Publications by authors named "Stefan Lienenklaus"

91 Publications

PD-1 Blockade Aggravates Epstein-Barr Virus Post-Transplant Lymphoproliferative Disorder in Humanized Mice Resulting in Central Nervous System Involvement and CD4 T Cell Dysregulations.

Front Oncol 2020 12;10:614876. Epub 2021 Jan 12.

Laboratory of Regenerative Immune Therapies Applied, REBIRTH - Research Center for Translational Regenerative Medicine, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.

Post-transplant lymphoproliferative disorder (PTLD) is one of the most common malignancies after solid organ or allogeneic stem cell transplantation. Most PTLD cases are B cell neoplasias carrying Epstein-Barr virus (EBV). A therapeutic approach is reduction of immunosuppression to allow T cells to develop and combat EBV. If this is not effective, approaches include immunotherapies such as monoclonal antibodies targeting CD20 and adoptive T cells. Immune checkpoint inhibition (ICI) to treat EBV PTLD was not established clinically due to the risks of organ rejection and graft--host disease. Previously, blockade of the programmed death receptor (PD)-1 by a monoclonal antibody (mAb) during infection of mononuclear cells with the EBV/M81 strain showed lower xenografted lymphoma development in mice. Subsequently, fully humanized mice infected with the EBV/B95-8 strain and treated with a PD-1 blocking mAb showed aggravation of PTLD and lymphoma development. Here, we evaluated in fully humanized mice after EBV/B95-8 or EBV/M81 infections the effects of a clinically used PD-1 blocker. Fifteen to 17 weeks after human CD34 stem cell transplantation, Nod.Rag.Gamma mice were infected with two types of EBV laboratory strains expressing firefly luciferase. Dynamic optical imaging analyses showed systemic EBV infections and this triggered vigorous human CD8 T cell expansion. Pembrolizumab administered from 2 to 5 weeks post-infections significantly aggravated EBV systemic spread and, for the M81 model, significantly increased the mortality of mice. ICI promoted Ki67CD30CD20EBERPD-L1 PTLD with central nervous system (CNS) involvement, mirroring EBV CNS PTLD in humans. PD-1 blockade was associated with lower frequencies of circulating T cells in blood and with a profound collapse of CD4 T cells in lymphatic tissues. Mice treated with pembrolizumab showed an escalation of exhausted T cells expressing TIM-3, and LAG-3 in tissues, higher levels of several human cytokines in plasma and high densities of FoxP3 regulatory CD4 and CD8 T cells in the tumor microenvironment. We conclude that PD-1 blockade during acute EBV infections driving strong CD8 T cell priming decompensates T cell development towards immunosuppression. Given the variety of preclinical models available, our models conferred a cautionary note indicating that PD-1 blockade aggravated the progression of EBV PTLD.
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http://dx.doi.org/10.3389/fonc.2020.614876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837057PMC
January 2021

Lipid-Polyglutamate Nanoparticle Vaccine Platform.

ACS Appl Mater Interfaces 2021 Feb 28;13(5):6011-6022. Epub 2021 Jan 28.

Department of Pharmaceutics, Ghent University, 9000 Ghent, Belgium.

Peptide-based subunit vaccines are attractive in view of personalized cancer vaccination with neo-antigens, as well as for the design of the newest generation of vaccines against infectious diseases. Key to mounting robust antigen-specific immunity is delivery of antigen to antigen-presenting (innate immune) cells in lymphoid tissue with concomitant innate immune activation to promote antigen presentation to T cells and to shape the amplitude and nature of the immune response. Nanoparticles that co-deliver both peptide antigen and molecular adjuvants are well suited for this task. However, in the context of peptide-based antigen, an unmet need exists for a generic strategy that allows for co-encapsulation of peptide and molecular adjuvants due to the stark variation in physicochemical properties based on the amino acid sequence of the peptide. These properties also strongly differ from those of many molecular adjuvants. Here, we devise a lipid nanoparticle (LNP) platform that addresses these issues. Key in our concept is poly(l-glutamic acid) (PGA), which serves as a hydrophilic backbone for conjugation of, respectively, peptide antigen (Ag) and an imidazoquinoline (IMDQ) TLR7/8 agonist as a molecular adjuvant. Making use of the PGA's polyanionic nature, we condensate PGA-Ag and PGA-IMDQ into LNP by electrostatic interaction with an ionizable lipid. We show in vitro and in vivo in mouse models that LNP encapsulation favors uptake by innate immune cells in lymphoid tissue and promotes the induction of Ag-specific T cells responses both after subcutaneous and intravenous administration.
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http://dx.doi.org/10.1021/acsami.0c20607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116839PMC
February 2021

Corticosteroids and cellulose purification improve, respectively, the in vivo translation and vaccination efficacy of sa-mRNAs.

Mol Ther 2021 04 21;29(4):1370-1381. Epub 2021 Jan 21.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium; Cancer Research Institute (CRIG), Ghent University, 9000 Ghent, Belgium. Electronic address:

Synthetic mRNAs are an appealing platform with multiple biomedical applications ranging from protein replacement therapy to vaccination. In comparison with conventional mRNA, synthetic self-amplifying mRNAs (sa-mRNAs) are gaining interest because of their higher and longer-lasting expression. However, sa-mRNAs also elicit an innate immune response, which may complicate their clinical application. Approaches to reduce the innate immunity of sa-mRNAs have not been studied in detail. Here we investigated, in vivo, the effect of several innate immune inhibitors and a novel cellulose-based mRNA purification approach on the type I interferon (IFN) response and the translation and vaccination efficacy of our formerly developed sa-mRNA vaccine against Zika virus. Among the investigated inhibitors, we found that corticosteroids and especially topical application of clobetasol at the sa-mRNA injection site was the most efficient in suppressing the type I IFN response and increasing the translation of sa-mRNA. However, clobetasol prevented formation of antibodies against sa-mRNA-encoded antigens and should therefore be avoided in a vaccination context. Residual dsRNA by-products of the in vitro transcription reaction are known inducers of immediate type I IFN responses. We additionally demonstrate a drastic reduction of these dsRNA by-products upon cellulose-based purification, reducing the innate immune response and improving sa-mRNA vaccination efficacy.
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http://dx.doi.org/10.1016/j.ymthe.2021.01.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058483PMC
April 2021

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Lipid Amphiphile Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine*.

