Abstract Glioblastomas are incurable and lethal brain cancers. Immunotherapies offer new and promising treatment options for glioblastoma patients, but the highly immunosuppressive nature of these cancers presents a challenging clinical obstacle. Glioblastoma immune evasion is driven by cell-cell interactions in the tumor microenvironment and recent studies have identified astrocytes as important contributors to immune silencing [1, 2]. Cell plasticity is a key feature of reactive astrocytes that drives heterogeneous, pro- or anti-inflammatory states [3], but the molecular regulators of astrocyte-immune interactions remain incompletely understood. Here, we investigate whether cell plasticity of glioblastoma-associated astrocytes promotes or opposes tumor progression and show that loss of astrocyte plasticity results in T-cell recruitment and immune activation. We evaluate how astrocytic cell plasticity contributes to immune functions in the glioblastoma microenvironment using single cell sequencing from preclinical models, in vivo genetic perturbations and in vitro mouse and human experimental systems. We show that astrocytes surrounding glioblastoma express the stem cell-associated transcription factor, ZEB1, and that conditional-inducible astrocytic deletion of Zeb1 remarkably reduces glioblastoma growth and extends survival. Increased recruitment and activation of T cells in astrocytic Zeb1 -deficient mouse models is linked to increased expression of the immunoattractant cytokine CXCL14, and viral delivery of CXCL14 in experimental glioblastoma models increases survival. Our data support that CXCL14 is a candidate therapeutic target for reprogramming the tumor microenvironment that can restrict and reduce glioblastoma growth and progression.

Infections have long been implicated as causative factors in Alzheimer's disease (AD). Multiple studies have further suggested a key role for herpesviruses, such as cytomegalovirus (CMV). Using transgenic 3xTg-AD mice, we demonstrate that systemic infection with the β-herpesvirus murine CMV (MCMV) accelerates the development of cognitive decline, tauopathy and synaptic loss in the hippocampus, all of which are key features of AD. Accelerated disease progression after infection was associated with substantial lymphocyte infiltration into the brain, dominated by MCMV-specific effector memory CD8+ T cells expressing CXCR3. T cell receptor analyses revealed that clonally diverse virus-specific CD8+ T cells were selectively recruited into the brain during the development of AD. T cell depletion or treatment with the antiviral drug valganciclovir during chronic infection reduced lymphocytic infiltrates in the brain and reversed cognitive decline. These data provide a mechanistic link between chronic viral infections and the development of AD.

Human cytomegalovirus (HCMV) is a ubiquitous member of the herpesvirus family, of significant clinical importance, and highly adapted to its host, resulting from millions of years of co-evolution. As a result, the virus systematically subverts almost all aspects of antiviral immune defence to successfully establish a lifelong persistent infection, and in the process, dramatically reshapes the phenotype and function of host immunity to both HCMV and other diseases. Natural killer (NK) cells are a critical component of successful herpesvirus control. Here, we discuss their role in modulating HCMV disease and the multitude of ways that HCMV has evolved to prevent and manipulate this process. We also consider how antibody-dependent cellular cytotoxicity by NK cells directed against HCMV might overcome NK immune evasion mechanisms and be useful therapeutically.

ABSTRACT There are no licensed vaccines for human cytomegalovirus (HCMV), and current antiviral drugs that target viral proteins are toxic and prone to resistance. Targeting host pathways essential for virus replication provides an alternate strategy that may reduce opportunities for drug resistance to occur. Oxidative stress is triggered by numerous viruses including HCMV. Peroxynitrite is a reactive nitrogen species that is formed during oxidative stress. Herein, we identified that HCMV rapidly induces the generation of intracellular peroxynitrite upon infection in a manner partially dependent upon xanthine oxidase generation. Peroxynitrite promoted HCMV infection in both cell-free and cell-associated infection systems in multiple cell types. Inhibiting peroxynitrite within the first 24 hours of infection prevented HCMV replication and peroxynitrite promoted cell entry and pp65 translocation into the host cell nuclei. Furthermore, using the murine cytomegalovirus model, we demonstrated that antagonizing peroxynitrite significantly reduces cytomegalovirus replication and pathogenesis in vivo . Overall, our study highlights a proviral role for peroxynitrite in CMV infection and implies that RNS and/or the mechanisms that induce their production could be targeted as a novel strategy to inhibit HCMV infection. IMPORTANCE Human cytomegalovirus (HCMV) causes significant disease in individuals with impaired or immature immune systems, such as transplant patients and after congenital infection. Antiviral drugs that target the virus directly are toxic and are susceptible to antiviral drug resistance due to virus mutations. An alternate strategy is to target processes within host cells that are required by the virus for replication. Herein, we show that HCMV infection triggers a highly reactive molecule, peroxynitrite, during the initial stages of infection. Peroxynitrite was required for the initial entry of the virus into the cell and promotes virus replication in multiple cell types, suggesting a broad pro-viral function. Importantly, targeting peroxynitrite dramatically inhibited cytomegalovirus replication in cells in the laboratory and in mice, suggesting that therapeutic targeting of this molecule and/or the cellular functions it regulates could represent a novel strategy to inhibit HCMV infection.

