Ingo Drexler

Modified Vaccinia Virus Ankara Triggers Type I IFN Production in Murine Conventional Dendritic Cells via a cGAS/STING-Mediated Cytosolic DNA-Sensing Pathway

Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus that has been engineered as a vaccine against infectious agents and cancers. Our goal is to understand how MVA modulates innate immunity in dendritic cells (DCs), which can provide insights to vaccine design. In this study, using murine bone marrow-derived dendritic cells, we assessed type I interferon (IFN) gene induction and protein secretion in response to MVA infection. We report that MVA infection elicits the production of type I IFN in murine conventional dendritic cells (cDCs), but not in plasmacytoid dendritic cells (pDCs). Transcription factors IRF3 (IFN regulatory factor 3) and IRF7, and the positive feedback loop mediated by IFNAR1 (IFN alpha/beta receptor 1), are required for the induction. MVA induction of type I IFN is fully dependent on STING (stimulator of IFN genes) and the newly discovered cytosolic DNA sensor cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase). MVA infection of cDCs triggers phosphorylation of TBK1 (Tank-binding kinase 1) and IRF3, which is abolished in the absence of cGAS and STING. Furthermore, intravenous delivery of MVA induces type I IFN in wild-type mice, but not in mice lacking STING or IRF3. Treatment of cDCs with inhibitors of endosomal and lysosomal acidification or the lysosomal enzyme Cathepsin B attenuated MVA-induced type I IFN production, indicating that lysosomal enzymatic processing of virions is important for MVA sensing. Taken together, our results demonstrate a critical role of the cGAS/STING-mediated cytosolic DNA-sensing pathway for type I IFN induction in cDCs by MVA. We present evidence that vaccinia virulence factors E3 and N1 inhibit the activation of IRF3 and the induction of IFNB gene in MVA-infected cDCs.

Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1β production

Double-stranded DNA (dsDNA) in the cytoplasm triggers the production of interleukin 1β (IL-1β) as an antiviral host response, and deregulation of the pathways involved can promote inflammatory disease. Here we report a direct cytosolic interaction between the DNA-damage sensor Rad50 and the innate immune system adaptor CARD9. Transfection of dendritic cells with dsDNA or infection of dendritic cells with a DNA virus induced the formation of dsDNA-Rad50-CARD9 signaling complexes for activation of the transcription factor NF-κB and the generation of pro-IL-1β. Primary cells conditionally deficient in Rad50 or lacking CARD9 consequently exhibited defective DNA-induced production of IL-1β, and Card9−/− mice had impaired inflammatory responses after infection with a DNA virus in vivo. Our results define a cytosolic DNA-recognition pathway for inflammation and a physical and functional connection between a conserved DNA-damage sensor and the innate immune response to pathogens.

Brain-resident memory T cells represent an autonomous cytotoxic barrier to viral infection

During viral infections, brain tissue–resident memory T cells (bTRM) prevent fatal brain infection after acquiring perforin- and IFN-γ–dependent effector functions through a pathway that involves presentation of cognate antigen on MHC-I.

Antigen-dependent competition shapes the local repertoire of tissue-resident memory CD8+ T cells

Muschaweckh et al. show that antigen presentation in the skin regulates the generation of tissue-resident memory T (TRM) cells by orchestrating local competition of antiviral CD8+ T cells, revealing a mechanism to fine-tune the repertoire of regional pools of TRM cells.

