Abstract
NK cells have potent antitumor capacity. They are enriched in the human liver, with a large subset specialized for tissue-residence. The potential for liver-resident versus liver-infiltrating NK cells to populate, and exert antitumor functions in, human liver tumors has not been studied. We examined liver-resident and liver-infiltrating NK cells directly ex vivo from human hepatocellular carcinomas (HCCs) and liver colorectal (CRC) metastases, compared with matched uninvolved liver tissue. We found that NK cells were highly prevalent in both HCC and liver CRC metastases, although at lower frequencies than unaffected liver. Up to 79% of intratumoral NK cells had the CXCR6+CD69+ liver-resident phenotype. Direct ex vivo staining showed that liver-resident NK cells had increased NKG2D expression compared to their non-resident counterparts, but both subsets had NKG2D downregulation within liver tumors compared to uninvolved liver. Proliferation of intratumoral NK cells (identified by Ki67) was selectively impaired in those with the most marked NKG2D downregulation. Human liver tumor NK cells were functionally impaired, with reduced capacity for cytotoxicity and production of cytokines, even when compared to the hypo-functional tissue-resident NK cells in unaffected liver. Coculture of human liver NK cells with the human hepatoma cell line PLC/PRF/5, or with autologous HCC, recapitulated the defects observed in NK cells extracted from tumors, with downmodulation of NKG2D, cytokine production, and target cell cytotoxicity. Transwells and conditioned media confirmed a requirement for cell contact with PLC/PRF/5 to impose NK cell inhibition. IL-15 was able to recover antitumor functionality in NK cells inhibited by in vitro exposure to HCC cell lines or extracted directly from HCC. In summary, our data suggest that the impaired antitumor function of local NK cells reflects a combination of the tolerogenic features inherent to liver-resident NK cells together with additional contact-dependent inhibition imposed by HCC itself. The demonstration that IL-15 can recover hepatic NK cell function following tumor exposure supports its inclusion in immunotherapy strategies.
Highlights
Hepatocellular carcinoma (HCC) remains a very difficult to treat tumor with an exceptionally poor prognosis
Cells were identified by multiparameter flow cytometry of single cell suspensions derived from liver and tumor tissue. In both primary liver tumors (HCC) and secondary liver tumors arising from CRC metastases NK cells were prevalent, accounting for around 25% of the CD45+ lymphocytes, with a frequency intermediate between blood and uninvolved liver
We use direct ex vivo analysis of freshly isolated human tumor and liver lymphocytes to show that HCC and CRC metastases are populated by high frequencies of NK cells with liver-resident and liver-infiltrating phenotypes
Summary
Hepatocellular carcinoma (HCC) remains a very difficult to treat tumor with an exceptionally poor prognosis. We and others have recently discovered that a variable fraction (up to 80%) of intrahepatic NK cells are liverresident, characterized by expression of CXCR6 and CD69, with a distinct transcriptional and functional signature [18,19,20] These tissue-resident NK cells survive long term in the human liver and are unable to recirculate, while the remaining liver-infiltrating fraction have the transcriptional profile of peripheral NK cells [20, 21]. We use direct ex vivo analysis of freshly isolated human tissue lymphocytes to compare the contribution of liver-resident and liver-infiltrating NK cells to the composition and functional features of the intratumoral pool. We probe the capacity of HCC to further impair tolerogenic liver NK cells via NKG2D downregulation and the potential for cytokine-mediated rescue as an immunotherapeutic strategy in this setting
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