ReviewAdjuvant activity mediated by iNKT cells
Introduction
Invariant natural killer T (iNKT) cells are characterized by the expression of an invariant antigen receptor encoded by Vα14Jα18 in mice and Vα24Jα18 in humans and also by the rapid production of both Th1 and Th2 cytokines after stimulation with their ligands [1], [2], [3]. An exogenous glycolipid, α-galactosylceramide (α-GalCer), has been identified as a ligand for mouse iNKTcells and is presented to these T cells by the monomorphic CD1d molecule. Particular CD1d amino acids (Ser76, Arg79, Asp80, Glu83, and Gln150) that are important for binding with either α-GalCer or the iNKTcell receptor are well conserved among species such as mouse, rat, sheep, and human [4], [5], [6], [7]. In addition, the first four amino acids (Asp94, Arg95, Gly96, and Ser97) in the Jα18 region of the iNKTcell receptor important for the binding with α-GalCer and the CD1d molecule are also conserved in mice and humans. Thus, α-GalCer identified can be used to activate both human and mouse iNKT cells. Although α-GalCer is an exogenous ligand, the existence of endogenous self-ligands has been speculated based on the observation that iNKT cells appear to be persistently activated in vivo; freshly isolated iNKT cells express activation markers such as CD69 and CD44. Moreover, because no iNKT cells develop in the absence of CD1d, it appears that developing iNKT cells recognize self-ligands presented by CD1d and are positively selected.
Because of their apparent self-reactivity and ability to quickly release large quantities of cytokines such as interferon-γ (IFN-γ), iNKT cells have been demonstrated to play important roles in the initiation of protective immune responses. In fact, iNKT cells freshly isolated from tissues express large amounts of mRNA for IFN-γ and IL-4, although their apparent self-reactivity does not elicit any iNKT cell effector functions in vivo. However, the recognition of self-ligands and the subsequent weak responses of iNKT cells are essential during the initial phase of protective immunity.
In an immune response against pathogens, one of the first cells to be activated is the dendritic cell (DC) of the innate system. This activation is mediated by Toll-like receptors (TLR) on the DCs, leading to the production of pro-inflammatory cytokines and IL-12 and the up-regulation of co-stimulatory molecules (e.g., CD40, CD80, and CD86). Most importantly, IL-12 has been shown to be essential for the activation of iNKT cells, because only iNKT cells, and not other cells such as naïve T cells or NK cells, express substantial amounts of the mature form of the IL-12 receptor (IL-12R), and iNKT cells have been shown to be the primary targets for IL-12.
Weak responses by iNKT cells to self-ligands are further augmented by IL-12 secreted by DCs in response to TLR activation, resulting in the production of IFN-γ by the iNKT cells. Thus, both inherent self-ligand activation and extrinsic IL-12-induced signaling are necessary to initiate iNKT cell-mediated protective immune responses. Although some pathogen-derived glycolipids can directly activate iNKT cells, in most cases the recognition of pathogen products is not required for iNKT cell activation. Thus, pathogen products only seem to play a role in stimulating DCs through TLR-signaling to produce IL-12.
IL-12 alone does not activate iNKT cells in the absence of DCs, and the recognition of self-ligands by the invariant NKT cell receptor is required for IL-12-mediated iNKT cell activation. By contrast, α-GalCer recognition induces signals that activate iNKT cells efficiently even in the absence of IL-12. Thus, the molecular mechanism underlying iNKT cell activation under physiological conditions appears to be different from that induced by strong non-self-ligands, such as α-GalCer.
After activation by α-GalCer or pathogens, mouse and human iNKT cells exhibit strong adjuvant effects on protective responses by MHC-dependent and -independent activation of various effector cells in vitro and in vivo[4], [8], [9], [10], [11]. IFN-γ produced by activated iNKT cells in turn activates various other effector cells, including DCs, NK cells, and neutrophils in the innate immune system, and CD4 Th1 and CD8 T cells in the acquired immune system, which is characterized by memory and secondary antigen-specific immune responses [1], [12], [13]. Thus, iNKT cells link the two arms of the immune system, forming a bridge between innate and acquired immunity.
