A brief review of clinical trials involving manipulation of invariant NKT cells as a promising approach in future cancer therapies

Introduction

Among the broad and heterogenic population of lymphocytes, recently the natural killer T-cells (NKT) have generated a lot of interest. These cells are rare and generally represent only from 0.001% up to less than 1% of the human peripheral blood lymphocytes [1, 2], although for some individuals this amount may be higher, reaching as much as 3% of peripheral blood lymphocytes [3, 4]. The basis for such variability in the range of NKT frequencies is not clear, but is unquestionably connected with the influence of genetic factors [2]. Despite their small amount in human blood they significantly affect the immune system [5, 6]. Ongoing studies have revealed their involvement in immune responses, especially during infections, allergies, autoimmune disease, or even cancer. Most of the available information on NKT cells comes from research conducted on mice. However, both phenotypic and functional features of the mouse and human NKT cells appear to be similar [7, 8]. NKT gained their name after a unique cell surface feature: they express receptor characteristic to T lymphocytes (-TCR) and lineage markers of Natural Killer cells (NK) (e.g. human CD161, mouse NK1.1) [7, 5, 9-11]. In light of current data, the above definition seems to be insufficient and incomplete. The expression of NK-markers is not necessarily needed to identify a cell as an NKT cell. Additionally, the level of NK cell markers expressed on NKT cells seems to vary in accordance with their maturation, activation state, and presumably their genetic background [12, 13]. NK1.1 together with other NK-typical molecules may be acquired by CD8+ T lymphocytes upon appropriate stimulation or during a viral infection [14]. NKT cells nowadays are defined by the restriction of their TCR receptors. While the TCR receptors on T cells react with peptide antigens in the context of major histocompatibility complex (MHC) class I or II molecules, the NKT cells, with use of their TCR receptors, recognise the lipid and glycolipid antigens presented by non-polymorphic MHC class I-like glycoprotein, known as CD1d (Fig. 1) [15]. The NKT cells can react with both endogenous and exogenous antigens, regulating a variety of autoimmune diseases, but also providing protection against different pathogens like Sphingomonas spp. or Borrelia burgdorferi [11, 16, 17]. In order to be recognised, and to stimulate the NKT cells, the aforementioned antigens have to be at first internalised, processed, and finally presented through the cells containing CD1d. These molecules are constitutively expressed on antigen-presenting cells (APC): dendritic cells (DCs), B lymphocytes, and macrophages [18].
Some tumour cells also highly express CD1d molecules and are able to present endogenous tumorous glycolipids. By recognising them, the NKT cells can directly conduct lysis on the sensitive CD1d+ tumour cells. This observation indicates the NKT cells as improvers of antitumor response, thus encouraging investigators to further examine ways to strengthen the immune response against cancer in future therapies. The CD1d restriction, relative to the NKT cell reagents, allows the detection, enumeration, and characterisation of functions, as well as examination of the responsiveness of these cells [12, 17, 19]. In this review, an outline of anti-tumour properties of the NKT cells has been presented. It also presents the clinical approaches concerning this unique population of cells as possible enhancers in future anti-cancer strategies.

