Abstract
Human Vγ9Vδ2 T cells recognize pyrophosphates produced by microbes and transformed cells and play a role in anti-infective immunity and tumor surveillance. Toll-like receptors (TLR) are ...pattern recognition receptors in innate immune cells which sense microbial structures including nucleic acids. Given that γδ T cells are in clinical development for application in cellular cancer immunotherapy and TLR ligands have potent adjuvant activity, we investigated the co-stimulatory role of selected TLR ligands in γδ T-cell activation. Here we have used recently described RNA ligands for TLR7 and TLR8 together with Vγ9Vδ2 T-cell specific pyrophosphate antigens to analyze the rapid cytokine induction in Vδ2 T cells as well as the accessory cell requirements. While TLR8- as well as TLR7/8-specific RNA did not induce IFN-γ in Vδ2 T cells on their own, they provided strong co-stimulation for Vδ2 T cells within peripheral blood mononuclear cells in the presence of additional T-cell receptor activation. In contrast, TLR7 ligands were ineffective. Purified γδ T cells did not directly respond to TLR8 co-stimulation but required the presence of monocytes. Further experiments revealed a critical role of IL-1β and IL-18, and to a slightly lesser extent of IL-12p70, in the co-stimulation of Vδ2 T cells by TLR8 and TLR7/8 RNA ligands. Results of intracellular cytokine expression were validated by ELISA analysis of cytokines in cell culture supernatants. The cell context-dependent adjuvant activity of TLR8 and TLR7/8 RNA ligands described here might be important for the future optimization of γδ T-cell based cancer immunotherapy.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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.
•Endolysosomal RNase 2 and RNase T2 cooperatively process RNA into TLR8 ligands•RNase 2 and RNase T2 synergistically release uridine from RNA ligands•RNase T2-hypomorphic patients have an impaired to TLR8 response to pathogen RNA•Sensing of live pathogens by TLR8 requires both RNase 2 and RNase T2
Toll-like receptor 8 (TLR8) is an endosomal RNA sensor that induces a robust T-helper 1 response downstream of pathogen recognition. Ostendorf and colleagues identify two RNases, RNase 2 and RNase T2, which synergistically process pathogen RNA into TLR8 ligands and are critically required for the sensing of live bacteria and plasmodia.
Antiviral immunity is triggered by immunorecognition of viral nucleic acids. The cytosolic helicase RIG-I is a key sensor of viral infections and is activated by RNA containing a triphosphate at the ...5′ end. The exact structure of RNA activating RIG-I remains controversial. Here, we established a chemical approach for 5′ triphosphate oligoribonucleotide synthesis and found that synthetic single-stranded 5′ triphosphate oligoribonucleotides were unable to bind and activate RIG-I. Conversely, the addition of the synthetic complementary strand resulted in optimal binding and activation of RIG-I. Short double-strand conformation with base pairing of the nucleoside carrying the 5′ triphosphate was required. RIG-I activation was impaired by a 3′ overhang at the 5′ triphosphate end. These results define the structure of RNA for full RIG-I activation and explain how RIG-I detects negative-strand RNA viruses that lack long double-stranded RNA but do contain blunt short double-stranded 5′ triphosphate RNA in the panhandle region of their single-stranded genome.
RATIONALE:Endothelial dysfunction and atherosclerosis are chronic inflammatory diseases characterized by activation of the innate and acquired immune system. Specialized protein receptors of the ...innate immune system recognize products of microorganisms and endogenous ligands such as nucleic acids. Toll-like receptor 3 (TLR3), for example, detects long double-stranded RNA and is abundantly expressed in endothelial cells. Whether innate immunity contributes to atherogenic mechanisms in endothelial cells is poorly understood.
OBJECTIVE:We sought to determine the effects of TLR3 activation in endothelial cells.