Similar mechanisms may be operative in other protective responses, including rejection of tumor cells and prevention of tumor metastasis. iNKT cells involved in tumor immunity appear to be activated through the recognition of endogenous self-ligands in the presence of IL-12, rather than directly by tumor products. Moreover, in the absence of iNKT cells, unstimulated NK cells and conventional T cells both fail to produce IFN-γ even after direct injection of IL-12 and appear functionally impaired. IFN-γ produced by iNKT cells activates both CD4 T helper cell responses and CD8 T cell-mediated cytotoxic responses against tumor targets and also activates the innate NK cells and neutrophils, thus facilitating inflammatory responses toward tumor targets. Activated NK cells, neutrophils, as well as iNKT cells themselves launch a coordinated cytotoxic attack against the tumor cells.
In general, tumors contain MHC+ and MHC− cells. MHC+ tumor cells are eliminated by CD8 killer T cells, because CD8 T cells can recognize tumor antigen in conjunction with MHC I. On the other hand, MHC negative tumor cells are killed by innate immune cells, such as NK cells, because one type of NK receptors delivers negative signals that inhibit NK cytotoxic activity in the presence of an MHC I molecule, the ligand for this class of NK receptor. Thus, NK cells but not CD8 T cells can kill tumor cells that have lost expression of MHC I on their surface. For optimal therapeutic purposes both MHC+ and MHC− target tumor cells should be eliminated at the same time, thus activation of the iNKT cell-dependent cellular cascade is an important strategy for treatment of cancer (Fig. 1). These are adjuvant effects of iNKT cells, and without the iNKT cell system, the protective immune responses against tumors could be impaired. The critical initial event for iNKT cell activation is mediated by the recognition of self-ligands and IL-12 receptor signaling. Thus, the manipulation of DCs to produce IL-12 is a promising strategy for treatment of cancer patients to selectively trigger protective immune responses through the iNKT cell system.
Section snippets
iNKT cell-mediated adjuvant effects on innate immunity
DCs in the steady state are immature; they are able to capture antigens, but fail to stimulate T cell immunity (Fig. 1). However, iNKT cells can be activated by immature DCs, which is different from conventional T cell activation by peptide/MHC [14], [15]. Thus, in the initial step in the iNKT cell–DC interaction, α-GalCer presented on immature DCs activates iNKT cells to proceed to maturation step of DCs.
Concerning maturation of DCs, both co-stimulatory molecule- and cytokine-mediated signals
iNKT cell-mediated adjuvant effects on acquired immunity
Similar to their effects on the innate immunity, iNKT cell-mediated adjuvant activities also affect T cell immunity (Fig. 1). As mentioned in the previous section, most DCs in vivo are functionally immature in terms of their capacity to elicit acquired immunity in the OVA antigen mouse model [24], [25]. However, OVA-specific T cell responses do develop in mice immunized with OVA when it is administered together with α-GalCer. DC transfer experiments from mice given OVA plus α-GalCer into naïve
iNKT cell-mediated anti-tumor adjuvant activity using α-GalCer-loaded DC
For effective iNKT cell activation, α-GalCer-loaded DCs (α-GalCer-DCs) have distinct advantages over an injection of free α-GalCer [16], [17]. A single injection of α-GalCer-DCs induces a burst of IFN-γ production by iNKT cells that lasts for 2 weeks, whereas the response to soluble α-GalCer is rapid, but disappears by day 2. In keeping with their effective Th1 cytokine production, α-GalCer-DCs induce significant expansion of iNKT cells in the tissues and inhibit in vivo tumor growth in a
Adjuvant iNKT cell therapy by co-administration of antigen with α-GalCer
As discussed above, α-GalCer treatment leads to the full maturation of DCs that efficiently induce OVA-specific T cell immunity when co-administered with OVA. The successful induction of both innate and acquired immunity with co-administration of antigen plus α-GalCer allows extending this approach to several therapeutic models.
