NKT cells in liver diseases

Abstract

Natural killer T cells are innate-like and tissue-resident lymphocytes, which recognize lipid antigens and are enriched in the liver. Natural killer T cells play important roles in infections, tumors, autoimmune diseases, and metabolic diseases. In this study, we summarize recent findings on biology of natural killer T cells and their roles in hepatitis B virus and hepatitis C virus infection, autoimmune liver diseases, alcoholic liver disease, nonalcoholic fatty liver disease, and hepatocellular carcinoma. Controversial results from previous studies are discussed, and indicate the dynamic alteration in the role of natural killer T cells during the progression of liver diseases, which might be caused by changes in natural killer T subsets, factors skewing cytokine responses, and intercellular crosstalk between natural killer T cells and CD1d-expressing cells or bystander cells.

Keywords

natural killer T cells / hepatitis B virus and hepatitis C virus infection / autoimmune liver diseases / alcoholic liver disease / nonalcoholic fatty liver disease / hepatocellular carcinoma

Introduction

Natural killer T (NKT) cells were first named in 1995 as such because they coexpress T cell receptor (TCR) and natural killer (NK) cell receptor (NK1.1) [¹]. Different from conventional T cells, which recognize peptide antigens presented by MHC molecules, NKT cells recognize lipid antigens presented by CD1d molecules [²,³]. CD1d molecules are highly conserved in mammals [⁴]. Based on the TCR chain bias and antigen specificity, NKT cells can be categorized into type I NKT cells and type II NKT cells [⁵]. Type I NKT cells are known as iNKT cells, which express semi-invariant TCR in human and mouse (Vα14-Jα18 paired with Vβ8.2, Vβ7 or Vβ2 in mice, Vα24-Jα18 paired with Vβ11 in human) [⁶], whereas type II NKT cells possess a more diverse TCR repertoire. The lipid ligands for type II NKT cells are largely unknown; therefore, identifying type II NKT cells are difficult, and thus, these cells are less characterized [⁵,⁷]. Here, we only review the type I NKT cells without further statement. Due to their innate-like phenotype, NKT cells response rapidly to stimuli and regulate downstream immune cells either directly or indirectly [⁸]. Consistent with their important immunoregulatory functions, NKT cells are involved in most known diseases in animal models and humans. In this review, we discuss the role of NKT cells in liver diseases.

Biology of NKT cells

Immunoregulatory role of NKT cells

NKT cells are innate-like T lymphocytes that possess an effector/memory phenotype [⁹]. Upon activation, NKT cells rapidly produce large amounts of chemokines, Th1/Th2 cytokines [³,¹⁰,¹¹], such as IFN-γ, TNF-α, IL-2, IL-4, IL-13, and IL-5, regulate the functions of dendritic cells (DCs), macrophages, B cells, T cells, and NK cells [¹²–¹⁷]. α-Galactosylceramide (α-GalCer) from marine sponges is the most effective and commonly used lipid ligand to activate NKT cells [⁶,¹⁸]. Moreover, α-glycuronosylceramides and α-galactosyl-diacylglycerols from microbes, as well as self-lipids, isoglobotrihexosylceramide (iGb3) and β-glucosylceramide, have been identified as NKT cell ligands [¹³,¹⁹–²³]. In addition to exogenous microbial lipid antigens, inflammatory signals also activate NKT cells and promote IFN-γ production [²⁴]. Agonists of toll-like receptor 4 (TLR4), TLR7 and TLR9 activate NKT cells in a way depending on both cytokines (IL-12 or type-I IFN) and self-lipids, whereas the combination of IL-12 and IL-18 drives the activation of NKT cells independently of self-lipids [¹³,²⁵–³¹]. Several factors that influence NKT-cell-mediated immune responses have been reported [³²–³⁹]. For example, ligand variants with different structures would polarize NKT-cell-mediated immune responses toward either Th1 (α-GalCer, PBS57, C-glycoside) or Th2 (OCH, ac C8:0, ac C20:2) [³²,³³,³⁶], and these ligand variants have been used to inhibit infectious diseases [⁴⁰–⁴²], tumors [⁴³,⁴⁴], and autoimmune diseases [³²,⁴⁵,⁴⁶]. Furthermore, different types of antigen-presenting cells (APCs) can also shift NKT-cell-mediated immune responses. Changes of APCs in the adipose tissues alter the NKT-cell-mediated immune responses during the progression of obesity [⁴⁷]. Additionally, functional versatility implies the complicated roles of NKT cells in diseases.

