The evolutionary appearance of the vertebrate immune system equipped complex organisms with the ability to resist invasion by pathogenic microbes and to sense and respond to a loss of tissue integrity due to infection, aberrant cell growth, or mechanical injury. As organisms became increasingly more complex and lived beyond their fecund years, a finer ability to discriminate self from non-self was required (1, 2). Thus, the maintenance of homeostasis in such organisms requires the concerted action of multiple cell types that stand poised to respond to a hostile world filled with a seemingly endless array of infectious agents, toxic chemicals, and biologics. The first responders in this elaborate defensive network have historically been classified as members of the more archaic, multi-modular innate immune system. Should the innate defenses prove insufficient, the evolutionarily younger, adaptive immune system—consisting of B and T lymphocytes—is recruited to restore the homeostatic state. The quick-acting cells of the innate immune system senses an altered homeostatic state with pattern recognition receptors to detect conserved molecular structures shared by many pathogens alike (3, 4). By contrast, the slow-responding, adaptive immune system senses alterations in homeostasis by using diverse, clonally distributed B cell receptors (BCR and their secreted counterparts, antibodies), and T cell receptors (TCRs), respectively.

Bridging the gap between innate and adaptive immune responses are the innate-like B and T lymphocytes. These are a group of cells that express a relatively restricted repertoire of receptors generated through somatic recombination, yet unlike conventional T and B cells, exhibit innate-like recognition principles and functional responses (5). Innate-like lymphocytes include both T cells (γδ T cells, natural killer T cells, mucosal-associated invariant T lymphocytes, and CD8αα-expressing intestinal intraepithelial lymphocytes) and B cells (B-1 B cells and marginal zone B cells). The evolutionary appearance of this group of immune cells, including natural killer T cells (NKT cells) endowed upon vertebrates the capacity to initiate and amplify both the innate and adaptive immune responses. By virtue of their immunoregualtory functions, innate-like lymphocytes can fine-tune the nature and magnitude of these immune responses (6). Although each immune module plays a specific role, it is the controlled integration of multiple modules that results in an effective inflammatory response that is essential in maintaining a stable milieu intérieur (7).

NKT cells—originally defined as cells that co-express key natural killer (NK) cell surface markers and a conserved αβ TCR repertoire—are thymus-derived, innate-like T lymphocytes. The functions of NKT cells are controlled by self and non-self-lipid agonists presented by CD1d molecules (8). The majority of NKT cells (type I, invariant NKT) express an invariant TCR α-chain (Vα14Jα18 in mice; Vα24Jα18 in humans). The invariant α-chain pairs predominantly with Vβ8.2, Vβ7, or Vβ2 in mouse NKT cells, or Vβ11 almost exclusively in human NKT cells. A small NKT cell population—referred to as type II NKT cells—expresses a more diverse TCR repertoire and recognizes a distinct group of lipid antigens; these, however, are the focus of other reviews (9–14). The recognition of lipid agonists rapidly activates NKT cells, which respond just as quickly by secreting a variety of cytokines and chemokines, and upregulate costimulatory molecules. By acting promptly, NKT cells alert and regulate the effector functions of myeloid and lymphoid cells. In so doing, NKT cells play a critical role in controlling microbial and tumor immunity as well as autoimmune and inflammatory diseases (6, 15–17).

The functions of NKT cells are controlled by CD1d molecules. CD1d molecules bind to and present a variety of lipid ligands to reactive T cells (18). Numerous in vitro and in vivo studies using the synthetic lipid α-galactosylceramide (αGalCer, KRN7000) and its analogs (Table 1 and references therein) has led to our current understanding of NKT cell biology. αGalCer is a natural product isolated from the marine sponge, A. mauritianus. The gut bacterium, Bacteroides fragilis, and the fungus, Aspergillus fumigatus, also biosynthesise αGalCers and/or related compounds (Table 1 and references therein). Hence, αGalCer and related compounds may be more prevalent in nature than previously thought and the NKT cell biology so gleaned may be highly relevant.

αGalCer is a potent NKT cell agonist, which when presented by CD1d molecules directly activates NKT cells in a TCR-dependent manner without need for additional signals. This activation mechanism is considered TCR-dominated mode of NKT cell activation (Figure 1).

