviral genes but not of cytokines, while hPBMCs responded only to IFNα. The restriction of both IFNλ responsiveness and productive IAV replication to pulmonary epithelia allows IFNλ to limit IAV spread through antiviral gene induction in relevant cells without overstimulating the immune system and driving immunopathology. We propose IFNλ as a non-inflammatory and hence superior treatment option for human IAV infection.

HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression.

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.

Cellular Mechanism for Impaired Hepatitis C Virus Clearance by Interferon Associated with IFNL3 Gene Polymorphisms Relates to Intrahepatic Interferon-λ Expression.

The single nucleotide polymorphism located within the IFNL3 (also known as IL28B) promoter is one of the host factors associated with hepatitis C virus (HCV) clearance by interferon (IFN)-α therapy; however the mechanism remains unknown. We investigated how IL28B gene polymorphism influences HCV clearance with infected primary human hepatocytes, liver biopsies, and hepatoma cell lines. Our study confirms that the rs12979860-T/T genotype has a strong correlation with ss469415590-ΔG/ΔG single nucleotide polymorphism that produces IFN-λ4 protein. We found that IFN-α and IFN-λ1 antiviral activity against HCV was impaired in IL28B T/T infected hepatocytes compared with C/C genotype. Western blot analysis showed that IL28B TT genotype hepatocytes expressed higher levels of IFN-λ proteins (IL28B, IL-29), preactivated IFN-stimulated gene (ISG) expression, and impaired Stat phosphorylation when stimulated with either IFN-α or IFN-λ1. Furthermore, we showed that silencing IFN-λ1 in T/T cell line reduced basal ISG expression and improved antiviral activity. Likewise, overexpression of IFN-λ (1 to 4) in C/C cells induced basal ISG expression and prevented IFN-α antiviral activity. We showed that IFN-λ4, produced at low level only in T/T cells induced expression of IL28B and IL-29 and prevented IFN-α antiviral activity in HCV cell culture. Our results suggest that IFN-λ4 protein expression associated with the IL28B-T/T variant preactivates the Janus kinase-Stat signaling, leading to impaired HCV clearance by both IFN-α and IFN-λ.

Antiviral Activities of Different Interferon Types and Subtypes against Hepatitis E Virus Replication.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genusOrthohepevirusin the familyHepeviridae HEV infections are the common cause of acute hepatitis but can also take chronic courses. Ribavirin is the treatment of choice for most patients, and type I interferon (IFN) has been evaluated in a few infected transplant patientsin vivo In this study, the antiviral effects of different exogenously administered interferons were investigated by using state-of-the-art subgenomic replicon and full-length HEV genome cell culture models. Hepatitis C virus (HCV) subgenomic replicons based on the genotype 2a JFH1 isolate served as the reference. The experiments revealed that HEV RNA replication was inhibited by the application of all types of IFN, including IFN-α (type I), IFN-γ (type II), and IFN-λ3 (type III), but to a far lesser extent than HCV replication. Simultaneous determination of interferon-stimulated gene (ISG) expression levels for all IFN types demonstrated efficient downregulation by HEV. Furthermore, different IFN-α subtypes were also able to block viral replication in combination with ribavirin. The IFN-α subtypes 2a and 2b exerted the strongest antiviral activity against HEV. In conclusion, these data demonstrate for the first time moderate anti-HEV activities of types II and III IFNs and different IFN-α subtypes. As HEV employed a potent anti-interferon mechanism by restricting ISG expression, exogenous application of IFNs as immunotherapy should be carefully assessed.