Natural killer cells in hepatocellular carcinoma: current status and perspectives for future immunotherapeutic approaches

Abstract

Hepatocellular carcinoma (HCC) is currently the fifth most common malignancy and the third leading cause of cancer-related mortalities worldwide. In the last few years, treatments for HCC have significantly improved from a mere surgical resection to a series of minimally invasive therapies and targeted drugs. However, recurrence frequently occurs even upon curative therapeutics, and drug therapies generally produce disappointing results, with the overall prognosis dismal. This challenging clinical scenario warrants new effective and life-prolonging strategies for patients with HCC. Compelling evidence suggests that NK cells play a critical role in the immune function of the liver and in the immune defenses against HCC, indicating that HCC might be an ideal target for NK cell-based immunotherapies. To obtain comprehensive insights into the putative influence of NK cells on HCC, this paper summarizes current knowledge on NK cells in HCC and discusses the usefulness and prospects of NK cell-based immunotherapies. Critical issues that require consideration for the successful clinical translation of NK cell-based therapies are also addressed. If appropriately used and further optimized, NK cell-based therapies could dominate important roles in the future immunotherapeutic market of HCC.

Keywords

natural killer cell / hepatocellular carcinoma / immunotherapy

Introduction

Given its poor prognosis, hepatocellular carcinoma (HCC) is currently the fifth most common malignancy and the third leading cause of cancer-related mortalities worldwide [1]. The development of modern imaging technologies and the establishment of surveillance protocols for high-risk individuals have facilitated the diagnosis of many HCC cases at the early stages, thereby providing opportunities for curative treatments, including carcinectomy and ablation [2]. However, recurrence frequently occurs even upon curative therapeutics, and outcomes are generally dismal [2]. Transcatheter arterial chemoembolization (TACE) offers limited palliative benefits to patients with unresectable intermediate-stage HCC [3,4], and systematic chemotherapy lacks benefits. Sorafenib is still the only targeted drug therapy that is clinically recommended for advanced HCC; however, it can only extend the overall survival (OS) by 3 months, and subsequent efforts of drug development have failed [5]. This challenging clinical scenario warrants new effective and life-prolonging strategies for patients with HCC.

Current insights into tumor biology and immunology have opened new perspectives to treat human malignant tumors; therapeutic strategies are aimed at harnessing the patient’s immune system to attack malignant cells [6]. Natural killer (NK) cells, characterized in humans as CD56-positive and CD3-negative lymphocytes, constitute the major players of innate immunity involved in the early defense against both cancers and certain virus-infected cells [7]. Unlike their counterpart T cells that require somatic gene rearrangements to produce highly antigen-specific receptors, NK cells are innately equipped with germline-encoded activating and inhibitory receptors that determine NK cell activation [7,8]. NK cells deliver cytotoxic granules, secrete effector cytokines, and engage death-inducing receptors, such as Fas ligand and tumor necrosis factor (TNF)-related apoptosis-induced ligand, to stimulate target cell apoptosis. In addition, NK cells are the main mediators of antibody-dependent cell-mediated cytotoxicity (ADCC) [9–12]. The properties of NK cell candidates being rigorously pursued in different settings of caner immunotherapies and preceding clinical results using NK cells have yielded compelling positive responses [13–15].

NK cells are enriched in human livers, forming up to 30%–50% of the intrahepatic lymphocytes, the proportion of which is approximately two to five times as high as that of peripheral NK cells. Moreover, NK cells within a healthy liver have unique phenotypic features and functional properties, displaying significantly higher cytotoxic activity against tumor cells compared with circulating NK cells [16,17]. In patients with HCC, tumor infiltrating lymphocytes (TILs) and peritumor lymphocytes are primarily T and NK cells [18]. Patients with low intratumoral NK cells among TILs have shorter disease-free survival (DFS) and OS, and a restoration of NK cell activity after curative surgery has been associated with recurrence-free survival (RFS) [18–20]. The immune surveillance exerted by NK cells is pivotal in the immune functions of the liver and in the immune defense against HCC, suggesting that HCC is an ideal target for NK cell-based immunotherapies. To obtain comprehensive insights into the putative influence of NK cells on HCC, this paper summarizes the biological relevance of NK cells to cancer immunotherapy and discusses the usefulness and prospects of manipulating NK cells in HCC treatment. Critical issues that require consideration for the successful clinical translation of these therapies are also addressed. If appropriately used and further optimized, NK cell-based therapies could dominate important roles in the future immunotherapeutic market of HCC.

NK cells in the context of HCC

The etiological factors of HCC are hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, alcoholism, nonalcoholic steatohepatitis, and diabetes. These factors cause chronic inflammation, contribute to fibrosis, and eventually result in cirrhosis, a precancerous condition of HCC [21]. Evidence in the last few decades suggests that NK cells play an important role in controlling viral hepatitis, liver fibrosis, and carcinogenesis but also contribute to liver damage in viral and inflammatory diseases [22]. Deciphering the dynamic alterations of NK cells is important to identify the roles of these cells in different stages. Accordingly, this paper summarizes previous findings on the functions of NK cells in HCC.

A genetically engineered mouse model was used to study the onset and progression of HCC and detect the persistent deregulation of numerous NK cell-related genes in the early stages of the disease; results suggest that the disruption of NK cell-mediated immune surveillance contributes to the early onset of hepatocarcinogenesis [23]. Patients with HCC have impaired or decreased number of peripheral and intrahepatic NK cells; these features are correlated with post-surgical recurrence and prognosis [18,24]. In addition, these patients show altered distributions of NK sub-populations, with a significant reduction in the CD56-dim NK subset (more mature and cytotoxic) both peripherally and intratumorally [25]. Moreover, residual CD56-dim NK cells present reduced interferon-g production and cytotoxicity [24]. Increased frequencies of NK cells expressing high levels of activating and reduced levels of inhibitory NK receptors, together with increased functional activity, participate in controlling HCC [26]. However, rare experimental studies that explored the anti-tumor activity of NK cells found that they are efficient in killing different HCC cell lines, inhibiting the engraftment of intraportally injected HCC cells, and eliminating small HCC lesions and metastases in vivo [27]. Clinical trials showing the adoptive infusion of lymphokine-activated killer (LAK) cells containing mostly NK cells also demonstrated the therapeutic safety and efficacy of these cells in patients with advanced HCC [28–30]. Although somewhat unilateral, these current studies demonstrate the effects of NK cells on HCC and the potential of NK cell-based immunotherapies against HCC.

NK recognition of HCC

NK cells express a broad range of activating and inhibitory receptors, and the engagement balance determines NK cell activation [31,32]. Activating receptors bind ligands on the transformed cells and trigger target cell lysis, whereas inhibitory receptors recognize major histocompatibility complex (MHC) class I molecules and inhibit cytotoxicity by overruling the activity of the activating receptors [33]. Normal self cells abundantly express MHC-I molecules and are protected from NK cell attack. Transformed cells inductively express stress ligands (“induced-self”) and reduce MHC-I expression (“missing-self”), rendering these cells susceptible to NK cell-mediated attack [33,34]. Along with tumor progression, the expression of these receptors on NK cells is frequently altered; tumor variants also constantly upregulate/lose ligands for inhibitory/activating receptors (immunoediting), both representing the mainstay hallmarks to evade NK cell responses [35].