Subsets of natural killer T-cells

Invariant natural killer T-cells

Depending on the composition of T-cell receptors and the type of molecule presenting the identified antigens to which they respond, the NKT cells are currently classified into three main groups: type I, type II, and NKT-like cells. The TCRs of most NKT cells in human bodies are formed by a canonical  chain, comprised of V24 and J18 gene segments, and a  chain of limited variability – usually V11. Such NKT cells are known as type I or invariant NKT (iNKT). Homologous V14-J18 and V8.2, V7, V2 create TCRs of the iNKT in mice bodies [15, 20-22]. Such TCRs restrict the gamut of recognised antigens and facilitate reactivity to CD1d. Despite iNKT cell populations using conserved TCRs, they differ essentially. The differences may concern the phenotype, localisation, or functions of both murine and human iNKT cells. The V24+ NKT cells can be further classified as: CD4+CD8, CD4CD8+, and double negative (DN) for CD4 and CD8 expression [6, 23]; murine iNKT are either CD4+CD8 or DN [8, 24]. Particular subpopulations differ potentially from each other in terms of cytokine production. The human iNKT cells with CD4 molecule produce a mixture of Th1 and Th2 cytokines, whereas the DN produces only Th1-type cytokines [6]. Such differences are less evident for mice [6, 9]. The discrepancies in cytokine release seem to depend on the tissue localisation, which can at least partially explain why the iNKT cells obtained from different organs present different functions [25]. The iNKT cells occur mainly in the blood and various tissues including the thymus, spleen, liver, and bone marrow. Studies on mice have shown that the liver-resident iNKT cells are the most prevalent [10]. Their protection against tumours is also stronger than in thymus or spleen [25]. Strangely enough, the number of the iNKT cells in most human organs is lower than for mice, and their appearance seems to vary substantially among individuals [26].
Recent studies have revealed the existence of the iNKT subpopulations that differ in terms of production of particular cytokines and expression of specific transcription factors. This connotes with the classification of conventional T lymphocytes. Mature iNKT cells may be divided into three dominant groups: NKT1, NKT2, and NKT17. These cells express transcription factors that typically regulate the cell lines of Th1, Th2, and Th17 (T-bet, GATA3, and RORt, respectively); they also produce similar cytokines to the foregoing subsets of T lymphocytes [27-29]. The T-bet NKT1 release large amounts of IFN- – a cytokine involved in antiviral and antitumor response. The NKT2 seem to take part in the course of diseases with Th2 dominance through the secretion of IL-4, IL-9, IL-10 or IL-13 [30]. At present, a lot of focus has been placed on the NKT17, which release IL-17A upon stimulation and promote the inflammation state [29]. Researchers have also identified a group of iNKT cells with FoxP3+ expression, which act similarly to regulatory T lymphocytes by inhibiting the proliferation of other cells. A novel subpopulation of iNKT cells named NKT10 was discovered in 2014. They were observed to produce IL-10 under steady-state conditions in mice and human bodies, in this way impairing anti-tumour response [31].

Variant natural killer T-cells

The second identified subset of NKT cells is called variant NKT (vNKT), or more commonly type II NKT. They are more heterogeneous than type I in both  and  chain usage [7]. Their TCRs form diverse combinations on the cells’ surface. Table 1 shows their main features in comparison to the iNKT. This subset often plays an opposite or cross-regulating role with the iNKT, especially under conditions of immune dysregulation such as cancer. Like the iNKT, they are present in human and mice organisms [32]. Since their discovery in 1995 [33], only a limited number of studies have reported their physiological functions and our knowledge about them is still quite limited [34].

The natural killer T-like cells

All of the other other NKT cells form the group of the NKT-like cells – the most heterogeneous compartment of NKT cells. They constitute a subset of T lymphocytes that express some natural killer cell receptors. However, they are not restricted by CD1d molecules, so they are also called CD1d independent NKT (CD1dind NKT) [35]. Scientists have confirmed a highly specialised effector-memory phenotype of these lymphocytes, thus their percentage in peripheral blood increases with age. In comparison, the amount of human iNKT in peripheral blood decreases with age [36]. The majority of NKT-like cells are CD16-, and CD8 dominates over the expression of CD4 [37]. The functionally mature CD3+CD56+ NKT-like cells have been observed to show high tumour-killing abilities against many tumour cell targets [38-40]. They hold high levels of granzyme and can produce substantial amounts of proinflammatory cytokines like IFN- and TNF­ [41, 42]. The frequency of CD3+CD56+ NKT-like cells has been reported to decrease significantly among patients with progressive chronic lymphocytic leukaemia [43], which suggests their protective role against cancer. These cells can be generated when cultured in vitro as one of the cytokine-induced killer (CIK) cells [44].
Given that far more is known about the iNKT cells and their antitumor activity, this review will focus predominantly on these cells and recent immunological approaches based on implementing them into cancer treatment.