Tissue-specific distribution of NKT cell subsets

NKT cells accumulate in the vasculature of the liver and lung and are mainly located in the red pulp and marginal zone of the spleen [⁴⁸–⁵²]. The location of NKT cells favors their rapid activation by blood-borne antigens. However, NKT cells are tissue-resident lymphocytes; therefore, they rarely enter the circulatory system. Studies with parabiotic congenic mice, which formed capillary anastomoses between two vascular systems and allowed the exchange of circulating peripheral blood cells, showed that only blood NKT cells are distributed equally, whereas NKT cells in the spleen, liver, lung, and bone marrow do not exchange between the two joined mice [⁴⁸,⁵³,⁵⁴]. Tissue-resident and innate-like phenotypes indicate an important role of NKT cells in maintaining tissue homeostasis. Interestingly, distinct subsets of NKT cells have been reported to have tissue-specific distributions and contribute to the functional versatility of NKT cells in addition to those factors mentioned above. According to the transcription factors and cytokine profiles, NKT cells can be classified as NKT1, NKT2, NKT10, and NKT17 [⁵⁵] (Fig. 1). The branch points of differentiation emerge at the early stage of development in the thymus. Moreover, hepatic and splenic NKT cells are defined as NKT1, which are IL-17RB⁻T-bet^high and secrete IFN-γ and IL-4. Most of our current knowledge of NKT cells is attributed to studies on NKT1, which is the predominant subset in vivo, especially in the liver and spleen. GATA3^high PLZF^high NKT2 cells and ROR γt⁺ NKT17 cells accumulate in the lung and lymph node, respectively, and both are IL-17RB⁺ [⁵⁶–⁵⁸]. Large amounts of NKT2 cells have been detected in the lungs of BALB/c mice and drive hypersensitive inflammatory responses by producing Th2 cytokines and recruiting mast cells and eosinophils [⁵⁹,⁶⁰]. Thus, NKT2 cells are closely related to the pathogenesis of airway diseases. NKT17 cells exert their immunomodulatory activities through IL-17 production, and contribute to airway hyperreactivity and collagen-induced arthritis [⁶¹–⁶³]. Recently, a novel population of NKT cells in adipose tissues has been defined as NKT10, which expresses the transcription factor E4BP4 but low promyelocytic leukemia zinc finger (PLZF). These NKT10 cells polarize macrophages toward an anti-inflammatory phenotype and promote the accumulation of Treg cells through IL-10 production in adipose tissues [⁵³]. Studies have suggested that the differentiation of NKT10 is controlled by the distinct TCR signaling threshold in the thymus [⁶⁴]. Interestingly, a recent study has demonstrated that a fraction of Vα14⁺ NKT cells are generated from CD4⁻CD8⁻ (double negative, DN) thymocytes and bypass the CD4⁺CD8⁺ (double positive, DP) stage. Moreover, these DN NKT cells of DN-stage origin are mainly in the liver and account for almost half of the liver DN NKT cells. However, the DN NKT cells in the spleen are almost exclusively DP-stage origin. Furthermore, DN NKT cells of DN-stage origin show greater cytotoxicity and higher expression of liver-homing factors, including S1PR1, S1PR5, ITGA1, and ITGA4, than their counterparts of DP-stage origin [⁶⁵]. These NKT cells of DN-stage origin could indicate a liver-specific subset, and the mechanisms controlling their development are still unclear. Liver-specific microenvironments may provide the appropriate milieu required for the differentiation and accumulation of these DN-stage origin cells. In humans, the differentiation and distribution of NKT subsets have not been reported. However, previous studies have indicated biased Th2 cytokines from human CD4⁺ NKT cells and biased Th1 cytokines from human CD4⁻ NKT cells [⁶⁶]. Overall, functional subsets and extrinsic factors in tissue microenvironments have shaped the immunoregulatory role of NKT cells.