Activating receptors, such as the NK group 2D (NKG2D), natural cytotoxicity receptors (NCRs), cluster of differentiation (CD)-226, and Fcg receptor IIIA (CD16), are significant in cancer immunosurveillance [33]. NKG2D recognizes multiple molecules, including polymorphic MHC class I chain-related molecules (MIC)A/B and the cytomegalovirus UL-16 protein (ULBP1-6), which are poorly expressed on healthy cells but are inductively upregulated on transformed hepatocytes [33,36,37]. The upregulation may tip the balance of NK cells from inhibition to activation and trigger target cell lysis [33]. However, tumors develop mechanisms (i.e., downregulation of ligands on target cells or downmodulation of NKG2D on effector cells due to shedding ligands) to overcome the NKG2D-mediated immune response, which is associated with poor prognosis in many human cancers, including HCC [38–40]. Other activating receptors are less defined than NKG2D despite the growing evidence of their collaborative or equally significant roles in NK cell functions. The three known NCRs are NKp30, NKp46, and NKp46. Among the NCRs, only NKp30 has identified tumor antigens (B7-H6), and its downregulation has been observed in HCC patients and correlated with disease progression [41]. CD226 is another important activating receptor. Its ligands, CD155 (poliovirus receptor) and CD112 (nectin-2), are expressed on various malignancies. Low expression levels of CD112 and CD155 have been associated with resistance to NK cytotoxicity and poor prognosis in patients with HCC [42]. CD16 endows NK cells with ADCC property, which is the primary killing mechanism for some monoclonal antibodies (mAbs) [9–12]. Clinical trials have shown that HCC patients with a high affinity FcgRIII polymorphic variant experience a better outcome in response to mAb, which favors the desirable role of CD16 in HCC [12].

Inhibitory receptors, such as killer-cell immunoglobulin-like receptors (KIRs) and the receptor complex CD94-NKG2A groups, activate NK cells by interacting with different MHC I molecules/human leukocyte antigens (HLAs) [32,33]. Unlike activating receptors, inhibitory receptors (particularly KIRs) form a polymorphic immunogenetic profile with their ligands where NK cell functions vary and HCC outcomes differ; therefore, incorporating the KIR ligand interactions into a biological model and studying the immunogenetic background that impacts NK cell activity are highly recommended. The results are presented in the section below [43].

KIR/HLA compound genotypes on HCC

Both KIRs and their cognates are highly diverse and polymorphic, encoded by multigenic and multiallelic families of genes that are independently inherited [44]. The binding affinity of KIR/HLA interactions varies, and a specific KIR may be expressed in the absence of the cognate HLA molecule. Therefore, various KIR/HLA combinations with potentially variegated NK cell activity are generated [44,45]. The binding of high-affinity inhibitory KIRs to their cognates exerts a counteractive effect on NK cell activation; however, the “licensing” or “education” occurs during the maturation of competent NK cells [46]. NK cells become functionally competent once their KIRs interact with the cognate molecules. This change causes the high-affinity genotype compound to produce educated NK cells with vigorous cytotoxicity [31,46]. In support of the “licensing” model, an immunogenetic study found that the concurrent presence of KIR2DL2 and HLA-C1 correlates with prolonged RFS of patients with HCC after therapeutic ablation (C1+/C1‒). Individuals with HLA–Bw4I80 (high-affinity) have an analogous prognosis as compared with those with HLA-Bw4T80 (low affinity) [43,47].

Multiple basic studies have shown a synergistic effect of compound genotypes on licensing, but its clinical relevance remained vague until Tanimine et al. have recently demonstrated that the presence of each functional KIR/HLA compound genotype does not affect HCC recurrence; the multiplicity of at least three compound genotypes is significantly associated with reduced HCC recurrence [43,48]. Inhibitory receptors that drive the education of NK cells are not always helpful because litigation of inhibitory receptors might offset education benefits in the context of HLA-expressing cells. However, both studies compared NK cells that expressed HLA-specific inhibitory receptors with those that did not and focused on the impact of NK cell education while ignoring that of “non-beneficial” inhibition. Previous studies demonstrated that unlicensed NK cells form the dominant subset of NK cells with potent ADCC due to lack of inhibitory receptors for self cells. Therefore, the impact of inhibitory KIR/HLA function on HCC progression needs further studies. Moreover, both studies are based on HCV-related HCC patients, where NK cells exhibit protective effects against HCC development. For patients with HBV infection, the opposite role of NK cell activation has been suggested for HCC development, and the immunogenetic type equipped with high NK cell functional potential has been associated with HCC progression [49–51]. The apparent discrepancy is derived from their different virological characteristics and immune responses. HBV is not a potent inducer of innate immune response but allows potent adaptive immunity to control the infection, as demonstrated by more than 90% spontaneous resolution in infected adults [52,53]. In patients who are unable to eliminate the virus, an enhanced phenotype of NK function contributes to tissue damage and persistent inflammation condition, both of which promote HCC development [51,54]. Contrary to HBV infection, HCV generally replicates at a very high level and induces good innate response. An impairment of NK cell function influences not only HCV virus load but also the risk of HCC progression [53,55]. Although not yet fully defined, HCC progression is partly due to the dysfunction of NK cells, and the immunogenetic profile of KIR/HLA is associated with NK cell activity, which influences the outcomes of HCC patients.

Tumor microenvironment in shaping NK cell functions

Once the tumor is established, mutual interactions between tumor cells and host immune system create a strong intrinsic inhibitory network that favors tumor survival [56]. This network is one of the major obstacles hindering the success of cancer immunotherapy, as displayed by the partially, if not totally, mitigated in vivo efficacy of the remarkable in vitro anti-tumor effect. This situation is applied to NK cell-based immunotherapies. Understanding how their attack might be compromised is essential in exploring clinically feasible approaches to orchestrate cancer cell-based immunotherapies (Fig. 1).

TGF-β and IL-10, the most potent soluble immunosuppressors, inversely correlate with clinical outcome in patients with HCC [57–60]. These immunosuppressors exert various effects on NK cells, including inhibition of activation, cytotoxicity, and cytokine production, which might contribute to HCC progression [61–64]. Some of the mechanisms involved have been suggested by indirect evidence; direct effects on NK cells should be further confirmed in HCC models [63,64]. Multiple cellular components within the liver, including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), immature dendritic cells, and macrophages polarized toward the immunoregulatory M2 phenotype, pose a detrimental impact in cancer development through promoting immune tolerance [65]. Tregs usually infiltrate HCC, and their levels have been correlated with HCC stages [66,67]. They compete with NK cells for IL-2 availability and impair NK cell responses through membrane-bound TGF-β [68]. MDSCs are immature myeloid cells with immunosuppressive activity [69]. They accumulate in tumor-bearing humans with different types of cancer, including HCC [69,70]. Recent data have suggested that MDSCs from patients with HCC could inhibit autologous NK cell cytotoxicity and cytokine production, and the suppression is mainly dependent on NKp30 from NK cells [41]. Hypoxic stress, a prominent feature of solid tumors, favors the emergence of tumor variants with increased metastatic and invasive potentials [71]. Nonetheless, accumulating evidence has shown that hypoxic stress also modulates the functions of immune effectors, including NK cells, but the underlying mechanisms remain to be completely elucidated [72,73]. These studies suggest that the milieu within tumors usually poses a formidable challenge to NK cell efficacy; studies aiming at establishing a favorable tumor microenvironment to boost NK cell cytotoxicity would be promising.

Immunotherapies of HCC based on NK cells

Knowledge on the roles of NK cells in HCC was briefly reviewed to evaluate and develop innovative approaches in the field. Recent studies and clinical trials have employed NK cell-based strategies, including the administration of immunomodulatory drugs that activate NK cell functions, the adoptive transfer of activated NK cells, and the use of monoclonal antibodies that block the interaction between inhibitory receptors and their ligands. Rather than summarizing all available HCC therapies that are associated with NK cells, this paper presents an overview of HCC studies that employed NK cell-based immunotherapeutic approaches that are clinically relevant and have been intensely pursued (Fig. 2).

Off-target effects on NK cells

In the past decades, radiofrequency ablation and TACE have become the most popular or standard modalities for curative or palliative treatments in patients with HCC. Both therapies are efficient in controlling tumor progression and improving prognosis [1,2]. Apart from the straightforward anti-tumor effects through thermodestruction or chemoembolization, multiple evidence from both mouse models and clinical settings has shown increased tumor susceptibility to host immunity, including NK cell-mediated immune response after minimally invasive therapies, to which the preferable outcomes should be attributed at least partly [74–76]. Unfortunately, the status usually lasts for a short time. Adoptive immunotherapies are thus being explored to consolidate the effects after these therapies, which would be detailed in the next section.