For example, when irradiated tumor cells (J558 plasmacytoma cells) are used as cellular vaccine along with α-GalCer, vaccinated mice are protected against tumor cells
iNKT cell-mediated anti-tumor adjuvant therapy using tumor cells loaded with α-GalCer
The iNKT cell-mediated adjuvant effects induced by α-GalCer-DCs have turned out to induce the most powerful stimulation of iNKT and innate NK cell responses in vivo[16]. In order to efficiently augment T cell immunity by the iNKT cell-mediated adjuvant effect, the use of a combination of α-GalCer and peptide-pulsed DCs has long been considered. In fact, Stober et al. have reported that α-GalCer-loaded DCs pulsed with MHC I binding peptides stimulate CD8+ T cells better than DCs pulsed with
Phase I/II clinical trial of adjuvant iNKT cell-targeted therapy for advanced lung cancer
The administration of α-GalCer-pulsed DCs activates iNKT cells in vivo, which mediates strong adjuvant activity and leads to the eradication of established metastatic tumor foci in the liver or lung in a therapeutic experimental animal model [39]. Therefore, similar anti-tumor effects may be expected when α-GalCer-pulsed DCs are administered into humans.
A phase I/II clinical trial of iNKT cell-targeted immunotherapy was performed in patients with lung cancer to evaluate the safety, feasibility,
Future directions: in vitro generation of functional iNKT cells
Although an iNKT cell-targeted adjuvant therapy is a promising approach, it can only be applied in cancer patients with more than 10 iNKT cells per ml of peripheral blood according to the patient eligibility criteria. Thus, only one-third of the patients in the case of our lung cancer clinical trials were eligible for this therapy.
To overcome this problem, it will be necessary to establish in vitro methods for generation of functional iNKT cells, which then can be transferred into the patients.
Summary and discussion
iNKT cells bridge innate and adaptive immunity. Thus, iNKT cells are important potential therapeutic targets. Clinical studies of iNKT cell-targeted adjuvant therapy using α-GalCer-loaded patient DCs have demonstrated clinical safety and efficacy. Lung cancer patients receiving this therapy had prolonged stable disease with increased MST. Although IFN-γ production by NK and iNKT cells may be a good biomarker to judge the efficacy of this treatment and prognosis, we need to establish a
Acknowledgment
The authors are grateful to Dr. Peter Burrows for helpful comments and constructive criticisms in the preparation of the manuscript.
References (49)
- et al.
IFN-γ-mediated inhibition of tumor angiogenesis by natural killer T-cell ligand, α-galactosylceramide
Blood
(2002) Exploiting dendritic cells and natural killer T cells in immunotherapy against malignancies
Trends Immunol
(2008)- et al.
A single intranasal immunization with inactivated influenza virus and α-galactosylceramide induces long-term protective immunity without redirecting antigen to the central nervous system
Vaccine
(2007) - et al.
Mechanism of NKT cell activation by intranasal coadministration of α-galactosylceramide, which can induce cross-protection against influenza viruses
Mucosal Immunol
(2008) - et al.
α-C-galactosylceramide as an adjuvant for a live attenuated influenza virus vaccine
Vaccine
(2009) - et al.
Enhancement of HIV DNA vaccine immunogenicity by the NKT cell ligand, α-galactosylceramide
Vaccine
(2008) - et al.
Sequential production of interferon-γ by NK1.1+ T cells and natural killer cells is essential for the antimetastatic effect of α-galactosylceramide
Blood
(2002) Revisions in the International System for Staging Lung Cancer
Chest
(1997)- et al.
