cGAS-like receptors sense RNA and control 3'2'-cGAMP signalling in Drosophila.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger cG[2'-5']pA[3'-5']p (2'3'-cGAMP) and controls activation of innate immunity in mammalian cells1-5. Animal genomes typically encode multiple proteins with predicted homology to cGAS6-10, but the function of these uncharacterized enzymes is unknown. Here we show that cGAS-like receptors (cGLRs) are innate immune sensors that are capable of recognizing divergent molecular patterns and catalysing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signalling core shared with cGAS and a diversified primary ligand-binding surface modified with notable insertions and deletions. We demonstrate that surface remodelling of cGLRs enables altered ligand specificity and used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. We show that RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3'-5']pA[2'-5']p (3'2'-cGAMP). A crystal structure of Drosophila stimulator of interferon genes (dSTING) in complex with 3'2'-cGAMP explains selective isomer recognition, and 3'2'-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, our results establish cGLRs as a diverse family of metazoan pattern recognition receptors.

Selective reconstitution of IFN­Î³ gene function in Ncr1+ NK cells is sufficient to control systemic vaccinia virus infection.

IFN-γ is an enigmatic cytokine that shows direct anti-viral effects, confers upregulation of MHC-II and other components relevant for antigen presentation, and that adjusts the composition and balance of complex cytokine responses. It is produced during immune responses by innate as well as adaptive immune cells and can critically affect the course and outcome of infectious diseases, autoimmunity, and cancer. To selectively analyze the function of innate immune cell-derived IFN-γ, we generated conditional IFN-γOFF mice, in which endogenous IFN-γ expression is disrupted by a loxP flanked gene trap cassette inserted into the first intron of the IFN-γ gene. IFN-γOFF mice were intercrossed with Ncr1-Cre or CD4-Cre mice that express Cre mainly in NK cells (IFN-γNcr1-ON mice) or T cells (IFN-γCD4-ON mice), respectively. Rosa26RFP reporter mice intercrossed with Ncr1-Cre mice showed selective RFP expression in more than 80% of the NK cells, while upon intercrossing with CD4-Cre mice abundant RFP expression was detected in T cells, but also to a minor extent in other immune cell subsets. Previous studies showed that IFN-γ expression is needed to promote survival of vaccinia virus (VACV) infection. Interestingly, during VACV infection of wild type and IFN-γCD4-ON mice two waves of serum IFN-γ were induced that peaked on day 1 and day 3/4 after infection. Similarly, VACV infected IFN-γNcr1-ON mice mounted two waves of IFN-γ responses, of which the first one was moderately and the second one profoundly reduced when compared with WT mice. Furthermore, IFN-γNcr1-ON as well as IFN-γCD4-ON mice survived VACV infection, whereas IFN-γOFF mice did not. As expected, ex vivo analysis of splenocytes derived from VACV infected IFN-γNcr1-ON mice showed IFN-γ expression in NK cells, but not T cells, whereas IFN-γOFF mice showed IFN-γ expression neither in NK cells nor T cells. VACV infected IFN-γNcr1-ON mice mounted normal cytokine responses, restored neutrophil accumulation, and showed normal myeloid cell distribution in blood and spleen. Additionally, in these mice normal MHC-II expression was detected on peripheral macrophages, whereas IFN-γOFF mice did not show MHC-II expression on such cells. In conclusion, upon VACV infection Ncr1 positive cells including NK cells mount two waves of early IFN-γ responses that are sufficient to promote the induction of protective anti-viral immunity.

TRADD protein is an essential component of the RIG-like helicase antiviral pathway.

Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif (also known as IPS-1, MAVS, or VISA), the activation of the transcription factors NF-kappaB and IRF3, which collaborate to induce an antiviral type I interferon (IFN) response. FADD and RIP1, known as mediators of death-receptor signaling, are implicated in this antiviral pathway; however, the link between death-receptor and antiviral signaling is not known. Here we showed that TRADD, a crucial adaptor of tumor necrosis factor receptor (TNFRI), was important in RIG-like helicase (RLH)-mediated signal transduction. TRADD is recruited to Cardif and orchestrated complex formation with the E3 ubiquitin ligase TRAF3 and TANK and with FADD and RIP1, leading to the activation of IRF3 and NF-kappaB. Loss of TRADD prevented Cardif-dependent activation of IFN-beta, reduced the production of IFN-beta in response to RNA viruses, and enhanced vesicular stomatitis virus replication. Thus, TRADD is not only an essential component of proinflammatory TNFRI signaling, but is also required for RLH-Cardif-dependent antiviral immune responses.