Sphingomonas spp. biosynthesises an αGalCer-related compound, α-galacturonosylceramide (αGalACer). Other weak NKT cell agonists include microbial glycosphingolipid [GSL; e.g., αGalCer-related asparamide B (A fumigatus)], diacylglycerolipids [e.g., α-galactosyl- (Borrelia burgdorferi—the agent of Lyme disease) and α-glucosyl-diacylglycerol (Streptococcus pneumoniae)] and cholesteryl-α-glycoside [e.g., cholesteryl-6-O-acyl α-glucoside (Helicobacter pylori)] (Table 1 and references therein). Being a weak agonist, NKT cell activation by these microbial glycolipids requires a second activation signal from inflammatory cytokines. Such inflammatory cytokines result from dendritic cells (DCs) that are activated through their pattern recognition receptors (45–47). This activation mechanism is considered TCR- and cytokine-mediated mode of NKT cell activation (Figure 1)—a feature that is important for NKT cell activation by weak microbial and self-lipid agonists.

NKT cells react to CD1d molecules presenting self-lipids on host APCs in the presence of a second signal (6, 48). The inability to activate NKT cell hybridomas by using artificial APCs lacking βGlcCer synthase (49) and impaired NKT cell development in mice lacking βGlcCer synthase in their thymocytes (50), suggested that a cellular, βGlcCer-derived GSL is an endogenous mouse NKT cell agonist (49, 50). Several microbes—bacteria (e.g., Staphylococcus aureus, Salmonella typhimurium, Listeria monocytogenes, etc.), fungi (e.g., A. fumigatus) and viruses—activate NKT cells but do not biosynthesise NKT cell agonists. Such microbes induce the biosynthesis and/or presentation of self-lipids, which are thought to be mammalian αGalCer and perhaps iGb3 (19, 28, 35). As self-lipids are weak NKT cell agonists, NKT cell activation is bolstered by IL-12 secreted by DCs activated through dectin-1 DCs (31, 47) or toll-like receptor (TLR)-4 (45, 46). This activation mechanism is a variation on the TCR- and cytokine-mediated mode of NKT cell activation and a feature that is important for NKT cell activation by microbes that do not themselves biosynthesise an NKT cell agonist.

Type I interferon (IFN)—produced by DCs activated by the TLR9 ligand CpG—can serve as a second signal for NKT cell activation in conjunction with the presentation of sialylated cellular glycolipids by CD1d molecules (51). This finding is significant because almost all viral infections induce type I IFN response. Even though viruses do not biosynthesise NKT cell agonists, or any lipid for that matter, viral infections also activate NKT cells (52–62). Perchance, in such a circumstance, NKT cell activation occurs via the recognition of a self-lipid(s) presented by CD1d in the presence of inflammatory signals relayed by type I IFNs.

NKT cells are activated by the combined actions of IL-12 and IL-18. Under such conditions, NKT cell activation does not require the recognition of a CD1d-restricted agonist (63–65). This latter mechanism is considered cytokine-driven NKT cell activation (Figure 1). This mechanism is important for immunity to cytomegalovirus (65). Summarily, these multiple modes of activation suggest that NKT cells have evolved many different mechanisms to sense an altered homeostatic state caused by microbial infections. How activated NKT cells steer downstream innate and adaptive immune responses is described below.

NKT cells form immune synapses upon recognition of lipid agonists presented by CD1d molecules displayed on APCs or planar membranes. The kinetics NKTCR/ligand interactions determine the functional outcome (66). Positive cooperative engagement of CD1d-lipid agonistic complexes by the NKTCR allows NKT cells to recognize subtle changes in cellular lipid content and to actuate a response (67). Upon activation, NKT cells rapidly polarize IFN-γ and lytic granules to the immune synapse to transmit an effector response (66, 68, 69). The synaptic transmission of effector molecules controls downstream innate and adaptive immune responses as described below.