The regulatory role of Vα14 NKT cells in innate and acquired immune response
Annu Rev Immunol
(2003) - et al.
NKT cells: what's in a name?
Nat Rev Immunol
(2004)
The biology of NKT cells
Annu Rev Immunol
CD1d-restricted and TCR-mediated activation of Vα14 NKT cells by glycosylceramides
Science
CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells
J Exp Med
CD1d-mediated recognition of an α-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution
J Exp Med
CD1: antigen presentation and T cell function
Annu Rev Immunol
Cutting edge: cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells
J Immunol
Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells
Eur J Immunol
Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining
J Exp Med
Activation of natural killer T cells by α-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a co-administered protein
J Exp Med
NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells
J Immunol
Tumor cells loaded with α-galactosylceramide induce innate NKT and NK cell-dependent resistance to tumor implantation in mice
J Immunol
Prolonged interferon-g producing NKT response induced with α-galactosylceramide-loaded dendritic cells
Nat Immunol
The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation
J Exp Med
Mouse Vα14i natural killer T cells are resistant to cytokine polarization in vivo
Proc Natl Acad Sci USA
Cited by (31)
Design and evaluation of surface and adjuvant modified PLGA microspheres for uptake by dendritic cells to improve vaccine responses
2015, International Journal of PharmaceuticsCitation Excerpt :Similarly, when MPLA was encapsulated into PLGA nanoparticles it upregulated MHC class II molecules and CD86 markers on DCs (Elamanchili et al., 2004). Finally, α-GalCer interacts and activates iNKT (invariant NKT) cells (Kawano et al., 1997), playing an important role on the control of bacterial, viral, and parasite infections as well as regulating self-tolerance and tumor surveillance (Cerundolo et al., 2010) by the release of IL4, IL12 and IFN-γ (Fujii et al., 2010). One of the main drawbacks of soluble α-GalCer is that it induces anergy of NKT cells.
Ex-vivo α-Galactosylceramide activation of NKT cells in humans and macaques
2012, Journal of Immunological MethodsCitation Excerpt :NKT cells are lymphocytes of the innate immune system that are important in viral and antitumor immunity through their ability to be rapidly activated and express a wide range of effector molecules (Lindqvist et al., 2009; Motohashi et al., 2009; Brigl and Brenner, 2010; Fujii et al., 2010).
Combination of immune stimulating adjuvants with poly(lactide-co-glycolide) microspheres enhances the immune response of vaccines
2012, VaccineCitation Excerpt :By influencing the activation of APCs, iNKT cells play an important role in the control of bacterial, viral, and parasitic infections and take part in the regulation of processes such as self-tolerance, tumor surveillance, and antimicrobial responses [16]. Briefly, α-GalCer presenting DCs activate iNKT cells, which induce maturation of DC, in addition of releasing IL-4, IL-12 and IFN-γ [17]. One of the problems of soluble α-GalCer is its induction of anergy of NKT cells due to the non-selective presentation by B cells.
C-galactosylceramide: Synthesis and immunology
2012, Comptes Rendus ChimieAn α-GalCer analogue with branched acyl chain enhances protective immune responses in a nasal influenza vaccine
2011, VaccineCitation Excerpt :These cells recognize glycolipid antigens presented by APCs in the context of the non-polymorphic MHC class I-like molecule CD1d [8]. Upon activation by their agonists, iNKT cells mediate strong adjuvant activity on protective responses by inducing the production of large amounts of Th1 and Th2 cytokines that, in turn, activate various effector cells in innate and adaptive immune systems [9,10]. The glycolipid α-galactosylceramide (α-GalCer), which was originally isolated from marine sponges for its antitumor activity, was the first discovered and most potent ligand of both mouse and human iNKT cells reported to date [11].
Progress in Natural Killer T Cell–Based Immunotherapy for Cancer: Use of Allogeneic and Gene-Edited Cells
2024, Critical Reviews in Oncogenesis