Comparative transcriptomics of porcine liver-resident CD8αdim, liver CD8αhigh and circulating blood CD8αhigh NK cells reveals an intermediate phenotype of liver CD8αhigh NK cells.

Liver-resident NK (lrNK) cells have been studied in humans as well as in mice. Unfortunately, important differences have been observed between murine and human lrNK cells, complicating the extrapolation of data obtained in mice to man. We previously described two NK cell subsets in the porcine liver: A CD8αhigh subset, with a phenotype much like conventional CD8αhigh NK cells found in the peripheral blood, and a specific liver-resident CD8αdim subset which phenotypically strongly resembles human lrNK cells. These data suggest that the pig might be an attractive model for studying lrNK cell biology. In the current study, we used RNA-seq to compare the transcriptome of three porcine NK cell populations: Conventional CD8αhigh NK cells from peripheral blood (cNK cells), CD8αhigh NK cells isolated from the liver, and the liver-specific CD8αdim NK cells. We found that highly expressed transcripts in the CD8αdim lrNK cell population mainly include genes associated with the (adaptive) immune response, whereas transcripts associated with cell migration and extravasation are much less expressed in this subset compared to cNK cells. Overall, our data indicate that CD8αdim lrNK cells show an immature and anti-inflammatory phenotype. Interestingly, we also observed that the CD8αhigh NK cell population that is present in the liver appears to represent a population with an intermediate phenotype. Indeed, while the transcriptome of these cells largely overlaps with that of cNK cells, they also express transcripts associated with liver residency, in particular CXCR6. The current, in-depth characterization of the transcriptome of porcine liver NK cell populations provides a basis to use the pig model for research into liver-resident NK cells.

ß-Glucan-Induced IL-10 Secretion by Monocytes Triggers Porcine NK Cell Cytotoxicity.

Beta-glucans are naturally occurring polysaccharides present in cell walls of fungi, yeast, bacteria, cereals, seaweed, and algae. These microbe-associated molecular patterns (MAMPs) possess immunomodulatory properties. In human, it has been suggested that NK cells can be activated by ß-glucans. Here, we aimed to elucidate whether ß-glucans modulate porcine NK cell responses in vitro and if so, how these effects are mediated. We investigated the effect of two ß-glucans, Macrogard and Curdlan, which differ in solubility and structure. Direct addition of ß-glucans to purified porcine NK cells did not affect cytotoxicity of these cells against K562 target cells. However, when using PBMC instead of purified NK cells, ß-glucan addition significantly increased NK cell-mediated cytotoxicity. This effect depended on factors secreted by CD14+ monocytes upon ß-glucan priming. Further analysis showed that monocytes secrete TNF-α, IL-6, and IL-10 upon ß-glucan addition. Of these, IL-10 turned out to play a critical role in ß-glucan-triggered NK cell cytotoxicity, since depletion of IL-10 completely abrogated the ß-glucan-induced increase in cytotoxicity. Furthermore, addition of recombinant IL-10 to purified NK cells was sufficient to enhance cytotoxicity. In conclusion, we show that ß-glucans trigger IL-10 secretion by porcine monocytes, which in turn leads to increased NK cell cytotoxicity, and thereby identify IL-10 as a potent stimulus of porcine NK cell cytotoxicity.

Pseudorabies Virus Infection Causes Downregulation of Ligands for the Activating NK Cell Receptor NKG2D.

Herpesviruses display a complex and carefully balanced interaction with important players in the antiviral immune response of immunocompetent natural hosts, including natural killer (NK) cells. With regard to NK cells, this delicate balance is illustrated on the one hand by severe herpesvirus disease reported in individuals with NK cell deficiencies and on the other hand by several NK cell evasion strategies described for herpesviruses. In the current study, we report that porcine cells infected with the porcine alphaherpesvirus pseudorabies virus (PRV) display a rapid and progressive downregulation of ligands for the major activating NK cell receptor NKG2D. This downregulation consists both of a downregulation of NKG2D ligands that are already expressed on the cell surface of an infected cell and an inhibition of cell surface expression of newly expressed NKG2D ligands. Flow cytometry and RT-qPCR assays showed that PRV infection results in downregulation of the porcine NKG2D ligand pULBP1 from the cell surface and a very substantial suppression of mRNA expression of pULBP1 and of another potential NKG2D ligand, pMIC2. Furthermore, PRV-induced NKG2D ligand downregulation was found to be independent of late viral gene expression. In conclusion, we report that PRV infection of host cells results in a very pronounced downregulation of ligands for the activating NK cell receptor NKG2D, representing an additional NK evasion strategy of PRV.

Identification of a Porcine Liver EomeshighT-betlow NK Cell Subset That Resembles Human Liver Resident NK Cells.

Natural killer (NK) cells are cells of the innate immunity and play an important role in the defense against viral infections and cancer, but also contribute to shaping adaptive immune responses. Long-lived tissue-resident NK cells have been described in man and mouse, particularly in the liver, contributing to the idea that the functional palette of NK cells may be broader than originally thought, and may include memory-like responses and maintaining tissue homeostasis. Remarkably, liver resident (lr)NK cells in man and mouse show substantial species-specific differences, in particular reverse expression patterns of the T-box transcription factors Eomesodermin (Eomes) and T-bet (EomeshighT-betlow in man and vice versa in mouse). In pig, compared to blood NK cells which are CD3-CD8αhigh cells, the porcine liver contains an abundant additional CD3-CD8αdim NK cell subpopulation. In the current study, we show that this porcine CD3-CD8αdim liver NK population is highly similar to its human lrNK counterpart and therefore different from mouse lrNK cells. Like human lrNK cells, this porcine NK cell population shows an EomeshighT-betlow expression pattern. In addition, like its human counterpart, the porcine liver NK population is CD49e- and CXCR6+. Furthermore, the porcine EomeshighT-betlow liver NK cell population is able to produce IFN-γ upon IL-2/12/18 stimulation but lacks the ability to kill K562 or pseudorabies virus-infected target cells, although limited degranulation could be observed upon incubation with K562 cells or upon CD16 crosslinking. All together, these results show that porcine EomeshighT-betlow NK cells in the liver strongly resemble human lrNK cells, and therefore indicate that the pig may represent a unique model to study the function of these lrNK cells in health and disease.