Innate and adaptive natural killer cells in the liver and peripheral blood

Background and Aims: Natural Killer (NK) cells are innate-like lymphocytes, yet selected murine populations display antigen-specific ‘memory’ towards haptens and viruses. Liver-specific adhesion molecules CXCR6 and CD49a act as surface markers. Cytokines can also generate NK cells with memory-like behaviour. I aimed to identify CXCR6+ and CD49a+ NK cells in humans, define their phenotype and function and examine the role of cytokines in driving their differentiation and proliferation. Secondly, following the discovery of an association between reduced STAT4 expression and NK cell tolerance, I aimed to investigate if overactivation of the IL-12/STAT4 axis, linked to susceptibility of the autoimmune liver disease Primary Biliary Cholangitis (PBC), may generate hyperfunctional autologous NK cells in this condition, and whether NK cells with a liverresident or memory-like phenotype play a role in mediating this.Results: CD49a+ and CXCR6+ NK cells comprised 7.8% and 59.5% of intrahepatic NK cells (both <5% in the circulation). CD49a+ NK cells were present at high frequencies in only 10% of individuals. They harboured higher frequencies of KIR+ and NKG2C+ NK cells compared to their CD49a- counterparts, suggestive of clonal expansion and licensing and produced high quantities of Th1 cytokines. Conversely CXCR6+ NK cells were found universally and were immature (CD56brightCD16low) with upregulation of markers of tissue-residency and cell adhesion. Liverresident CD49a+ NK cells underwent rapid expansion following culture with all activating cytokines, in contrast to CXCR6+ NK cells, however both phenotypes can be induced in the peripheral blood using IL-12 and IL-15. Cytokine-induced CD49a+CXCR6+ NK cells expressed markers of adaptive behaviour (NKG2C, IFNγ) and tissue-residency (CD69). Furthermore I present the first functional correlate of the PBC GWAS studies, identifying overactivation of the IL-12/STAT4 axis in NK cells in PBC. This is manifest by elevated resting levels of pSTAT4 and the generation of CXCR6+ and CD49a+ NK cells which produce high quantities of IFNγ in response to low doses of IL-12. Hyperfunctional NK cells may home to the biliary tree via their interaction with CXCL16 and potentially lyse autologous BECs.Conclusion: Human hepatic CD49a+ NK cells display features consistent with adaptive behaviour, whereas CXCR6 is a marker of liver-residency. Cytokine stimulation generates hyperfunctional, potentially adaptive-like, CD49a+ NK cells in the blood and may induce their migration to the liver via CXCR6, linking peripheral activation of NK cells with liver-homing. These findings may have important implications for the development of locally acting immunotherapies for viral hepatitis and hepatocellular carcinoma. The identification of overactivation of the IL-12/STAT4 signalling cascade implicates NK cells in the pathogenesis of PBC and suggests a role for the newly discovered CXCR6+ and CD49a+ liver-resident NK cell subsets in autoimmunity. Inhibition of this pathway may provide a new therapeutic target for PBC.