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Allergic asthma is a chronic inflammatory disease of the conducting airways characterized by the presence of allergen-specific IgE, Th2 cytokine production, eosinophilic airway inflammation, bronchial hyperreactivity, mucus overproduction, and structural changes in the airways. Investigators have tried to mimic these features of human allergic asthma in murine models. Whereas the surrogate allergen ovalbumin has been extremely valuable for unravelling underlying mechanisms of the disease, murine asthma models depend nowadays on naturally occurring allergens, such as house dust mite (HDM), cockroach, and Alternaria alternata. Here we describe a physiologically relevant model of acute allergic asthma based on sensitization and challenge with HDM extracts, and compare it with the ovalbumin/alum-induced asthma model. Moreover, we propose a detailed readout of the asthma phenotype, determining the degree of eosinophilia in bronchoalveolar lavage fluids by flow cytometry, visualizing goblet cell metaplasia, and measuring Th cytokine production by lung-draining mediastinal lymph node cells restimulated with HDM. © 2016 by John Wiley & Sons, Inc.

Summary The phenotypic and functional dichotomy between IRF8+ type 1 and IRF4+ type 2 conventional dendritic cells (cDC1s and cDC2s, respectively) is well accepted; it is unknown how robust this dichotomy is under inflammatory conditions, when additionally monocyte-derived cells (MCs) become competent antigen-presenting cells (APCs). Using single-cell technologies in models of respiratory viral infection, we found that lung cDC2s acquired expression of the Fc receptor CD64 shared with MCs and of IRF8 shared with cDC1s. These inflammatory cDC2s (inf-cDC2s) were superior in inducing CD4+ T helper (Th) cell polarization while simultaneously presenting antigen to CD8+ T cells. When carefully separated from inf-cDC2s, MCs lacked APC function. Inf-cDC2s matured in response to cell-intrinsic Toll-like receptor and type 1 interferon receptor signaling, upregulated an IRF8-dependent maturation module, and acquired antigens via convalescent serum and Fc receptors. Because hybrid inf-cDC2s are easily confused with monocyte-derived cells, their existence could explain why APC functions have been attributed to MCs., Graphical Abstract, Highlights • Type I interferon drives differentiation of inf-cDC2s that closely resemble MCs • Inf-cDC2s prime CD4+ and CD8+ T cells, whereas MCs lack APC function • Inf-cDC2s internalize antibody-complexed antigen via Fc receptors • IRF8 controls maturation gene module in inf-cDC2s, The dichotomy between type 1 and 2 conventional DCs under steady-state conditions is well defined. Bosteels et al. demonstrate that, upon inflammation, cDC2s acquire a hybrid inf-cDC2 phenotype, sharing phenotype, gene expression, and function with cDC1s and monocyte-derived cells, to optimally boost CD4 and CD8 immunity via Fc receptors.

The study of Vetters et al. identifies the ubiquitin-modifying enzyme A20 as a critical regulator of mTOR. Loss of A20 unleashes mTOR activity and induces NK cell death, underscoring the need for a tightly controlled mTOR pathway for proper NK cell homeostasis., The ubiquitin-editing enzyme A20 is a well-known regulator of immune cell function and homeostasis. In addition, A20 protects cells from death in an ill-defined manner. While most studies focus on its role in the TNF-receptor complex, we here identify a novel component in the A20-mediated decision between life and death. Loss of A20 in NK cells led to spontaneous NK cell death and severe NK cell lymphopenia. The few remaining NK cells showed an immature, hyperactivated phenotype, hallmarked by the basal release of cytokines and cytotoxic molecules. NK-A20−/− cells were hypersensitive to TNF-induced cell death and could be rescued, at least partially, by a combined deficiency with TNF. Unexpectedly, rapamycin, a well-established inhibitor of mTOR, also strongly protected NK-A20−/− cells from death, and further studies revealed that A20 restricts mTOR activation in NK cells. This study therefore maps A20 as a crucial regulator of mTOR signaling and underscores the need for a tightly balanced mTOR pathway in NK cell homeostasis.