Angew Chem Int Ed Engl 2021 04 11;60(17):9467-9473. Epub 2021 Mar 11.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. It is water-soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.
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http://dx.doi.org/10.1002/anie.202015362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014308PMC
April 2021

Imidazoquinoline-Conjugated Degradable Coacervate Conjugate for Local Cancer Immunotherapy.

ACS Biomater Sci Eng 2020 09 11;6(9):4993-5000. Epub 2020 Aug 11.

Department of Pharmaceutics, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 Wenhuaxi Road, Jinan, Shandong Province 250012, P. R. China.

Strategies that can reduce the harmful side effects of potent immunomodulatory drugs are in high demand to facilitate clinical translation of the newest generation of immunotherapy. Indeed, uncontrolled triggering of the immune system can lead to life-threatening cascade reactions, such as e.g. cytokine storm. In particular, drug formulations that combine simplicity and degradability are of formidable relevance. Imidazoquinolines are an excellent example of such small molecule immunomodulatory drugs that exhibit in unformulated form a highly undesirable pharmacokinetic profile. Imidazoquinolines are potent inducers of type I interferons that are of great interest in the context of anticancer and antiviral therapy through triggering of Toll like receptors 7 and 8. In this work we aimed to alter the pharmacokinetic profile of imidazoquinolines using a simple, yet efficient, strategy that holds high potential for clinical translation. Hereto, we conjugated an imidazoquinoline to the backbone of poly(aspartate) and further formulated this into a degradable coacervate through complex coacervation with a nontoxic degradable polycation. The intrinsic TLR activity of the imidazoquinoline was well preserved and our formulation strategy offered spatial control over its biological activity in vivo.
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http://dx.doi.org/10.1021/acsbiomaterials.0c00485DOI Listing
September 2020

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Modified Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine.

bioRxiv 2020 Oct 23. Epub 2020 Oct 23.

Department of Microbiology, Icahn School of Medicine at Mount Sinai New York, NY, USA.

The search for vaccines that protect from severe morbidity and mortality as a result of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Several vaccine candidates are currently being tested in the clinic. Inactivated virus and recombinant protein vaccines can be safe options but may require adjuvants to induce robust immune responses efficiently. In this work we describe the use of a novel amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile). This amphiphile is water soluble and exhibits massive translocation to lymph nodes upon local administration, likely through binding to albumin. IMDQ-PEG-CHOL is used to induce a protective immune response against SARS-CoV-2 after single vaccination with trimeric recombinant SARS-CoV-2 spike protein in the BALB/c mouse model. Inclusion of amphiphilic IMDQ-PEG-CHOL in the SARS-CoV-2 spike vaccine formulation resulted in enhanced immune cell recruitment and activation in the draining lymph node. IMDQ-PEG-CHOL has a better safety profile compared to native soluble IMDQ as the former induces a more localized immune response upon local injection, preventing systemic inflammation. Moreover, IMDQ-PEG-CHOL adjuvanted vaccine induced enhanced ELISA and in vitro microneutralization titers, and a more balanced IgG2a/IgG1 response. To correlate vaccine responses with control of virus replication in vivo, vaccinated mice were challenged with SARS-CoV-2 virus after being sensitized by intranasal adenovirus-mediated expression of the human angiotensin converting enzyme 2 (ACE2) gene. Animals vaccinated with trimeric recombinant spike protein vaccine without adjuvant had lung virus titers comparable to non-vaccinated control mice, whereas animals vaccinated with IMDQ-PEG-CHOL-adjuvanted vaccine controlled viral replication and infectious viruses could not be recovered from their lungs at day 4 post infection. In order to test whether IMDQ-PEG-CHOL could also be used to adjuvant vaccines currently licensed for use in humans, proof of concept was also provided by using the same IMDQ-PEG-CHOL to adjuvant human quadrivalent inactivated influenza virus split vaccine, which resulted in enhanced hemagglutination inhibition titers and a more balanced IgG2a/IgG1 antibody response. Enhanced influenza vaccine responses correlated with better virus control when mice were given a lethal influenza virus challenge. Our results underscore the potential use of IMDQ-PEG-CHOL as an adjuvant to achieve protection after single immunization with recombinant protein and inactivated virus vaccines against respiratory viruses, such as SARS-CoV-2 and influenza viruses.
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http://dx.doi.org/10.1101/2020.10.23.344085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587831PMC
October 2020

Non-Invasive Luciferase Imaging of Type I Interferon Induction in a Transgenic Mouse Model of Biomaterial Associated Bacterial Infections: Microbial Specificity and Inter-Bacterial Species Interactions.

Microorganisms 2020 Oct 21;8(10). Epub 2020 Oct 21.

Department of Prosthetic Dentistry and Biomedical Materials Science, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.

The performance of biomaterials is often compromised by bacterial infections and subsequent inflammation. So far, the conventional analysis of inflammatory processes in vivo involves time-consuming histology and biochemical assays. The present study employed a mouse model where interferon beta (IFN-β) is monitored as a marker for non-invasive rapid detection of inflammation in implant-related infections. The mouse model comprises subcutaneous implantation of morphologically modified titanium, followed by experimental infections with four taxonomically diverse oral bacteria: and (as mono culture or selected mixed-culture). IFN-β expression increased upon infections depending on the type of pathogen and was prolonged by the presence of the implant. IFN-β expression kinetics reduced with two mixed species infections when compared with the single species. Histological and confocal microscopy confirmed pathogen-specific infiltration of inflammatory cells at the implant-tissue interface. This was observed mainly in the vicinity of infected implants and was, in contrast to interferon expression, higher in infections with dual species. In summary, this non-invasive mouse model can be used to quantify longitudinally host inflammation in real time and suggests that the polymicrobial character of infection, highly relevant to clinical situations, has complex effects on host immunity.
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http://dx.doi.org/10.3390/microorganisms8101624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589032PMC
October 2020

CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease.

Mol Ther Oncolytics 2020 Sep 8;18:504-524. Epub 2020 Aug 8.

Laboratory of Regenerative Immune Therapies Applied, Hannover Medical School, 30625 Hannover, Germany.