Regulatory T (T-reg) cells contribute to immune homeostasis but suppress immune responses to cancer. Strategies to disrupt T-reg cell-mediated cancer immunosuppression have been met with limited clinical success, but the underlying mechanisms for treatment failure are poorly understood. By modeling T-reg cell-targeted immunotherapy in mice, we find that CD4(+) Foxp3(-) conventional T (T-conv) cells acquire suppressive function upon depletion of Foxp3(+) T-reg cells, limiting therapeutic efficacy. Foxp3(-) T-conv cells within tumors adopt a T-reg cell-like transcriptional profile upon ablation of T-reg cells and acquire the ability to suppress T cell activation and proliferation ex vivo. Suppressive activity is enriched among CD4(+) T-conv cells marked by expression of C-C motif receptor 8 (CCR8), which are found in mouse and human tumors. Upon T-reg cell depletion, CCR8(+) T-conv cells undergo systemic and intratumoral activation and expansion, and mediate IL-10-dependent suppression of antitumor immunity. Consequently, conditional deletion of Il10 within T cells augments antitumor immunity upon T-reg cell depletion in mice, and antibody blockade of IL-10 signaling synergizes with T-reg cell depletion to overcome treatment resistance. These findings reveal a secondary layer of immunosuppression by T-conv cells released upon therapeutic T-reg cell depletion and suggest that broader consideration of suppressive function within the T cell lineage is required for development of effective T-reg cell-targeted therapies.

CD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation. Expression of these CD8 variants in model systems preferentially enhanced pMHCI antigen recognition in the context of low-affinity TCRs. A similar effect was observed using primary CD4+ T cells transduced with cancer-targeting TCRs. The introduction of high-affinity CD8 variants also enhanced the functional sensitivity of primary CD8+ T cells expressing cancer-targeting TCRs, but comparable results were obtained using exogenous wild-type CD8. Specificity was retained in every case, with no evidence of reactivity in the absence of cognate antigen. Collectively, these findings highlight a generically applicable mechanism to enhance the sensitivity of low-affinity pMHCI antigen recognition, which could augment the therapeutic efficacy of clinically relevant TCRs.

Inhibitory CD4 + T cells have been linked with suboptimal immune responses against cancer and pathogen chronicity. However, the mechanisms that underpin the development of these regulatory cells, especially in the context of ongoing antigen exposure, have remained obscure. To address this knowledge gap, we undertook a comprehensive functional, phenotypic, and transcriptomic analysis of interleukin (IL)-10-producing CD4 + T cells induced by chronic infection with murine cytomegalovirus (MCMV). We identified these cells as clonally expanded and highly differentiated T H 1-like cells that developed in a T-bet-dependent manner and coexpressed arginase-1 (Arg1), which promotes the catalytic breakdown of L -arginine. Mice lacking Arg1-expressing CD4 + T cells exhibited more robust antiviral immunity and were better able to control MCMV. Conditional deletion of T-bet in the CD4 + lineage suppressed the development of these inhibitory cells and also enhanced immune control of MCMV. Collectively, these data elucidated the ontogeny of IL-10-producing CD4 + T cells and revealed a previously unappreciated mechanism of immune regulation, whereby viral persistence was facilitated by the site-specific delivery of Arg1.

