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Antigen-specific priming of T cells results in the activation of T cells that exert effector functions by interaction of their T-cell receptor (TCR) with the corresponding self-MHC molecule presenting a peptide on the surface of a target cell. Such antigen-specific T cells potentially can also interact with peptide-MHC complexes that contain peptides from unrelated antigens, a phenomenon that often is referred to as heterologous immunity. For example, some individuals that were pre-immunized against an allergen, could subsequently mount better anti-viral T-cell responses than non-allergic individuals. So far only few peptide pairs that experimentally have been shown to provoke heterologous immunity were  identified, and available prediction tools that can identify potential candidates are imprecise. We developed the MORITS algorithm to rapidly screen large lists of peptides for sequence similarities, while giving enhanced consideration to peptide residues presented by MHC that are particularly relevant for TCR interactions. In combination with established peptide-MHC binding prediction tools, the MORITS algorithm revealed peptide similarities between the SARS-CoV-2 proteome and certain allergens. The method outperformed previously published workflows and may help to identify novel pairs of peptides that mediate heterologous immune responses.

Conventional Ly6C hi monocytes have developmental plasticity for a spectrum of differentiated phagocytes. Here we show, using conditional deletion strategies in a mouse model of Toll-like receptor (TLR) 7-induced inflammation, that the spectrum of developmental cell fates of Ly6C hi monocytes, and the resultant inflammation, is coordinately regulated by TLR and Notch signaling. Cell-intrinsic Notch2 and TLR7-Myd88 pathways independently and synergistically promote Ly6C lo patrolling monocyte development from Ly6C hi monocytes under inflammatory conditions, while impairment in either signaling axis impairs Ly6C lo monocyte development. At the same time, TLR7 stimulation in the absence of functional Notch2 signaling promotes resident tissue macrophage gene expression signatures in monocytes in the blood and ectopic differentiation of Ly6C hi monocytes into macrophages and dendritic cells, which infiltrate the spleen and major blood vessels and are accompanied by aberrant systemic inflammation. Thus, Notch2 is a master regulator of Ly6C hi monocyte cell fate and inflammation in response to TLR signaling.

Human cytomegalovirus (HCMV) infections of healthy individuals are mostly unnoticed and result in viral latency. However, HCMV can also cause devastating disease, e.g., upon reactivation in immunocompromised patients. Yet, little is known about human immune cell sensing of DNA-encoded HCMV. Recent studies indicated that during viral infection the cyclic GMP/AMP synthase (cGAS) senses cytosolic DNA and catalyzes formation of the cyclic di-nucleotide cGAMP, which triggers stimulator of interferon genes (STING) and thus induces antiviral type I interferon (IFN-I) responses. We found that plasmacytoid dendritic cells (pDC) as well as monocyte-derived DC and macrophages constitutively expressed cGAS and STING. HCMV infection further induced cGAS, whereas STING expression was only moderately affected. Although pDC expressed particularly high levels of cGAS, and the cGAS/STING axis was functional down-stream of STING, as indicated by IFN-I induction upon synthetic cGAMP treatment, pDC were not susceptible to HCMV infection and mounted IFN-I responses in a TLR9-dependent manner. Conversely, HCMV infected monocyte-derived cells synthesized abundant cGAMP levels that preceded IFN-I production and that correlated with the extent of infection. CRISPR/Cas9- or siRNA-mediated cGAS ablation in monocytic THP-1 cells and primary monocyte-derived cells, respectively, impeded induction of IFN-I responses following HCMV infection. Thus, cGAS is a key sensor of HCMV for IFN-I induction in primary human monocyte-derived DC and macrophages.

