These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defence against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, Infectious Bronchitis Virus (IBV) activates autophagy. A screen of individual IBV non-structural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses Mouse Hepatitis Virus, and Severe Acute Respiratory Syndrome Virus, and the equivalent nsp5-7 of the arterivirus Porcine Reproductive and Respiratory Syndrome Virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II-positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double FYVE-domain containing protein (DFCP) indicating localised concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signalling, activation of sirtuin1 or induction of ER stress.

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Over the last few years, we have seen an increasing interest and demand for pigs in biomedical research. Domestic pigs (Sus scrofa domesticus) are closely related to humans in terms of their anatomy, genetics, and physiology, and often are the model of choice for the assessment of novel vaccines and therapeutics in a preclinical stage. However, the pig as a model has much more to offer, and can serve as a model for many biomedical applications including aging research, medical imaging, and pharmaceutical studies to name a few. In this review, we will provide an overview of the innate immune system in pigs, describe its anatomical and physiological key features, and discuss the key players involved. In particular, we compare the porcine innate immune system to that of humans, and emphasize on the importance of the pig as model for human disease.

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So far little is known about natural killer (NK) cells in the pig due to the lack of NK cell-specific markers. In this study, we identified the activating receptor NKp46 (CD335) in swine with newly developed monoclonal antibodies (mAbs) for more detailed studies on NK cells in this species. The NKp46 mAbs showed a specific reactivity with a distinct population of perforin + CD2 + CD3 − CD8α + CD16 + lymphocytes. In spleen and liver, an additional subset of CD8α dim/− lymphocytes with increased NKp46 expression was observed. Surprisingly, we could identify NKp46 − cells with an NK cell phenotype in all animals analyzed. These lymphocytes showed comparable cytolytic activity against xenogeneic and allogeneic target cells as NKp46 + NK cells. In contrast, NKp46 + NK cells produced several fold higher levels of interferon-γ (IFN-γ) than the NKp46 − cells after cytokine stimulation. Furthermore, an activation-dependent induction of NKp46 expression in formerly NKp46 − cells after stimulation with interleukin-2 (IL-2), IL-12, and IL-18 could be shown. In summary, our data indicate that NKp46 is not expressed by all porcine NK cells and that NKp46 discriminates porcine NK cells differing in regard to cytokine production, which challenges the paradigm of NKp46 as a comprehensive marker for NK cells across different mammalian species.Keywords: NK cell · NKp46 · Swine Supporting Information available online IntroductionNatural killer (NK) cells are a highly specialized subpopulation of lymphocytes that play a crucial role in the early phase of immune responses. So far little is known about NK cells in swine. The phenotype of porcine NK cells was recently described as perforin + CD2 + CD3 − CD4 − CD5 − CD6 − CD8α + CD8ß − CD11b + CD16 + and it was observed that these cells exhibit natural cytotoxicity against NK-susceptible targets [1][2][3][4]. Likewise, the Correspondence: Dr. Armin Saalmüller e-mail: armin.saalmueller@vetmeduni.ac.at involvement of porcine NK cells has been reported in immune responses against parasitic and viral infections [5][6][7][8][9]. But since porcine NK cells share several phenotypic markers with other cell populations such as NKT cells, TCR-γδ T cells, and myeloid cells [3,4], the precise detection, isolation, and more detailed functional characterization of porcine NK cells has been thus far difficult. Thus, the identification of a discrete and unifying marker for this cell population in swine would be highly beneficial.NKp46 (CD335, NCR1), a member of the natural cytotoxicity receptor (NCR) family, is specifically expressed on NK cells, * These authors contributed equally to this work. , the triggering receptor NKp46 might also be a good candidate molecule for the specific identification of NK cells in swine. Porcine NKp46 was previously described as being encoded in the porcine leukocyte receptor complex on chromosome 6 [24]. The first expression analysis at the mRNA level revealed that the overall sequence and tissue distribution of porcine NKp46 was comparable with that of other species [2...

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