The means of tumour cell recognition by invariant natural killer T-cells

The progress in the characterisation of iNKT that has occurred in recent years has allowed us to form a belief about how they recognise tumour cells and disallow them to evade an immune response [45]. Research indicates participation of CD1d in this process. These molecules are expressed on cells of the monocytic lineage like monocytes, macrophages, and dendritic cells [46-50], as well as on B lymphocytes. They are also present on malignant human haematopoietic cells, originating from the corresponding tissues, e.g. a few types of leukaemia cells of patients with acute myeloid leukaemia (M4 or M5 AML and juvenile myelomonocytic leukaemia) [51], malignancies originating from Langerhans cells, or interdigitating dendritic cells [46]. Tumour cells of patients with B-cell malignancies are also CD1d-positive, like B-precursor acute lymphoblastic leukaemia with MLL/AF4 gene rearrangement and chronic lymphocytic leukaemia (CLL) [51]. Studies conducted by Metelitsa et al. [46] confirmed that CD1d+ myelomonocytic leukaemia cells trigger a cytotoxic feedback via the NKT cells. The above-mentioned path of activating iNKT cells is called a “direct activation” (Fig. 2A); it constitutes one of the possibilities of recognising and eliminating tumour cells. Similarly, Renukaradhya et al. [52] revealed that the expression of CD1d molecules on B cell lymphoma NSO cells for mice correlates with iNKT cell-mediated lysis. Still, the role played by CD1d and their part in triggering cytotoxic mechanisms has not been fully established yet. Further investigations need to be conducted to determine how the CD1d of malignant cells can be distinguished from the other ones. The fact that tumours try to overcome iNKT-mediated immunity by losing the expression of CD1d on their surface or by repressing a synthesis of ligands for the TCR of iNKTs, should also be remembered [53].
Although direct recognition of CD1d-positive malignant haematopoietic cells is effective, most solid tumours and cell line models do not express CD1d, or its expression is very low [14, 51]. Still, iNKT cells may become activated indirectly (Fig. 2B). This mechanism involves APCs that are able to present CD1d-restricted antigen derived from tumour cells, and in this way activate the iNKT. In turn, the iNKT cells promote activation of NK cells. Thereby, the tumour cell elimination is conducted indirectly by activating the NK cells [54]. Wu et al. [55] conducted the first analysis of a ganglioside precursor disialohematoside (GD3) – a natural compound that is recognised with the above-described mechanism by our immune system. GD3 is a ganglioside highly expressed by many types of tumour, such as melanoma, sarcoma, or neuroblastoma, but very poorly by normal human or mice cells. Using mice immunised with human melanoma SK-MEL-28 cell line, the authors observed a response from the NKT cells. Since this tumour cell line is CD1d, the GD3 must be cross-presented by murine APC to the NKT cells. The cross-presentation of tumour-derived glycolipids seems to play an especially important role in the recognition of CD1d tumours [52, 55].
Another suggested way of reducing the growth of tumours is connected with tumour-associated macrophages (TAMs). Perhaps, the activated iNKT and NK cells could suppress the production of pro-angiogenic factors produced by these macrophages (Fig. 2C). This thesis, however, requires further research for confirmation [54].

Invariant natural killer T-cells-mediated anti-tumour response

Since the discovery of iNKT cells, many studies have tried to describe their activity with the help of potent exogenous stimulators such as IL-12 or -GalCer. Soon, they were followed by reports concerning the role of iNKT in tumour surveillance in physiological conditions (without stimulation) [56]. Smyth et al. [57] performed a study involving the use of knockout mice lacking the J18 gene. Such a change in the DNA resulted in a deficiency of murine iNKT cells, which correlated with an increased tendency for spontaneous development of methylcholanthrene-induced cancers like sarcomas and B16F10 melanoma tumours [39, 40]. They are characterised by early appearance and high frequency [57]. Subsequently, Crowe et al. [58] proved that this effect could be reversed after administrating the liver-derived iNKT cells in the early phase of tumour growth. However, the transfer of thymic or splenic iNKT was not as potent, which suggested functional discrepancies between subsets of iNKT cells [58]. Swan et al. [59] performed a study on mice deficient in p53 – an important suppressor of tumorigenesis in mice and human organisms. The lack of iNKT cells resulted in increased tumour occurrence for these animals. The above-mentioned studies confirmed the undeniable role of classic NKT cells in antitumor immunity and suggested endogenous ligands as effective activators of these cells. The antitumor properties questioned seem to derive from combining a number of different lines of action. After the antigen recognition, the activated iNKT cells rapidly release cytokines and chemokines, through which they affect many immune cells, or may present a direct cytolytic effect.

