Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide and the third leading cause of cancer-related mortality (1). The major risk factors for HCC involve chronic hepatitis B and hepatitis C infection, alcohol, and metabolic liver disease (2). Natural killer (NK) cells, characterized as CD3^-CD56⁺ lymphocytes, are mainly involved in the early defense against virus infections and play major roles in eliminating tumor cells (3). NK cells account for only about 5–20% of the circulating lymphocytes in the peripheral blood. In contrast, NK cells are abundant in human liver, accounting for almost half of intrahepatic lymphocytes (4), which lays foundation for the powerful role of NK cells in the liver tumor microenvironment.

Human NK cells are divided into two major subpopulations based on the surface density of CD56 antigen (5). CD56^dim NK cells display a mature phenotype, accounting for approximately 90% of all NK cells and mediating the cytolytic response, while immature CD56^bright NK cells account for 5%-15% of total NK cells and are regarded as cytokine producers (6). Another surface marker is CD57, which is a marker for differentiated and highly cytotoxic NK cells, and is described as a phenotypically stable NK cells marker (7).

The regulation of NK cell function is mediated by a series of activated or inhibitory surface receptors. The major activated receptors involved in target cell killing are NK group 2 member D (NKG2D) and natural cytotoxic receptors (NCRs). NCRs mainly consist of NKp44, NKp46 and NKp30 (8), and can recognize ligands from different sources, including viral, parasitic, bacterial, as well as cellular ligands, such as HLA-B-associated transcript 3/Bcl-2-associated athanogene 6 (BAT3/BAG6), mixed lineage leukemia 5 (MLL5), proliferating cell nuclear antigen (PCNA) and B7 homolog 6 (B7-H6) (9, 10). In contrast, NKG2D mainly binds to the major histocompatibility complex class I chain-related protein A and B (MICA and MICB) and UL16-binding proteins (ULBPs). After binding, it can activate NK cells to produce cytotoxic substances to kill harmful and tumor cells (11). Other activated receptors include CD16, NKp88, CD244, CD226 and cytokine receptors such as interleukin (IL)-2R, IL-12R, IL-28R, IL-18R, IL-1R8, IL-15R, IL-10R, interferon receptor (IFNR) and tumor growth factor-β receptor (TGF-βR) (12–14).

The major inhibitory receptors involved in target cell killing are NKG2A, CD96, killer immunoglobulin-like receptors (KIRs), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) and T cell immunoglobulin domain and mucin domain-3 (TIM-3) (12). Other inhibitory receptors include programmed cell death-1 (PD-1), lymphocyte activation gene-3 (LAG3), leukocyte-associated immunoglobulin-like receptors (LAIRs), adenosine 2A receptor (A2AR) and immunoglobulin-like transcripts (ILTs) (15). PD-1 is primarily expressed by activated T lymphocytes, but may also be expressed by NK cells in tumor patients. PD-1/Programmed cell death ligand-1 (PD-L1) interactions can inactivate T cells and NK cells, allowing tumor cells to escape immune surveillance (16). Human histocompatibility leucocyte antigen E (HLA-E) is the main ligand of NKG2A, and is generally upregulated in cancer patients and predicts poor prognosis (17–19). The major histocompatibility complex class I (MHC-I) is the main ligand of KIRs, and is expressed on healthy hepatocytes. It interacts with inhibitory receptors on NK cells to prevent the activation of NK cells (14).

Until now, the correlation of NK cells and their receptors/ligands with the prognosis of HCC remains controversial (20–28). The purpose of this meta-analysis and review is to evaluate the prognostic value of NK cells and their receptors/ligands in HCC.

NK cells are innate lymphocytes that can kill virus-infected or cancer cells, and have a vital role in early hepatocarcinogenesis (51, 52). Different to T cells which require somatic gene rearrangement to produce highly antigen-specific receptors (53), NK cells are innately equipped with germline-encoded activating and inhibitory receptors that can directly determine whether NK cells are activated or inhibited (12, 54). NK cells can deliver cytotoxic granules, secrete effector cytokines, and are involved in death receptor induced apoptosis (55). NK cells can also rapidly produce cytokines with anti-tumor effects, such as IFN-γ, to exert their killing effects in the early stage of disease (56). In addition, NK cells can bind to target cells through surface CD16 and kill them through exerting antibody-dependent cell-mediated cytotoxicity (ADCC) (57). These results imply that NK cells play an essential role in the body’s immune process in the defense against HCC.

In this study, we found that high NK cell level could predict better survival for patients with HCC. Similar results were previously reported in a meta-analysis of solid tumors (58). In subgroup analysis, CD57⁺ NK cells had better prognostic value over CD56⁺ NK cells. On the one hand, it may be that CD56⁺ NK cells, accounting for the majority of circulating NK cells, also expressed inhibitory molecules, which may strive for a dynamic balance between activating and inhibitory molecules. Moreover, lower IFN-γ production was also described in HCC, in accordance with the decreased cytotoxicity of NK cells (59). On the other hand, acquisition of CD57 represents a shift toward a higher cytotoxic capacity, greater responsiveness to signaling via CD16 and natural NCRs (60). The same result was also observed in Hu et al.’s study (61). In addition, compared to peripheral NK cells, NK cells from intratumor had better prognostic value for prognosis of HCC patients, possibly because NK cells are abundant in human liver.

