The role of natural killer cells in tumor recurrence after curative surgery

Abstract

Liver transplantation (LT) and liver resection provide the curative treatment for selected hepatocellular carcinoma (HCC) patients. However, tumor recurrence after curative surgery remains a significant problem, leading to a poor prognosis. Natural killer (NK) cells play an essential role in immune surveillance against HCC dependent on their inhibitory and activating surface receptors. However, the contributions to immune evasion from NK cell mediated immunosurveillance in tumor recurrence remains unclear. Elucidating the underlying mechanisms will be crucial to explore NK cell immunotherapy against tumor recurrence.

To investigate the clinical relevance of NK cells, 389 HCC adult patients receiving LT in Queen Mary Hospital, Hong Kong, from 1995 to 2020 were included in the current study for risk factor screening, univariate, and multivariate analyses. The blood samples were collected before and after LT for NK cell analysis and plasma cytokine detection. Another cohort of 100 HCC patient underwent liver resection from 2019 to 2020 were recruited for analyzing the clinical correlation between NKG2A+ NK cells and tumor recurrence. The blood and tumor tissues were collected. The frequency and function of NKG2A+ NK cells were detected by flow cytometry. RT-PCR was performed to examine HLA-E and TLR4 mRNA expressions in tumor and non-tumor tissues. An orthotopic liver cancer model was established in TLR4 knockout (KO) and wild type mice. To investigate the role of NK cells in hepatic ischemia-reperfusion (IR) injury, TLR4-KO, CXCR3-KO, CXCL10-KO, and wild type mice were subject to hepatic IR injury and the NK cells were analyzed by flow cytometry.

GWR (the ratio of graft volume to estimated standard liver volume) < 60% was an independent risk factor for tumor recurrence after LT. The frequency of circulating NK cells was significantly lower in the GWR < 60% patients, along with increased expression of inhibitory receptors and decreased production of IFN-γ. NKG2A+ NK cells were increased in HCC patients, associated with tumor recurrence after liver resection. The numbers of intra-tumor NKG2A+ NK cells and the mRNA expression of HLA-E, the ligand of NKG2A, were higher than those in the non-tumor tissues. Moreover, NKG2A inhibited the cytotoxicity and cytokine production (IFN-γ and Granzyme B) abilities of NK cells independent of PD1. TLR4 deficiency could hinder tumor growth and decrease NKG2A+ NK cells in vivo. Liver-infiltrated, CXCR3+, and NKG2A+ NK cells were increased in hepatic IR injury. The serum ALT level and hepatic inflammatory gene expressions were downregulated after NK cell depletion. Knockout of TLR4 or CXCL10 could significantly protect liver function and decrease CXCR3+ NK cells after hepatic IR injury.

In summary, LT with GWR < 60% provided a favorable immune microenvironment for late-phase tumor recurrence, characterized by the reduced number and impaired function of NK cells. TLR4 deficiency decreased intra-tumor NKG2A+ NK cells and inhibited tumor development. More CXCR3+ liver-resident NK cells and liver-infiltrated NK cells were correlated to enhanced hepatic IR injury. Understanding the broad spectrum of NK cell functional diversity would help to explore NK cell-based immunotherapy against tumor recurrence and hepatic IR injury.