Akin to the cells of the innate immune system (e.g., neutrophils, Mϕ, DCs, and NK cells), NKT cells respond within the first several hours of agonist recognition and secrete copious amounts of effector cytokines and chemokines (Figure 2). The nature of the activating NKT cell agonist controls the nature of the cytokine response (see Table 1). For example, the synthetic agonist αGalCer, within 30–90 min, elicits a wide variety of cytokines (Figure 2). Nonetheless, αGalCer variants containing different lipid chain length or unsaturation typically induce an IL-4 cytokine response (24, 25). By contrast, other αGalCer variants that have an altered glycosidic linkage, a chemically modified acyl-chain, or a modified sphingoid base, potently induce an IFN-γ response (Table 1 and references therein). Thus, it is possible to steer desirable immune responses against cancers by harnessing lipid agonists that induce therapeutic cytokine responses. This feature of αGalCer variants is further accentuated by the ability of activated NKT cell responses to transactivate cells of the innate and adaptive immune systems as narrated briefly below (see Figure 2).

Dendritic cells, especially CD8α⁺ DCs, which are a major producer of IL-12 (71), play a critical role in glycolipid agonist presentation and NKT cell activation (72–78). Activated NKT cells reciprocate by activating the interacting DCs. DCs so activated rapidly mature. Hence, they upregulate costimulatory molecules CD40, CD80, and CD86; several molecules critical for protein antigen capture and peptide presentation, such as DEC205 and MHC class II molecules (79); and induce the production of IFN-γ, tumor necrosis factor (TNF)-α, and IL-12 (80–83). IFN-γ produced by activated NKT cells coupled with CD154 (CD40 ligand on NKT cells) and CD40 (on DCs) mediate the NKT-DC crosstalk (81, 84). This crosstalk steers multiple downstream immune responses: (1) the number and phenotype of DCs after tumor induction (85). (2) IL-12 and IL-18 resulting from NKT-DC crosstalk transactivates NK cells to produce IFN-γ (82). (3) NKT-DC crosstalk can result in IL-4, IL-6, IL-13, and IL-21, which together can enhance B cells responses to protein antigens by B cells (86–93). (4) NKT-DC cross talk licenses DCs for antigen cross-presentation to CD8⁺ T cells (94–96), and the activation and differentiation of CD4 and CD8 T cells (79, 95–97). Through these bidirectional interactions, NKT cells and DCs cooperate to amplify and direct both innate and adaptive immune responses. Hence, NKT cells are an attractive target for cancer immunotherapies (98–102).

NKT cells have long represented an attractive target for tumor immunotherapy (103, 104). Numerous studies in both humans and mice have demonstrated their ability to directly target CD1d-expressing tumor cells (105–108), recruit and activate anti-tumor effector cells of the innate and adaptive immune systems (100, 109–114), and control the activity of immunosuppressive cells in the tumor microenvironment. After in vivo administration of αGalCer, NKT-DC cross-talk-mediated NK cell activation results in IFN-γ response (82) and, potentially, the anti-tumor effect of αGalCer (85, 115).

The potent anti-metastatic activity of αGalCer in mice (20, 116), which is NKT cell mediated (22), prompted investigations in the role of NKT cells in natural immunity against tumors. Such investigations include chemically induced tumors, transplanted tumors, and tumors arising in genetically engineered animals (115). The outcomes of these studies have been promising because NKT cells exhibit natural immunity against different cancer models. Independent studies have sometimes reported conflicting results as to the importance of NKT cells in the anti-tumor response, particularly with carcinomas induced by the topical carcinogen methylcholanthrene (117, 118). Such conflicting results were likely due to unknown environmental and/or genetic factors present in the mice used as controls in similar experiments by different groups (117). Studies in mice revealed that αGalCer variants that induce type I inflammatory response (see Table 1) were protective against tumor metastases. The mechanistic basis of this anti-metastatic effect remains elusive. Nonetheless, the ability of NKT cells activated by αGalCer variants to steer desirable downstream effector functions, such as NK cells, cytotoxic T cells, Th1 and Th17 cells, γδ T cells, IFN-γ, and direct lysis of myeloid lineage cells may underlie the outcome (100, 115). The anti-tumor activities of NKT cell agonists have already been exploited in a variety of clinical trials. The outcomes of these trials have also been promising (103, 104, 119–121).

NKT cell activation results in rapid secretion of pro-inflammatory and regulatory cytokines and chemokines. This property in conjunction with the capacity to transactivate a variety of innate and adaptive immune cells—see subsection on Transactivation—allows NKT cells to steer downstream immune responses. NKT cells are heterogeneous, consisting of at least four distinct subsets—NKT1, NKT2, NKT10, and NKT17. In addition, at least one induced subset, NKTfh, is also recognized. As with conventional CD4⁺ T cell subsets, NKT cell subsets are characterized by prototypic cytokine responses and subset-specific transcription factors (Figure 5). Each subset is represented at different proportions in various mouse strains (151–155).