NK cells express activating and inhibitory receptors in order to perceive signals and display their activity. Depending on the received signal, NK cells can be activated or restricted (62). In this study, activating receptors/ligands mainly contain NKG2D, NKp30, and their ligands. NKG2D ligands mainly consist of ULBPs and MICA/B. MICA/B molecules expressed on HCC cells are recognized by NKG2D to induce ubiquitination-mediated endocytosis of the NKG2D-DAP10 complex, thereby activating NK cells to kill HCC (46, 57). NKp30 contains two ligands. One is BAT3, a nuclear protein that induces apoptosis in target cells by interacting with P53. The other is B7-H6, a newly discovered member of the B7 family that is expressed on the surface of tumor cells (45). B7-H6/NKp30 pathway is involved in the NK cell-mediated immune responses, and NK cells can recognize and eliminate B7-H6-expressing tumors, including HCC. However, tumors can also impair NK cell function by shedding B7-H6 membranes or decreasing NKp30 expression, leading to tumor immune escape and tumor progression (63). In addition, hypoxia, some soluble forms of NCRs ligands, or soluble factors produced by tumor/tumor-associated cells, can induce a decrease in both NCR expression and function (10, 64, 65), and protect tumor from NK cell-mediated cytotoxicity (44, 66). Until now, the prognostic value of activating receptors remained inconsistent from different studies and deserved further investigation.

Inhibitory receptors/ligands mainly contain NKG2A, CD96, CD158b, TIGIT and TIM-3, and HLA-E. The NKG2A is expressed approximately in half of the peripheral blood NK cells, and is also expressed on CD8⁺ T cells (67). The inhibitory signals induced by NKG2A engagement can result in decreased capacity of NK cells and CD8⁺ T cells to lyse target cells (68), and enhanced expression of the HLA-E on tumor may result in resistance and immune escape by binding to NKG2A (68). Therefore, blocking the interaction of NKG2A with HLA-E has shown promising therapeutic effects in animal study (69). In addition, there is increasing evidence that PD-1 is also expressed on the surface of NK cells and exerts a suppressive function on T cell responses (16). In this study, though we found that inhibitory receptors of NK cells may be a good predictor for recurrence of HCC, however, their prognostic value in predicting survival was unclear. Therefore, more high-quality prospective studies are needed to explore the prognostic value of NK cells and their receptors/ligands for HCC.

The strength of this study is that it explored for the first time the prognostic value of NK cells and their receptors/ligands in HCC. Almost all relevant articles that could be collected were included and a comprehensive analysis was provided. However, the following limitations should also be considered. First, some of the data were not obtained directly from the included studies. HRs and 95% CIs were calculated using survival curves or 95% CIs were calculated from known P values and HRs, which may result in data inaccuracy to some extent. Second, although most studies used median as the cut-off value for NK cells level, these values were complex and related to the clinicopathological characteristics of the HCC patients. Third, although DFS/RFS/TTR/PFS of HCC patients are considered as composite outcome indicators, there are still slightly difference between them. Fourth, NK cells receptors/ligands are diverse. The pooled results may exist in bias to some extent. Fifth, this was an aggregate data rather than an individual data meta-analysis, and the data were various between studies, which somehow diminished the significance of the study. Finally, some surface markers were expressed not only in NK cells, but also in other immune cells, such as T cells, Dendritic cells (DCs), etc. Moreover, liver NK cells are composed of several subsets including conventional, type 1 innate lymphoid cells (ILC1) like, and liver-resident NK cells. All of them can express similar molecules and potentially be responsible for the clinical benefit observed (70).

In summary, we concluded that NK cells could be a good predictor for survival of HCC. More importantly, CD57⁺ NK cells may have better prognostic value over CD56⁺ NK cells, and intratumor NK cells have better prognostic value over peripheral NK cells. Inhibitory receptors of NK cell may be a good predictor for recurrence of HCC, but the value of activating and inhibitory receptors in predicting the survival of HCC was unclear. More high-quality prospective studies are essential to evaluate the prognostic value of NK cells and their receptors/ligands for HCC.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

J-SX and TL were responsible for designing the study. J-SX, Z-ND, and G-XM conducted the systematic search and performed the screening. J-SX, Z-ND, G-XM, L-JY, HL, H-CL, S-YY, B-WT, J-GH, Z-RD, Z-QC, and D-XW primarily performed the quality assessment as well as supervision. J-SX analyzed, interpreted the data, and drafted the manuscript. TL revised the manuscript. All data and material analyzed during this study were included in this article. All authors have read and approved the final version of the manuscript.

This work was supported by the grants from the Taishan Scholars Program for Young Expert of Shandong Province (Grant No. tsqn20161064), National Natural Science Foundation of China (Grant No. 82073200 & 81874178), funds for Independent Cultivation of Innovative Team from Universities in Jinan (Grant No. 2020GXRC023), and Major basic research of Shandong Provincial Natural Science Foundation (Grant No. ZR202105070027).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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