Relevance of MET activation and genetic alterations of KRAS and E-cadherin for cetuximab sensitivity of gastric cancer cell lines

PurposeThe therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated. Reliable biomarkers for the identification of patients who are likely to benefit from the treatment are not available. The aim of the study was to examine the drug sensitivity of five gastric cancer cell lines towards cetuximab as a single agent and to establish predictive markers for chemosensitivity in this cell culture model. The effect of a combination of cetuximab with chemotherapy was compared between a sensitive and a nonsensitive cell line.MethodsEGFR expression, activation and localisation, the presence and subcellular localisation of the cell adhesion molecule E-cadherin as well as MET activation were examined by Western blot analysis, flow cytometry and immunofluorescence staining. Cells were treated with varying concentrations of cetuximab and cisplatin and 5-fluorouracil in tumour-relevant concentrations. The biological endpoint was cell viability, which was measured by XTT cell proliferation assay. Response to treatment was evaluated using statistical methods.ResultsWe assessed the activity of cetuximab in five gastric cancer cell lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two cell lines, MKN1 and MKN28, was significantly reduced by cetuximab treatment. High EGFR expression and low levels of receptor activation were associated with cetuximab responsiveness. MET activation as well as mutations of KRAS and CDH1 (gene encoding E-cadherin) was associated with cetuximab resistance.ConclusionThese data indicate that our examinations may be clinically relevant, and the candidate markers should therefore be tested in clinical studies.

Modified Vaccinia Virus Ankara-Infected Dendritic Cells Present CD4+ T-Cell Epitopes by Endogenous Major Histocompatibility Complex Class II Presentation Pathways

ABSTRACT CD4+ T lymphocytes play a central role in the immune system and mediate their function after recognition of their respective antigens presented on major histocompatibility complex II (MHCII) molecules on antigen-presenting cells (APCs). Conventionally, phagocytosed antigens are loaded on MHCII for stimulation of CD4+ T cells. Certain epitopes, however, can be processed directly from intracellular antigens and are presented on MHCII (endogenous MHCII presentation). Here we characterized the MHCII antigen presentation pathways that are possibly involved in the immune response upon vaccination with modified vaccinia virus Ankara (MVA), a promising live viral vaccine vector. We established CD4+ T-cell lines specific for MVA-derived epitopes as tools for in vitro analysis of MHCII antigen processing and presentation in MVA-infected APCs. We provide evidence that infected APCs are able to directly transfer endogenous viral proteins into the MHCII pathway to efficiently activate CD4+ T cells. By using knockout mice and chemical inhibitory compounds, we further elucidated the molecular basis, showing that among the various subcellular pathways investigated, proteasomes and autophagy are key players in the endogenous MHCII presentation during MVA infection. Interestingly, although proteasomal processing plays an important role, neither TAP nor LAMP-2 was found to be involved in the peptide transport. Defining the molecular mechanism of MHCII presentation during MVA infection provides a basis for improving MVA-based vaccination strategies by aiming for enhanced CD4+ T-cell activation by directing antigens into the responsible pathways. IMPORTANCE This work contributes significantly to our understanding of the immunogenic properties of pathogens by deciphering antigen processing pathways contributing to efficient activation of antigen-specific CD4+ T cells. We identified autophagosome formation, proteasomal activity, and lysosomal integrity as being crucial for endogenous CD4+ T-cell activation. Since poxvirus vectors such as MVA are already used in clinical trials as recombinant vaccines, the data provide important information for the future design of optimized poxviral vaccines for the study of advanced immunotherapy options.

α4-integrins control viral meningoencephalitis through differential recruitment of T helper cell subsets

IntroductionNatalizumab blocks α4-integrins and is a prototypic agent for a series of anti-inflammatory drugs that impair trafficking of immune cells into the CNS. However, modulation of the access of immune cells to the CNS is associated with impaired immune surveillance and detrimental viral infections of the CNS. Here, we explored the potency of cellular immune responses within the CNS to protect against viral encephalitis in mice with T cell conditional disruption of VLA-4 integrin (α4β1) expression.ResultsWhile VLA-4 expression in virus specific Th1 cells is non-redundant for their ability to access the CNS, α4-integrin deficient Th17 cells enter the CNS compartment and generate an inflammatory milieu upon intrathecal vaccinia virus (VV) infection. However, in contrast to Th1 cells that can adopt direct cytotoxic properties, Th17 cells fail to clear the virus due to insufficient Eomes induced perforin-1 expression.ConclusionThe quality of the intrathecal cellular antiviral response under conditions of impaired VLA-4 function jeopardizes host protection. Our functional in vivo data extend our mechanistic understanding of anti-viral immunity in the CNS and help to estimate the risk potential of upcoming therapeutic agents that target the trafficking of immune cells into distinct anatomical compartments.