NKT cells and liver diseases

Liver is an immune organ and has a predominant innate immunity. About 50% of the total lymphocytes are NK cells and NKT cells in mice, and the percentage of NKT cells in liver is 20 to 100 folds higher than that in other organs. In contrast to the abundant NKT cells in mice, human hepatic NKT cells are significantly fewer but are more diverse in phenotype and function. Moreover, hepatic NKT cells in humans are potent producers of Th1 (IFN-γ and TNF-α) but not Th2 (IL-4) cytokines [⁶⁷]. Although both human and mouse NKT cells can be divided into CD4⁺ and CD4⁻ populations, CD8⁺ NKT cells, as the most potent inducers of Th1 immunity, are only found in humans [⁶⁸,⁶⁹]. As important immunoregulatory cells bridging innate and adaptive immunity, NKT cells are closely related to liver diseases in animal models and humans [⁷⁰–⁷²].

HBV and HCV infection

Hepatitis B and hepatitis C viruses (HBV and HCV) are two major hepatophilic viruses that threaten human health [⁷³–⁷⁵]. They can effectively escape the immune system, replicate persistently in hosts, and eventually develop chronic infections. Most adults can eliminate HBV by their own immune system; however, about 5% of adults could not effectively clear HBV and thus become chronic carriers. Additionally, due to an incomplete immune system, up to 90% of newborns are prone to become carriers after HBV infection. About 2 billion people worldwide have been infected and 350 million people are HBV carriers currently [⁷⁶,⁷⁷].

In the woodchuck model of hepadnavirus infection, a model for research on HBV, NKT cells were activated within hours after virus injection, whereas CD4⁺ and CD8⁺ T cell responses were induced 5 to 6 weeks later when histological evidence on hepatitis were developed [⁷⁸]. Similar kinetics was observed in HBV-infected humans [⁷⁹,⁸⁰]. Interestingly, Jiang et al. revealed that the frequencies of circulating NKT cells decreased in chronic hepatitis B (CHB) patients, and the low frequency recovered after antiviral therapy [⁸¹]. Other studies showed no difference in the numbers of circulating NKT cells between chronic HBV patients and healthy controls (HCs); however, they reported the enrichment of intrahepatic NKT cells in chronic viral hepatitis patients and reduced circulating NKT cells in chronic hepatitis B (CHB) and cirrhosis patients compared with those in immune-tolerant (IT) patients [⁸²,⁸³]. These studies suggest a critical role of NKT cells in controlling HBV infection. Consistently, activation of NKT cells by α-GalCer inhibits HBV replication via inducing IFN-γ and IFN-α/β production, and strongly promotes the activation of NK cells and HBV surface antigen-specific cytolytic T lymphocytes (CTLs) [⁸⁴,⁸⁵]. Moreover, HBV-infected human hepatocytes activate human NKT cell lines in a CD1d-dependent manner, indicating a similar role of NKT cells in human HBV clearance [⁸⁶]. However, clinical trials using α-GalCer as a monotherapy for HBV patients fail to impair HBV replication and are poorly tolerated in patients [⁸⁷]. Therefore, more strategies and precautions should be considered for targeting NKT cells as an alternative treatment. Furthermore, side effects, such as inflammatory responses induced by α-GalCer injection, should be carefully examined.