MOUSE NKT1 CELLS are marked by either the expression of CD4 or the absence of CD4/CD8 co-receptors. NKT1 cell activation results in a Th1-like cytokine response. The majority of mouse splenic and hepatic NKT cells are NKT1 subset, especially in the C57Bl/6 strain. NKT1 cell differentiation depends on T-bet (Tbx21) and IL-15 but less on GATA3 (151, 152, 187, 189–191). Unlike HDAC3 depletion in DP thymocytes, NKT cell lineage-specific deletion of Hdac3 (derived with the use if Zbtb-Cre) results in selective impairment in NKT1 cell development. The selective absence of HDAC3 in NKT cells resulted from reduced autophagy (193–195)—a cytoplasm recycling process essential to T and NKT cell development —and decreased GLUT1, CD71, and CD98 nutrient receptor expression (196). Moreover, the anti-tumor effect of αGalCer (109) is potentially mediated by IFNγ- and TNFα-producing NKT1 cells.

MOUSE NKT2 CELLS express the CD4 co-receptor. NKT2 cell actiation results in a Th2-like cytokine and chemokine response. This subset is enriched in mouse lungs and the intestine (152). IL-13 and IL-4 as well as CCL17, CCL22, CCL10/CCL6, and eosinophil chemotactic factor-L secreted by activated NKT2 cells may mediate airway hyperresponsiveness (151, 197–200). This Th2-type response recruits Mϕs, eosinophils, neutrophils, and lymphocytes into the lungs to incite tissue damage (197). Coincidently, in BALB/c mouse that is sensitive to airway hyperresponsiveness, NKT2 cells predominate (152).

NKT cells constitutively express Il4 and Ifng transcripts. This constitutively expressed cytokine genes may explain the rapid NKT cell response to agonistic stimulation in vivo (201). Epigenetic changes in the two cytokine genes control their transcription. For example, the conserved non-coding sequence (CNS) 2 located downstream of the mouse Il4 locus is constitutively active in NKT cells, which thereby constitutively transcribe the Il4 gene. CNS 2 activity depends on NOTCH and Rbp-j (recombination signal binding protein for immunoglobulin kappa J region)—a transcriptional regulator of NOTCH signaling. Hence, DP thymocyte-specific deletion of Rbp-j abolished CNS 2 activity and the ability to transcribe Il4 (202).

A similar epigenetic control of the human Ifng locus using CNS-30 and CNS +18–20 transcribes the Ifng locus in NKT cells (203, 204). Consistent with this finding, NKT cells showed acetylated histone 4 marks upstream and downstream of the Ifng coding region only when activated by weak (self agonists) or strong signals (phorbolmyristate acetate + ionomycin) but not in resting NKT cells. Furthermore, NKT cells rested after stimulation returned the Ifng locus to an unmarked state (205). H4 acetylation occurs at CNS +18–20, a site essential for human Ifng transcription in NKT cells and conserved within the mouse Ifng locus (203, 205). These findings notwithstanding, it is unclear whether human NKT cells constitutively transcribe the Ifng locus and how mouse NKT cells constitutively transcribe its Ifng locus.

MOUSE NKT17 CELLS do not express CD4 or CD8 co-receptors. They are enriched in the lungs, skin, and peripheral lymph nodes, and are poorly represented in the spleen and liver (206–208). These cells require IL-7, not IL-15, for survival (151, 209). The development of NKT17 cells also requires mTORC2 signaling and the transcription factors Runx1 and NKAP (168, 210–213). Thus, NKT cell-specific Runx1 deletion results in decreased IL-7Rα, BATF, and c-Maf expression against the backdrop of increased Lef and Bcl11b expression (211). On the other hand, how NKAP controls NKT17 cell development is not understood, but appears not to require mTOR, IL-7, and TGF-β signaling (210).

