SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA.

Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here, we show that human SAM-domain- and HD-domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3'exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic-acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.

Immune Sensing Mechanisms that Discriminate Self from Altered Self and Foreign Nucleic Acids.

All lifeforms have developed highly sophisticated systems equipped to detect altered self and non-self nucleic acids (NA). In vertebrates, NA-sensing receptors safeguard the integrity of the organism by detecting pathogens, dyshomeostasis and damage, and inducing appropriate responses to eliminate pathogens and reconstitute homeostasis. Effector mechanisms include i) immune signaling, ii) restriction of NA functions such as inhibition of mRNA translation, and iii) cell death pathways. An appropriate effector response is necessary for host defense, but dysregulated NA-sensing can lead to devastating autoimmune and autoinflammatory disease. Their inherent biochemical similarity renders the reliable distinction between self NA under homeostatic conditions and altered or exogenous NA particularly challenging. In this review, we provide an overview of recent progress in our understanding of the closely coordinated and regulated network of innate immune receptors, restriction factors, and nucleases to effectively respond to pathogens and maintain host integrity.

Immune Sensing of Synthetic, Bacterial, and Protozoan RNA by Toll-like Receptor 8 Requires Coordinated Processing by RNase T2 and RNase 2.

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.

Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.

Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2',5')pA and A(3',5')pG phosphodiester linkages, designated c[G(2',5')pA(3',5')p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2',5')pA(3',5')p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5'-pppG(2',5')pA prior to cyclization to c[G(2',5')pA(3',5')p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2',5')pA(3',5')p] as a founding member of a family of metazoan 2',5'-containing cyclic heterodinucleotide second messengers distinct from bacterial 3',5' cyclic dinucleotides.

Structure-function analysis of STING activation by c[G(2',5')pA(3',5')p] and targeting by antiviral DMXAA.

Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of the metazoan second messenger c[G(2',5')pA(3',5')p], which binds the signaling protein STING with subsequent activation of the interferon (IFN) pathway. We show that human hSTING(H232) adopts a "closed" conformation upon binding c[G(2',5')pA(3',5')p] and its linkage isomer c[G(2',5')pA(2',5')p], as does mouse mSting(R231) on binding c[G(2',5')pA(3',5')p], c[G(3',5')pA(3',5')p] and the antiviral agent DMXAA, leading to similar "closed" conformations. Comparing hSTING to mSting, 2',5'-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all-3',5'-linkage isomer. Guided by structural information, we identified a unique point mutation (S162A) placed within the cyclic-dinucleotide-binding site of hSTING that rendered it sensitive to the otherwise mouse-specific drug DMXAA, a conclusion validated by binding studies. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands within a small-molecule pocket created by the dimerization of STING.

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Inflammasome-Dependent Induction of Adaptive NK Cell Memory.

Monobenzone is a pro-hapten that is exclusively metabolized by melanocytes, thereby haptenizing melanocyte-specific antigens, which results in cytotoxic autoimmunity specifically against pigmented cells. Studying monobenzone in a setting of contact hypersensitivity (CHS), we observed that monobenzone induced a long-lasting, melanocyte-specific immune response that was dependent on NK cells, yet fully intact in the absence of T- and B cells. Consistent with the concept of "memory NK cells," monobenzone-induced NK cells resided in the liver and transfer of these cells conferred melanocyte-specific immunity to naive animals. Monobenzone-exposed skin displayed macrophage infiltration and cutaneous lymph nodes showed an inflammasome-dependent influx of macrophages with a tissue-resident phenotype, coinciding with local NK cell activation. Indeed, macrophage depletion or the absence of the NLRP3 inflammasome, the adaptor protein ASC or interleukin-18 (IL-18) abolished monobenzone CHS, thereby establishing a non-redundant role for the NLRP3 inflammasome as a critical proinflammatory checkpoint in the induction of hapten-dependent memory NK cells.

A conserved isoleucine in the binding pocket of RIG-I controls immune tolerance to mitochondrial RNA.

RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5'triphosphorylated and 5'base-paired RNA(dsRNA). Here, we found that, while 5'unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5'-monophosphate(5'p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5'p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5'p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5'p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.

A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N1-2'O-Methylated Self RNA.