A specific CD4 epitope bound by tregalizumab mediates activation of regulatory T cells by a unique signaling pathway.

CD4(+)CD25(+) regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing.

Histamine H1- and H4-receptor expression in human colon-derived cell lines.

In previous studies, we demonstrated the involvement of H4R in inflammatory bowel disease (IBD) and IBD-associated colon cancer in mice and could ascribe H4R-mediated histamine function to colon epithelial cells. The transferability of obtained data to humans is however lacking. Functional expression of H4R on colon epithelial cells is a prerequisite to pursue the hypothesis of involvement of H4R in carcinogenesis. Thus, we here compared the expression of histamine receptor subtypes in a series of cell lines. Out of these, three colon-derived cell lines displaying different combinations of H1R and H4R expression were submitted to functional analyses. Human hematopoietic HMC-1, HL-60, and U937, lung-derived A549 and Calu-3, and colorectal LoVo, SW 480, Caco-2, HT-29, and HCT116 cells were included in the study. mRNA expression was quantified by RT-qPCR. For functional analyses, Caco-2, HT-29, and HCT116 cells were treated by incubation with 1 - 10 µM histamine in the presence or absence of selective histamine receptor antagonists. Calcium mobilization, cAMP accumulation, and cell proliferation were measured by fluorimetry, mass spectrometry, and real-time bioimpedance measurements, respectively. Histamine receptor expression was heterogeneous in the cell lines tested. In most cell lines, we detected H1R mRNA while H4R mRNAs were found only occasionally. The colon-derived epithelial cell lines LoVo, SW480, and HT-29 expressed H1R mRNA exclusively, while in HCT116 cells H1R and H4R mRNAs and in CaCo-2 H2R mRNA were detectable. Subsequent functional analyses in HT29, Caco-2, and HCT116 cells, however, indicated that only HT-29 responded to histamine stimulation, by means of H1R. For a detailed analysis of histamine receptor function, esp. that of H1R and H4R, in human colon-derived cell lines, the cell lines tested here are not fully convenient unless genetically modified.

Type I IFN-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection.

The swift production of type I IFNs is one of the fundamental aspects of innate immune responses against viruses. Plasmacytoid dendritic cell-derived type I IFNs are of prime importance for the initial control of highly cytopathic viruses such as the mouse hepatitis virus (MHV). The aim of this study was to determine the major target cell populations of this first wave of type I IFNs. Generation of bone marrow-chimeric mice expressing the type I IFN receptor (IFNAR) on either hemopoietic or non-bone marrow-derived cells revealed that the early control of MHV depended mainly on IFNAR expression on hemopoietic cells. To establish which cell population responds most efficiently to type I IFNs, mice conditionally deficient for the IFNAR on different leukocyte subsets were infected with MHV. This genetic analysis revealed that IFNAR expression on LysM+ macrophages and CD11c+ dendritic cells was most important for the early containment of MHV within secondary lymphoid organs and to prevent lethal liver disease. This study identifies type I IFN-mediated cross-talk between plasmacytoid dendritic cells on one side and macrophages and conventional dendritic cells on the other, as an essential cellular pathway for the control of fatal cytopathic virus infection.

The Dual Role of a Polyvalent IgM/IgA-Enriched Immunoglobulin Preparation in Activating and Inhibiting the Complement System.