Many conventional chemotherapeutic drugs and targeted agents reportedly act on host immunocytes, including NK cells. This study provides a concise overview of some NK cell-based mechanisms exploited by sorafenib and several other potential drugs for HCC. Sorafenib is the only clinically approved targeted drug for advanced HCC [77]. Aside from its direct effects on tumor cells, sorafenib exerts an immune stimulatory function. This drug activates hepatic NK cells through activation of tumor-associated macrophages; it also enhances the NK sensitivity of HCC cells by increasing MICA expression despite its direct inhibitory effects on the proliferation and activation of NK cells [78–81]. However, a recent study has found that expanded NK cells considerably enhance the anti-tumor cytotoxicity of sorafenib and that NK cell cytotoxicity is unaffected. This finding suggests that a combination of sorafenib and activated NK cells would exert additive anti-tumor effects on HCC [82]. Signal transducer and activation of transcription 3 (STAT3) is highly activated in a wide variety of cancers. Blockade of STAT3 inhibits tumor growth in several types of cancers, including HCC [83]. A recent study has reported that STAT3 blockade potently enhances NK cell-mediated anti-tumor immunity by reducing inhibitory cytokines; this study has also initiated the potential use of STAT3 in patients with advanced HCC [84]. Other studies have suggested that proteasome inhibitors, such as bortezomib and MG132, and conventional chemotherapeutic agents, including cisplatin, render HCC and other tumors susceptible to NKG2D or TRAIL-mediated lysis [39,85,86]. These findings provided valuable information and prompted investigations on the possible combinations of immunotherapies and treatments that can improve clinical efficacy.

Autologous NK cell transfer

Initially, autologous NK cells were explored to combat against tumors. Highly purified NK cells are ideal candidates, but technical barriers in the past precluded. Therefore, LAK cells whose anti-tumor ability is mediated primarily by activated NK cells were employed in clinical studies of HCC [87]. The efficacy of LAK cells as adjuvant therapies for patients who have undergone curative treatments has been investigated in case reports, case series, and several randomized controlled trials, as well as in few case or series reports involving inoperable end-stage patients [88,89]. The safety and therapeutic efficacies of adoptive infusion of LAK cells have been reported. Three successive randomized trials confirmed the beneficial outcomes, although the benefits might be limited to certain objects or subgroups [28–30]. Nevertheless, the enthusiasm of studies on LAK cells for HCC treatment rapidly faded as increasing reports on other cancers indicated that clinical responses of LAK cells have been infrequent/transient and that severe toxicity of high doses of IL-2 cannot be avoided.

Thereafter, cytokine-induced killer (CIK) cells (a heterogeneous subset of ex vivo expanded lymphocytes that primarily presented a mixed T-NK phenotype) emerged and predominated scientific studies exploring efficient adjuvant therapies for HCC treatment [90,91]. Previous studies demonstrated that adjuvant immunotherapies with activated CIK cells are well tolerated and associated with improved DFS in HCC patients after curative treatment, as confirmed by several integrated analysis studies [91–93]. Notably, a recent multicenter, randomized, open-label, phase III trial study revealed that the median RFS of the therapy group (44 months) is significantly prolonged compared with that of the control group (30 months) [94]. Therefore, the clinical response following infusion of CIK cells in HCC treatment after curative therapeutics has been actually supported by accumulating evidence despite the fact that adoptive immunotherapies were otherwise thought of as outdated and useless. The unremovable microscopic lesions possibly benefited from the adjuvant therapies as displayed by the reduction in early recurrence rather than late recurrence [94]. Experimental studies supported the assumption that small tumors or micrometastases are eliminated by NK cells, whereas large established solid tumors usually do not respond. Although non-specific immunotherapies are ineffective in solid mass, they are feasible for HCC treatments after curative therapies, even if faced with the risk of signal silence by self-MHC.

After overcoming the technical challenges to effectively expand NK cells, clinical-grade highly-purified primary NK cells have become available [95,96]. Clinical trials (NCT02725996) using autologous purified NK cells have recently started to prevent HCC recurrence after curative therapy. This experiment was performed despite the recent shift in focus to the activation of allogeneic NK cells from KIR/HLA mismatch [97]. However, the prerequisite for preconditioning and the risk of rejection were dispensed; with their well-documented effectiveness for HCC treatment after curative therapies, strategies-based autologous NK cells could serve in HCC treatment.

Allogeneic NK cell transfer

Since the discovery of the renowned KIR/HLA mismatch, allogeneic NK cells were increasingly pursued for adoptive cellular therapies [98]. Immunotherapies using allogeneic NK cells alone or in association with hematopoietic stem cell transplantation, especially those exploiting KIR/HLA allogeneicity, are effective in the treatment of several hematological malignancies [14,15,99]. The beneficial outcomes could be extrapolated to solid tumors. Safety and efficacy of adoptive allogeneic NK cell infusion were established in patients with metastatic melanoma and renal cell carcinoma in phase I/II studies [13]. Case studies on HCC have found positive response associated with allogeneic NK cells, with a significant decrease in a-fetoprotein content [100]. However, in this study, no mismatch of KIR/HLA pairs was found. An alternative explanation might be based on the elevated expression levels of activating NK cell receptors. Two clinical trials (NCT02399735 and NCT02008929) have recently investigated the infusion of allogeneic NK cells to prevent HCC recurrence after curative resection or transplantation. Results revealed the potential effectiveness of allogeneic NK cell-based strategies in HCC treatment.

Liver NK cells inductively express TRAIL and demonstrate stronger activity against HCC compared with circulating NK cells, and the attack of IL-2-stimulated liver NK cells against self normal tissue is negligible; therefore, a novel type of adjuvant immunotherapy based on liver NK cells was proposed [16,19,101]. In this study, transplant recipients were intravenously injected with activated NK cells from liver allografts. The results of the clinical phase I trial revealed the feasibility and safety of this immune therapy [102]. To define the long-term benefits of this approach in terms of the control of HCC recurrence after liver transplantation, a phase II trial is currently under consideration (NCT02008929). This trial aims to investigate the influence of KIR/HLA genotypes on donors and recipients of liver transplants. Immunotherapies based on liver NK cells might be particularly prosperous in the transplantation setting. Next to primary NK cells, human NK cell lines can also be used for allogeneic NK cell therapy; NK-92 cells are highly toxic to a wide range of malignancies, including HCC, and have entered clinical trials successfully [103]. Despite their continuous and unlimited proliferation capacity, NK-92 cells have demonstrated their safety in clinical trials with some clinical response in some cancer patients.

Immune checkpoint blockade

Checkpoint blockade is characterized by blockades of programmed dealth-1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA-4), targeting regulatory pathways in T cells to overcome immune resistance. Checkpoint blockade has achieved robust responses in patients with melanoma and non-small cell lung cancer [104]. Evidence from preclinical studies also supported the use of this approach in HCC, and clinical testing is currently underway in advanced HCC patients [105]. Safety and early signs for clinical activity have been reported, although the efficacy warrants further investigation in large clinical trials [105]. PD-1 is expressed in a novel NK cell sub-population, which is characterized by fully mature phenotype and quantitatively increased in some cancer patients [106]. These cells display impaired anti-tumor activity that could be partially restored by PD-1 blockade, highlighting that manipulation of this pathway could circumvent tumor escape both from T/NK cell-mediated immunosurveillance.