METHODS AND RESULTS:We first investigated whether stimulation of TLR3 influences endothelial biology in mice. Intravenous injection of polyinosine polycytidylic acid, a synthetic double-stranded RNA analog and TLR3 ligand, impaired endothelium-dependent vasodilation, increased vascular production of reactive oxygen species, and reduced reendothelialization after carotid artery injury in wild-type mice compared with controls but had no effect in TLR3 animals. TLR3 stimulation not only induced endothelial dysfunction but also enhanced the formation of atherosclerotic plaques in apolipoprotein E–deficient mice. In vitro incubation of endothelial cells with polyinosine polycytidylic acid induced production of the proinflammatory cytokines interleukin-8 and interferon-γ–induced protein 10, increased formation of reactive oxygen species, diminished proliferation, and increased apoptosis, which suggests that endothelial cells are able to directly detect and respond to TLR3 ligands. Neutralization of interleukin-8 and interferon-γ–induced protein 10 antagonizes the observed negative effects of polyinosine polycytidylic acid. We found elevated levels of circulating endothelial progenitor cells in polyinosine polycytidylic acid–treated mice, although they displayed increased endothelial dysfunction. Stimulation of TLR3 in cultured endothelial progenitor cells, however, led to increased formation of reactive oxygen species, increased apoptosis, and reduced migration. Injection of endothelial progenitor cells that had been incubated with polyinosine polycytidylic acid ex vivo hindered reendothelialization after carotid artery injury. Therefore, endothelial progenitor cell function was affected by TLR3 stimulation. Finally, apolipoprotein E–deficient/TLR3-deficient mice exhibited improved endothelial function compared with apolipoprotein E–deficient/TLR3 littermates.
CONCLUSIONS:Immunorecognition of long double-stranded RNA by endothelial cells may be an important mechanism involved in endothelial cell activation and development of endothelial dysfunction.
Activation of the innate immune receptor retinoic acid‐inducible gene I (RIG‐I) by its specific ligand 5′‐triphosphate RNA (3pRNA) triggers anti‐tumor immunity, which is dependent on natural killer ...(NK) cell activation and cytokine induction. However, to date, RIG‐I expression and the functional consequences of RIG‐I activation in NK cells have not been examined. Here, we show for the first time the expression of RIG‐I in human NK cells and their activation upon RIG‐I ligand (3pRNA) transfection. 3pRNA‐activated NK cells killed melanoma cells more efficiently than NK cells activated by type I interferon. Stimulation of RIG‐I in NK cells specifically increased the surface expression of membrane‐bound TNF‐related apoptosis‐inducing ligand (TRAIL) on NK cells, while activated NK cell receptors were not affected. RIG‐I‐induced membrane‐bound TRAIL initiated death‐receptor‐pathway‐mediated apoptosis not only in allogeneic but also in autologous human leukocyte antigen (HLA) class I‐positive and HLA class I‐negative melanoma cells. These results identify the direct activation of RIG‐I in NK cells as a novel mechanism for how RIG‐I can trigger enhanced NK cell killing of tumor cells, underscoring the potential of RIG‐I activation for tumor immunotherapy.
What's new?
Activation of the innate immune receptor RIG‐I triggers anti‐tumor immunity, which is dependent on natural killer (NK) cell activation and cytokine induction. However, a mechanism explaining how RIG‐I activates NK cells remains elusive. Our study demonstrates that naïve NK cells express RIG‐I and are activated upon RIG‐I ligand transfection. RIG‐I activation induces TRAIL expression on NK cells, which, in turn, induces cell death in melanoma cells. TRAIL‐dependent killing via RIG‐I is induced not only in allogeneic but also in autologous melanoma cells. Direct RIG‐I activation in NK cells thus emerges as a novel, hitherto unappreciated, functional component of RIG‐I‐mediated immunotherapy.
