Dysfunctional Natural Killer cells expanded from a liver with Hepatocellular Carcinoma show reduced killing against a unique HCC cell line

Abstract

BackgroundThe liver has one of the largest reserves of NK cells in the body. NK cells in the liver are key defenses against viral hepatitis, liver fibrosis, and tumorigenesis. In the healthy liver, they exhibit higher cytotoxicity and secrete more cytokines compared to those isolated from peripheral blood. Hepatocellular carcinoma (HCC) is one of the most rapidly increasing cancers worldwide. Previously, NK cells have been shown to be dysfunctional in HCC. Additionally, NK cells are reduced in number intratumorally and peripherally and there is a decrease in the cytotoxic CD56dim subpopulation. Many studies have shown a downregulation of key signaling molecules involved in peripheral NK cell cytotoxic activity. We aimed to further investigate the level of dysfunction of NK cells isolated from HCC livers to document the nature of dysfunction of liver‐resident NK cells in eliminating HCC cell lines.MethodsBriefly, samples of tissue were collected from explanted livers and made into single cell suspensions. Next, lymphocytes were isolated using density‐based centrifugation. The resulting fraction was placed into RPMI‐1640 with IL‐2 and IL‐15. After two weeks, samples were taken to determine the number and purity of NK cells by flow cytometry. Target cell lines were labeled with chromium‐51 to test the killing capacities of NK cells isolated from healthy donor PBMC’s or HCC patient livers. NK cells were prepared in serial dilutions and in triplicate to generate a killing curve. After 4 hours of coincubation of effector NK cells with target cells, the released radioactive particles were measured and analyzed.ResultsTwo cell lines, Hep3B (ATCC) and HCO2 (generated in our lab), were used as target cells. Figures 1 and 2 depict the results of separate killing assays using the different target HCC cell lines. The effector cells in each assay consist of NK cells sourced from either healthy donor PBMC’s, cirrhotic patient PBMC’s, or HCC liver. Notably, the results show that even under the constant stimulation of IL‐2 and IL‐15, which are known interleukins that are involved in the proliferation and activation of NK cells and T‐cells, there is a large dysfunction in NK cells sourced from diseased livers. More strikingly, our results show that there is a specific inability of NK cells sourced from HCC livers to eliminate HCO2 cells which have been previously shown in our lab to be sorafenib‐resistant. Our hypothesis is that there is an innate dysfunction that is present in NK cells isolated from diseased livers that is not recoverable even when cultured in the presence of activating cytokines. Additionally, HCO2 cells possess a unique resistance to killing by NK cells isolated and expanded from an HCC liver.ConclusionHere we show that NK cells can be isolated and expanded, ex vivo, from human diseased livers. Additionally, we show that despite being stimulated for over 2 weeks with cytokines, expanded NK cells still show a marked reduction in killing capacity against a variety of HCC cell lines compared to NK cells expanded from PBMC’s from healthy and diseased donors. Our results confirm that there is an innate dysfunction that is present within NK cells isolated from diseased livers that is not recoverable even when cultured in the presence of activating cytokines.HCC cell lines (Hep3B, in circles, HCO2, in triangles) were labeled with Chromium‐51 for 2 hours. Target cells were rinsed with media and cocultured with NK cells at various effector‐to‐target ratios. NK cells were sourced from either PBMC’s from a cirrhotic patient (PBNK, in blue) or a liver with hepatocellular carcinoma (LNK, in green) and expanded ex vivo with IL‐2 and IL‐15.Figure 1