NK cells also express surface inhibitory receptors, including the KIR family and CD94/NKG2A heterodimer. IPH2101, a novel anti-inhibitory KIR antibody that relieves educated NK cells from MHC-mediated inhibition, has entered a phase II clinical trial for the treatment of smoldering multiple myeloma [107]. However, no clinical response was found, and the treatment reduced NK cell function mediated through monocyte trogocytosis [108]. The clinical trials were prematurely terminated and raised concerns that checkpoint paradise might be compromised by unexpected biological events [109]. IPH2201, an antibody directing against the CD94/NKG2A receptor, enhances the cytolytic activity of NK cells in preclinical studies, and clinical trials with anti-human NKG2A are ongoing in patients with tumor types that express HLA-E [110]. Previous studies have suggested that HLA-E is differently unregulated in HCC patients, highlighting the possibility of this approach for HCC treatment [111].

NK mediated ADCC

An expanding panel of mAbs was established as targeted therapies for malignancies. Different mechanisms were utilized to destroy cancer cells, one of which was ADCC, by which tumor cell death is evoked when IgG-coated target cells bind to CD16 (or FcgRIII) on the surface of cytotoxic immune cells, mostly NK cells [112]. Clinically approved targeted mAbs against HCC remain unavailable to date. Glypican-3 (GPC3) is an emerging therapeutic target being intensely pursued for HCC treatment [113]. Preclinical data have demonstrated the anti-tumor activity of GC33 (a humanized high-affinity anti-GPC3 mAb) in several human liver cancer xenograft models, and the efficacy is mainly attributable to NK cell-mediated ADCC [114]. Clinical data suggested that high exposure of GC33 with FcgRIIIA-158V polymorphism (higher CD16 expression) correlates with strong efficacy, reinforcing the clinical significance of ADCC [115]. However, no clinical response was observed, and alternative strategies were required to achieve optimal therapeutic benefit [115]. These strategies include increasing the binding affinity of the Fc domain, enhancing immune activation, using inhibitors preventing FcgRIII shedding, and infusing ex vivo activated NK cells. Notably, these approaches combined with tumor-specific antibodies are still being explored in preclinical models. In addition, various combinations that optimize the cure are evaluated and warrant further evaluation.

Another strategy that uses NK cells to target tumor cells involves the novel single-chain variable fragment (scFv) recombinant reagents termed bispecific and trispecific killer cell engagers (BiKEs and TriKEs) [116]. They also trigger degranulation and cytokine production via ADCC; however, BiKE and TriKE (due to higher affinity of scFv than Fc fragment) enhance ADCC as compared with conventional mAbs [117]. TriKE, which incorporates modified IL15 linker, has prolonged NK cell expansion and persistence, heralding a new generation of immunotherapeutics [118,119]. In this respect, these therapies hold great promise for cancer therapies; however, no relevant drugs have been developed for HCC treatment. In the future, a greater effort might be exerted to develop BiKE/TriKE against HCC, but further characteristics of the proliferative and functional responses of NK cells under various TriKE dosing regimens remain to be elucidated.

Genetically modified therapies

Recent studies have highlighted that manipulation of NK cell activation can be used in cancer immunotherapy; in addition, various genetically reprogrammed approaches can be undertaken to improve and broaden the use of NK cell-based immunotherapies [7,35]. Genetically engineered NK cells are commonly used in clinical trials. Previous studies have demonstrated that NK cells modified with cytokines have boosted NK cell cytotoxicity and persistence, successfully inhibiting HCC in xenograft models and prolonging the survival of tumor-bearing mice [120–122]. This and similar strategies are important because tumor regression following NK cell infusion depends on their ability to expand in vivo [35]. Various endogenous activating receptors on NK cells have been additionally introduced and demonstrated superior anti-tumor activity [123]. Modifying high-affinity CD16 substantially enhances NK cell tumorlytic capacity based on the available mAbs and compels the combination of treatment strategies.

NK cells are modified by chimeric antigen receptors (CARs) to increase NK cell activity and recognition specificity toward tumor cells [124]. Compared with T cells, NK cells have a shorter lifespan and do not trigger severe adverse effects, such as the fatal “cytokine release syndrome.” These cells do not cause graft-versus-host disease and can be used in allogeneic transplants. Moreover, these cells trigger spontaneous cytotoxicity more than CAR-restricted mechanism [125]. Multiple centers are gearing up to test whether CAR-modified NK cells can keep up with their T cell counterparts. Preclinical investigations of CAR-expressing NK cells have shown fairly promising results, and two clinical studies employing CAR-expressing NK cells are underway [35,126–128]. Despite the great promise of CAR-modified NK cells in cancer treatments, their therapeutic efficacy has never been explored in HCC. Thus, GPC3 has been suggested as a suitable target for CAR T cells in HCC, and NK cells bear several advantages as CAR drivers; these strategies will be incorporated in clinical NK cell cancer immunotherapy [129]. Careful investigations focusing on the toxicities and effectiveness as well as the industrialization criterion are warranted before these approaches can reach clinic.

Conclusions and perspectives

In the past two decades, the NK cell field has reached an encouraging stage, in which significant advances have been achieved in understanding of NK cell biology; increasingly proficient methodologies have been established for their purification/expansion, and the updated knowledge is rapidly being attempted to manipulate NK cell activation. Nonetheless, the efficacy of NK cell-based immunotherapies was largely determined in hematological malignancies. As regards HCC, most attempts are being explored in mouse models or in phase I/II clinical trials; in addition, further studies connecting the basic and the clinic are urgently needed to promote the translational stride.

Many issues need to be overcome to refine the NK cell-based treatment strategies, which have been well reviewed in previous publications. Herein, we highlighted several points for which we should particularly pay attention to HCC treatment. First, the influence of KIR/HLA immunogeneity on HCC outcomes has been confirmed after curative hepatectomy; therefore, the selection of NK cells with a predominant activating profile (KIR/HLA mismatched) might be critical for delivering a successful treatment. Second, only selected patients are able to receive these immunotherapies. Future immune-based trials on HCC should be conducted to validate predictive biomarkers and select patients for a specific NK cell-based regimen. Third, intrahepatic NK cells demonstrate higher cytotoxic activity against tumors and potential spontaneous homing to the liver; these cells represent an attractive source of NK cells for HCC treatment, particularly in liver transplants. Moreover, minimally invasive therapies have been widely used in patients with HCC. Incorporation of additional immunotherapeutic strategies with those established standard of care treatment is therefore expected to generate durable and powerful anti-tumor activity. Furthermore, the milieu of solid tumors generally poses a formidable challenge to NK cell efficacy while simultaneously hindering both infiltration and activation of NK cells at tumor nests; therefore, reprogramming of the immune microenvironment in tumors would be promising for improving the efficacy of NK cell-based immunotherapies. Additional knowledge of the complex microenvironment should be gained. Finally, with CAR-modified T lymphocytes being a major breakthrough in anti-cancer immunotherapy and the rapid developments in clinically compliant techniques to genetically manipulate NK cells, genetic engineering is depicted as an obligatory pathway to exploit the full potential of adoptive NK cell immunotherapy in patients with HCC. Eventually, additional studies, including basic and clinical research, are urgently required to employ the novel treatment approach.