Immune Protection by a Cytomegalovirus Vaccine Vector Expressing a Single Low-Avidity Epitope

Experimental CMV-based vaccine vectors expressing a single MHC class I–restricted high-avidity epitope provided strong, T cell–dependent protection against viruses or tumors. In this study we tested the low-avidity epitope KCSRNRQYL, and show that a mouse CMV (MCMV) vector provides complete immune control of recombinant vaccinia virus expressing the same epitope if KCSRNRQYL is expressed within the immediate-early MCMV gene ie2. The same epitope expressed within the early M45 gene provided no protection, although MCMV vectors expressing the high-avidity epitope SSIEFARL induced protective immunity irrespective of gene expression context. Immune protection was matched by Ag-induced, long-term expansion of effector memory CD8 T cells, regardless of epitope avidity. We explained this pattern by observing regularities in Ag competition, where responses to high-avidity epitopes outcompeted weaker ones expressed later in the replicative cycle of the virus. Conversely, robust and early expression of a low-avidity epitope compensated its weak intrinsic antigenicity, resulting in strong and sustained immunity and immune protection.

Gene Expression Driven by a Strong Viral Promoter in MVA Increases Vaccination Efficiency by Enhancing Antibody Responses and Unmasking CD8 + T Cell Epitopes

Viral vectors are promising tools for vaccination strategies and immunotherapies. However, CD8+ T cell responses against pathogen-derived epitopes are usually limited to dominant epitopes and antibody responses to recombinant encoded antigens (Ags) are mostly weak. We have previously demonstrated that the timing of viral Ag expression in infected professional Ag-presenting cells strongly shapes the epitope immunodominance hierarchy. T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses. In this work we evaluate the effect of overexpressing the recombinant Ag using the modified vaccinia virus early/late promoter H5 (mPH5). Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced. The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags. Moreover, the increase in Ag-secretion favors the induction of strong antibody responses. Our findings provide the rationale to develop new strategies for fine-tuning the responses elicited by recombinant modified vaccinia virus Ankara by using selected promoters to improve the performance of this viral vector.

Impact of the TAP-like transporter in antigen presentation and phagosome maturation

Cross-presentation is thought to require transport of proteasome-generated peptides by the TAP transporters into MHC class I loading compartments for most antigens. However, a proteasome-dependent but TAP-independent pathway has also been described. Depletion of the pool of recycling cell surface MHC class I molecules available for loading with cross-presented peptides might partly or largely account for the critical role of TAP in cross-presentation of phagocytosed antigens. Here we examined a potential role of the homodimeric lysosomal TAP-like transporter in cross-presentation and in presentation of endogenous peptides by MHC class II molecules. We find that TAP-L is strongly recruited to dendritic cell phagosomes at a late stage, when internalized antigen and MHC class I molecules have been degraded or sorted away from phagosomes. Cross-presentation of a receptor-targeted antigen in vitro and of a phagocytosed antigen in vivo, as well as presentation of a cytosolic antigen by MHC class II molecules, is not affected by TAP-L deficiency. However, accumulation in vitro of a peptide optimally adapted to TAP-L selectivity in purified phagosomes is abolished by TAP-L deficiency. Unexpectedly, we find that TAP-L deficiency accelerates phagosome maturation, as reflected in increased Lamp2b recruitment and enhanced proteolytic degradation of phagocytosed antigen and in vitro transported peptides. Although additional experimentation will be required to definitely conclude on the role of TAP-L in transport of peptides presented by MHC class I and class II molecules, our data suggest that the principal role of TAP-L in dendritic cells may be related to regulation of phagosome maturation.