Invariant natural killer T-cells-cytotoxic activity

Classic NKT cells have their own lytic activity. They produce cytotoxic molecules able to eradicate tumour cells. Up to 85% of these compounds consist of perforin and granzyme B, accompanied by Fas ligand (FASL), tumour necrosis factor  (TNF-), and TNF-related apoptosis-inducing ligand (TRAIL). Metelitsa et al. [46] examined the NKT cells for cytotoxicity against CD1d-positive myelomonocytic leukaemia cells. The cultured NKT cells demonstrated constitutive expression of high levels of mRNA for perforin and granzyme B. The stimulation with use of -GalCer led to a further increase in their level. In turn, the concentration of TNF- ligands was very low in the resting state. The iNKT cells transcribed them later, after -GalCer stimulation. The authors concluded that perforin together with granzyme B are responsible for the early cytolytic activity of iNKT cells, while ligands belonging to the TNF- family provide additional cytotoxicity.
This study also pointed to the presence of CD56 molecules in iNKT cells. The CD56-positive cells were noted to be more cytotoxic, more frequently expressing perforin without stimulation. Such dissimilarity was not observed under the influence of -GalCer [46]. The engagement of the direct-killing mechanism seems to be variable and depends on the employed tumours model [60].

Multimodal activity of released cytokines

Above and beyond the described cell-death-inducing mechanism, the reaction of V24 NKT with antigens (e.g. -GalCer) results also in the release of cytokines [61]. Initially, only secretion of IL-4 and INF- was detected [62], arising within hours of the activation. This observation was later partially explained after the finding of constitutive expression of mRNA for these cytokines in iNKT [13, 63, 64]. Subsequent studies have revealed that the pool of produced cytokines varies considerably as it also includes IL-2, IL-5, IL-6, IL-10, IL-13, IL-17, IL-21, IL-22, TNF-, transforming growth factor  (TGF-), and granulocyte-macrophage colony-stimulating factor (GM-CSF) [29, 62, 65, 66]. Thereby, the iNKT cells can have an impact on several other types of cells of an innate and adaptive immune system [13] including DC, macrophages, neutrophils, NK cells, conventional T and B lymphocytes, lymphocytes T, or type II NKT cells. A secretion of both Th1 and Th2 cytokines makes iNKT cells multimodal in their action, hence it is more difficult to predict the consequences of their actions in vivo [57]. Referring to cancer, the iNKT cells can launch an antitumor response through proinflammatory Th1 cytokine cascade, triggering “adjuvant effects” (activation of other antitumor cytolytic cells), and through revealing direct cytotoxicity. However, the role played by the NKT is far more complex because they may act on the contrary through IL-13 or the mentioned type II NKT [52]. This functional heterogeneity should be further explored in order to create future strategies that promote anti-tumour effects.
Taking a closer look into the process leading to the cytokine release, it all starts from the recognition of an appropriate antigen (e.g. -GalCer). Activated iNKT cells up-regulate CD40L molecules on their surface, to which DCs respond by remodelling their markers (enhancement of costimulatory molecules: CD40, CD80, and CD86). The described interaction between iNKT and DCs induces the maturation of the latter. DCs activated in this way start to secrete IL-12 [67], while production of IL-23 is inhibited [68]. The IL-12 acts on cells that possess corresponding receptors on their surface. The iNKT have substantial amounts of the mature form of these receptors (IL-12R), becoming the main recipient of a released cytokine. By binding it, it activates the iNKT. The activation signal can also be transmitted by the reaction between CXCR6 receptor on the iNKT and CXCL16 ligand on APCs [69]. Fully activated iNKT cells secrete large amounts of IFN- and IL-2, through which they influence e.g. NK and CD8+ T cells to express cytotoxic functions [5, 70, 71]. Activated NK cells start to secrete their own IFN-. Thus, the IFN- is first produced by the NKT cells, and later by the NK cells. The adjuvant effect of iNKT cells is emerging this way [72]. Furthermore, the DCs matured in a response to iNKT cells cross-present the tumour-derived antigens to CD4+ and CD8+ T lymphocytes and exert a significant influence on the efficacy of immune responses [73, 74]. A study by Hermans et al. [73] implies that the enhanced T-cell responses that they observed were caused by conditioning of DC by the iNKT cells. This observation additionally developed the ‘adjuvant effect’ of the iNKT cells. The described interactions are illustrated in Figure 3. IFN- seems to play a critical role in the tumour rejection. It mediates the emergence of the ‘adjuvant effect’. The loss of this activity has been observed in a study ran by Gonzales et al. [75], who used mice lacking the receptor of IFN-. Other studies conducted on patients with different cancer types (colon cancer, head and neck cancer, breast cancer, renal cancer, and melanoma) have shown, besides a decrease in the iNKT, a maintained ability to produce the IFN-. However, in patients with lung cancer, advanced prostate cancer, or other undefined advanced cancers except from glioma, the iNKT responded poorly, even under stimulation with -GalCer [76].
The influence of iNKT involves also B lymphocytes, which respond by an increase in secreted levels of IgG, thereby participating in the fight against tumour cells by antibody-dependent cellular cytotoxicity (ADCC) mechanism [76].