The cytosolic helicase retinoic acid-inducible gene-I (RIG-I) initiates immune responses to most RNA viruses by detecting viral 5'-triphosphorylated RNA (pppRNA). Although endogenous mRNA is also 5'-triphosphorylated, backbone modifications and the 5'-ppp-linked methylguanosine ((m7)G) cap prevent immunorecognition. Here we show that the methylation status of endogenous capped mRNA at the 5'-terminal nucleotide (N1) was crucial to prevent RIG-I activation. Moreover, we identified a single conserved amino acid (H830) in the RIG-I RNA binding pocket as the mediator of steric exclusion of N1-2'O-methylated RNA. H830A alteration (RIG-I(H830A)) restored binding of N1-2'O-methylated pppRNA. Consequently, endogenous mRNA activated the RIG-I(H830A) mutant but not wild-type RIG-I. Similarly, knockdown of the endogenous N1-2'O-methyltransferase led to considerable RIG-I stimulation in the absence of exogenous stimuli. Studies involving yellow-fever-virus-encoded 2'O-methyltransferase and RIG-I(H830A) revealed that viruses exploit this mechanism to escape RIG-I. Our data reveal a new role for cap N1-2'O-methylation in RIG-I tolerance of self-RNA.

A novel serum extracellular vesicle protein signature to monitor glioblastoma tumor progression.

Detection of tumor progression in patients with glioblastoma remains a major challenge. Extracellular vesicles (EVs) are potential biomarkers and can be detected in the blood of patients with glioblastoma. In our study, we evaluated the potential of serum-derived EVs from glioblastoma patients to serve as biomarker for tumor progression. EVs from serum of glioblastoma patients and healthy volunteers were separated by size exclusion chromatography and ultracentrifugation. EV markers were defined by using a proximity-extension assay and bead-based flow cytometry. Tumor progression was defined according to modified RANO criteria. EVs from the serum of glioblastoma patients (n = 67) showed an upregulation of CD29, CD44, CD81, CD146, C1QA and histone H3 as compared to serum EVs from healthy volunteers (P value range: <.0001 to .08). For two independent cohorts of glioblastoma patients, we noted upregulation of C1QA, CD44 and histone H3 upon tumor progression, but not in patients with stable disease. In a multivariable logistic regression analysis, a combination of CD29, CD44, CD81, C1QA and histone H3 correlated with RANO-defined tumor progression with an AUC of 0.76. Measurement of CD29, CD44, CD81, C1QA and histone H3 in serum-derived EVs of glioblastoma patients, along with standard MRI assessment, has the potential to improve detection of true tumor progression and thus could be a useful biomarker for clinical decision making.

Induction of Interferon-Stimulated Genes Correlates with Reduced Growth of Influenza A Virus in Lungs after RIG-I Agonist Treatment of Ferrets.

Intracellular RIG-I receptors represent key innate sensors of RNA virus infection, and RIG-I activation results in the induction of hundreds of host effector genes, including interferon-stimulated genes (ISGs). Synthetic RNA agonists targeting RIG-I have shown promise as antivirals against a broad spectrum of viruses, including influenza A virus (IAV), in both in vitro and mouse models of infection. Herein, we demonstrate that treatment of a ferret airway epithelial (FRL) cell line with a RIG-I agonist rapidly and potently induced expression of a broad range of ISGs and resulted in potent inhibition of growth of different IAV strains. In ferrets, a single intravenous injection of RIG-I agonist was associated with upregulated ISG expression in peripheral blood mononuclear cells and lung tissue, but not in nasal tissues. In a ferret model of viral contact transmission, a single treatment of recipient animals 24 h prior to cohousing with IAV-infected donors did not reduce virus transmission and shedding but did result in reduced lung virus titers 6 days after treatment. A single treatment of the IAV-infected donor animals also resulted in reduced virus titers in the lungs 2 days later. Thus, a single intravenous treatment with RIG-I agonist prior to infection or to ferrets with an established IAV infection can reduce virus growth in the lungs. These findings support further development of RIG-I agonists as effective antiviral treatments to limit the impact of IAV infections, particularly in reducing virus replication in the lower airways. IMPORTANCE RIG-I agonists have shown potential as broad-spectrum antivirals in vitro and in mouse models of infection. However, their antiviral potential has not been reported in outbred animals such as ferrets, which are widely regarded as the gold standard small animal model for human IAV infections. Herein, we demonstrate that RIG-I agonist treatment of a ferret airway cell line resulted in ISG induction and inhibition of a broad range of human influenza viruses. A single intravenous treatment of ferrets also resulted in systemic induction of ISGs, including in lung tissue, and when delivered to animals prior to IAV exposure or to animals with established IAV infection treatment resulted in reduced virus replication in the lungs. These data demonstrate the effectiveness of single RIG-I treatment against IAV in the ferret model and highlight the importance of future studies to optimize treatment regimens and delivery routes to maximize their ability to ameliorate IAV infections.

Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) originating from the central nervous system (CNS) can enter the blood stream and carry molecules characteristic of disease states. Therefore, circulating CNS-derived EVs have the potential to serve as liquid-biopsy markers for early diagnosis and follow-up of neurodegenerative diseases and brain tumors. Monitoring and profiling of CNS-derived EVs using multiparametric analysis would be a major advance for biomarker as well as basic research. Here, we explored the performance of a multiplex bead-based flow-cytometry assay (EV Neuro) for semi-quantitative detection of CNS-derived EVs in body fluids. METHODS: EVs were separated from culture of glioblastoma cell lines (LN18, LN229, NCH82) and primary human astrocytes and measured at different input amounts in the MACSPlex EV Kit Neuro, human. In addition, EVs were separated from blood samples of small cohorts of glioblastoma (GB), multiple sclerosis (MS) and Alzheimer's disease patients as well as healthy controls (HC) and subjected to the EV Neuro assay. To determine statistically significant differences between relative marker signal intensities, an unpaired samples t-test or Wilcoxon rank sum test were computed. Data were subjected to tSNE, heatmap clustering, and correlation analysis to further explore the relationships between disease state and EV Neuro data. RESULTS: Glioblastoma cell lines and primary human astrocytes showed distinct EV profiles. Signal intensities were increasing with higher EV input. Data normalization improved identification of markers that deviate from a common profile. Overall, patient blood-derived EV marker profiles were constant, but individual EV populations were significantly increased in disease compared to healthy controls, e.g. CD36+EVs in glioblastoma and GALC+EVs in multiple sclerosis. tSNE and heatmap clustering analysis separated GB patients from HC, but not MS patients from HC. Correlation analysis revealed a potential association of CD107a+EVs with neurofilament levels in blood of MS patients and HC. CONCLUSIONS: The semi-quantitative EV Neuro assay demonstrated its utility for EV profiling in complex samples. However, reliable statistical results in biomarker studies require large sample cohorts and high effect sizes. Nonetheless, this exploratory trial confirmed the feasibility of discovering EV-associated biomarkers and monitoring circulating EV profiles in CNS diseases using the EV Neuro assay. Video Abstract.

Extracellular vesicles (EVs) are tiny particles released by cells, carrying unique biomolecules specific to their cell of origin. EVs from the central nervous system (CNS) can reach the blood, where they could serve as liquid-biopsy markers for diagnosing brain diseases like neurodegenerative disorders and tumors. This study evaluated a flow cytometry platform (here termed EV Neuro assay), which can detect multiple EV-associated markers simultaneously, to assess its potential for identifying CNS-derived EVs and disease-specific markers in complex samples including the blood. The study compared different sample materials and methods for isolating EVs. We found distinct EV profiles in EVs derived from glioblastoma and human astrocytes, with signal intensities increasing as more EVs were present. Analyzing serum or plasma from patients with brain diseases and healthy individuals, we observed that EV marker intensities were varying between individuals. Importantly, data normalization improved the identification of disease-specific markers, such as CD36⁺EVs in glioblastoma and GALC⁺EVs in multiple sclerosis, which were significantly higher in disease compared to healthy controls. Advanced clustering analysis techniques effectively distinguished glioblastoma patients from controls. Furthermore, a potential correlation between CD107a⁺EVs and neurofilament levels in multiple sclerosis patients was discovered. Overall, the semi-quantitative EV Neuro assay proved useful for profiling EVs in complex samples. However, for more reliable results in biomarker studies, larger sample cohorts and higher effect sizes are necessary. Nonetheless, this initial trial confirmed the potential of the EV Neuro assay for discovering disease-associated EV markers and monitoring circulating EV profiles in CNS diseases.

Absence of cGAS-mediated type I IFN responses in HIV-1-infected T cells.