Background The emergence of the SARS-CoV-2 virus has highlighted the importance of genomic epidemiology in understanding the evolution of pathogens and guiding public health interventions. The Omicron variant in particular has underscored the role of epistasis in the evolution of lineages with both higher infectivity and immune escape, and therefore the necessity to update surveillance pipelines to detect them early on. Results In this study, we apply a method based on mutual information between positions in a multiple sequence alignment, which is capable of scaling up to millions of samples. We show how it can reliably predict known experimentally validated epistatic interactions, even when using as little as 10,000 sequences, which opens the possibility of making it a near real-time prediction system. We test this possibility by modifying the method to account for the sample collection date and apply it retrospectively to multiple sequence alignments for each month between March 2020 and March 2023. We detected a cornerstone epistatic interaction in the Spike protein between codons 498 and 501 as soon as seven samples with a double mutation were present in the dataset, thus demonstrating the method’s sensitivity. We test the ability of the method to make inferences about emerging interactions by testing candidates predicted after March 2023, which we validate experimentally. Conclusions We show how known epistatic interaction in SARS-CoV-2 can be detected with high sensitivity, and how emerging ones can be quickly prioritized for experimental validation, an approach that could be implemented downstream of pandemic genome sequencing efforts.

Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

Human cytomegalovirus (HCMV) is a widespread pathogen that in immunocompromised hosts can cause life-threatening disease. Studying HCMV-exposed monocyte-derived dendritic cells by single-cell RNA sequencing, we observe that most cells are entered by the virus, whereas less than 30% of them initiate viral gene expression. Increased viral gene expression is associated with activation of the stimulator of interferon genes (STING) that usually induces anti-viral interferon responses, and with the induction of several pro- ( RHOB, HSP1A1, DNAJB1 ) and anti-viral ( RNF213, TNFSF10, IFI16 ) genes. Upon progression of infection, interferon-beta but not interferon-lambda transcription is inhibited. Similarly, interferon-stimulated gene expression is initially induced and then shut off, thus further promoting productive infection. Monocyte-derived dendritic cells are composed of 3 subsets, with one being especially susceptible to HCMV. In conclusion, HCMV permissiveness of monocyte-derived dendritic cells depends on complex interactions between virus sensing, regulation of the interferon response, and viral gene expression. Human cytomegalovirus (HCMV) is a ubiquitous pathogen associated with morbidity and mortality in the immunocompromised or immunonaive context. Here the authors show that HCMV exploits STING signalling and subverts the interferon response to support infection of monocyte derive dendritic cells.

Dendritic cells (DCs) are indispensable for defense against pathogens but may also contribute to immunopathology. Activation of DCs upon the sensing of pathogens by Toll-like receptors (TLRs) is largely mediated by pattern recognition receptor/nuclear factor-κB (NF-κB) signaling and depends on the appropriate ubiquitination of the respective signaling molecules. However, the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood. Here, we reveal that the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) is upregulated in DCs upon murine Toxoplasmagondii infection and lipopolysaccharide challenge. Stimulation of DCs with the TLR11/12 ligand T. gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-κB activation in OTUB1-competent cells, resulting in elevated interleukin-6 (IL-6), IL-12, and tumor necrosis factor (TNF) production, which was also observed upon the specific stimulation of TLR2, TLR3, TLR7, and TLR9. Mechanistically, OTUB1 promoted NF-κB activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13, resulting in increased K63-linked ubiquitination of IRAK1 (IL-1 receptor-associated kinase 1) and TRAF6 (TNF receptor-associated factor 6). Consequently, DC-specific deletion of OTUB1 impaired the production of cytokines, in particular IL-12, by DCs over the first 2 days of T. gondii infection, resulting in the diminished production of protective interferon-γ (IFN-γ) by natural killer cells, impaired control of parasite replication, and, finally, death from chronic T.encephalitis, all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection. In contrast, impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology. Collectively, these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.