Pneumonia virus of mice (PVM) infection has been widely used as a rodent model to study the closely related human respiratory syncytial virus (hRSV). While T cells are indispensable for viral clearance, they also contribute to immunopathology. To gain more insight into mechanistic details, novel tools are needed that allow to study virus-specific T cells in C57BL/6 mice as the majority of transgenic mice are only available on this background. While PVM-specific CD8 T cell epitopes were recently described, so far no PVM-specific CD4 T cell epitopes have been identified within the C57BL/6 strain. Therefore, we set out to map H2-IAb-restricted epitopes along the PVM proteome. By means of in silico prediction and subsequent functional validation, we were able to identify a MHCII-restricted CD4 T cell epitope, corresponding to amino acids 37–47 in the PVM matrix protein (M37–47). Using this newly identified MHCII-restricted M37–47 epitope and a previously described MHCI-restricted N339–347 epitope, we generated peptide-loaded MHCII and MHCI tetramers and characterized the dynamics of virus-specific CD4 and CD8 T cell responses in vivo. The findings of this study can provide a basis for detailed investigation of T cell-mediated immune responses to PVM in a variety of genetically modified C57BL/6 mice.

Highlights ► NK cells and CD8+ T-cells expand relatively late following pneumovirus infection. ► Memory CD8+ T-cells support type 1 skewing of pneumovirus-specific responses. ► Memory CD8+ T-cells prevent pneumovirus-induced immunopathology. ► CD8+ T-cell targeted immunization protects against pneumovirus-induced disease., Pneumoviruses such as pneumonia virus of mice (PVM), bovine respiratory syncytial virus (bRSV) or human (h)RSV are closely related pneumoviruses that cause severe respiratory disease in their respective hosts. It is well-known that T-cell responses are essential in pneumovirus clearance, but pneumovirus-specific T-cell responses also are important mediators of severe immunopathology. In this study we determined whether memory- or pre-existing, transferred virus-specific CD8+ T-cells provide protection against PVM-induced disease. We show that during infection with a sublethal dose of PVM, both natural killer (NK) cells and CD8+ T-cells expand relatively late. Induction of CD8+ T-cell memory against a single CD8+ T-cell epitope, by dendritic cell (DC)-peptide immunization, leads to partial protection against PVM challenge and prevents Th2 differentiation of PVM-induced CD4 T-cells. In addition, adoptively transferred PVM-specific CD8+ T-cells, covering the entire PVM-specific CD8+ T-cell repertoire, provide partial protection from PVM-induced disease. From these data we infer that antigen-specific memory CD8+ T-cells offer significant protection to PVM-induced disease. Thus, CD8+ T-cells, despite being a major cause of PVM-associated pathology during primary infection, may offer promising targets of a protective pneumovirus vaccine.

NK cells play an important role in the early defense against invading pathogens. Although it is well established that infection leads to a substantial, local increase in NK cell numbers, little is known about the mechanisms that trigger their proliferation and migration. In this study, we investigated the dynamics of NK cell responses after intranasal respiratory virus infection. We show that NK cell numbers increased in the airways after influenza virus infection but find no evidence of proliferation either at the site of infection or in the draining lymph nodes. Instead, we find that the bone marrow (BM) is the primary site of proliferation of both immature and mature NK cells during infection. Using an adoptive transfer model, we demonstrate that peripheral, long-lived and phenotypically mature NK cells migrate back to the BM and proliferate there, both homeostatically and in response to infection. Thus, the BM is not only a site of NK cell development but also an important site for proliferation of long-lived mature NK cells.

CD4 Th2 lymfocytes producing IL-4, IL-5 and IL-13 are common controllers of type 2 immunity. They respond to allergens and helminthes only when antigen is presented by professional antigen presenting cells like dendritic cells. Dendritic cells express many pattern recognition receptors that can be triggered by type 2 antigens, leading to direct DC activation and Th2 polarization. Alternatively, type 2 stimuli can first trigger barrier epithelial cells that subsequently activate the DCs via release of TSLP, IL-33 and IL-25. It is clear now that also other innate immune cells involved in type 2 immunity such as basophils and ILC2s help DCs to polarize CD4 T cells towards the Th2 direction. In addition to the roles of DCs in priming CD4 Th2 responses, they also control recall responses of memory CD4 Th2 cells to allergens, identifying these cells as important targets for intervention in allergic inflammatory diseases like asthma, dermatitis and rhinitis.