Studies on NKT cells in HCV infections are rare and mainly focus on cell frequencies and phenotypes rather than functions. Moreover, their results are conflicting. Some studies report no difference in the numbers of peripheral NKT cells between HCV infected patients and healthy people [⁸⁸,⁸⁹], whereas reduced frequencies of circulating NKT cells in the blood of patients with HCV and decreased proportions of intrahepatic NKT cells in patients with cirrhotic HCV have been observed by other studies [⁹⁰]. Furthermore, upregulation of CD69 expression in hepatic NKT cells has been observed, thereby suggesting activation by viral infection [⁹¹]. The low NKT cell numbers in HCV patients might be caused by activation-induced cell death. Additionally, activation of NKT cells has been observed in healthcare workers who had been exposed to HCV but did not develop infections, and this phenomenon is correlated to the subsequent HCV-specific T cell responses [⁹²]. Moreover, a recent study with humanized mice demonstrated that IFN-α treatment promotes IFN-γ production in NKT cells, which enhances the antiviral effect against HCV [⁹³]. Thus, NKT cells may contribute to antiviral immunity as a bridge between innate immunity and adaptive immunity. Additionally, Mark et al. found a large number of CD3⁺CD161⁺ non-classical NKT cells in human livers. These cells show a strong Th1 bias that mainly produce IFN-γ in the mild stage of chronic HCV infection, thereby inhibit HCV infection; whereas in the stage of severe fibrotic or cirrhotic, these non-classical NKT cells can promote disease progression by increasing Th2 cytokine production. Therefore, type II NKT cells are considered to serve protective and pathogenic roles during HCV infection [⁹⁴,⁹⁵].

In addition to the antiviral functions, a pathogenic role of NKT cells has been indicated. NKT cells enrich in HBV or HCV chronically infected livers and promote progression of hepatic fibrosis to cirrhosis through profibrotic cytokine IL-4 and IL-13 [⁸²]. Thus, NKT cells might play different roles depending on the disease stage. In a recent study, researchers categorized chronic HBV infection into three stages, namely, an immune tolerance phase (HBV-IT), an immune clearance phase (HBV-IC), and an inactive carrier phase (HBV-IA), and revealed dynamic changes of peripheral NKT cell numbers and cytokine profiles. In the same study, authors demonstrated that IFN-γ production is reversely correlated with serum HBV DNA load, whereas IL-4 production is positively correlated with liver injury [⁹⁶]. Moreover, IL-4 has been suggested to exacerbate, whereas IFN-γ has been suggested to prevent, α-GalCer-induced liver injury via modulating hepatic neutrophil survival and infiltration [⁹⁷]. Therefore, modification of IFN-γ and IL-4 production could be useful in controlling NKT cell-mediated liver injury.

Autoimmune liver diseases

Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are common autoimmune liver diseases that cause bile duct destruction [⁹⁸]. Patients with PSC develop inflammation, fibrosis, and destruction of the intrahepatic and extrahepatic bile ducts; and patients with PBC develop destruction of small bile ducts, thereby leading to bile extravasation and subsequent fibrosis [⁹⁹]. In early PBC, livers show increased CD1d expression and NKT cell frequencies [¹⁰⁰,¹⁰¹], whereas the NKT cell frequencies in patients with PSC remain unknown [¹⁰²]. However, other studies have demonstrated decreased expression of CD1d in the biliary epithelium of late PSC and PBC patients [¹⁰³]. These discrepancies might be due to the different stages of the disease, and the factors regulating the CD1d expression remain enigmatic. Interestingly, the engagement of intestinal epithelial CD1d can suppress colitis by promoting IL-10 production [¹⁰⁴]. Thus, CD1d expression on the biliary epithelium may function similarly in suppressing biliary inflammation. Nevertheless, NKT cells are widely considered to be critical in exacerbating PBC. α-GalCer injection increases cell infiltration in dnTGFRII mice, and exacerbates autoimmune cholangitis in mice immunized with 2-octynoic acid coupled with bovine serum albumin (2-OA-BSA) [¹⁰³,¹⁰⁵]. Additionally, in the Novosphingobium aromaticivorans infection model, which develops chronic bile duct lesions similar to that in PBC, NKT cells play a pathogenic role by initiating the diseases [¹⁰⁶]. N. aromaticivorans expresses conserved pyruvate dehydrogenase complex E2-component (PDC-E2) epitopes; thus, NKT cells activated by glycosphingolipids of the bacteria can further promote anti-PDC-E2 antibody production through cognate NKT-B cell interactions. In this model, NKT cells are indispensable at the initiation stage of chronic liver inflammation; however, T cells are more necessary at the late chronic phase of the immunopathological process rather than NKT cells. The precise mechanisms by which NKT cells regulate autoimmune liver diseases remain ill-defined, and their contributions might depend on the types of CD1d-expressing cells and bystander cells.

