6.1.1 NKT
Natural Killer T (NKT) cells are unique lymphocytes characterized by their expression of a single invariant antigen receptor encoded by Vα14Jα18 in mice and Vα24Jα18 in humans, which recognizes glycolipid antigens in association with the monomorphic CD1d molecule.NKT cells mediate adjuvant activity to activate both CD8T cells to kill MHC-positive tumor cells and NK cells to eliminate MHC-negative tumor at the same time in patients, resulting in the complete eradication of tumors without relapse.Therefore, the NKT cell-targeted therapy can be applied to any type of tumor and anyone individual, regardless of HLA type [2].
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.Numerous in vitro and in vivo studies using the synthetic lipid α-galactosylceramide (αGalCer, KRN7000) and its analogs 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 biosynthesize αGalCers and/or related compounds.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 [3].
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.Sphingomonas spp.biosynthesizes an αGalCer-related compound, α-galacturonosyl ceramide(αGalACer).Other weak NKT cell agonists include microbial glycosphingolipid, diacylglycerolipids and cholesteryl-αglycoside.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.The activation mechanism is considered TCR and cytokine-mediated mode of NKT cell activation—a feature that is important for NKT cell activation by weak microbial and self-lipid agonists [4].
NKT cells react to CD1d molecules presenting self-lipids on host APCs in the presence of a second signal.The inability to activate NKT cell hybridomas by using artificial APCs lacking βGlcCer synthase and impaired NKT cell development in mice lacking βGlcCer synthase in their thymocytes, suggested that a cellular, βGlcCer-derived GSL is an endogenous mouse NKT cell agonist.Several microbes—bacteria, fungi and viruses—activate NKT cells but do not biosynthesize NKT cell agonists.Such microbes induce the biosynthesis and/or presentation of self-lipids, which are thought to be mammalian αGalCer and perhaps iGb3.As self-lipids are weak NKT cell agonists,NKT cell activation is bolstered by IL-12 secreted by DCs activated through dectin-1 DCs or toll-like receptor (TLR)-4.This activation mechanism is a variation on the TCR- and cytokinemediated mode of NKT cell activation and a feature that is important for NKT cell activation by microbes that do not themselves biosynthesize an NKT cell agonist [5].
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.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.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 [6,7,8,9].
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.This latter mechanism is considered cytokine-driven NKT cell activation.This mechanism is important for immunity to cytomegalovirus.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 in Figure 6.1 [10,11,12].
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Figure 6.1 Th ree distinct strategies activate mouse N KT cells.Potent NKT cell agonists—such as αGalCer—directly activate NKT cells without the need for a second signal, in a T cell receptor(TCR)-signaling dominated fashion (left panel).Alternatively, microbes containing toll-like receptor(TLR) ligands such as LPS activate NKT cells by inducing IL-12 production by DCs, which amplifies weak responses elicited upon the recognition of CD1d bound with self-glycolipids by the NKTCR.Several endogenous lipid agonists have been identified and characterized.Some microbes such as Sphingomonas capsulata, which are α-Proteobacteria, synthesize α-anomeric glycolipids for their cell walls.These glycolipids, when presented by CD1d, weakly activate NKT cells directly.In the presence of a second signal—generally a pro-inflammatory cytokine such as IL-12—such weak agonists strongly activate NKT cells (middle panel).Intriguingly, NKT cells can be activated solely by cytokines—mainly IL-12—in a TCR-independent manner (right panel).(Please scan the QR code on the Preface to get original color figures.)
Source: Kumar A, Suryadevara N, Hill TM, Bezbradica JS, Van Kaer L, Joyce S.“Natural Killer T Cells: An Ecological Evolutionary Developmental Biology Perspective”.FrontiersinImmunol, 2017, 8:1858.
For the development of anti-cancer immunotherapy, the following three issues have to be generally considered.The first is that, for an optimal therapy, both MHC-positive and -negative tumor cells should be eliminated simultaneously to avoid tumor relapse, because tumors in general contain both cell types.The second important point is that tumor cells do not usually contain any endogenous adjuvant materials because, unlike pathogens, they are derived from autologous cells, so that it is difficult to mount efficient anti-tumor immune responses in patients without adjuvant.The third point is that it is essential to convert DCs from an immature to mature state in the tumor patients to induce efficient protective responses, because most advanced cancer patients are immune deficient[13].