Summary Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. Early symptoms include the loss of memory and mild cognitive ability; however, as the disease progresses, these symptoms can present with increased severity manifesting as mood and behaviour changes, disorientation, and a loss of motor/body control. AD is one of the leading causes of death in the UK, and with an ever-increasing ageing society, patient numbers are predicted to rise posing a significant global health emergency. AD is a complex neurophysiological disorder where pathology is characterized by the deposition and aggregation of misfolded amyloid-beta (Aβ)-protein that in-turn promotes excessive tau-protein production which together drives neuronal cell dysfunction, neuroinflammation, and neurodegeneration. It is widely accepted that AD is driven by a combination of both genetic and immunological processes with recent data suggesting that adaptive immune cell activity within the parenchyma occurs throughout disease. The mechanisms behind these observations remain unclear but suggest that manipulating the adaptive immune response during AD may be an effective therapeutic strategy. Using immunotherapy for AD treatment is not a new concept as the only two approved treatments for AD use antibody-based approaches to target Aβ. However, these have been shown to only temporarily ease symptoms or slow progression highlighting the urgent need for newer treatments. This review discusses the role of the adaptive immune system during AD, how microbial infections may be contributing to inflammatory immune activity and suggests how adaptive immune processes can pose as therapeutic targets for this devastating disease.

Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that limits viral pathogenesis and exerts poorly understood immunoregulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 promotes MyD88-dependent, TLR-mediated IL-6 production following exposure to cytomegalovirus (CMV). IFITM3 also restricts IL-6 production in response to influenza and SARS-CoV-2. In dendritic cells, IFITM3 binds to the reticulon 4 isoform Nogo-B and promotes its proteasomal degradation. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesis in vivo, and in the case of TLR2 responses, this process involves alteration of TLR2 cellular localization. Nogo-B deletion abrogates inflammatory cytokine responses and associated disease in virus-infected IFITM3-deficient mice. Thus, we uncover Nogo-B as a driver of viral pathogenesis and highlight an immunoregulatory pathway in which IFITM3 fine-tunes the responsiveness of myeloid cells to viral stimulation.

SummaryInterferon induced transmembrane protein 3 (IFITM3) is an important viral restriction factor in viral pathogenesis that also exhibits poorly understood immune regulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 regulates MyD88-dependent TLR-mediated cytokine production following dendritic cell exposure to cytomegalovirus (CMV), and this process limits viral pathogenesisin vivo. IFITM3 also restricted pro-inflammatory (IL-6) cytokine production in response to influenza. IFITM3 bound to and promoted ubiquitination and proteasomal degradation of the reticulon 4 isoform Nogo-B. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesisin vivo, and this process involved alteration of TLR dynamics. The anti-inflammatory function of IFITM3 was intrinsically linked to its ability to regulate Nogo-B. Thus, we uncover Nogo-B as an unappreciated driver of viral pathogenesis and highlight a novel immune regulatory pathway where IFITM3 fine-tunes TLR responsiveness of myeloid cells to viral stimulation.

CD8+ T cells are inherently cross-reactive and recognize numerous peptide antigens in the context of a given major histocompatibility complex class I (MHCI) molecule via the clonotypically expressed T cell receptor (TCR). The lineally expressed coreceptor CD8 interacts coordinately with MHCI at a distinct and largely invariant site to slow the TCR/peptide-MHCI (pMHCI) dissociation rate and enhance antigen sensitivity. However, this biological effect is not necessarily uniform, and theoretical models suggest that antigen sensitivity can be modulated in a differential manner by CD8. We used two intrinsically controlled systems to determine how the relationship between the TCR/pMHCI interaction and the pMHCI/CD8 interaction affects the functional sensitivity of antigen recognition. Our data show that modulation of the pMHCI/CD8 interaction can reorder the agonist hierarchy of peptide ligands across a spectrum of affinities for the TCR.

Cytomegalovirus (CMV) induction of large frequencies of highly functional memory T-cells has attracted much interest in the utility of CMV-based vaccine vectors, with exciting preclinical data obtained in models of infectious diseases and cancer. However, pathogenesis of human CMV (HCMV) remains a concern. Attenuated CMV-based vectors, such as replication- or spread-deficient viruses potentially offer an alternative to fully replicating vectors. However, it is not well-understood how CMV attenuation impacts vector immunogenicity, in particularly when administered via relevant routes of immunization such as the skin. Herein we used the murine cytomegalovirus (MCMV) model to investigate the impact of vector attenuation on T-cell memory formation following subcutaneous administration. We found that the spread deficient virus (ΔgL-MCMV) was impaired in its ability to induce memory CD8 + T-cells reactive to some (M38, IE1) but not all (IE3) viral antigens. Impaired memory T-cell development was associated with a preferential and pronounced loss of polyfunctional (IFN-γ + TNF-α + ) T-cells, and was not rescued by increasing the dose of replication-defective MCMV. Finally, whilst vector attenuation reduced dendritic cell (DC) recruitment to skindraining lymph nodes, systematic depletion of multiple DC subsets during acute subcutaneous MCMV infection had a negligible impact on T-cell memory formation, implying that attenuated responses induced by replication-deficient vectors were likely not a consequence of impaired initial DC activation. Thus, overall, these data imply that the choice of antigen and/or cloning strategy of exogenous antigen in combination with the route of immunization may influence the ability of attenuated CMV vectors to induce robust functional T-cell memory.