Epstein-Barr virus (EBV) is a latent and oncogenic human herpesvirus. Lytic viral protein expression plays an important role in EBV-associated malignancies. The EBV envelope glycoprotein 350 (gp350) is expressed abundantly during EBV lytic reactivation and sporadically on the surface of latently infected cells. Here we tested T cells expressing gp350-specific chimeric antigen receptors (CARs) containing scFvs derived from two novel gp350-binding, highly neutralizing monoclonal antibodies. The scFvs were fused to CD28/CD3ζ signaling domains in a retroviral vector. The produced gp350CAR-T cells specifically recognized and killed gp350 293T cells . The best-performing 7A1-gp350CAR-T cells were cytotoxic against the EBV B95-8 cell line, showing selectivity against gp350 cells. Fully humanized Nod.Rag.Gamma mice transplanted with cord blood CD34 cells and infected with the EBV/M81/fLuc lytic strain were monitored dynamically for viral spread. Infected mice recapitulated EBV-induced lymphoproliferation, tumor development, and systemic inflammation. We tested adoptive transfer of autologous CD8gp350CAR-T cells administered protectively or therapeutically. After gp350CAR-T cell therapy, 75% of mice controlled or reduced EBV spread and showed lower frequencies of EBER B cell malignant lymphoproliferation, lack of tumor development, and reduced inflammation. In summary, CD8gp350CAR-T cells showed proof-of-concept preclinical efficacy against impending EBV lymphoproliferation and lymphomagenesis.
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http://dx.doi.org/10.1016/j.omto.2020.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479496PMC
September 2020

Altered bone development with impaired cartilage formation precedes neuromuscular symptoms in spinal muscular atrophy.

Hum Mol Genet 2020 09;29(16):2662-2673

Pediatric Orthopedics, Department of Trauma, Orthopedic and Plastic Surgery, University Medical Center Goettingen, Goettingen, Germany.

Spinal muscular atrophy (SMA) is a fatal neurodegenerative disease of newborns and children caused by mutations or deletions of the survival of motoneuron gene 1 resulting in low levels of the SMN protein. While neuromuscular degeneration is the cardinal symptom of the disease, the reduction of the ubiquitously expressed SMN additionally elicits non-motoneuron symptoms. Impaired bone development is a key feature of SMA, but it is yet unknown whether this is an indirect functional consequence of muscle weakness or caused by bone-intrinsic mechanisms. Therefore, we radiologically examined SMA patients in a prospective, non-randomized cohort study characterizing bone size and bone mineral density (BMD) and performed equivalent measurements in pre-symptomatic SMA mice. BMD as well as lumbar vertebral body size were significantly reduced in SMA patients. This growth defect but not BMD reduction was confirmed in SMA mice by μCT before the onset of neuromuscular symptoms indicating that it is at least partially independent of neuromuscular degeneration. Interestingly, the number of chondroblasts in the hypertrophic zone of the growth plate was significantly reduced. This was underlined by RNAseq and expression data from developing SMA mice vertebral bodies, which revealed molecular changes related to cell division and cartilage remodeling. Together, these findings suggest a bone intrinsic defect in SMA. This phenotype may not be rescued by novel drugs that enhance SMN levels in the central nervous system only.
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http://dx.doi.org/10.1093/hmg/ddaa145DOI Listing
September 2020

Potent and Prolonged Innate Immune Activation by Enzyme-Responsive Imidazoquinoline TLR7/8 Agonist Prodrug Vesicles.

J Am Chem Soc 2020 07 30;142(28):12133-12139. Epub 2020 Jun 30.

Institute for Experimental Molecular Imaging, Uniklinik RWTH Aachen and Helmholtz Institute for Biomedical Engineering, Faculty of Medicine, RWTH Aachen University, 52074 Aachen, Germany.

Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node .
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http://dx.doi.org/10.1021/jacs.0c01928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116109PMC
July 2020

CXCR4/MIF axis amplifies tumor growth and epithelial-mesenchymal interaction in non-small cell lung cancer.

Cell Signal 2020 09 16;73:109672. Epub 2020 May 16.

Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany; German Center for Lung Research, BREATH, Hannover, Germany; Department of Pulmonology, Hannover Medical School, Hannover, Germany. Electronic address:

Overexpression of C-X-C chemokine receptor type 4 (CXCR4) has been shown in several cancers, including non-small cell lung cancer (NSCLC) and is linked to early metastasis and worse prognosis. The crosstalk between cancer cells and tumor stroma promotes the growth and metastasis and CXCR4 signaling is a key element of this crosstalk. To test the effects of CXCR4 overexpression (CXCR4-OE), we transduced the human NSCLC cell line A549 by using a lentiviral vector. A 3D cell culture model showed generations of tumorspheres and the effects derived by the co-culturing of lung fibroblasts. Using a xenograft mouse model, we also studied the effects of CXCR4-OE in pulmonary cell engraftment and tumor burden in vivo. Our data indicate that CXCR4-OE leads to increased tumorsphere formation and epithelial-mesenchymal transition (EMT). CXCR4-OE by A549 cells resulted in a significant increase in the production of the CXCR4-ligand macrophage migration inhibitory factor (MIF) compared to those transduced with an empty vector (EV) or in which the CXCR4 expression was deleted (KO). In our in vitro system, we did not detect any production of the canonical CXCR4 ligand CXCL12. Autocrine MIF production and CXCR4 signaling are part of a self-perpetuating loop that amplifies tumor growth and EMT. Co-culture with lung fibroblasts further increased tumorsphere formation, partially driven by an increase in IL-6 production. When A549 cells were injected into murine lungs, we observed more abundant and significantly larger tumor lesions in recipients of CXCR4-OE A549 cells compared to those receiving EV or KO cells, consistent with our in vitro findings. Treatment of mice with the MIF antagonist ISO-1 resulted in significantly less tumor burden. In conclusion, our data highlight the role of the CXCR4-OE/MIF/IL-6 axis in epithelial mesenchymal crosstalk and NSCLC progression.
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http://dx.doi.org/10.1016/j.cellsig.2020.109672DOI Listing
September 2020

Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells.

Cell 2020 05 6;181(5):1080-1096.e19. Epub 2020 May 6.

Immunopathology Unit, Institute of Clinical Chemistry and Clinical Pharmacology, Medical Faculty, University Hospital Bonn, University of Bonn, 53127 Bonn, Germany.

Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.
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http://dx.doi.org/10.1016/j.cell.2020.04.022DOI Listing
May 2020

Potent Lymphatic Translocation and Spatial Control Over Innate Immune Activation by Polymer-Lipid Amphiphile Conjugates of Small-Molecule TLR7/8 Agonists.

Angew Chem Int Ed Engl 2019 10 12;58(43):15390-15395. Epub 2019 Sep 12.