Metabolic liver disease

Alcoholic liver disease (ALD)

Alcoholic liver disease (ALD) is one of the most common causes of death worldwide. Chronic alcohol abuse leads to a broad range of liver diseases from hepatosteatosis to hepatocellular carcinoma (HCC) [¹⁰⁷]. An original concept believes that alcohol itself is the main cause of ALD. However, various factors, including oxidative stress, metabolism, apoptosis, and inflammation, have now been shown to regulate the initiation and progression of ALD [¹⁰⁸]. Recently, several reports have shown that immune cells, such as Kupffer cells, dendritic cells, neutrophils, and T cells, also contribute to ALD by inducing inflammatory responses [¹⁰⁹–¹¹⁴]. Alcohol consumption activates NKT cells and increases NKT cell numbers in the liver [¹¹⁵–¹¹⁷], which may partially be due to the enhanced lipid antigen loading facilitated by alcohol [¹¹⁸]. Importantly, activation of NKT cells by α-GalCer injection significantly exacerbates liver injury and cell death in a Fas and TNF-α receptor-1 dependent manner [¹¹⁵], whereas the depletion of NKT cells delays ALD progression [¹¹⁶,¹¹⁷,¹¹⁹]. All-trans retinoic acid (ATRA) and RARγ agonist tazarotene, which inhibit the proliferation and functions of NKT cells, significantly decrease NKT cell accumulation and blunt liver damage in animal models of ALD [¹¹⁷]. Moreover, the crosstalk between NKT cells and other immune cells have been explored. Neutrophil infiltration, which is correlated with liver injury in ALD, is promoted by NKT cells, as indicated by studies with Jα18^−/− mice, and possibly in a TNF-α dependent manner [¹¹⁶,¹¹⁹,¹²⁰]. Furthermore, crosstalk between Kupffer cells and NKT cells has been shown to exacerbate the liver injury in ALD [¹¹⁹,¹²¹]. Upon excessive alcohol consumption, IL-1β from Kupffer cells leads to accumulation and activation of NKT cells in the liver [¹¹⁹], which in turn produce granulocyte-macrophage colony-stimulating factor (GM-CSF) and further promote IL-1β production from Kupffer cells, thereby aggravating ALD progression [¹²¹]. The mechanisms by which NKT cells regulate the development of ALD are not fully understood. The crosstalk between NKT cells and other cells involved in ALD, including immune cells, parenchymal cells, and non-parenchymal cells, may also influence ALD progression.