Akin to Th17 cells, NKT17 cells constitutively express RORγt (206), rapidly produce IL-17A in response to certain bacterial infections, and induce airway neutrophilia when challenged with synthetic glycolipid or LPS (37, 206, 214). NKT17 cells may contribute to ozone-induced airway hypersensitivity (215), the development of experimental autoimmune encephalomyelitis (214), and the pathogenesis of acute hepatitis in mice (216).

MOUSE NKT10 CELLS, the PLZF-independent subset (154), are found in low frequency in unchallenged mice and in human peripheral blood mononuclear cells (PBMCs). Upon re-activation, NKT10 cells that previously responded to αGalCer in vivo, secrete IL-10 (155). IL-10 produced by activated NKT10 is thought to maintain immune-privilege sites. This NKT cell subset may also control Treg cell functions in adipose tissues (154).

Mouse NKT cells can provide cognate (lipid antigens) or non-cognate (protein antigens) help to B cells and regulate antibody responses (89, 90, 92, 217, 218). Upon immunization with αGalCer a subset of NKT cells acquire a phenotype similar to T follicular helper T cells (Tfh) referred to as NKT follicular helper (NKTfh) cells (218–220). NKTfh are characterized by the expression of CXCR5, ICOS, PD1, Bcl6, and BTLA. Their development is dependent on same factors that drive Tfh development (219). NKTfh cells induce rapid production of germinal centers through IL-21 production that yields detectable levels of antigen-specific IgG (91, 219, 220). Nonetheless, NKTfh cell-induced antibody responses are short-lived and inferior to Tfh cell-induced responses (91, 219, 220). NKTfh cells may play a role in antibody responses against human pathogens such as Borrelia hermsii, Streptococcus pneumoniae, and Plasmodium falciparum (91, 219, 220). NKTfh and Tfh cells can act synergistically to induce robust antigen-specific antibody responses underscoring the use of αGalCer as a vaccine adjuvant (218).

Human NKT cell responses are as diverse as those of mouse (221), yet NKT cell subsets have not been formalized in humans. Functional dichotomy has been reported in human CD4⁺ and DN NKT cell subsets: activated human CD4⁺ NKT cells secrete IL-4. A pathological role has been attributed to human CD4⁺ NKT cells, which accumulate in the lungs of chronic asthmatic patients and produce IL-4 and IL-13 (222). Hence, human CD4⁺ NKT cell resembles the mouse NKT2 cell subset. On the other hand, the activated DN NKT cells secrete IFN-γ and TNF-α. Furthermore, both CD4 and DN human NKT cell subsets upregulate perforin in the presence of inflammatory signals. The DN NKT cells also upregulate NKG2D expression, which together with perforin may mediate cytotoxicity against infected cells and cancer cells (223, 224). These functions of human NKT cells resemble those of mouse NKT1 cells. Activated human NKT cells can also secrete IL-17 (221), suggesting the presence of an NKT17-like subset.

In summary, mouse NKT cells divide labor into four subsets. Global and single cell transcriptome analyzes demonstrated that the thymic NKT1, NKT2, and NKT17 cells were distinct subsets (156, 225). Even though not formalized, human NKT cells also have the potential to mirror mouse NKT cell subsets, but this requires further investigation. That the tissue environment plays a role in the differentiation of NKT cell subsets is supported by the finding that NKT17 differentiation required mammalian target of rapamycin complex-2 (213) or is suppressed by Tet enzymes that modify 5-methylcytosine in DNA by controlling the expression of Tbet and ThPOK transcription factors (226). Another study using somatic cell nuclear transfer to generate mice with monoclonal NKT cell populations demonstrated that tissue homing pattern, and, to a lesser extent, TCR avidity governed NKT cell subset differentiation (208). That NKT1, NKT2, and NKT17 cells differentiated within peripheral tissues of each of the three monoclonal mouse lines, derived from somatic cell nuclear transfer, suggests that the subsets are perhaps NKT cell “reaktionsnorm [German for reaction norm or norm of reaction; Woltereck 1909 cited in Ref. (227)]” induced by the tissue-specific environment, potentially by local cytokine/chemokine milieu in conjunction with the host microbiota.

An intriguing property of NKT cells is their frequency variation observed in lymphoid tissues of different inbred strains of similar age: low in 129 and NOD, intermediate in C57Bl/6, and high in BALB/c, CBA, and DBA/2 mice (152, 153, 228–230). Likewise, NKT cells show striking frequency variation that can range from as little as 0.001% to 5–10% within human PBMCs (221, 231, 232).