Invariant natural killer T-cells in relation to TAMs

As mentioned earlier, iNKT cells cannot kill CD1d-negative tumour cells directly [77]. Although the iNKT activate NK cells and cause augmentation of their cytotoxicity [78], the NK cell migration to the established tumours in human bodies is inferior [79]. For example, their level in neuroblastoma (tumour without CD1d expression) is significantly low [80]. This finding suggests that the iNKT may act by another mechanism, much more sophisticated than NK mediation, involving the control of tumour initiation and metastatic process. The iNKT cells infiltrate primary and metastatic human tumours, where they interact with neoplastic cells or other immune cells present in the tumour microenvironment. According to Song et al. [53], who conducted research on neuroblastoma, the accumulation of the iNKT cells occurs due to the expression of high levels of CCL2 by the tumour cells. The same cytokine also attracts monocytes and other immature myelomonocytic precursors of TAMs. TAMs are precisely the crucial attachment point of a proposed novel antitumor mechanism. They develop under the influence of IL-6 – a tumour growth-promoting cytokine secreted by neuroblastoma cells [53]. TAM cells promote angiogenesis [81], protect tumour cells from chemotherapy-induced apoptosis [82], facilitate the metastasis of tumour [83], and cause T-cell death through the expression of programmed-death ligand-1 [53]. As TAMs are the dominant CD1d-positive cells in the examined tumour microenvironment, by cross-presenting neuroblastoma-derived antigens, they enable the iNKT cells to destroy the tumour in a CD1d-dependent way. Killing the TAMs would terminate their growth support for tumour cells [51, 53, 84]. The presented mechanism may partially explain the affiliation of the iNKT on CD1d-negative tumours with better disease outcome in neuroblastoma and other types of cancer [53].

Invariant natural killer T-cells in relation to myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) incorporate immature macrophages, granulocytes, DCs, monocytes, and other myeloid cells at early stages of differentiation [85]. A low incidence of these occur for healthy individuals [86]. Growing tumours release factors (e.g. IL-6, IL-10) that disrupt the normal differentiation of myeloid-line cells and lead to their accumulation in blood, spleen, or undifferentiated bone marrow cells [87]. MDSCs enhance the tumour progression in those tissues. Their main functions are immunosuppression and constitution of a tumour suppressive environment. They have been observed to inhibit CD4+ and CD8+ T lymphocytes, B lymphocytes, and NK cells and induce CD4+CD25+FoxP3+ regulatory T lymphocytes [86]. There have been reports suggesting iNKT as key cells in overcoming immune suppression mediated by the MDSC [88, 89]. Studies performed on mice prove that iNKT cells convert CD11b+ Gr1+ MDSC into stimulatory APCs or suppress them in association with a CD1d molecule [88]. The NKT cells resisted the MDSCs effects and were observed to reverse their effect on T-cell proliferation [90]. The interaction of iNKT cells with MDSCs in human organisms has been incompletely understood. In one model, the iNKT cells interact with CD1d on the surface of MDSCs, which results in an increase in CD80/86, CD70 and ICAM-1 expression, and transformation of the MDSCs into the DC phenotype. The MDSCs converted through CD80 and CD70 molecules react with those on the T cells (CD28 and CD27), supporting their response against cancer [91]. Cancer immunotherapy designed to strengthen the anti-tumour activity of iNKT cells may not only remain effective in the presence of MDSCs, but also erase the suppression of MDSCs on immune responses.