Nonalcoholic fatty liver disease (NAFLD)

Nonalcoholic fatty liver disease (NAFLD) has become a major health problem worldwide [¹²²,¹²³]. Similar to ALD, NAFLD is also associated with abnormal fat accumulation in the liver and encompasses various stages of liver disease, ranging from simple steatosis, nonalcoholic steatohepatitis (NASH), to fibrosis and cirrhosis [¹²⁴,¹²⁵]. Nearly 30% of adults and 10% of children in western countries suffer from NAFLD due to the high rate of obesity [¹²⁶]. Additionally, a number of genetic, environmental, and inflammatory factors are involved in the etiopathogenesis of NAFLD. Inflammatory cell infiltration contributes to liver injury, and thus leads to NASH [¹²⁷,¹²⁸]. Studies on NAFLD demonstrated decreased NKT cells in the livers of leptin-deficient ob/ob mice [¹²⁹–¹³¹] and mice on high-fat diet (HFD) [¹³²–¹³⁴] or on choline-deficient diet (CDD) [¹³⁵,¹³⁶]. Impaired self-lipid presentation [³⁸], decreased CD1d expression in hepatocytes [¹³¹], and Kupffer cell-derived IL-12 [¹³⁶] are responsible for the loss of NKT cells in fatty livers. Additionally, decreased Kupffer cell-derived IL-15 and low norepinephrine (NE) activity in ob/ob mice also promote the depletion of hepatic NKT cells. Moreover, the injection of IL-15, leptin, or norepinephrine restores NKT cells in ob/ob mice [¹³⁰,¹³⁷]. The findings that HFD polarizes NKT cells toward Th2 responses and reduced CD1d in hepatocytes causes increased Th1 responses, imply an anti-inflammatory role of NKT cells [¹³¹,¹³⁸]. Transferring NKT cells into ob/ob mice or activating NKT cells with α-GalCer in HFD mice significantly reduces inflammation and hepatic fat accumulation and ameliorates non-alcoholic steatohepatitis [¹³⁹,¹⁴⁰]. Additionally, deficiency of NKT cells in Jα18^−/− mice exacerbates fat accumulation in the liver [¹³⁹]. These studies demonstrate an important role of NKT cells in inhibiting hepatic steatosis. This protective role of NKT cells can be accomplished via in situ protection and non-in situ protection. Moreover, hepatocytes with CD1d expression may activate NKT cells and induce anti-inflammatory responses [¹³¹]. On the other hand, activation of NKT10 cells in adipose tissues has been shown to inhibit systemic inflammation and reduce fat accumulation in the liver [¹³⁹]. Interestingly, other groups have reported controversial results. Wu et al. reported that activation of NKT cells by α-GalCer promotes hepatic steatosis in mice on HFD by increasing Th1 cytokine responses, and deficiency of NKT cells ameliorates hepatic steatosis in CD1d^−/− and Jα18^−/− mice [¹⁴¹]. Moreover, another study indicated that type II but not type I NKT cells contribute to steatohepatitis due to the different outcomes from CD1d^−/− and Jα18^−/− mice [¹⁴²]. Different animal models also caused conflicting results. Mice on methionine choline-deficient (MCD) diets develop NASH-fibrosis, a more advanced stage of NAFLD. In these mice, NKT cells are depleted in steatosis; however, they accumulate and drive fibrogenesis during NASH by upregulating hedgehog (Hh) and osteopontin, which activate hepatic stellate cells [¹⁴³,¹⁴⁴]. Consistently, patients with hepatic steatosis show increased amount of NKT cells in the liver [¹⁴⁵–¹⁴⁷].

Factors causing these discrepancies remain unclear. Stage of disease would influence the NKT-cell-mediated immune responses due to the potential differences in antigens, APCs, cytokines, and metabolic products. Moreover, NKT cells change their functions in adipose tissues due to the alteration of APCs during the progression of obesity [⁴⁷]. Activation of distinct bystander cells would also shift the immune responses and the role of NKT cells through intercellular crosstalk. Furthermore, whether distinct NKT subsets are related to different stages of NAFLD remains unclear. Thus, researchers should be careful with their animal models and NAFLD stages.

Hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common liver cancer and has a high recurrence rate, which is associated with chronic liver inflammation [¹⁴⁸–¹⁵⁰]. NKT cells are enriched in the liver, but their role in HCC remains unclear. Recently, Wolf et al. revealed a pathogenic role of NKT cells in promoting NASH-HCC transition. Significantly increased numbers of light-expressing NKT cells and CD8⁺ T cells were observed in the livers of NASH and NASH-related HCC patients. NKT cells efficiently enhanced fatty acid uptake in the hepatocytes through light-LTβR signaling and exacerbated liver damage, NASH, and HCC development, with the cooperation of CD8⁺ T cells [¹⁴⁵]. However, the approaches in this study were unable to distinguish type I NKT cells from type II NKT cells. On the other hand, NKT cells have been suggested as a good target for immunotherapy against tumors due to their ability to mount direct and indirect antitumor responses. α-GalCer administration activates NKT cells, which promote the activation of NK cells, and thus, elicit tumor rejection in murine liver [¹⁵¹]. Moreover, the adoptive transfer of NKT cells experienced “ex vivo education” by HCC-derived antigen pulsed DCs efficiently suppresses HCC growth in mice [¹⁵²]. Furthermore, NKT cell-based immunotherapies have shown beneficial effects in patients with multiple myelomas, non-small cell lung cancers [¹⁵³,¹⁵⁴], head and neck cancers [¹⁵⁵,¹⁵⁶], and other solid tumors [¹⁵⁷]. Additionally, the expression of chimeric antigen receptors (CARs) further expands the types of tumor antigen-activating NKT cells. Furthermore, CARs with costimulatory domains have been shown to polarize NKT cells toward Th1 responses [¹⁵⁸]. Using engineered artificial antigen-presenting cells (aAPCs), Tian et al. expanded CD62L⁺ CAR-NKT cells in vitro, which exhibited prolonged persistence and superior antitumor activity in vivo [¹⁵⁹]. These studies indicate the potential of using CAR-modified NKT cells in therapies for HCC.

Importantly, the dual role of NKT cells in tumor immunity has been reported. They promote tumor rejection by Th1 cytokines and favor tumor growth by Th2 cytokines. The reduced cell frequency and diminished IFN-γ production of NKT cells have not been consistently detected in all cancer patients [⁴³,⁸²,¹⁶⁰–¹⁶²]; however, clinical studies have demonstrated a correlation between IFN-γ production and anti-tumor effect of NKT cells [¹⁶²]. Moreover, the intratumoral NKT cells and IFN-γ production are correlated with HCC prognosis after curative resection, thus suggesting a new way to predict HCC recurrence and survival [¹⁶³]. In HCC patients, CD4⁺ NKT cells are enriched in tumors and produce more Th2 cytokines than CD4⁻ NKT cells [¹⁶⁴], which might suppress the anti-tumor responses. Additionally, lactic acid accumulation in tumor microenvironments has been shown to inhibit IFN-γ production; however, lactic acid accumulation has less effect on IL-4 production [³⁹]. Thus, changes in NKT cell subsets and tumor microenvironments can alter the role of NKT cells in tumor immunity, which should also be considered in NKT cell-based immunotherapies.

Future perspectives

The roles of NKT cells vary between different liver diseases. The stage of disease also influences the functions of NKT cells by skewing their cytokine production and modulating their crosstalk with other cell types (Fig. 2). Thus, demonstrating the mechanisms by which factors in the liver microenvironments regulate NKT-cell-mediated immune responses, and exploring the crosstalk between NKT cells and other immune cells or non-immune cells are important. Hepatic NKT cells are predominantly NKT1 and are similar to splenic NKT cells; however, whether the liver contains tissue-specific NKT cell subsets remains unknown, and this might contribute to liver diseases. Recently identified DN NKT cells of DN-stage origin are enriched in the liver and exhibit tissue-specific distribution and superior cytotoxicity. However, their roles in liver diseases remain unclear. A growing interest in using NKT cell-based immunotherapies has emerged. However, most of the knowledge on the role of NKT cells in liver diseases comes from studies on animal models. More clinical studies are required in the future. Additionally, the stage of diseases and influence of tissue microenvironments on NKT cell functions should also be considered.

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