The number of markers measured in both flow and mass cytometry keeps increasing steadily. Although this provides a wealth of information, it becomes infeasible to analyze these datasets manually. When using 2D scatter plots, the number of possible plots increases exponentially with the number of markers and therefore, relevant information that is present in the data might be missed. In this article, we introduce a new visualization technique, called FlowSOM, which analyzes Flow or mass cytometry data using a Self-Organizing Map. Using a two-level clustering and star charts, our algorithm helps to obtain a clear overview of how all markers are behaving on all cells, and to detect subsets that might be missed otherwise. R code is available at https://github.com/SofieVG/FlowSOM and will be made available at Bioconductor.

Because of its air-exchange function, the lung is continuously exposed to foreign particles such as microbes. Viral infections such as influenza A virus, respiratory syncytial virus, human metapneumovirus, and rhinovirus are common and the lung is a very vulnerable organ that needs to preserve gas exchange during antiviral defense. To recognize these threats and at the same time maintain tissue homeostasis, the lung is equipped with an elaborate network of innate and adaptive immune cells. The innate mucosal immune response to respiratory viruses is made up of natural killer cells and B cells producing low-affinity natural antibodies. Dendritic cells are the sentinels of the immune system linking sensing by ancient innate immune system receptors to activation of adaptive immunity to respiratory viruses. These cells also induce the polarization of virus-specific CD4 and CD8 T cell responses, necessary for viral clearance. Memory responses to viral antigens are found in the CD4 and CD8 compartment of T cells, and these cells often home back to the lung mucosa to become tissue-resident memory cells, which produce copious amounts of cytokines upon renewed viral encounter. Following respiratory viral infection, there frequently is induction of tertiary lymphoid organs such as bronchus-associated lymphoid tissues that similarly play important roles in maintaining antiviral immunity.

The lungs are constantly exposed to antigens, most of which are non-pathogenic and do not require the induction of an immune response. Dendritic cells (DCs) are situated at the basolateral site of the lungs and continuously scan the environment to detect the presence of pathogens and subsequently initiate an immune response. They are a heterogeneous population of antigen-presenting cells that exert specific functions. Compelling evidence is now provided that DCs are both sufficient and necessary to induce allergic responses against several inhaled harmless allergens. How various DC subsets exactly contribute to the induction of allergic asthma is currently a subject of intense investigation. We here review the current progress in this field.

Recently we have shown that liposomes can be used as artificial microbes for the production and delivery of DNA-encoded antigens. These so-called antigen-expressing immunostimulatory liposomes (AnExILs) were superior in inducing antigen-specific antibodies compared to conventional liposomal protein or DNA vaccines when tested in mice after i.m. immunization. In this study, we investigated the capacity of AnExILs to induce T-cell responses. By using a plasmid vector encoding a model antigen under control of both the prokaryotic T7 and the eukaryotic CMV promoter we hypothesized that antigen production could lead to CTL activation via two distinct routes: i. production of antigens inside the AnExILs with subsequent cross-presentation after processing by APCs and ii. endogenous production of antigens after AnExIL-mediated transfection of the pDNA. Although we were not able to demonstrate transfection-mediated expression of luc-NP in mice, i.m. injection of AnExILs producing luc-NP resulted in T-cell responses against the encoded NP epitope, as determined by tetramer staining. T-cell responses were comparable to the responses obtained after i.m. injection of naked pDNA. In order to find out whether CTL activation was caused by cross-presentation of the exogenous antigens produced inside AnExILs or by endogenous antigen production from transfection with the same pDNA source a second study was initiated in which the contribution of each of these effects could be separately determined. These results demonstrate that the observed T-cell responses were not exclusively caused by cross-presentation of the AnExIL-produced antigens alone, but were rather a combination of dose-dependent antigen cross-presentation and low levels of endogenous antigen production. (c) 2012 Elsevier B. V. All rights reserved.