Department of Pharmaceutics, Ghent University, Ghent, Belgium.

Uncontrolled systemic inflammatory immune triggering has hampered the clinical translation of several classes of small-molecule immunomodulators, such as imidazoquinoline TLR7/8 agonists for vaccine design and cancer immunotherapy. By taking advantage of the inherent serum-protein-binding property of lipid motifs and their tendency to accumulate in lymphoid tissue, we designed amphiphilic lipid-polymer conjugates that suppress systemic inflammation but provoke potent lymph-node immune activation. This work provides a rational basis for the design of lipid-polymer amphiphiles for optimized lymphoid targeting.
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http://dx.doi.org/10.1002/anie.201905687DOI Listing
October 2019

STING induces early IFN-β in the liver and constrains myeloid cell-mediated dissemination of murine cytomegalovirus.

Nat Commun 2019 06 27;10(1):2830. Epub 2019 Jun 27.

TWINCORE-Centre for Experimental and Clinical Infection Research, a joint venture between the Hanover Medical School and the Helmholtz Centre for Infection Research, Institute for Experimental Infection Research, 30625, Hanover, Germany.

Cytomegalovirus is a DNA-encoded β-herpesvirus that induces STING-dependent type 1 interferon responses in macrophages and uses myeloid cells as a vehicle for dissemination. Here we report that STING knockout mice are as resistant to murine cytomegalovirus (MCMV) infection as wild-type controls, whereas mice with a combined Toll-like receptor/RIG-I-like receptor/STING signaling deficiency do not mount type 1 interferon responses and succumb to the infection. Although STING alone is dispensable for survival, early IFN-β induction in Kupffer cells is STING-dependent and controls early hepatic virus propagation. Infection experiments with an inducible reporter MCMV show that STING constrains MCMV replication in myeloid cells and limits viral dissemination via these cells. By contrast, restriction of viral dissemination from hepatocytes to other organs is independent of STING. Thus, during MCMV infection STING is involved in early IFN-β induction in Kupffer cells and the restriction of viral dissemination via myeloid cells, whereas it is dispensable for survival.
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http://dx.doi.org/10.1038/s41467-019-10863-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597531PMC
June 2019

Self-assembled peptide-poloxamine nanoparticles enable in vitro and in vivo genome restoration for cystic fibrosis.

Nat Nanotechnol 2019 03 28;14(3):287-297. Epub 2019 Jan 28.

Department of Pediatrics, Ludwig Maximilian University of Munich, Munich, Germany.

Developing safe and efficient non-viral delivery systems remains a major challenge for in vivo applications of gene therapy, especially in cystic fibrosis. Unlike conventional cationic polymers or lipids, the emerging poloxamine-based copolymers display promising in vivo gene delivery capabilities. However, poloxamines are invalid for in vitro applications and their in vivo transfection efficiency is still low compared with viral vectors. Here, we show that peptides developed by modular design approaches can spontaneously form compact and monodisperse nanoparticles with poloxamines and nucleic acids via self-assembly. Both messenger RNA and plasmid DNA expression mediated by peptide-poloxamine nanoparticles are greatly boosted in vitro and in the lungs of cystic fibrosis mice with negligible toxicity. Peptide-poloxamine nanoparticles containing integrating vectors enable successful in vitro and in vivo long-term restoration of cystic fibrosis transmembrane conductance regulator deficiency with a safe integration profile. Our dataset provides a new framework for designing non-viral gene delivery systems qualified for in vivo genetic modifications.
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http://dx.doi.org/10.1038/s41565-018-0358-xDOI Listing
March 2019

Spatiotemporally Skewed Activation of Programmed Cell Death Receptor 1-Positive T Cells after Epstein-Barr Virus Infection and Tumor Development in Long-Term Fully Humanized Mice.

Am J Pathol 2019 03 26;189(3):521-539. Epub 2018 Dec 26.

Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; Laboratory of Regenerative Immune Therapies Applied, Excellence Cluster REBIRTH and German Centre for Infection Research, Partner Site Hannover, Hannover, Germany. Electronic address:

Humanized mice developing functional human T cells endogenously and capable of recognizing cognate human leukocyte antigen-matched tumors are emerging as relevant models for studying human immuno-oncology in vivo. Herein, mice transplanted with human CD34 stem cells and bearing endogenously developed human T cells for >15 weeks were infected with an oncogenic recombinant Epstein-Barr virus (EBV), encoding enhanced firefly luciferase and green fluorescent protein. EBV-firefly luciferase was detectable 1 week after infection by noninvasive optical imaging in the spleen, from where it spread rapidly and systemically. EBV infection resulted into a pronounced immunologic skewing regarding the expansion of CD8 T cells in the blood outnumbering the CD4 T and CD19 B cells. Furthermore, within 10 weeks of infections, mice developing EBV-induced tumors had significantly higher absolute numbers of CD8 T cells in lymphatic tissues than mice controlling tumor development. Tumor outgrowth was paralleled by an up-regulation of the programmed cell death receptor 1 on CD8 and CD4 T cells, indicative for T-cell dysfunction. Histopathological examinations and in situ hybridizations for EBV in tumors, spleen, liver, and kidney revealed foci of EBV-infected cells in perivascular regions in close association with programmed cell death receptor 1-positive infiltrating lymphocytes. The strong spatiotemporal correlation between tumor development and the T-cell dysfunctional status seen in this viral oncogenesis humanized model replicates observations obtained in the clinical setting.
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http://dx.doi.org/10.1016/j.ajpath.2018.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902117PMC
March 2019

Signatures of T and B Cell Development, Functional Responses and PD-1 Upregulation After HCMV Latent Infections and Reactivations in Nod.Rag.Gamma Mice Humanized With Cord Blood CD34 Cells.

Front Immunol 2018 22;9:2734. Epub 2018 Nov 22.

Clinic of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.