Glioblastoma is a devastating disease, for which biomarkers allowing a prediction of prognosis are urgently needed. microRNAs have been described as potentially valuable biomarkers in cancer. Here, ...we studied a panel of microRNAs in extracellular vesicles (EVs) from the serum of glioblastoma patients and evaluated their correlation with the prognosis of these patients. The levels of 15 microRNAs in EVs that were separated by size-exclusion chromatography were studied by quantitative real-time PCR, followed by CD44 immunoprecipitation (SEC + CD44), and compared with those from the total serum of glioblastoma patients (
= 55) and healthy volunteers (
= 10). Compared to total serum, we found evidence for the enrichment of miR-21-3p and miR-106a-5p and, conversely, lower levels of miR-15b-3p, in SEC + CD44 EVs. miR-15b-3p and miR-21-3p were upregulated in glioblastoma patients compared to healthy subjects. A significant correlation with survival of the patients was found for levels of miR-15b-3p in total serum and miR-15b-3p, miR-21-3p, miR-106a-5p, and miR-328-3p in SEC + CD44 EVs. Combining miR-15b-3p in serum or miR-106a-5p in SEC + CD44 EVs with any one of the other three microRNAs in SEC + CD44 EVs allowed for a prognostic stratification of glioblastoma patients. We have thus identified four microRNAs in glioblastoma patients whose levels, in combination, can predict the prognosis for these patients.
Purpose
To evaluate the use of 2 mg intravitreal aflibercept for treatment of choroidal neovascularization (CNV) secondary to angioid streaks in patients with pseudoxanthoma elasticum (PXE).
Methods
...In this 12-month prospective, open-label, uncontrolled, non-randomized interventional clinical trial, 15 PXE patients with CNV (mean age: 53 years, range 22–65) received one initial intravitreal injection of 2 mg aflibercept. Further injections were based on CNV activity at monthly examinations. The primary endpoint was change of best corrected visual acuity (BCVA) after 12 months. Secondary outcomes were change of central retinal thickness (CRT), leakage from CNV, retinal sensitivity, and vision-related quality of life.
Results
BCVA improved from 75.0 ± 10.8 (± SD, Snellen equivalent 20/32) to 79.3 ± 7.3 ETDRS letters (20/32) at final visit (
p
= 0.083). CRT decreased from 317 ± 81 to 279 ± 51 μm (
p
= 0.004). Retinal sensitivity on microperimetry changed from 17.8 ± 4.5 to 18.5 ± 4.3 dB (
p
= 0.103) and vision-related quality of life from a VQF-25 score of 80.7 ± 10.4 to 83.5 ± 14.5 (
p
= 0.554). The mean number of injections was 6.7 ± 2.6, and 5 participants had persistent or reactivated CNV activity at final visit. The observed adverse events were comparable with studies on aflibercept for other indications.
Conclusion
The results of this study indicate that intravitreal aflibercept is a treatment option for CNV secondary to PXE.
Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, ...comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma.
Persistent infections of the skin with the human papillomavirus of genus beta (β-HPV) in immunocompetent individuals are asymptomatic, but in immunosuppressed patients, β-HPV infections exhibit much ...higher viral loads on the skin and are associated with an increased risk of skin cancer. Unlike with HPV16, a high-risk α-HPV, the impact of β-HPV early genes on the innate immune sensing of viral nucleic acids has not been studied. Here, we used primary skin keratinocytes and U2OS cells expressing HPV8 or distinct HPV8 early genes and well-defined ligands of the nucleic-acid-sensing receptors RIG-I, MDA5, TLR3, and STING to analyze a potential functional interaction. We found that primary skin keratinocytes and U2OS cells expressed RIG-I, MDA5, TLR3, and STING, but not TLR7, TLR8, or TLR9. While HPV16-E6 downregulated the expression of RIG-I, MDA5, TLR3, and STING and, in conjunction with HPV16-E7, effectively suppressed type I IFN in response to MDA5 activation, the presence of HPV8 early genes showed little effect on the expression of these immune receptors, except for HPV8-E2, which was associated with an elevated expression of TLR3. Nevertheless, whole HPV8 genome expression, as well as the selective expression of HPV8-E1 or HPV8-E2, was found to suppress MDA5-induced type I IFN and the proinflammatory cytokine IL-6. Furthermore, RNA isolated from HPV8-E2 expressing primary human keratinocytes, but not control cells, stimulated a type I IFN response in peripheral blood mononuclear cells, indicating that the expression of HPV8-E2 in keratinocytes leads to the formation of stimulatory RNA ligands that require the active suppression of immune recognition. These results identify HPV8-E1 and HPV8-E2 as viral proteins that are responsible for the immune escape of β-HPV from the innate recognition of viral nucleic acids, a mechanism that may be necessary for establishing persistent β-HPV infections.
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