References

[1] Maluccio M, Covey A. Recent progress in understanding, diagnosing, and treating hepatocellular carcinoma. CA Cancer J Clin 2012; 62(6): 394–399

[2] Bruix J, Reig M, Sherman M. Evidence-based diagnosis, staging, and treatment of patients with hepatocellular carcinoma. Gastroenterology 2016; 150(4): 835–853

[3] Forner A, Gilabert M, Bruix J, Raoul JL. Treatment of intermediate-stage hepatocellular carcinoma. Nat Rev Clin Oncol 2014; 11(9): 525–535

[4] Sieghart W, Hucke F, Peck-Radosavljevic M. Transarterial chemoembolization: modalities, indication, and patient selection. J Hepatol 2015; 62(5): 1187–1195

[5] Llovet JM, Villanueva A, Lachenmayer A, Finn RS. Advances in targeted therapies for hepatocellular carcinoma in the genomic era. Nat Rev Clin Oncol 2015; 12(7): 408–424

[6] Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature 2011; 480(7378): 480–489

[7] Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol 2008; 9(5): 503–510

[8] Martinet L, Smyth MJ. Balancing natural killer cell activation through paired receptors. Nat Rev Immunol 2015; 15(4): 243–254

[9] Herberman RB, Reynolds CW, Ortaldo JR. Mechanism of cytotoxicity by natural killer (NK) cells. Annu Rev Immunol 1986; 4(1): 651–680

[10] Orange JS. Formation and function of the lytic NK-cell immunological synapse. Nat Rev Immunol 2008; 8(9): 713–725

[11] de Saint Basile G, Ménasché G, Fischer A. Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules. Nat Rev Immunol 2010; 10(8): 568–579

[12] Wang W, Erbe AK, Hank JA, Morris ZS, Sondel PMNK. NK cell-mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy. Front Immunol 2015; 6: 368

[13] Igarashi T, Wynberg J, Srinivasan R, Becknell B, McCoy JP Jr, Takahashi Y, Suffredini DA, Linehan WM, Caligiuri MA, Childs RW. Enhanced cytotoxicity of allogeneic NK cells with killer immunoglobulin-like receptor ligand incompatibility against melanoma and renal cell carcinoma cells. Blood 2004; 104(1): 170–177

[14] Willemze R, Rodrigues CA, Labopin M, Sanz G, Michel G, Socié G, Rio B, Sirvent A, Renaud M, Madero L, Mohty M, Ferra C, Garnier F, Loiseau P, Garcia J, Lecchi L, Kögler G, Beguin Y, Navarrete C, Devos T, Ionescu I, Boudjedir K, Herr AL, Gluckman E, Rocha V; Eurocord-Netcord and Acute Leukaemia Working Party of the EBMT.KIR-ligand incompatibility in the graft-versus-host direction improves outcomes after umbilical cord blood transplantation for acute leukemia. Leukemia 2009; 23(3): 492–500

[15] Rubnitz JE, Inaba H, Ribeiro RC, Pounds S, Rooney B, Bell T, Pui CH, Leung W. NKAML: a pilot study to determine the safety and feasibility of haploidentical natural killer cell transplantation in childhood acute myeloid leukemia. J Clin Oncol 2010; 28(6): 955–959

[16] Takeda K, Hayakawa Y, Smyth MJ, Kayagaki N, Yamaguchi N, Kakuta S, Iwakura Y, Yagita H, Okumura K. Involvement of tumor necrosis factor-related apoptosis-inducing ligand in surveillance of tumor metastasis by liver natural killer cells. Nat Med 2001; 7(1): 94–100

[17] Shi FD, Ljunggren HG, La Cava A, Van Kaer L. Organ-specific features of natural killer cells. Nat Rev Immunol 2011; 11(10): 658–671

[18] Chew V, Chen J, Lee D, Loh E, Lee J, Lim KH, Weber A, Slankamenac K, Poon RT, Yang H, Ooi LL, Toh HC, Heikenwalder M, Ng IO, Nardin A, Abastado JP. Chemokine-driven lymphocyte infiltration: an early intratumoural event determining long-term survival in resectable hepatocellular carcinoma. Gut 2012; 61(3): 427–438

[19] Ishiyama K, Ohdan H, Ohira M, Mitsuta H, Arihiro K, Asahara T. Difference in cytotoxicity against hepatocellular carcinoma between liver and periphery natural killer cells in humans. Hepatology 2006; 43(2): 362–372

[20] Chew V, Tow C, Teo M, Wong HL, Chan J, Gehring A, Loh M, Bolze A, Quek R, Lee VK, Lee KH, Abastado JP, Toh HC, Nardin A. Inflammatory tumour microenvironment is associated with superior survival in hepatocellular carcinoma patients. J Hepatol 2010; 52(3): 370–379

[21] Marengo A, Rosso C, Bugianesi E. Liver cancer: connections with obesity, fatty liver, and cirrhosis. Annu Rev Med 2016; 67: 103–117

[22] Tian Z, Chen Y, Gao B. Natural killer cells in liver disease. Hepatology 2013; 57(4): 1654–1662

[23] Coulouarn C, Factor VM, Conner EA, Thorgeirsson SS. Genomic modeling of tumor onset and progression in a mouse model of aggressive human liver cancer. Carcinogenesis 2011; 32(10): 1434–1440

[24] Cai L, Zhang Z, Zhou L, Wang H, Fu J, Zhang S, Shi M, Zhang H, Yang Y, Wu H, Tien P, Wang FS. Functional impairment in circulating and intrahepatic NK cells and relative mechanism in hepatocellular carcinoma patients. Clin Immunol 2008; 129(3): 428–437

[25] Gao B, Radaeva S, Park O. Liver natural killer and natural killer T cells: immunobiology and emerging roles in liver diseases. J Leukoc Biol 2009; 86(3): 513–528

[26] He L, Wang X, Montell DJ. Shining light on Drosophila oogenesis: live imaging of egg development. Curr Opin Genet Dev 2011; 21(5): 612–619

[27] Chirinda W, Chen H. Comparative study of disability-free life expectancy across six low- and middle-income countries. Geriatr Gerontol Int 2017; 17(4): 637–644

[28] Une Y, Kawata A, Uchino J. Adopted immunochemotherapy using IL-2 and spleen LAK cell—randomized study. Nihon Geka Gakkai Zasshi 1991; 92(9): 1330–1333 (in Japanese)

[29] Lygidakis NJ, Pothoulakis J, Konstantinidou AE, Spanos H. Hepatocellular carcinoma: surgical resection versus surgical resection combined with pre- and post-operative locoregional immunotherapy-chemotherapy. A prospective randomized study. Anticancer Res 1995; 15(2): 543–550

[30] Kawata A, Une Y, Hosokawa M, Wakizaka Y, Namieno T, Uchino J, Kobayashi H. Adjuvant chemoimmunotherapy for hepatocellular carcinoma patients. Adriamycin, interleukin-2, and lymphokine-activated killer cells versus adriamycin alone. Am J Clin Oncol 1995; 18(3): 257–262

[31] Long EO, Kim HS, Liu D, Peterson ME, Rajagopalan S. Controlling natural killer cell responses: integration of signals for activation and inhibition. Annu Rev Immunol 2013; 31(1): 227–258

[32] Beneker C. Report from Hannover. Family practitioners in emergency admission. MMW Fortschr Med 2015; 157(1): 8–9 (in German) 

[33] Lanier LL. NK cell recognition. Annu Rev Immunol 2005; 23(1): 225–274

[34] Woo SR, Corrales L, Gajewski TF. Innate immune recognition of cancer. Annu Rev Immunol 2015; 33(1): 445–474

[35] Guillerey C, Huntington ND, Smyth MJ. Targeting natural killer cells in cancer immunotherapy. Nat Immunol 2016; 17(9): 1025–1036

[36] Raulet DH. Roles of the NKG2D immunoreceptor and its ligands. Nat Rev Immunol 2003; 3(10): 781–790

[37] Eagle RA, Trowsdale J. Promiscuity and the single receptor: NKG2D. Nat Rev Immunol 2007; 7(9): 737–744

[38] Jinushi M, Takehara T, Tatsumi T, Hiramatsu N, Sakamori R, Yamaguchi S, Hayashi N. Impairment of natural killer cell and dendritic cell functions by the soluble form of MHC class I-related chain A in advanced human hepatocellular carcinomas. J Hepatol 2005; 43(6): 1013–1020

[39] Kamimura H, Yamagiwa S, Tsuchiya A, Takamura M, Matsuda Y, Ohkoshi S, Inoue M, Wakai T, Shirai Y, Nomoto M, Aoyagi Y. Reduced NKG2D ligand expression in hepatocellular carcinoma correlates with early recurrence. J Hepatol 2012; 56(2): 381–388