Human cytomegalovirus (HCMV) latency is typically harmless but reactivation can be largely detrimental to immune compromised hosts. We modeled latency and reactivation using a traceable HCMV laboratory strain expressing the luciferase reporter gene (HCMV/GLuc) in order to interrogate the viral modulatory effects on the human adaptive immunity. Humanized mice with long-term (more than 17 weeks) steady human T and B cell immune reconstitutions were infected with HCMV/GLuc and 7 weeks later were further treated with granulocyte-colony stimulating factor (G-CSF) to induce viral reactivations. Whole body bio-luminescence imaging analyses clearly differentiated mice with latent viral infections vs. reactivations. of vigorous viral reactivations were detectable in liver, lymph nodes and salivary glands. The number of viral genome copies in various tissues increased upon reactivations and were detectable in sorted human CD14, CD169, and CD34 cells. Compared with non-infected controls, mice after infections and reactivations showed higher thymopoiesis, systemic expansion of Th, CTL, Treg, and Tfh cells and functional antiviral T cell responses. Latent infections promoted vast development of memory CD4 T cells while reactivations triggered a shift toward effector T cells expressing PD-1. Further, reactivations prompted a marked development of B cells, maturation of IgG plasma cells, and HCMV-specific antibody responses. Multivariate statistical methods were employed using T and B cell immune phenotypic profiles obtained with cells from several tissues of individual mice. The data was used to identify combinations of markers that could predict an HCMV infection vs. reactivation status. In spleen, but not in lymph nodes, higher frequencies of effector CD4 T cells expressing PD-1 were among the factors most suited to distinguish HCMV reactivations from infections. These results suggest a shift from a T cell dominated immune response during latent infections toward an exhausted T cell phenotype and active humoral immune response upon reactivations. In sum, this novel humanized model combined with advanced analyses highlights a dynamic system clearly specifying the immunological spatial signatures of HCMV latency and reactivations. These signatures can be merged as predictive biomarker clusters that can be applied in the clinical translation of new therapies for the control of HCMV reactivation.
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http://dx.doi.org/10.3389/fimmu.2018.02734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6262073PMC
October 2019

Genetic models reveal origin, persistence and non-redundant functions of IL-17-producing γδ T cells.

J Exp Med 2018 12 19;215(12):3006-3018. Epub 2018 Nov 19.

Institute of Immunology, Hannover Medical School, Hannover, Germany

γδ T cells are highly conserved in jawed vertebrates, suggesting an essential role in the immune system. However, γδ T cell-deficient mice display surprisingly mild phenotypes. We hypothesized that the lack of γδ T cells in constitutive mice is functionally compensated by other lymphocytes taking over genuine γδ T cell functions. To test this, we generated a knock-in model for diphtheria toxin-mediated conditional γδ T cell depletion. In contrast to IFN-γ-producing γδ T cells, IL-17-producing γδ T cells (Tγδ17 cells) recovered inefficiently after depletion, and their niches were filled by expanding Th17 cells and ILC3s. Complementary genetic fate mapping further demonstrated that Tγδ17 cells are long-lived and persisting lymphocytes. Investigating the function of γδ T cells, conditional depletion but not constitutive deficiency protected from imiquimod-induced psoriasis. Together, we clarify that fetal thymus-derived Tγδ17 cells are nonredundant local effector cells in IL-17-driven skin pathology.
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http://dx.doi.org/10.1084/jem.20181439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279411PMC
December 2018

Human Teratoma-Derived Hematopoiesis Is a Highly Polyclonal Process Supported by Human Umbilical Vein Endothelial Cells.

Stem Cell Reports 2018 11 18;11(5):1051-1060. Epub 2018 Oct 18.

Institute of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Straße1, 30625 Hannover, Germany; REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Hematopoietic stem cells (HSCs) ensure a life-long regeneration of the blood system and are therefore an important source for transplantation and gene therapy. The teratoma environment supports the complex development of functional HSCs from human pluripotent stem cells, which is difficult to recapitulate in culture. This model mimics various aspects of early hematopoiesis, but is restricted by the low spontaneous hematopoiesis rate. In this study, a feasible protocol for robust hematopoiesis has been elaborated. We achieved a significant increase of the teratoma-derived hematopoietic population when teratomas were generated in the NSGS mouse, which provides human cytokines, together with co-injection of human umbilical vein endothelial cells. Since little is known about hematopoiesis in teratomas, we addressed localization and clonality of the hematopoietic lineage. Our results indicate that early human hematopoiesis is closely reflected in teratoma formation, and thus highlight the value of this model.
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http://dx.doi.org/10.1016/j.stemcr.2018.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234902PMC
November 2018

Lymph-Node-Targeted Immune Activation by Engineered Block Copolymer Amphiphiles-TLR7/8 Agonist Conjugates.

J Am Chem Soc 2018 10 17;140(43):14300-14307. Epub 2018 Oct 17.

Department of Pharmaceutics and Cancer Research Institute Ghent (CRIG) , Ghent University , Ghent 9000 , Belgium.

Small molecule immuno-modulators such as agonists of Toll-like receptors (TLRs) are attractive compounds to stimulate innate immune cells toward potent antiviral and antitumor responses. However, small molecules rapidly enter the systemic circulation and cause "wasted inflammation". Hence, synthetic strategies to confine their radius of action to lymphoid tissue are of great relevance, to both enhance their efficacy and concomitantly limit toxicity. Here, we demonstrate that covalent conjugation of a small molecule TLR7/8 agonist immunomodulatory to a micelle-forming amphiphilic block copolymer greatly alters the pharmacokinetic profile, resulting in highly efficient lymphatic delivery. Moreover, we designed amphiphilic block copolymers in such a way to form thermodynamically stable micelles through π-π stacking between aromatic moieties, and we engineered the block copolymers to undergo an irreversible amphiphilic to hydrophilic transition in response to the acidic endosomal pH.
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http://dx.doi.org/10.1021/jacs.8b08595DOI Listing
October 2018

Nanoparticle-Conjugate TLR7/8 Agonist Localized Immunotherapy Provokes Safe Antitumoral Responses.

Adv Mater 2018 Nov 1;30(45):e1803397. Epub 2018 Oct 1.

Department of Pharmaceutics, Cancer Research Institute Ghent (CRIG), Ghent University, 9000, Ghent, Belgium.