Activation of the complement system is important for efficient clearance of a wide variety of pathogens via opsonophagocytosis, or by direct lysis via complement-dependent cytotoxicity (CDC). However, in severe infections dysregulation of the complement system contributes to hyperinflammation. The influence of the novel IgM/IgA-enriched immunoglobulin preparation trimodulin on the complement pathway was investigated in in vitro opsonophagocytosis, binding and CDC assays. Immunoglobulin levels before and after trimodulin treatment were placed in relation to complement assessments in humans. In vitro, trimodulin activates complement and induces opsonophagocytosis, but also interacts with opsonins C3b, C4b and anaphylatoxin C5a in a concentration-dependent manner. This was not observed for standard intravenous IgG preparation (IVIg). Accordingly, trimodulin, but not IVIg, inhibited the downstream CDC pathway and target cell lysis. If applied at a similar concentration range in healthy subjects, trimodulin treatment resulted in C3 and C4 consumption in a concentration-dependent manner, which was extended in patients with severe community-acquired pneumonia. Complement consumption is found to be dependent on underlying immunoglobulin levels, particularly IgM, pinpointing their regulative function in humans. IgM/IgA provide a balancing effect on the complement system. Trimodulin may enhance phagocytosis and opsonophagocytosis in patients with severe infections and prevent excessive pathogen lysis and release of harmful anaphylatoxins.

Sfaira accelerates data and model reuse in single cell genomics.

Single-cell RNA-seq datasets are often first analyzed independently without harnessing model fits from previous studies, and are then contextualized with public data sets, requiring time-consuming data wrangling. We address these issues with sfaira, a single-cell data zoo for public data sets paired with a model zoo for executable pre-trained models. The data zoo is designed to facilitate contribution of data sets using ontologies for metadata. We propose an adaption of cross-entropy loss for cell type classification tailored to datasets annotated at different levels of coarseness. We demonstrate the utility of sfaira by training models across anatomic data partitions on 8 million cells.

Wild boars as an important reservoir for foodborne pathogens.

One hundred fifty-three wild boars shot in the canton of Geneva, Switzerland, were studied for the occurrence of foodborne pathogens. Tonsils and fecal samples of the animals were examined using real-time polymerase chain reaction, enzyme-linked fluorescent immunoassay, and cultural methods. The detection rate of Salmonella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, stx-positive Escherichia coli, and Listeria monocytogenes was 12%, 35%, 20%, 9%, and 17%, respectively, when tonsil samples were studied. Only Y. enterocolitica (5%) and L. monocytogenes (1%) were detected in fecal samples. None of the samples was positive for Campylobacter spp. Females (71%) and young animals (61%) carried more frequently one or more pathogens than males (53%) and older ones (44%). In total, 8 Salmonella spp., 14 Y. enterocolitica, 4 Y. pseudotuberculosis, and 26 L. monocytogenes strains were further characterized. Most of the Salmonella spp. strains were of serotype Salmonella Enteritidis (75%) followed by serotypes Salmonella Stourbridge (13%) and Salmonella Veneziana (13%). L. monocytogenes strains belonged to serotypes 1/2a (42%), 1/2b (19%), and 4b (38%). Serotypes O:3 (36%), O:5,27 (21%), and O:9 (29%) were identified among Y. enterocolitica strains and serotypes O:1 (75%) and O:2 (25%) among Y. pseudotuberculosis strains. This study shows that wild boars are frequent carriers of foodborne pathogens. High wild boar densities and increasing popularity of outdoor ranging of pigs may intensify the risk of transmission of these pathogens to fattening pigs.

Functional relevance of in vivo half antibody exchange of an IgG4 therapeutic antibody-drug conjugate.

An increasing number of monoclonal antibodies and derivatives such as antibody-drug conjugates (ADC) are of the IgG1 and IgG4 isotype with distinct structural and functional properties. In cases where antibody-mediated cytotoxicity is not desired, IgG4 is often used, as its Fc region is relatively poor at inducing antibody-dependent cell-mediated or complement-dependent cytotoxicity. IgG4 ADCs with highly cytotoxic drugs against proliferating target cells but which lack or have diminished antibody effector functions against quiescent cells may have a favorable safety profile compared to IgG1. Another unique property of the IgG4 subclass is the capability to exchange half antibodies in vivo creating randomly bispecific antibodies. To investigate the functional properties of process-derived antibody species, and determine the influence of shuffling on the therapeutic efficacy, several model antibodies on the basis of the anti-CD138 antibody-drug conjugate BT062 (Indatuximab ravtansine) were generated: (I) A wild type nBT062, (II) a stable nBT062 comprising mutations to prevent half-antibody exchange, (III) a half nBT062 lacking covalent binding between two heavy chains and (IV) a stabilized, bispecific nBT062-natalizumab antibody with a second, monovalent specificity against CD49d. All nBT062 model variants were capable of CD138-specific binding and antigen-mediated internalization into cells. Furthermore, all nBT062 models inhibited tumor growth in vitro after conjugation with the maytansinoid DM4. The in vivo effects of the different molecular variants were assessed in the MAXF1322 xenograft model. The bispecific nBT062-natalizumab-DM4 demonstrated the least efficacy and was only moderately active even without the co-administration of a human IgG preparation. Wild type, stable and half nBT062-DM4 models demonstrated great anti-tumor activities. The efficacy of wild type and half nBT062-DM4 was reduced in the presence of IgG, while stable nBT062-DM4 was only marginally influenced. These pre-clinical data demonstrate the advantage of introducing half-antibody exchange-preventing mutations into therapeutic IgG4-based antibody drug-conjugates.