[40] Gao J, Duan Z, Zhang L, Huang X, Long L, Tu J, Liang H, Zhang Y, Shen T, Lu F. Failure recovery of circulating NKG2D(+)CD56(dim)NK cells in HBV-associated hepatocellular carcinoma after hepatectomy predicts early recurrence. OncoImmunology 2016; 5(1): e1048061

[41] Hoechst B, Voigtlaender T, Ormandy L, Gamrekelashvili J, Zhao F, Wedemeyer H, Lehner F, Manns MP, Greten TF, Korangy F. Myeloid derived suppressor cells inhibit natural killer cells in patients with hepatocellular carcinoma via the NKp30 receptor. Hepatology 2009; 50(3): 799–807

[42] Qu P, Huang X, Zhou X, Lü Z, Liu F, Shi Z, Lü L, Wu Y, Chen Y. Loss of CD155 expression predicts poor prognosis in hepatocellular carcinoma. Histopathology 2015; 66(5): 706–714

[43] Tanimine N, Ohdan H. Impact of multiplicity of functional KIR-HLA compound genotypes on hepatocellular carcinoma. OncoImmunology 2015; 4(1): e983765

[44] Vilches C, Parham P. KIR: diverse, rapidly evolving receptors of innate and adaptive immunity. Annu Rev Immunol 2002; 20(1): 217–251

[45] Raulet DH, Vance RE, McMahon CW. Regulation of the natural killer cell receptor repertoire. Annu Rev Immunol 2001; 19(1): 291–330

[46] Kim S, Poursine-Laurent J, Truscott SM, Lybarger L, Song YJ, Yang L, French AR, Sunwoo JB, Lemieux S, Hansen TH, Yokoyama WM. Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature 2005; 436(7051): 709–713

[47] Cariani E, Pilli M, Zerbini A, Rota C, Olivani A, Zanelli P, Zanetti A, Trenti T, Ferrari C, Missale G. HLA and killer immunoglobulin-like receptor genes as outcome predictors of hepatitis C virus-related hepatocellular carcinoma. Clin Cancer Res 2013; 19(19): 5465–5473

[48] Tanimine N, Tanaka Y, Kobayashi T, Tashiro H, Miki D, Imamura M, Aikata H, Tanaka J, Chayama K, Ohdan H. Quantitative effect of natural killer-cell licensing on hepatocellular carcinoma recurrence after curative hepatectomy. Cancer Immunol Res 2014; 2(12): 1142–1147

[49] Singh R, Kaul R, Kaul A, Khan K. A comparative review of HLA associations with hepatitis B and C viral infections across global populations. World J Gastroenterol 2007; 13(12): 1770–1787

[50] Jamil KM, Khakoo SI. KIR/HLA interactions and pathogen immunity. J Biomed Biotechnol 2011; 2011: 298348

[51] Pan N, Jiang W, Sun H, Miao F, Qiu J, Jin H, Xu J, Shi Q, Xie W, Zhang J. KIR and HLA loci are associated with hepatocellular carcinoma development in patients with hepatitis B virus infection: a case-control study. PLoS One 2011; 6(10): e25682

[52] Guidotti LG, Chisari FV. Immunobiology and pathogenesis of viral hepatitis. Annu Rev Pathol 2006; 1(1): 23–61

[53] Shin EC, Sung PS, Park SH. Immune responses and immunopathology in acute and chronic viral hepatitis. Nat Rev Immunol 2016; 16(8): 509–523

[54] Pan N, Qiu J, Sun H, Miao F, Shi Q, Xu J, Jiang W, Jin H, Xie W, He Y, Zhang J. Combination of human leukecyte antigen and killer cell immunoglulin-like antigen and killer cell influences the onset age of hepatocellular carcinoma in male patients with hepatitis B virus infenction. Clin Dev Immunol  2013; 2013: 874514

[55] De Re V, Caggiari L, De Zorzi M, Repetto O, Zignego AL, Izzo F, Tornesello ML, Buonaguro FM, Mangia A, Sansonno D, Racanelli V, De Vita S, Pioltelli P, Vaccher E, Berretta M, Mazzaro C, Libra M, Gini A, Zucchetto A, Cannizzaro R, De Paoli P. Genetic diversity of the KIR/HLA system and susceptibility to hepatitis C virus-related diseases. PLoS One 2015; 10(2): e0117420

[56] Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating immunity’s roles in cancer suppression and promotion. Science 2011; 331(6024): 1565–1570

[57] Chan SL, Mo FK, Wong CS, Chan CM, Leung LK, Hui EP, Ma BB, Chan AT, Mok TS, Yeo W. A study of circulating interleukin 10 in prognostication of unresectable hepatocellular carcinoma. Cancer 2012; 118(16): 3984–3992

[58] Chen Z, Xie B, Zhu Q, Xia Q, Jiang S, Cao R, Shi L, Qi D, Li X, Cai L. FGFR4 and TGF-β1 expression in hepatocellular carcinoma: correlation with clinicopathological features and prognosis. Int J Med Sci 2013; 10(13): 1868–1875

[59] Makarova-Rusher OV, Medina-Echeverz J, Duffy AG, Greten TF. The yin and yang of evasion and immune activation in HCC. J Hepatol 2015; 62(6): 1420–1429

[60] Lin TH, Shao YY, Chan SY, Huang CY, Hsu CH, Cheng AL. High serum transforming growth factor-β1 levels predict outcome in hepatocellular carcinoma patients treated with sorafenib. Clin Cancer Res 2015; 21(16): 3678–3684

[61] Lippitz BE. Cytokine patterns in patients with cancer: a systematic review. Lancet Oncol 2013; 14(6): e218–e228

[62] Mouri H, Sakaguchi K, Sawayama T, Senoh T, Ohta T, Nishimura M, Fujiwara A, Terao M, Shiratori Y, Tsuji T. Suppressive effects of transforming growth factor-beta1 produced by hepatocellular carcinoma cell lines on interferon-γ production by peripheral blood mononuclear cells. Acta Med Okayama 2002; 56(6): 309–315

[63] Sui Q, Zhang J, Sun X, Zhang C, Han Q, Tian Z. NK cells are the crucial antitumor mediators when STAT3-mediated immunosuppression is blocked in hepatocellular carcinoma. J Immunol 2014; 193(4): 2016–2023

[64] Xu D, Han Q, Hou Z, Zhang C, Zhang J. miR-146a negatively regulates NK cell functions via STAT1 signaling. Cell Mol Immunol2016 Mar 21. [Epub ahead of print] doi: 10.1038/cmi.2015.113 

[65] Hernandez-Gea V, Toffanin S, Friedman SL, Llovet JM. Role of the microenvironment in the pathogenesis and treatment of hepatocellular carcinoma. Gastroenterology 2013; 144(3): 512–527

[66] Fu J, Xu D, Liu Z, Shi M, Zhao P, Fu B, Zhang Z, Yang H, Zhang H, Zhou C, Yao J, Jin L, Wang H, Yang Y, Fu YX, Wang FS. Increased regulatory T cells correlate with CD8 T-cell impairment and poor survival in hepatocellular carcinoma patients. Gastroenterology 2007; 132(7): 2328–2339

[67] Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS, Xu Y, Li YW, Tang ZY. Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. J Clin Oncol 2007; 25(18): 2586–2593

[68] Langhans B, Alwan AW, Krämer B, Glässner A, Lutz P, Strassburg CP, Nattermann J, Spengler U. Regulatory CD4⁺ T cells modulate the interaction between NK cells and hepatic stellate cells by acting on either cell type. J Hepatol 2015; 62(2): 398–404

[69] Parker KH, Beury DW, Ostrand-Rosenberg S. Myeloid-derived suppressor cells: critical cells driving immune suppression in the tumor microenvironment. Adv Cancer Res 2015; 128: 95–139

[70] Wan S, Kuo N, Kryczek I, Zou W, Welling TH. Myeloid cells in hepatocellular carcinoma. Hepatology 2015; 62(4): 1304–1312

