Anti-West Nile virus activity of in vitro expanded human primary natural killer cells

<p>Abstract</p> <p>Background</p> <p>Natural Killer (NK) cells are a crucial component of the host innate immune system with anti-viral and anti-cancer properties. However, the role of NK cells in West Nile virus (WNV) infection is controversial, with reported effects ranging from active suppression of virus to no effect at all. It was previously shown that K562-mb15-41BBL (K562D2) cells, which express IL-15 and 4-1BBL on the K562 cell surface, were able to expand and activate human primary NK cells of normal peripheral blood mononuclear cells (PBMC). The expanded NK cells were tested for their ability to inhibit WNV infection <it>in vitro</it>.</p> <p>Results</p> <p>Co-culture of PBMC with irradiated K562D2 cells expanded the NK cell number by 2-3 logs in 2-3 weeks, with more than 90% purity; upregulated NK cell surface activation receptors; downregulated inhibitory receptors; and boosted interferon gamma (IFN-γ) production by ~33 fold. The expanded NK (D2NK) cell has strong natural killing activity against both K562 and Vero cells, and killed the WNV infected Vero cells through antibody-dependent cellular cytotoxicity (ADCC). The D2NK cell culture supernatants inhibited both WNV replication and WNV induced cytopathic effect (CPE) in Vero cells when added before or after infection. Anti-IFN-γ neutralizing antibody blocked the NK supernatant-mediated anti-WNV effect, demonstrating a noncytolytic activity mediated through IFN-γ.</p> <p>Conclusions</p> <p>Co-culture of PBMC with K562D2 stimulatory cells is an efficient technique to prepare large quantities of pure and active NK cells, and these expanded NK cells inhibited WNV infection of Vero cells through both cytolytic and noncytolytic activities, which may imply a potential role of NK cells in combating WNV infection.</p

Different Patterns of Evolution in the Centromeric and Telomeric Regions of Group A and B Haplotypes of the Human Killer Cell Ig-Like Receptor Locus

The fast evolving human KIR gene family encodes variable lymphocyte receptors specific for polymorphic HLA class I determinants. Nucleotide sequences for 24 representative human KIR haplotypes were determined. With three previously defined haplotypes, this gave a set of 12 group A and 15 group B haplotypes for assessment of KIR variation. The seven gene-content haplotypes are all combinations of four centromeric and two telomeric motifs. 2DL5, 2DS5 and 2DS3 can be present in centromeric and telomeric locations. With one exception, haplotypes having identical gene content differed in their combinations of KIR alleles. Sequence diversity varied between haplotype groups and between centromeric and telomeric halves of the KIR locus. The most variable A haplotype genes are in the telomeric half, whereas the most variable genes characterizing B haplotypes are in the centromeric half. Of the highly polymorphic genes, only the 3DL3 framework gene exhibits a similar diversity when carried by A and B haplotypes. Phylogenetic analysis and divergence time estimates, point to the centromeric gene-content motifs that distinguish A and B haplotypes having emerged ∼6 million years ago, contemporaneously with the separation of human and chimpanzee ancestors. In contrast, the telomeric motifs that distinguish A and B haplotypes emerged more recently, ∼1.7 million years ago, before the emergence of Homo sapiens. Thus the centromeric and telomeric motifs that typify A and B haplotypes have likely been present throughout human evolution. The results suggest the common ancestor of A and B haplotypes combined a B-like centromeric region with an A-like telomeric region

Human intrahepatic ILC2 are IL-13^*positive Amphiregulin^*positive and their frequency correlates with Model of End stage Liver Disease score

Innate lymphoid cells (ILC) have been implicated in the initiation of inflammation and fibrosis in mice. However, ILC have not been characterized in inflamed human liver tissue.Human intrahepatic lymphocytes were isolated by mechanical digestion and phenotyped by flow cytometry. Conditioned medium from cultures of primary human biliary epithelial cells, stellate cells, fibroblasts and inflamed human liver tissue was used to model the effects of the inflammatory liver environment of ILC phenotype and function.All three ILC subsets were present in the human liver, with the ILC1 (CRTH2negCD117neg) subset constituting around 70% of intrahepatic ILCs. Both NCRpos (NKp44+) and NCRneg ILC3 (CRTH2negCD117pos) subsets were also detected. ILC2 (CRTH2pos) frequency correlated with disease severity measured by model of end stage liver disease (MELD) scoring leading us to study this subset in more detail. ILC2 displayed a tissue resident CD69+ CD161++ phenotype and expressed chemokine receptor CCR6 allowing them to respond to CCL20 secreted by cholangiocytes and stellate cells. ILC2 expressed integrins VLA-5 and VLA-6 and the IL-2 and IL-7 cytokine receptors CD25 and CD127 although IL-2 and IL-7 were barely detectable in inflamed liver tissue. Although biliary epithelial cells secrete IL-33, intrahepatic ILC2 had low expression of the ST2 receptor. Intrahepatic ILC2 secreted the immunoregulatory and repair cytokines IL-13 and amphiregulin.Intrahepatic ILC2 express receptors allowing them to be recruited to bile ducts in inflamed portal tracts. Their frequencies increased with worsening liver function. Their secretion of IL-13 and amphiregulin suggests they may be recruited to promote resolution and repair and thereby they may contribute to ongoing fibrogenesis in liver disease