Application in clinical trials

Strategies to combat tumours are among the most rapidly evolving areas of contemporary medicine. Even the development of chemotherapy, radiotherapy, and drug design have not sufficiently reduced the human morbidity and mortality caused by cancer. Tumours learn to develop different mechanisms that allow them to avoid the immune system. For this reason, over the last years, researchers have made efforts to strengthen human immune response and help the healthy cells to control cancer. Probably, manipulating the iNKT cells can act as such an immunotherapeutic tool because they recognise and destroy the tumour cells.
Researchers have observed that a decline in the number of iNKTs is often accompanied by their functional alternation in patients with many types of tumours, like prostate cancer or head and neck cancer [18, 53, 60]. A low level of iNKT cells in blood circulation has been found to correlate with poor prognosis in patients with acute myeloid leukaemia and head and neck cancer [18, 92, 93]. The impairment of the iNKT cell functions correlated with the clinical stage of tumours. iNKT cells have been observed to accumulate in the tumours (e.g. colorectal cancer), in the mechanism of a tumour tissue infiltration, which related to their improved survival [94, 95]. Therefore, actual therapeutic strategies focus on restoring the proper amount and functional condition of the iNKT cells, aiming especially at potentiating a Th1-type response.

Administration of soluble α-linked galactosylceramide

The discovery of -linked galactosylceramide (-GalCer) has been extremely helpful in extending our knowledge on iNKT biology. -GalCer had been obtained from marine sponges as one of the compounds having anti-tumour properties. Researchers have received encouraging data in terms of effectiveness and safety from in vitro studies and murine models [57, 96-98]. To verify if the activity of -GalCer for humans is relevant to the preclinical outcomes, the first in-man administration was performed. It was an open-label, non-randomised phase I study that included 24 patients with refractory solid tumours [99]. They were intravenously injected with a soluble -GalCer at a varying range of doses in four weekly cycles. Giaccone et al. [99] provided basic information about the pharmacokinetics of -GalCer in the human organism, indicating that it does not accumulate in the body and is not detectable in urine. It is safe when applied at a wide range of doses, and generally has been well tolerated. Its toxicity, assessed using the National Cancer Institute Common Toxicity Criteria, was minimal. Only one of the patients developed grade 3 symptoms: fever with vomiting and shivering. No serious adverse events were observed. Although previous studies conducted on mice have reported that -GalCer induced liver damage, such toxicity was not observed for humans [99]. Concerning the number of iNKT cells, their rapid (within 24 hours) decrease after -GalCer administration was noted, just like in the murine models [100]. Only later it occurred that their disappearance was connected with a TCR down-regulation at that time. Within ten days, most of the iNKT cells died by apoptosis, while the remaining part went into an anergy state. In this condition, the iNKT cells had become unresponsive to another administration of ligand for at least thirty days. It occurred that this mechanism depends on the interaction between programmed cell death-1 and its ligand [101]. The conducted study met with little success and did not show any clinical improvement, but the malignant disease of seven patients maintained at a stable level. Thus, other schemes should be taken into consideration in order to effectively increase the amount of iNKT cells for cancer patients [99].

Administration of α-linked galactosylceramide-pulsed immature dendritic cell

Murine models were also tested in terms of the iNKT reaction on -GalCer-pulsed dendritic cells instead of the -GalCer alone [14, 97, 102]. Based on the favourable results of these studies, many clinical trials have been undertaken using -GalCer combined with the variations in APC platform [103-106] (Table 2).
Nieda et al. [103] used autologous, immature, monocyte-derived DC pulsed with -GalCer. The study included a group of 12 patients with metastatic tumours. Injections were well tolerated, without any serious side effects. Most of the patients experienced only minor adverse events. Soon after the injection, a transient decrease in peripheral blood of the iNKT, but also NK, T, and B cells was observed, followed by a small increase of the iNKT cells within a few days. Consequently, a larger number of NK and T cells were activated, leading to an increase in the IFN- level. Just like in the previously described trial on mice model, Nieda et al. did not detect any reductions of tumour. However, in two of the patients tumour markers decreased, which indicated an antitumor effect of the -GalCer-pulsed, immature, monocyte-derived DCs [103].
Nicol et al. [107] conducted a phase I clinical trial using the same APCs as in Nieda’s study. In order to estimate the safety and tolerability, they used dose-escalation and different routes of administration: intravenous and intradermal. Twelve patients with different metastatic diseases, who could not be treated with standard therapy, took part in the study. Immature DCs (obtained from monocytes cultured with GM-CSF and IL-4) were pulsed with -GalCer one day before the administration. The surface of immature APCs proved to be richer in CD1d molecules than for the mature forms. Subsequently, contact with iNKT would lead to their maturation. The iNKT cells stimulated in the above-described way showed an adjuvant effect by activating other peripheral blood lymphoid cells, e.g. NK cells and T lymphocytes, and by causing an increase of the IFN- detected in the serum. Overall, the therapy was well tolerated. Most of the patients experienced temporary intensification of inflammation symptoms in the place where metastasis had been diagnosed (in connection with iNKT activation). These changes were more common after intravenous, rather than intradermal injections. The cause lies in the accuracy with which most of the -GalCer-pulsed, immature DCs administered intradermally stay in the skin, with only a small part moving into the lungs, liver, or spleen. The same cells injected intravenously encounter the iNKT cells without obstacles. The progression of the disease, as observed in six out of 12 cases, evolved into a stabilisation or minor objective improvement, and in three cases lasted up to one year [107].