[71] Denko NC. Hypoxia, HIF1 and glucose metabolism in the solid tumour. Nat Rev Cancer 2008; 8(9): 705–713

[72] Nizet V, Johnson RS. Interdependence of hypoxic and innate immune responses. Nat Rev Immunol 2009; 9(9): 609–617

[73] Hasmim M, Messai Y, Ziani L, Thiery J, Bouhris JH, Noman MZ, Chouaib S. Critical role of tumor microenvironment in shaping NK cell functions: implication of hypoxic stress. Front Immunol 2015; 6: 482

[74] Zerbini A, Pilli M, Penna A, Pelosi G, Schianchi C, Molinari A, Schivazappa S, Zibera C, Fagnoni FF, Ferrari C, Missale G. Radiofrequency thermal ablation of hepatocellular carcinoma liver nodules can activate and enhance tumor-specific T-cell responses. Cancer Res 2006; 66(2): 1139–1146

[75] Mizukoshi E, Yamashita T, Arai K, Sunagozaka H, Ueda T, Arihara F, Kagaya T, Yamashita T, Fushimi K, Kaneko S. Enhancement of tumor-associated antigen-specific T cell responses by radiofrequency ablation of hepatocellular carcinoma. Hepatology 2013; 57(4): 1448–1457

[76] Zerbini A, Pilli M, Laccabue D, Pelosi G, Molinari A, Negri E, Cerioni S, Fagnoni F, Soliani P, Ferrari C, Missale G. Radiofrequency thermal ablation for hepatocellular carcinoma stimulates autologous NK-cell response. Gastroenterology 2010; 138(5): 1931–1942

[77] Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I, Häussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J; SHARP Investigators Study Group. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008; 359(4): 378–390

[78] Tomuleasa C, Giannelli G, Cucuianu A, Aldea M, Paradiso A, Berindan-Neagoe I. Interplay between cancer cells, macrophages and natural killer cells may actually decide the outcome of therapy with sorafenib. Hepatology 2014; 60(1): 430

[79] Kohga K, Takehara T, Tatsumi T, Ishida H, Miyagi T, Hosui A, Hayashi N. Sorafenib inhibits the shedding of major histocompatibility complex class I-related chain A on hepatocellular carcinoma cells by down-regulating a disintegrin and metalloproteinase 9. Hepatology 2010; 51(4): 1264–1273

[80] Zhang QB, Sun HC, Zhang KZ, Jia QA, Bu Y, Wang M, Chai ZT, Zhang QB, Wang WQ, Kong LQ, Zhu XD, Lu L, Wu WZ, Wang L, Tang ZY. Suppression of natural killer cells by sorafenib contributes to prometastatic effects in hepatocellular carcinoma. PLoS One 2013; 8(2): e55945

[81] Sprinzl MF, Reisinger F, Puschnik A, Ringelhan M, Ackermann K, Hartmann D, Schiemann M, Weinmann A, Galle PR, Schuchmann M, Friess H, Otto G, Heikenwalder M, Protzer U. Sorafenib perpetuates cellular anticancer effector functions by modulating the crosstalk between macrophages and natural killer cells. Hepatology 2013; 57(6): 2358–2368

[82] Kamiya T, Chang YH, Campana D. Expanded and activated natural killer cells for immunotherapy of hepatocellular carcinoma. Cancer Immunol Res 2016; 4(7): 574–581

[83] Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer 2009; 9(11): 798–809

[84] Yu H, Kortylewski M, Pardoll D. Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment. Nat Rev Immunol 2007; 7(1): 41–51

[85] Armeanu S, Krusch M, Baltz KM, Weiss TS, Smirnow I, Steinle A, Lauer UM, Bitzer M, Salih HR. Direct and natural killer cell-mediated antitumor effects of low-dose bortezomib in hepatocellular carcinoma. Clin Cancer Res 2008; 14(11): 3520–3528

[86] Shi L, Lin H, Li G, Sun Y, Shen J, Xu J, Lin C, Yeh S, Cai X, Chang C. Cisplatin enhances NK cells immunotherapy efficacy to suppress HCC progression via altering the androgen receptor (AR)-ULBP2 signals. Cancer Lett 2016; 373(1): 45–56

[87] Prieto J, Melero I, Sangro B. Immunological landscape and immunotherapy of hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 2015; 12(12): 681–700

[88] Takeda T, Watanabe M, Umeshita K, Goto M, Monden M. Long-term prognosis of hepatocellular carcinoma patients treated with adoptive immunotherapy. Gan To Kagaku Ryoho 2004; 31(11): 1646–1648 (in Japanese)

[89] Hsieh KH, Chang JS, Wu HL, Chu CT. Interleukin 2 and lymphokine-activated killer cells in the treatment of childhood primary hepatocellular carcinoma—a preliminary report. Asian Pac J Allergy Immunol 1987; 5(1): 13–16

[90] Huang ZM, Li W, Li S, Gao F, Zhou QM, Wu FM, He N, Pan CC, Xia JC, Wu PH, Zhao M. Cytokine-induced killer cells in combination with transcatheter arterial chemoembolization and radiofrequency ablation for hepatocellular carcinoma patients. J Immunother 2013; 36(5): 287–293

[91] Xu L, Wang J, Kim Y, Shuang ZY, Zhang YJ, Lao XM, Li YQ, Chen MS, Pawlik TM, Xia JC, Li SP, Lau WY. A randomized controlled trial on patients with or without adjuvant autologous cytokine-induced killer cells after curative resection for hepatocellular carcinoma. OncoImmunology 2015; 5(3): e1083671

[92] Li X, Dai D, Song X, Liu J, Zhu L, Xu W. A meta-analysis of cytokine-induced killer cells therapy in combination with minimally invasive treatment for hepatocellular carcinoma. Clin Res Hepatol Gastroenterol 2014; 38(5): 583–591

[93] He G, Zheng C, Huo H, Zhang H, Zhu Z, Li J, Zhang H. TACE combined with dendritic cells and cytokine-induced killer cells in the treatment of hepatocellular carcinoma: a meta-analysis. Int Immunopharmacol 2016; 40: 436–442

[94] Lee JH, Lee JH, Lim YS, Yeon JE, Song TJ, Yu SJ, Gwak GY, Kim KM, Kim YJ, Lee JW, Yoon JH. Adjuvant immunotherapy with autologous cytokine-induced killer cells for hepatocellular carcinoma. Gastroenterology 2015; 148(7): 1383–91.e6

[95] Fujisaki H, Kakuda H, Shimasaki N, Imai C, Ma J, Lockey T, Eldridge P, Leung WH, Campana D. Expansion of highly cytotoxic human natural killer cells for cancer cell therapy. Cancer Res 2009; 69(9): 4010–4017

[96] Granzin M, Soltenborn S, Müller S, Kollet J, Berg M, Cerwenka A, Childs RW, Huppert V. Fully automated expansion and activation of clinical-grade natural killer cells for adoptive immunotherapy. Cytotherapy 2015; 17(5): 621–632

[97] Lim O, Jung MY, Hwang YK, Shin EC. Present and future of allogeneic natural killer cell therapy. Front Immunol 2015; 6: 286

[98] Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A, Posati S, Rogaia D, Frassoni F, Aversa F, Martelli MF, Velardi A. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295(5562): 2097–2100

[99] Miller JS, Soignier Y, Panoskaltsis-Mortari A, McNearney SA, Yun GH, Fautsch SK, McKenna D, Le C, Defor TE, Burns LJ, Orchard PJ, Blazar BR, Wagner JE, Slungaard A, Weisdorf DJ, Okazaki IJ, McGlave PB. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005; 105(8): 3051–3057

[100] Takayama T, Sekine T, Makuuchi M, Yamasaki S, Kosuge T, Yamamoto J, Shimada K, Sakamoto M, Hirohashi S, Ohashi Y, Kakizoe T. Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial. Lancet 2000; 356(9232): 802–807

