Investigating the phenotype and functional roles of human liver-resident natural killer (NK) cells

Abstract

The human liver is an immunologically complex organ populated with distinct resident immune cells including liver-resident CD56bright NK (lrNK) cells which have a distinct CD69+CXCR6+T-betloEomeshi phenotype. These phenotypical markers aid in distinguishing lrNK cells from blood counterparts but they don t provide insights into their effector function within the liver. In this study, the upregulation of genes encoding Ly9, CD160 and TIGIT, which were identified from an RNA-seq dataset, were validated by flow cytometry using matched liver tissue and perfusate samples (n=5). Expression of both novel and validated liver-residency markers were assessed on hepatic NK cell and T cell populations isolated from healthy hepatic healthy tissue (n=3-12), liver perfusate (n=9-25) and cirrhotic explant tissue (n=4-14). The majority of hepatic CD56bright NK cells co-expressed CXCR6, CD69, Ly9, TIGIT, NKG2D and lacked CD49a, in both liver perfusate and tissue (70±15.3% and 58.3±12.9% respectively). A CD49a+ CD56bright NK cell subset was confined to digested tissue samples (7.0±4.3% of total CD56bright NK cells; p<0.02). The receptors Ly9, CD160 and TIGIT, were also expressed by other hepatic immune cell subsets and may be used as liver-residency defining markers.

LrNK cells degranulate more potently than their blood counterparts in response to target cells or cytokine stimulation, and in this study we explored their role beyond tumour-directed cytotoxicity. Activated T cells can be targeted for clearance within the liver, and in the context of liver disease can mediate autoimmune disease and rejection of liver transplants. We investigated lrNK cell s ability to regulate T cell activation using an in vitro coculture system of liver perfusate-derived NK cells and blood-derived allogeneic activated T cells (n=10-13). Coculture of activated PBMCs with hepatic NK cells, but not blood NK cells, increased T cell death (p<0.003). CD8+ T cell death was significantly higher than CD4+ T cells (p<0.0001). Addition of an agonistic mAb against CD160 increased CD8+ T cell death in PBMC (p<0.03) and CD3+ T cell cocultures (p<0.01). Addition of the anti-CD160 mAb to FACS-sorted CD56brightCD16+/-, but not CD56dimCD16+ NK cells, increased overall T cell death (p<0.0221). We hypothesise that this lrNK cell-mediated killing may be involved in the prevention of autoimmune disease and in tolerance induction post-liver transplant.

LrNK cells are long-lived in the human liver and may be replenished from the circulation. We investigated whether hepatic-specific soluble factors or hepatocytes can mediate the replenishment of the lrNK cell niche from blood NK cells. Liver-conditioned medium reduced blood NK cells T-bet expression (p=0.04), which we show is mediated by TGF-&#61538; (p=0.03; n=3). An in vitro 3D liver model of HepG2 cells and PBMCs was optimised which resulted in the proliferation of blood NK cells and their induction of lrNK cell markers (n=4-6). This study is the first to identify human lrNK cells as mediators of hepatic immune tolerance and highlights the potential of targeting them for the treatment of autoimmune liver disease or the improvement of liver transplant outcomes.