Localized therapeutic modalities that subvert the tumor microenvironment from immune-suppressive to pro-immunogenic can elicit systemic antitumor immune responses that induce regression of directly treated as well as nontreated distal tumors. A key toward generating robust antitumor T cell responses is the activation of dendritic cells (DCs) in the tumor microenvironment. Treatment with agonists triggering various pattern recognition receptors is very efficient to activate DCs, yet suffers from the induction of serious immune-related adverse effects, which is closely linked to their unfavorable PK/PD profile causing systemic immune activation and cytokine release. Here, it is reported that nanoparticle conjugation of a highly potent TLR7/8 agonist restricts immune activation to the tumor bed and its sentinel lymph nodes without hampering therapeutic antitumor efficacy. On a mechanistic level, it is confirmed that localized treatment with a nanoparticle-conjugated TLR7/8 agonist leads to potent activation of DCs in the sentinel lymph nodes and promotes proliferation of tumor antigen-specific CD8 T cells. Furthermore, therapeutic improvement upon combination with anti-PDL1 checkpoint inhibition and Flt3L, a growth factor that expands and mobilizes DCs from the bone marrow, is demonstrated. The findings provide a rational base for localized tumor engineering by nanomedicine strategies that provide spatial control over immune-activation.
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http://dx.doi.org/10.1002/adma.201803397DOI Listing
November 2018

Dendritic Cell Targeting mRNA Lipopolyplexes Combine Strong Antitumor T-Cell Immunity with Improved Inflammatory Safety.

ACS Nano 2018 10 1;12(10):9815-9829. Epub 2018 Oct 1.

Laboratory of Molecular and Cellular Therapy, Department of Biomedical Sciences , Vrije Universiteit Brussel (VUB) , Brussels 1090 , Belgium.

In vitro transcribed mRNA constitutes a versatile platform to encode antigens and to evoke CD8 T-cell responses. Systemic delivery of mRNA packaged into cationic liposomes (lipoplexes) has proven particularly powerful in achieving effective antitumor immunity in animal models. Yet, T-cell responses to mRNA lipoplexes critically depend on the induction of type I interferons (IFN), potent pro-inflammatory cytokines, which inflict dose-limiting toxicities. Here, we explored an advanced hybrid lipid polymer shell mRNA nanoparticle (lipopolyplex) endowed with a trimannose sugar tree as an alternative delivery vehicle for systemic mRNA vaccination. Like mRNA lipoplexes, mRNA lipopolyplexes were extremely effective in conferring antitumor T-cell immunity upon systemic administration. Conversely to mRNA lipoplexes, mRNA lipopolyplexes did not rely on type I IFN for effective T-cell immunity. This differential mode of action of mRNA lipopolyplexes enabled the incorporation of N1 methyl pseudouridine nucleoside modified mRNA to reduce inflammatory responses without hampering T-cell immunity. This feature was attributed to mRNA lipopolyplexes, as the incorporation of thus modified mRNA into lipoplexes resulted in strongly weakened T-cell immunity. Taken together, we have identified lipopolyplexes containing N1 methyl pseudouridine nucleoside modified mRNA as potent yet low-inflammatory alternatives to the mRNA lipoplexes currently explored in early phase clinical trials.
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http://dx.doi.org/10.1021/acsnano.8b00966DOI Listing
October 2018

Interferon-beta expression and type I interferon receptor signaling of hepatocytes prevent hepatic necrosis and virus dissemination in Coxsackievirus B3-infected mice.

PLoS Pathog 2018 08 3;14(8):e1007235. Epub 2018 Aug 3.

Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research, Braunschweig, and the Hannover Medical School, Hannover, Germany.

During Coxsackievirus B3 (CVB3) infection hepatitis is a potentially life threatening complication, particularly in newborns. Studies with type I interferon (IFN-I) receptor (IFNAR)-deficient mice revealed a key role of the IFN-I axis in the protection against CVB3 infection, whereas the source of IFN-I and cell types that have to be IFNAR triggered in order to promote survival are still unknown. We found that CVB3 infected IFN-β reporter mice showed effective reporter induction, especially in hepatocytes and only to a minor extent in liver-resident macrophages. Accordingly, upon in vitro CVB3 infection of primary hepatocytes from murine or human origin abundant IFN-β responses were induced. To identify sites of IFNAR-triggering we performed experiments with Mx reporter mice, which upon CVB3 infection showed massive luciferase induction in the liver. Immunohistological studies revealed that during CVB3 infection MX1 expression of hepatocytes was induced primarily by IFNAR-, and not by IFN-III receptor (IFNLR)-triggering. CVB3 infection studies with primary human hepatocytes, in which either the IFN-I or the IFN-III axis was inhibited, also indicated that primarily IFNAR-, and to a lesser extent IFNLR-triggering was needed for ISG induction. Interestingly, CVB3 infected mice with a hepatocyte-specific IFNAR ablation showed severe liver cell necrosis and ubiquitous viral dissemination that resulted in lethal disease, as similarly detected in classical IFNAR-/- mice. In conclusion, we found that during CVB3 infection hepatocytes are major IFN-I producers and that the liver is also the organ that shows strong IFNAR-triggering. Importantly, hepatocytes need to be IFNAR-triggered in order to prevent virus dissemination and to assure survival. These data are compatible with the hypothesis that during CVB3 infection hepatocytes serve as important IFN-I producers and sensors not only in the murine, but also in the human system.
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http://dx.doi.org/10.1371/journal.ppat.1007235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107283PMC
August 2018

Hyaluronic Acid Conjugates of TLR7/8 Agonists for Targeted Delivery to Secondary Lymphoid Tissue.

Bioconjug Chem 2018 08 17;29(8):2741-2754. Epub 2018 Jul 17.

Department of Medicinal Chemistry , University of Minnesota , Minneapolis , Minnesota 55455 , United States.

Immunogens carried in lymphatic fluid drain via afferent vessels into regional lymph nodes and facilitate the efficient induction of appropriate immune responses. The lymphatic system possesses receptors recognizing hyaluronic acid (HA). Covalent conjugates of small-molecule TLR7/8 agonists with HA are entirely devoid of immunostimulatory activity in vitro. In murine models of immunization, however, such conjugates traffic to lymph nodes, where they are "unmasked", releasing the small molecule TLR7/8 agonist from the carrier polysaccharide. The resulting focal immunostimulation is manifested in potent adjuvantic effects with negligible systemic exposure. The efficient delivery of immunogens has been a major challenge in the development of subunit vaccines, and enhancing targeted delivery of immunogens to secondary lymphoid organs might be a promising approach for improving vaccine efficacy, as well as safety.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00386DOI Listing
August 2018

Type I interferon receptor signaling delays Kupffer cell replenishment during acute fulminant viral hepatitis.

J Hepatol 2018 04 21;68(4):682-690. Epub 2017 Dec 21.

Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Hanover Medical School and the Helmholtz Centre for Infection Research, Brunswick, Germany. Electronic address:

Background & Aim: Virus-induced fulminant hepatitis is a major cause of acute liver failure. During acute viral hepatitis the impact of type I interferon (IFN-I) on myeloid cells, including liver-resident Kupffer cells (KC), is only partially understood. Herein, we dissected the impact of locally induced IFN-I responses on myeloid cell function and hepatocytes during acute liver inflammation.

Methods: Two different DNA-encoded viruses, vaccinia virus (VACV) and murine cytomegalovirus (MCMV), were studied. In vivo imaging was applied to visualize local IFN-β induction and IFN-I receptor (IFNAR) triggering in VACV-infected reporter mice. Furthermore, mice with a cell type-selective IFNAR ablation were analyzed to dissect the role of IFNAR signaling in myeloid cells and hepatocytes. Experiments with Cx3cr1 mice revealed the origin of reconstituted KC. Finally, mixed bone marrow chimeric mice were studied to specifically analyze the effect of IFNAR triggering on liver infiltrating monocytes.

Results: VACV infection induced local IFN-β responses, which lead to IFNAR signaling primarily within the liver. IFNAR triggering was needed to control the infection and prevent fulminant hepatitis. The severity of liver inflammation was independent of IFNAR triggering of hepatocytes, whereas IFNAR triggering of myeloid cells protected from excessive inflammation. Upon VACV or MCMV infection KC disappeared, whereas infiltrating monocytes differentiated to KC afterwards. During IFNAR triggering such replenished monocyte-derived KC comprised more IFNAR-deficient than -competent cells in mixed bone marrow chimeric mice, whereas after the decline of IFNAR triggering both subsets showed an even distribution.

Conclusion: Upon VACV infection IFNAR triggering of myeloid cells, but not of hepatocytes, critically modulates acute viral hepatitis. During infection with DNA-encoded viruses IFNAR triggering of liver-infiltrating blood monocytes delays the development of monocyte-derived KC, pointing towards new therapeutic strategies for acute viral hepatitis.

Lay Summary: Viral infection can cause fulminant hepatitis, which in turn is a major cause of acute liver failure. Herein, we aimed to study the role of type 1 interferon responses in acute viral hepatitis. We identified that during infection with DNA-encoded viruses, type 1 interferon receptor triggering of blood monocytes delays the development of monocyte-derived Kupffer cells. This points to new therapeutic strategies for acute viral hepatitis.
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http://dx.doi.org/10.1016/j.jhep.2017.11.029DOI Listing
April 2018

Natural killer cell-intrinsic type I IFN signaling controls Klebsiella pneumoniae growth during lung infection.

PLoS Pathog 2017 Nov 7;13(11):e1006696. Epub 2017 Nov 7.

Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter (VBC), Vienna, Austria.

Klebsiella pneumoniae is a significant cause of nosocomial pneumonia and an alarming pathogen owing to the recent isolation of multidrug resistant strains. Understanding of immune responses orchestrating K. pneumoniae clearance by the host is of utmost importance. Here we show that type I interferon (IFN) signaling protects against lung infection with K. pneumoniae by launching bacterial growth-controlling interactions between alveolar macrophages and natural killer (NK) cells. Type I IFNs are important but disparate and incompletely understood regulators of defense against bacterial infections. Type I IFN receptor 1 (Ifnar1)-deficient mice infected with K. pneumoniae failed to activate NK cell-derived IFN-γ production. IFN-γ was required for bactericidal action and the production of the NK cell response-amplifying IL-12 and CXCL10 by alveolar macrophages. Bacterial clearance and NK cell IFN-γ were rescued in Ifnar1-deficient hosts by Ifnar1-proficient NK cells. Consistently, type I IFN signaling in myeloid cells including alveolar macrophages, monocytes and neutrophils was dispensable for host defense and IFN-γ activation. The failure of Ifnar1-deficient hosts to initiate a defense-promoting crosstalk between alveolar macrophages and NK cell was circumvented by administration of exogenous IFN-γ which restored endogenous IFN-γ production and restricted bacterial growth. These data identify NK cell-intrinsic type I IFN signaling as essential driver of K. pneumoniae clearance, and reveal specific targets for future therapeutic exploitations.
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http://dx.doi.org/10.1371/journal.ppat.1006696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675380PMC
November 2017

Escape of Tick-Borne Flavivirus from 2'--Methylated Nucleoside Antivirals Is Mediated by a Single Conservative Mutation in NS5 That Has a Dramatic Effect on Viral Fitness.

J Virol 2017 Nov 13;91(21). Epub 2017 Oct 13.

Department of Virology, Veterinary Research Institute, Brno, Czech Republic

Tick-borne encephalitis virus (TBEV) causes a severe and potentially fatal neuroinfection in humans. Despite its high medical relevance, no specific antiviral therapy is currently available. Here we demonstrate that treatment with a nucleoside analog, 7-deaza-2'--methyladenosine (7-deaza-2'-CMA), substantially improved disease outcomes, increased survival, and reduced signs of neuroinfection and viral titers in the brains of mice infected with a lethal dose of TBEV. To investigate the mechanism of action of 7-deaza-2'-CMA, two drug-resistant TBEV clones were generated and characterized. The two clones shared a signature amino acid substitution, S603T, in the viral NS5 RNA-dependent RNA polymerase (RdRp) domain. This mutation conferred resistance to various 2'--methylated nucleoside derivatives, but no cross-resistance was seen with other nucleoside analogs, such as 4'--azidocytidine and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187). All-atom molecular dynamics simulations revealed that the S603T RdRp mutant repels a water molecule that coordinates the position of a metal ion cofactor as 2'--methylated nucleoside analogs approach the active site. To investigate its phenotype, the S603T mutation was introduced into a recombinant TBEV strain (Oshima-IC) generated from an infectious cDNA clone and into a TBEV replicon that expresses a reporter luciferase gene (Oshima-REP-luc2A). The mutants were replication impaired, showing reduced growth and a small plaque size in mammalian cell culture and reduced levels of neuroinvasiveness and neurovirulence in rodent models. These results indicate that TBEV resistance to 2'--methylated nucleoside inhibitors is conferred by a single conservative mutation that causes a subtle atomic effect within the active site of the viral NS5 RdRp and is associated with strong attenuation of the virus. This study found that the nucleoside analog 7-deaza-2'--methyladenosine (7-deaza-2'-CMA) has high antiviral activity against tick-borne encephalitis virus (TBEV), a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. Treating mice infected with a lethal dose of TBEV with 7-deaza-2'-CMA resulted in significantly higher survival rates and reduced the severity of neurological signs of the disease. Thus, this compound shows promise for further development as an anti-TBEV drug. It is important to generate drug-resistant mutants to understand how the drug works and to develop guidelines for patient treatment. We generated TBEV mutants that were resistant not only to 7-deaza-2'-CMA but also to a broad range of other 2'--methylated antiviral medications. Our findings suggest that combination therapy may be used to improve treatment and reduce the emergence of drug-resistant viruses during nucleoside analog therapy for TBEV infection.
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http://dx.doi.org/10.1128/JVI.01028-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640847PMC
November 2017

CTLA-4-mediated posttranslational modifications direct cytotoxic T-lymphocyte differentiation.