The DNA sensor cGAS catalyzes the production of the cyclic dinucleotide cGAMP, resulting in type I interferon responses. We addressed the functionality of cGAS-mediated DNA sensing in human and murine T cells. Activated primary CD4+ T cells expressed cGAS and responded to plasmid DNA by upregulation of ISGs and release of bioactive interferon. In mouse T cells, cGAS KO ablated sensing of plasmid DNA, and TREX1 KO enabled cells to sense short immunostimulatory DNA. Expression of IFIT1 and MX2 was downregulated and upregulated in cGAS KO and TREX1 KO T cell lines, respectively, compared to parental cells. Despite their intact cGAS sensing pathway, human CD4+ T cells failed to mount a reverse transcriptase (RT) inhibitor-sensitive immune response following HIV-1 infection. In contrast, infection of human T cells with HSV-1 that is functionally deficient for the cGAS antagonist pUL41 (HSV-1ΔUL41N) resulted in a cGAS-dependent type I interferon response. In accordance with our results in primary CD4+ T cells, plasmid challenge or HSV-1ΔUL41N inoculation of T cell lines provoked an entirely cGAS-dependent type I interferon response, including IRF3 phosphorylation and expression of ISGs. In contrast, no RT-dependent interferon response was detected following transduction of T cell lines with VSV-G-pseudotyped lentiviral or gammaretroviral particles. Together, T cells are capable to raise a cGAS-dependent cell-intrinsic response to both plasmid DNA challenge or inoculation with HSV-1ΔUL41N. However, HIV-1 infection does not appear to trigger cGAS-mediated sensing of viral DNA in T cells, possibly by revealing viral DNA of insufficient quantity, length, and/or accessibility to cGAS.

Retinoic Acid-Inducible Gene I Activation Inhibits Human Respiratory Syncytial Virus Replication in Mammalian Cells and in Mouse and Ferret Models of Infection.

Infections caused by human respiratory syncytial virus (RSV) are associated with substantial rates of morbidity and mortality. Treatment options are limited, and there is urgent need for the development of efficient antivirals. Pattern recognition receptors such as the cytoplasmic helicase retinoic acid-inducible gene (RIG) I can be activated by viral nucleic acids, leading to activation of interferon-stimulated genes and generation of an "antiviral state." In the current study, we activated RIG-I with synthetic RNA agonists (3pRNA) to induce resistance to RSV infection in vitro and in vivo. In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication. Moreover, a single intravenous injection of 3pRNA 1 day before RSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues. These studies provide evidence that RIG-I agonists represent a promising antiviral drug for RSV prophylaxis.

Recessive NLRC4-Autoinflammatory Disease Reveals an Ulcerative Colitis Locus.

PURPOSE: NLRC4-associated autoinflammatory disease (NLRC4-AID) is an autosomal dominant condition presenting with a range of clinical manifestations which can include macrophage activation syndrome (MAS) and severe enterocolitis. We now report the first homozygous mutation in NLRC4 (c.478G > A, p.A160T) causing autoinflammatory disease with immune dysregulation and find that heterozygous carriers in the general population are at increased risk of developing ulcerative colitis. METHODS: Circulating immune cells and inflammatory markers were profiled and historical clinical data interrogated. DNA was extracted and sequenced using standard procedures. Inflammasome activation assays for ASC speck formation, pyroptosis, and IL-1ß/IL-18 secretion confirmed pathogenicity of the mutation in vitro. Genome-wide association of NLRC4 (A160T) with ulcerative colitis was examined using data from the IBD exomes portal. RESULTS: A 60-year-old Brazilian female patient was evaluated for recurrent episodes of systemic inflammation from six months of age. Episodes were characterized by recurrent low-grade fever, chills, oral ulceration, uveitis, arthralgia, and abdominal pain, followed by diarrhea with mucus and variable skin rash. High doses of corticosteroids were somewhat effective in controlling disease and anti-IL-1ß therapy partially controlled symptoms. While on treatment, serum IL-1ß and IL-18 levels remained elevated. Genetic investigations identified a homozygous mutation in NLRC4 (A160T), inherited in a recessive fashion. Increased ASC speck formation and IL-1ß/IL-18 secretion confirmed pathogenicity when NLRC4 (A160T) was analyzed in human cell lines. This allele is significantly enriched in patients with ulcerative colitis: OR 2.546 (95% 1.778-3.644), P = 0.01305. CONCLUSION: NLRC4 (A160T) can either cause recessively inherited autoinflammation and immune dysregulation, or function as a heterozygous risk factor for the development of ulcerative colitis.

Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.

Correlation between a quantitative anti-SARS-CoV-2 IgG ELISA and neutralization activity.

In the current COVID-19 pandemic, a better understanding of the relationship between merely binding and functionally neutralizing antibodies is necessary to characterize protective antiviral immunity following infection or vaccination. This study analyzes the level of correlation between the novel quantitative EUROIMMUN Anti-SARS-CoV-2 QuantiVac ELISA (IgG) and a microneutralization assay. A panel of 123 plasma samples from a COVID-19 outbreak study population, preselected by semiquantitative anti-SARS-CoV-2 IgG testing, was used to assess the relationship between the novel quantitative ELISA (IgG) and a microneutralization assay. Binding IgG targeting the S1 antigen was detected in 106 (86.2%) samples using the QuantiVac ELISA, while 89 (72.4%) samples showed neutralizing antibody activity. Spearman's correlation analysis demonstrated a strong positive relationship between anti-S1 IgG levels and neutralizing antibody titers (rs = 0.819, p < 0.0001). High and low anti-S1 IgG levels were associated with a positive predictive value of 72.0% for high-titer neutralizing antibodies and a negative predictive value of 90.8% for low-titer neutralizing antibodies, respectively. These results substantiate the implementation of the QuantiVac ELISA to assess protective immunity following infection or vaccination.

Turning tumors into vaccines: co-opting the innate immune system.

Immunotherapy of cancer must be effective in the pre-established disease; i.e., in the therapeutic rather than prophylactic setting. Here, we review novel immunotherapeutic approaches for targeting established cancers. In addition to novel checkpoint-blocking antibodies, recent insight into innate immune sensors may further improve cancer immunotherapy protocols and help to overcome the limitations of conventional therapeutic immunization strategies. Specifically, the local induction of IL-12 and IFNα turns the immunosuppressive tumor microenvironment into an immunosupportive tissue, which is attained, for example, by local Toll-like receptor or RIG-I-like receptor triggering. Notably, the latter are endogenously expressed in all tumor cells and have the advantage of turning tumors into tumor vaccines by inducing apoptosis and improving antigen presentation. Thus, immunostimulatory agents embody strong promise as a part of combinatorial cancer immunotherapies.

Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing.

Immune sensing of DNA is critical for antiviral immunity but can also trigger autoimmune diseases such as lupus erythematosus (LE). Here we have provided evidence for the involvement of a damage-associated DNA modification in the detection of cytosolic DNA. The oxidized base 8-hydroxyguanosine (8-OHG), a marker of oxidative damage in DNA, potentiated cytosolic immune recognition by decreasing its susceptibility to 3' repair exonuclease 1 (TREX1)-mediated degradation. Oxidizative modifications arose physiologically in pathogen DNA during lysosomal reactive oxygen species (ROS) exposure, as well as in neutrophil extracellular trap (NET) DNA during the oxidative burst. 8-OHG was also abundant in UV-exposed skin lesions of LE patients and colocalized with type I interferon (IFN). Injection of oxidized DNA in the skin of lupus-prone mice induced lesions that closely matched respective lesions in patients. Thus, oxidized DNA represents a prototypic damage-associated molecular pattern (DAMP) with important implications for infection, sterile inflammation, and autoimmunity.

RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate.

RNA is sensed by Toll-like receptor 7 (TLR7) and TLR8 or by the RNA helicases LGP2, Mda5 and RIG-I to trigger antiviral responses. Much less is known about sensors for DNA. Here we identify a novel DNA-sensing pathway involving RNA polymerase III and RIG-I. In this pathway, AT-rich double-stranded DNA (dsDNA) served as a template for RNA polymerase III and was transcribed into double-stranded RNA (dsRNA) containing a 5'-triphosphate moiety. Activation of RIG-I by this dsRNA induced production of type I interferon and activation of the transcription factor NF-kappaB. This pathway was important in the sensing of Epstein-Barr virus-encoded small RNAs, which were transcribed by RNA polymerase III and then triggered RIG-I activation. Thus, RNA polymerase III and RIG-I are pivotal in sensing viral DNA.