Activation of NKT cells with α-GalCer promotes production of IFNγ, which induces NKT cell-mediated adjuvant activity, activating both NK and CD8T cells and bridging innate and acquired immune systems and thus overcoming the problem of the tumor-relapse because it induces clonal expansion of antigen-specific CD8T cells and activation of NK cells as well as maturation of immature DCs in patients.Therefore, NKT cell-targeted therapy should be useful for treatment of advanced cancer patients, whose specific CD8T cells, even though they are present in patients, fail to expand, because DCs are usually immature due to the immune suppressive cytokines, such as interleukin (IL)-10 or TGFβ, produced by tumor cells [14].
Unlike other lymphocytes, only NKT cells have the ability to interact with immature DCs in the presence of α-GalCer, leading to the induction of full maturation of DCs through CD40-CD40L interactions, resulting in their robust production of IL-12 to further activate NKT cell production of IFN-γ.The final result of these cellular interactions is the activation of both CD8 cytotoxic T cells and NK cells to eliminate MHC-positive and -negative tumor cells, respectively, resulting in tumor eradication without relapse.In fact, it was experimentally demonstrated the strong adjuvant activity mediated by NKT cells is observed in a tumor model using OVA as an artificial tumor antigen.In this experiment, mice were immunized with OVA-loaded TAP-KO spleen cells together with α-GalCer;TAP-KO mice were used because they are unable to present peptide antigen to conventional CD8T cells.For the detection of IFN-γ production, we stimulated spleen cells with OVA peptide in vitro and observed significant production of IFN-γ by NK cells and CD8T cells , as well as a robust increase in the number of OVA-specific IFN-γ producing CD8T cells in vivo and, importantly, suppression of the OVA-expressing EL4 (EG7) tumor growth (Figure 6.2) [15,16,17].
Figure 6.2 N KT cell-mediated adjuvant effects in ant i-tumor immune responses.(A) Mechanisms of NKT cell-targeted anti-tumor adjuvant cell therapy: Upon NKT cell activation by α-GalCer-DCs,specific CD8 killer T cells and NK cells are activated by the NKT cell-mediated adjuvant activity, and kill both MHC-positive and -negative tumor cells in patients.NKT cells also interact with immature dendritic cells to induce maturation, resulting in recovery from the immunodeficiency state common in cancer patients.(B) Experimental model using OVA as an artificial tumor antigen to demonstrate the effects of NKT cell-mediated adjuvant activity.Mice were immunized with OVA-loaded TAP-deficient spleen cells treated with OVA under osmotic shock.A week later, CD8T cells and NK cells in these immunized mice were analyzed for IFN-γ production after re-stimulation with OVA antigen in vit ro(3C).A significant increase in the number of antigen-specific IFN-γ producing CD8T cells was also detected in mice ex vivo (3D).(C) NKT cell-mediated adjuvant effects on NK- and CD8T-cell production of IFN-γ after α-GalCer-DC stimulation.(D) Increase in the number of OVA-specific IFN-γ producing CD8T cells (21 fold) and NK cells (3.5 fold) after α-GalCer-DC administration in vivo.(E)Suppression of OVA-bearing tumor (EG7) growth by α-GalCer-DC administration in the OVA-model in vivo.
Source: Taniguchi M, Harada M, Dashtsoodol N, Kojo S.“Discovery of NKT Cells and Development of NKT Cell-Targeted Anti-Tumor Immunotherapy”.ProceedingsoftheJapanAcademy, Series B, PhysicalandBiologicalSciences, 2015, 91(7):292-304.
Therefore, NKT cell-targeted anti-tumor immunotherapy overcomes the major problem of tumor relapse usually observed with current anti-cancer immunotherapies, which target only one type of anti-tumor immune effector cells.For example, in the immunotherapy using tumor antigen peptides or antibodies against PD-1 or CTLA4, the target is a CD8T cell, which kills MHC-positive but not-negative tumor cells, resulting in the recurrence of an MHC-negative tumor.Similarly, in the artificial cells developed by the forced expression of NK receptor ligands, such as Rae1/H60/Mult-1 (NKG2D-L), or TNF ligand (CD70-L), the target cell is an NK cell, which eliminates only MHC-negative tumor cells, resulting in relapse with MHC-positive tumor cells.Only NKT cells, and not other effector cell types, simultaneously activate both CD8T and NK cells,resulting in the simultaneous elimination of both MHC-positive and -negative tumor cells.Therefore, NKT cell-targeted therapy is a promising treatment option for tumor treatment to overcome tumor relapse [13].