[101] Ochi M, Ohdan H, Mitsuta H, Onoe T, Tokita D, Hara H, Ishiyama K, Zhou W, Tanaka Y, Asahara T. Liver NK cells expressing TRAIL are toxic against self hepatocytes in mice. Hepatology 2004; 39(5): 1321–1331

[102] Nishida S, Levi DM, Tzakis AG. Liver natural killer cell inoculum for liver transplantation with hepatocellular carcinoma. Curr Opin Organ Transplant 2013; 18(6): 690–694

[103] Suck G, Odendahl M, Nowakowska P, Seidl C, Wels WS, Klingemann HG, Tonn T. NK-92: an ‘off-the-shelf therapeutic’ for adoptive natural killer cell-based cancer immunotherapy. Cancer Immunol Immunother 2016; 65(4): 485–492

[104] Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012; 12(4): 252–264

[105] Hato T, Goyal L, Greten TF, Duda DG, Zhu AX. Immune checkpoint blockade in hepatocellular carcinoma: current progress and future directions. Hepatology 2014; 60(5): 1776–1782

[106] Pesce S, Greppi M, Tabellini G, Rampinelli F, Parolini S, Olive D, Moretta L, Moretta A, Marcenaro E. Identification of a subset of human natural killer cells expressing high levels of programmed death 1: a phenotypic and functional characterization. J Allergy Clin Immunol 2017; 139(1): 335–346.e3

[107] Korde N, Carlsten M, Lee MJ, Minter A, Tan E, Kwok M, Manasanch E, Bhutani M, Tageja N, Roschewski M, Zingone A, Costello R, Mulquin M, Zuchlinski D, Maric I, Calvo KR, Braylan R, Tembhare P, Yuan C, Stetler-Stevenson M, Trepel J, Childs R, Landgren O. A phase II trial of pan-KIR2D blockade with IPH2101 in smoldering multiple myeloma. Haematologica 2014; 99(6): e81–e83

[108] Carlsten M, Korde N, Kotecha R, Reger R, Bor S, Kazandjian D, Landgren O, Childs RW. Checkpoint inhibition of KIR2D with the monoclonal antibody IPH2101 induces contraction and hyporesponsiveness of NK cells in patients with myeloma. Clin Cancer Res 2016; 22(21): 5211–5222

[109] Felices M, Miller JS. Targeting KIR blockade in multiple myeloma: trouble in checkpoint paradise? Clin Cancer Res 2016; 22(21): 5161–5163

[110] Ruggeri L, Urbani E, André P, Mancusi A, Tosti A, Topini F, Bléry M, Animobono L, Romagné F, Wagtmann N, Velardi A. Effects of anti-NKG2A antibody administration on leukemia and normal hematopoietic cells. Haematologica 2016; 101(5): 626–633

[111] Chen A, Shen Y, Xia M, Xu L, Pan N, Yin Y, Miao F, Shen C, Xie W, Zhang J. Expression of the nonclassical HLA class I and MICA/B molecules in human hepatocellular carcinoma. Neoplasma 2011; 58(5): 371–376

[112] Casadevall A, Pirofski LA. A new synthesis for antibody-mediated immunity. Nat Immunol 2011; 13(1): 21–28

[113] Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E, Filmus J. Glypican-3: a novel serum and histochemical marker  for hepatocellular carcinoma. Gastroenterology 2003; 125(1): 89–97

[114] Nakano K, Orita T, Nezu J, Yoshino T, Ohizumi I, Sugimoto M, Furugaki K, Kinoshita Y, Ishiguro T, Hamakubo T, Kodama T, Aburatani H, Yamada-Okabe H, Tsuchiya M. Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells. Biochem Biophys Res Commun 2009; 378(2): 279–284

[115] Zhu AX, Gold PJ, El-Khoueiry AB, Abrams TA, Morikawa H, Ohishi N, Ohtomo T, Philip PA. First-in-man phase I study of GC33, a novel recombinant humanized antibody against glypican-3, in patients with advanced hepatocellular carcinoma. Clin Cancer Res 2013; 19(4): 920–928

[116] Felices M, Lenvik TR, Davis ZB, Miller JS, Vallera DA. Generation of BiKEs and TriKEs to improve NK cell-mediated targeting of tumor cells. Methods Mol Biol 2016; 1441: 333–346

[117] Tay SS, Carol H, Biro M. TriKEs and BiKEs join CARs on the cancer immunotherapy highway. Hum Vaccin Immunother 2016; 12(11): 2790–2796

[118] Vallera DA, Felices M, McElmurry R, McCullar V, Zhou X, Schmohl JU, Zhang B, Lenvik AJ, Panoskaltsis-Mortari A, Verneris MR, Tolar J, Cooley S, Weisdorf DJ, Blazar BR, Miller JS. IL15 trispecific killer engagers (TriKE) make natural killer cells specific to CD33⁺ targets while also inducing persistence, in vivo expansion, and enhanced function. Clin Cancer Res 2016; 22(14): 3440–3450

[119] Schmohl JU, Felices M, Taras E, Miller JS, Vallera DA. Enhanced ADCC and NK cell activation of an anticarcinoma bispecific antibody by genetic insertion of a modified IL-15 cross-linker. Mol Ther 2016; 24(7): 1312–1322

[120] Mariani E, Meneghetti A, Tarozzi A, Cattini L, Facchini A. Interleukin-12 induces efficient lysis of natural killer-sensitive and natural killer-resistant human osteosarcoma cells: the synergistic effect of interleukin-2. Scand J Immunol 2000; 51(6): 618–625

[121] Jiang W, Zhang C, Tian Z, Zhang J. hIFN-α gene modification augments human natural killer cell line anti-human hepatocellular carcinoma function. Gene Ther 2013; 20(11): 1062–1069

[122] Jiang W, Zhang C, Tian Z, Zhang J. hIL-15 gene-modified human natural killer cells (NKL-IL15) augments the anti-human hepatocellular carcinoma effect in vivo. Immunobiology 2014; 219(7): 547–553

[123] Chang YH, Connolly J, Shimasaki N, Mimura K, Kono K, Campana D. A chimeric receptor with NKG2D specificity enhances natural killer cell activation and killing of tumor cells. Cancer Res 2013; 73(6): 1777–1786

[124] Hermanson DL, Kaufman DS. Utilizing chimeric antigen receptors to direct natural killer cell activity. Front Immunol 2015; 6: 195

[125] Klingemann H. Are natural killer cells superior CAR drivers? OncoImmunology 2014; 3(4): e28147

[126] Esser R, Müller T, Stefes D, Kloess S, Seidel D, Gillies SD, Aperlo-Iffland C, Huston JS, Uherek C, Schönfeld K, Tonn T, Huebener N, Lode HN, Koehl U, Wels WS. NK cells engineered to express a GD2-specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin. J Cell Mol Med 2012; 16(3): 569–581

[127] Boissel L, Betancur-Boissel M, Lu W, Krause DS, Van Etten RA, Wels WS, Klingemann H. Retargeting NK-92 cells by means of CD19- and CD20-specific chimeric antigen receptors compares favorably with antibody-dependent cellular cytotoxicity. OncoImmunology 2013; 2(10): e26527

[128] Zhang C, Burger MC, Jennewein L, Genßler S, Schönfeld K, Zeiner P, Hattingen E, Harter PN, Mittelbronn M, Tonn T, Steinbach JP, Wels WS. ErbB2/HER2-specific NK cells for targeted therapy of glioblastoma. J Natl Cancer Inst 2016; 108(5): djv375

[129] Gao H, Li K, Tu H, Pan X, Jiang H, Shi B, Kong J, Wang H, Yang S, Gu J, Li Z. Development of T cells redirected to glypican-3 for the treatment of hepatocellular carcinoma. Clin Cancer Res 2014; 20(24): 6418–6428

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