Tregalizumab - A Monoclonal Antibody to Target Regulatory T Cells.

Regulatory T cells (Tregs) represent a subpopulation of CD4(+) T cells, which are essential for the maintenance of immunological tolerance. The absence or dysfunction of Tregs can lead to autoimmunity and allergies. The restoration of functional Tregs and/or Treg cell numbers represents a novel and attractive approach for the treatment of autoimmune diseases, e.g., rheumatoid arthritis (RA). The CD4 cell surface receptor is a target for modulation of T cell function. Monoclonal antibodies (mAbs) against CD4 have previously been tested for the treatment of autoimmune diseases, including RA. Furthermore, in model systems, anti-CD4 antibodies are able to induce tolerance and mediate immunomodulatory effects through a variety of mechanisms. Despite the availability of innovative and effective therapies for RA, many patients still have persistently active disease or experience adverse events that can limit use. A growing body of evidence suggests that Treg modulation could offer a new therapeutic strategy in RA and other autoimmune disorders. Here, we describe tregalizumab (BT-061), which is a novel, non-depleting IgG1 mAb that binds to a unique epitope of CD4. Tregalizumab represents the first humanized anti-CD4 mAb that selectively induces Treg activation.

Vaccination against prion diseases.

Prion diseases are a group of fatal neurodegenerative conditions that affect humans and a wide variety of animals. To date there is no therapeutic or prophylactic approach against prion diseases available. The causative infectious agent is the prion, also termed PrP(Sc), which is a pathological conformer of the cellular prion protein PrP(C). As passive immunization studies with PrP(C)-specific antibodies indicated that immunotherapeutic strategies can prevent prion replication, researchers are now aiming at the development of active prion vaccines.

Pancreatic endoprostheses in chronic pancreatitis: criteria to predict stent occlusion.

BACKGROUND: Good clinical results of main pancreatic duct (MPD) stent placement in chronic pancreatitis (CP) are clouded by early stent occlusion. The aim of this study was to increase knowledge about stent occlusion and its effects on clinical symptoms, and to define criteria that enable the prediction of clogging. METHODS: A total of 100 pancreatic endoprostheses of 47 patients (32 men, 15 women; mean age, 53 years; standard deviation, 9 years) with CP were bench tested by simulating the pathophysiologically increased MPD pressure. The main study parameter was the reduction of water flow through clogged stents in comparison with native endoprostheses of identical type, length, and diameter. Major stent occlusion was defined as flow reduction by > or = 75%. The association between time to stent occlusion and stent- or patient-related variables was evaluated. RESULTS: Occlusion took place in nearly all endoprostheses (97%). No significant association of occlusion with clinical or blood parameters was found. Multifactorial analysis proved 4 risk factors for major stent occlusion: (A) stent diameter > 8.5F, (B) stent length > 8 cm, (C) female gender, (D) exocrine pancreatic insufficiency that required regular oral enzyme supplementation. According to the relative risk, these factors were given the following scores: A, 3 points; B to D, 2 points. Stents in patients with a score sum > 5 showed a significantly higher risk of major stent occlusion within 90 days. CONCLUSIONS: Stent clogging in CP seems to be an inevitable phenomenon. Because clinical and laboratory data do not reliably indicate clogging, stent removal or exchange should be performed in high-risk patients (score sum > 5) within 3 months.