Cell Death Differ 2017 10 23;24(10):1739-1749. Epub 2017 Jun 23.

Department of Experimental Pediatrics, University Hospital, Health Campus Immunology, Infectiology and Inflammation, Otto-von-Guericke-University, Magdeburg 39120, Germany.

The blockade of inhibitory receptors such as CTLA-4 (CD152) is being used as immune-checkpoint therapy, offering a powerful strategy to restore effective immune responses against tumors. To determine signal components that are induced under the control of CTLA-4 we analyzed activated murine CD8 T cells by quantitative proteomics. Accurate mass spectrometry revealed that CTLA-4 engagement led to central changes in the phosphorylation of proteins involved in T-cell differentiation. Beside other targets, we discovered a CTLA-4-mediated induction of the translational inhibitor programmed cell death-4 (PDCD4) as a result of FoxO1 nuclear re-localization. PDCD4 further bound a distinct set of mRNAs including Glutaminase, which points out a critical role for CTLA-4 in CD8 T-cell metabolism. Consequently, PDCD4-deficient cytotoxic T-lymphocytes (CTLs) expressed increased amounts of otherwise repressed effector molecules and ultimately led to superior control of tumor growth in vivo. These findings reveal a novel CTLA-4-mediated pathway to attenuate CTLs and indicate the importance of post-transcriptional mechanisms in the regulation of anti-tumor immune responses.
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http://dx.doi.org/10.1038/cdd.2017.102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5596418PMC
October 2017

Degradable magnesium implant-associated infections by bacterial biofilms induce robust localized and systemic inflammatory reactions in a mouse model.

Biomed Mater 2017 Sep 13;12(5):055006. Epub 2017 Sep 13.

Central Facility for Microscopy, Animal Experimental Unit, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany.

Biomaterial-associated Pseudomonas aeruginosa biofilm infections constitute a cascade of host immune reactions ultimately leading to implant failure. Due to the lack of relevant in vivo biofilm models, the majority of the studies report host immune responses to free-living or planktonic bacteria, while bacteria in clinical situations live more frequently as biofilm communities than as single cells. The present study investigated host immune responses to biomaterial-associated P. aeruginosa biofilms in a clinically relevant mouse model. Previously, we reported metallic magnesium, a prospective biodegradable implant, to be permissive for bacterial biofilm in vivo even though it exhibits antibacterial properties in vitro. Therefore, magnesium was employed as biomaterial to investigate in vivo biofilm formation and associated host immune responses by using two P. aeruginosa strains and two mouse strains. P. aeruginosa formed biofilm on subcutaneously implanted magnesium disks. Non-invasive in vivo imaging indicated transient inflammatory responses at control sites, whereas robust prolonged interferon-β (IFN-β) expression was observed from biofilm in a transgenic animal reporter. Furthermore, immunohistology and electron microscopic results showed that bacterial biofilms were located in 2D immediately on the implant surface and at a short distance in the adjacent tissue. These biofilms were surrounded by inflammatory cells (mainly polymorphonuclear cells) compared to the controls. Interestingly, even though the number of live bacteria in various organs remained below detectable levels, splenomegaly indicated systemic inflammatory processes. Overall, these findings confirmed the resistance of biofilm infections in vivo to potentially antibacterial properties of magnesium degradation products. In vivo imaging and histology indicated the induction of both local and systemic host inflammatory responses to P. aeruginosa biofilms. Even though the innate host immune defenses could not eliminate the local infection for up to two weeks, there was no apparent systemic bacteremia and all the animals investigated survived the infection.
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http://dx.doi.org/10.1088/1748-605X/aa7667DOI Listing
September 2017

The murine cytomegalovirus M35 protein antagonizes type I IFN induction downstream of pattern recognition receptors by targeting NF-κB mediated transcription.

PLoS Pathog 2017 May 25;13(5):e1006382. Epub 2017 May 25.

Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.

The type I interferon (IFN) response is imperative for the establishment of the early antiviral immune response. Here we report the identification of the first type I IFN antagonist encoded by murine cytomegalovirus (MCMV) that shuts down signaling following pattern recognition receptor (PRR) sensing. Screening of an MCMV open reading frame (ORF) library identified M35 as a novel and strong negative modulator of IFNβ promoter induction following activation of both RNA and DNA cytoplasmic PRR. Additionally, M35 inhibits the proinflammatory cytokine response downstream of Toll-like receptors (TLR). Using a series of luciferase-based reporters with specific transcription factor binding sites, we determined that M35 targets NF-κB-, but not IRF-mediated, transcription. Expression of M35 upon retroviral transduction of immortalized bone marrow-derived macrophages (iBMDM) led to reduced IFNβ transcription and secretion upon activation of stimulator of IFN genes (STING)-dependent signaling. On the other hand, M35 does not antagonize interferon-stimulated gene (ISG) 56 promoter induction or ISG transcription upon exogenous stimulation of the type I IFN receptor (IFNAR). M35 is present in the viral particle and, upon MCMV infection of fibroblasts, is immediately shuttled to the nucleus where it exerts its immunomodulatory effects. Deletion of M35 from the MCMV genome and hence from the viral particle resulted in elevated type I IFN transcription and secretion in vitro and in vivo. In the absence of M35, lower viral titers are observed during acute infection of the host, and productive infection in the salivary glands was not detected. In conclusion, the M35 protein is released by MCMV immediately upon infection in order to deftly inhibit the antiviral type I IFN response by targeting NF-κB-mediated transcription. The identification of this novel viral protein reinforces the importance of timely countermeasures in the complex relationship between virus and host.
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http://dx.doi.org/10.1371/journal.ppat.1006382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444856PMC
May 2017