Recognition of influenza A virus (IAV) by the innate immune system triggers pathways that restrict viral replication, activate innate immune cells, and regulate adaptive immunity. However, excessive innate immune activation can exaggerate disease. The pathways promoting excessive activation are incompletely understood, with limited experimental models to investigate the mechanisms driving influenza virus-induced inflammation in humans. Interferon regulatory factor 5 (IRF5) is a transcription factor that plays important roles in the induction of cytokines after viral sensing. In an in vivo model of IAV infection, IRF5 deficiency reduced IAV-driven immune pathology and associated inflammatory cytokine production, specifically reducing cytokine-producing myeloid cell populations in Irf5(-/-) mice but not impacting type 1 interferon (IFN) production or virus replication. Using cytometry by time of flight (CyTOF), we identified that human lung IRF5 expression was highest in cells of the myeloid lineage. To investigate the role of IRF5 in mediating human inflammatory responses by myeloid cells to IAV, we employed human-induced pluripotent stem cells (hIPSCs) with biallelic mutations in IRF5, demonstrating for the first time that induced pluripotent stem cell-derived dendritic cells (iPS-DCs) with biallelic mutations can be used to investigate the regulation of human virus-induced immune responses. Using this technology, we reveal that IRF5 deficiency in human DCs, or macrophages, corresponded with reduced virus-induced inflammatory cytokine production, with IRF5 acting downstream of Toll-like receptor 7 (TLR7) and, possibly, retinoic acid-inducible gene I (RIG-I) after viral sensing. Thus, IRF5 acts as a regulator of myeloid cell inflammatory cytokine production during IAV infection in mice and humans and drives immune-mediated viral pathogenesis independently of type 1 IFN and virus replication. IMPORTANCE The inflammatory response to influenza A virus (IAV) participates in infection control but contributes to disease severity. After viral detection, intracellular pathways are activated, initiating cytokine production, but these pathways are incompletely understood. We show that interferon regulatory factor 5 (IRF5) mediates IAV-induced inflammation and, in mice, drives pathology. This was independent of antiviral type 1 IFN and virus replication, implying that IRF5 could be specifically targeted to treat influenza virus-induced inflammation. We show for the first time that human iPSC technology can be exploited in genetic studies of virus-induced immune responses. Using this technology, we deleted IRF5 in human myeloid cells. These IRF5-deficient cells exhibited impaired influenza virus-induced cytokine production and revealed that IRF5 acts downstream of Toll-like receptor 7 and possibly retinoic acid-inducible gene I. Our data demonstrate the importance of IRF5 in influenza virus-induced inflammation, suggesting that genetic variation in the IRF5 gene may influence host susceptibility to viral diseases.

Human cytomegalovirus (HCMV) is a pathogenic beta-herpesvirus that establishes a lifelong infection in hosts. It causes significant morbidity and mortality in the immunocompromised and is associated with a range of birth defects following congenital infection. Current therapeutic approaches that target key viral proteins are toxic and antiviral drug resistance is common. Thus, targeting host genes and cellular pathways essential for HCMV infection offers an alternative strategy for the development of antivirals. Here we show that host oxidative/nitrosative stress responses to CMV are critical for virus replication. Oxidative/nitrosative stress occurs due to accumulation of reactive oxygen/nitrogen species (ROS/RNS). Using a range of ROS/RNS scavengers, we identified that peroxynitrite, a powerful oxidant and nitrating agent, dramatically promoted virus replication in both in vitro and in vivo models of CMV infection. HCMV rapidly induced production of intracellular peroxynitrite upon infection. Inhibition of peroxynitrite within the first 24 hours alleviates efficient HCMV infection in both cell-free and cell-associated infection systems, indicating that peroxynitrite may influence pathways necessary for HCMV entry and/or replication. Furthermore, peroxynitrite inhibition also inhibited HCMV reactivation from latency. Interestingly, the neurotransmitter and naturally-occurring peroxynitrite antagonist 5-hydroxytryptamine, commonly known as serotonin, also impinged on HCMV-induced peroxynitrite production and exhibited anti-HCMV activity. Thus, overall, our study demonstrates a novel role for intracellular peroxynitrite in CMV pathogenesis and implies that peroxynitrite could be targeted as a novel approach to inhibiting CMV infection.