Mice show inter-strain variation in thymic NKT cell subset numbers (152). C57Bl/6 mice have high proportion of NKT1 cells and low frequency of NKT2 cells, whereas BALB/c have high frequency of NKT2 and NKT17 suggesting an inverse correlation between frequency of NKT1 cells versus NKT2 cells and mouse strains. Curiously, mouse strains that have a high frequency of NKT2 cells (BALB/c, CBA, and DBA/2) showed high numbers of eomesodermin-expressing memory-like CD8⁺ thymocytes (152) which was attributed to the steady-state production of IL-4 by the expanded NKT2 population in these mice. In an effort to understand whether genetic polymorphisms between mouse strains controlled NKT cell frequency, recombinant inbred and co-isogenic strains begotten from NOD (low NKT cell frequency) X C57Bl/10 (intermediate NKT cell frequency) crosses were analyzed. The outcomes of several such studies indicated that NKT cell frequency segregated with the genetic background of the mouse (153, 229, 230). Whereas this outcome suggests that NKT cell frequency is under genetic control, whether this control is direct or indirect remains to be ascertained.

NKT cells surveil barrier mucosae such as that of the small and large intestine (233, 234). The number, phenotype, and functional maturation of NKT cells in the gut epithelium and lamina propria are controlled by neonatal colonization of the gut by bacterial symbionts. Thus, germ-free (GF) mice have high numbers of NKT cells in the gut epithelium and lamina propria that are immature and, hence, hypo-responsive to αGalCer (233). Curiously, reconstitution of young, but not adult mouse gut by bacteria that biosynthesise αGalCer or related compounds reverses the hypo-responsiveness of NKT cells found in GF intestinal mucosae (234). Similarly, GF mice also harbor high hepatic and pulmonary, but not thymic and splenic NKT cell frequencies (234). Additional evidence implicates the CXCR6 ligand CXCL16, whose expression is under the control of gut microbiota, in regulating gut NKT cell frequency and maturation (234, 235). Furthermore, αGalCer compounds (see Table 1) synthesized by the bacterial symbiont Bacteriodes fragilis, exert either an inhibitory effect preventing proliferation, or are stimulatory on developing NKT cells (26, 27). As the gut microbiota varies between individuals of different genetic, ethnic, and geographic backgrounds (236), the above findings in mice suggest the intriguing possibility that the human symbionts may impart an epistatic control over human NKT cell frequency and maturation as well. Because the frequency and functional status are environmentally controlled even though the genotype of the differentiating NKT cells remains the same, NKT cell frequency and proper maturation are potentially polyphenic (227, 237) properties.

Early-life microbial ecology has implications for health. Thus, GF mice are prone to severe airway hypersensitivity and dextran sodium sulfate-induced colitis (233–235). The latter phenotype is obviated by the interaction of NKT cells with B. fragilis-derived glycosphingolipid(s) during early life (26). Not surprisingly, NKT cells can, in turn, control gut microbial ecology and gut physiology (238). Whether similar reciprocal interactions between NKT cells and the gut microbiota occur in humans currently remains unknown.

Microbial ecology has emerged as an important deterministic factor in the outcome of chemotherapy, radiation therapy, and immunotherapy against cancers (239). NKT cells have been targeted in the clinic for immunotherapy (see Implications for Cancer Immunotherapy), but how each of these therapies impact NKT cells is not known. It is noteworthy that a fraction of NKT cells are radiation resistant (130). This feature can be exploited for NKT cell-targeted immunotheraphy against lymphomas and leukemias. Clinical trials have shown that the outcome of NKT cell-targeted immunotherapy varied between recipients (103, 104). Hence, what roles the gut microbiota played in the outcome is worthy of investigation. So also, considering that NKT cells can impact microbial ecology (238), what roles NKT cells play in tumorigenesis and metastasis are also worthy of investigation. Insights into how the microbial community assembles and forms the host–symbiont ecosystem will facilitate an essential understanding of the molecular underpinnings that govern reciprocal interactions between the host and its internal ecosystem. These new insights can, in turn, impact the way by which new cancer therapies are designed, developed, and refined.

All authors wrote and edited the manuscript.

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.