Age‐related macular degeneration (AMD) is a leading cause of vision loss among the elderly. AMD pathogenesis involves chronic activation of the innate immune system including complement factors and microglia/macrophage reactivity in the retina. Here, we show that lack of interferon‐β signaling in the retina accelerates mononuclear phagocyte reactivity and promotes choroidal neovascularization (CNV) in the laser model of neovascular AMD. Complete deletion of interferon‐α/β receptor (Ifnar) using Ifnar 1 −/− mice significantly enhanced early microglia and macrophage activation in lesion areas. This triggered subsequent vascular leakage and CNV at later stages. Similar findings were obtained in laser‐treated Cx3cr1 Cre ER : Ifnar1 fl/fl animals that allowed the tamoxifen‐induced conditional depletion of Ifnar in resident mononuclear phagocytes only. Conversely, systemic IFN‐β therapy of laser‐treated wild‐type animals effectively attenuated microgliosis and macrophage responses in the early stage of disease and significantly reduced CNV size in the late phase. Our results reveal a protective role of Ifnar signaling in retinal immune homeostasis and highlight a potential use for IFN‐β therapy in the eye to limit chronic inflammation and pathological angiogenesis in AMD. Synopsis Innate immune activation is a hallmark of age‐related macular degeneration (AMD). Here, interferon‐beta signaling was identified as an intrinsic regulator of retinal mononuclear phagocytes and therefore as potential therapeutic target for AMD. Loss of Ifnar1 signaling triggers retinal microglia and macrophage reactivity and promotes angiogenesis in a laser‐induced model for AMD. Specific knockdown of Ifnar1 in mononuclear phagocytes affects their immunomodulatory potential and exacerbates choroidal neovascularization. IFN‐β therapy attenuates microgliosis and macrophage reactivity and thereby reduces choroidal neovascularization. Graphical Abstract Innate immune activation is a hallmark of age‐related macular degeneration (AMD). Here, interferon‐beta signaling was identified as an intrinsic regulator of retinal mononuclear phagocytes and therefore as potential therapeutic target for AMD.

The cytokine tumor necrosis factor (TNF) critically regulates the intertwined cell death and pro-inflammatory signaling pathways of dendritic cells (DCs) via ubiquitin modification of central effector molecules, but the intrinsic molecular switches deciding on either pathway are incompletely defined. Here, we uncover that the ovarian tumor deubiquitinating enzyme 7b (OTUD7b) prevents TNF-induced apoptosis of DCs in infection, resulting in efficient priming of pathogen-specific CD8 + T cells. Mechanistically, OTUD7b stabilizes the E3 ligase TNF-receptor-associated factor 2 (TRAF2) in human and murine DCs by counteracting its K48-ubiquitination and proteasomal degradation. TRAF2 in turn facilitates K63-linked polyubiquitination of RIPK1, which mediates activation of NF-κB and MAP kinases, IL-12 production, and expression of anti-apoptotic cFLIP and Bcl-xL. We show that mice with DC-specific OTUD7b-deficiency displayed DC apoptosis and a failure to induce CD8 + T cell-mediated brain pathology, experimental cerebral malaria, in a murine malaria infection model. Together, our data identify the deubiquitinating enzyme OTUD7b as a central molecular switch deciding on survival of human and murine DCs and provides a rationale to manipulate DC responses by targeting their ubiquitin network downstream of the TNF receptor pathway.

Activation of the DNA-sensing STING axis by RNA viruses plays a role in antiviral response through mechanisms that remain poorly understood. Here, we show that the STING pathway regulates Nipah virus (NiV) replication in vivo in mice. Moreover, we demonstrate that following both NiV and measles virus (MeV) infection, IFNγ-inducible protein 16 (IFI16), an alternative DNA sensor in addition to cGAS, induces the activation of STING, leading to the phosphorylation of NF-κB p65 and the production of IFNβ and interleukin 6. Finally, we found that paramyxovirus-induced syncytia formation is responsible for loss of mitochondrial membrane potential and leakage of mitochondrial DNA in the cytoplasm, the latter of which is further detected by both cGAS and IFI16. These results contribute to improve our understanding about NiV and MeV immunopathogenesis and provide potential paths for alternative therapeutic strategies.