The acute antiviral response is mediated by a family of interferon-stimulated genes (ISGs), providing cell-intrinsic immunity. Mutations in genes encoding these proteins are often associated with increased susceptibility to viral infections. One family of ISGs with antiviral function is the interferon-inducible transmembrane proteins (IFITMs), of which IFITM3 has been studied extensively. In contrast, IFITM1 has not been studied in detail. Since IFITM1 can localize to the plasma membrane, we investigated its function with a range of enveloped viruses thought to infect cells by fusion with the plasma membrane. Overexpression of IFITM1 prevented infection by a number of Paramyxoviridae and Pneumoviridae, including respiratory syncytial virus (RSV), mumps virus, and human metapneumovirus (HMPV). IFITM1 also restricted infection with an enveloped DNA virus that can enter via the plasma membrane, herpes simplex virus 1 (HSV-1). To test the importance of plasma membrane localization for IFITM1 function, we identified blocks of amino acids in the conserved intracellular loop (CIL) domain that altered the subcellular localization of the protein and reduced antiviral activity. By screening reported data sets, 12 rare nonsynonymous single nucleotide polymorphisms (SNPs) were identified in human IFITM1, some of which are in the CIL domain. Using an Ifitm1(-/-) mouse, we show that RSV infection was more severe, thereby extending the range of viruses restricted in vivo by IFITM proteins and suggesting overall that IFITM1 is broadly antiviral and that this antiviral function is associated with cell surface localization. IMPORTANCE Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon-stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is interferon-inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can inhibit infection with a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localize to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo, and Ifitm1(-/-) mice were more susceptible to viral lung infection. These data contribute to our understanding of how ISGs prevent viral infections.

Candidate vaccines designed to generate T cell-based immunity are typically vectored by nonpersistent viruses, which largely fail to elicit durable effector memory T cell responses. This limitation can be overcome using recombinant strains of CMV. Proof-of-principle studies have demonstrated the potential benefits of this approach, most notably in the SIV model, but safety concerns require the development of nonreplicating alternatives with comparable immunogenicity. In this study, we show that IL-33 promotes the accumulation and recall kinetics of circulating and tissue-resident memory T cells in mice infected with murine CMV. Using a replication-deficient murine CMV vector, we further show that exogenous IL-33 boosts vaccine-induced memory T cell responses, which protect against subsequent heterologous viral challenge. These data suggest that IL-33 could serve as a useful adjuvant to improve the efficacy of vaccines based on attenuated derivatives of CMV.

Human cytomegalovirus (HCMV) is a β-herpesvirus with high sero-prevalence within the human population. Primary HCMV infection and life-long carriage are typically asymptomatic. However, HCMV is implicated in exacerbation of chronic conditions and associated damage in individuals with intact immune systems. Furthermore, HCMV is a significant cause of morbidity and mortality in the immunologically immature and immune-compromised where disease is associated with tissue damage. Infection-induced inflammation, including robust cytokine responses, is a key component of pathologies associated with many viruses. Despite encoding a large number of immune-evasion genes, HCMV also triggers the induction of inflammatory cytokine responses during infection. Thus, understanding how cytokines contribute to CMV-induced pathologies and the mechanisms through which they are regulated may inform clinical management of disease. Herein, we discuss our current understanding based on clinical observation and in vivo modeling of disease of the role that cytokines play in CMV pathogenesis. Specifically, in the context of the different tissues and organs in which CMV replicates, we give a broad overview of the beneficial and adverse effects that cytokines have during infection and describe how cytokine-mediated tissue damage is regulated. We discuss the implications of findings derived from mice and humans for therapeutic intervention strategies and our understanding of how host genetics may influence the outcome of CMV infections.