Administration of α-linked galactosylceramide-pulsed mature dendritic cell

Another phase I clinical study, run by Chang et al. [105], evaluated the efficacy of -GalCer-loaded mature DCs administered to patients with advanced cancer. Some of the earlier findings indicated that DCs were preferable to monocytes in presenting the -GalCer. The maturation process of DCs correlated with an increase in their ability to activate iNKT cells [108], mainly because of more effective co-stimulation and cytokine production (IL-2, IL-7, IL-12, IL-15) [110]. Chang et al. [105] obtained a reliable intensification of the iNKT cells’ in vivo expansion for humans. After injections with -GalCer-loaded mature DCs, all patients experienced a substantial growth in the number of iNKT cells (more than 100-fold). In some cases, this state persisted for more than six months after injection. In comparison, after using -GalCer-loaded immature DCs or -GalCer alone, the increase was barely transient. The authors also detected higher amounts of cytokines associated with maturation of the DCs. Three patients experienced clinically relevant decreases in urine and serum M protein, and one patient showed disease stabilisation [105].
The -GalCer-loaded, monocyte-derived mature DCs (obtained in a similar way as in the previous study) have also been used in a trial conducted by Richter et al. [110]. However, in this case the investigators decided to apply those cells accompanied with an immunomodulatory drug, lenalidomide. They assumed that such a means of treatment might enhance the anti-tumour response, as lenalidomide co-stimulates human T lymphocytes, including the iNKT cells [111]. An important factor in choosing the patients for the trial was a CD1d+ character of myeloma cells, thus sensitive to the iNKT cell lysis. The effects of the administered therapy differed from those observed earlier with -GalCer monocyte-derived DCs alone. A short-term exposure of six patients with asymptomatic myeloma provided clear data on a decline in the amount of iNKT cells, and a broad immune activation involving iNKT cells, NK cells, monocytes, and eosinophils. The NK cells up-regulated CD56 and NKG2D and increased their number. No toxicity was observed, nor intolerance. The study pointed out promising possibilities of using medicines in future therapies to help with harnessing the immunological response against tumours [110].

Administration of α-linked galactosylceramide-pulsed antigen-presenting cells

Other researches have modified the administration approach by choosing APC cells, obtained from peripheral blood mononuclear cells in the presence of GM-CSF and IL-2, and pulsing them with the -GalCer. Ishikawa et al. [104] applied this strategy for 11 patients with advanced and recurrent non-small-cell lung cancer. The intravenously injected -GalCer APCs seemed to migrate to the lung and activate the iNKT cells in situ [112]. These results are very promising because the above approach could be implicated especially for non-small-cell lung cancer patients after radical surgery. More than half of such patients experience local or distant micro-metastases that are impossible to remove during surgery. Applying such postsurgical immunotherapy could suppress the growth of the micro-metastases. Authors of the study have not observed any major toxicity or severe side effects. In five cases the disease has remained stable [104].
In 2009, Motohashi et al. [113] published the results of a phase I-II study with administration of similar APCs. The study included 23 patients with advanced non-small cell lung cancer, but only 17 of them completed the treatment. For most patients (in 10 out of 17 cases), the injection induced a noticeable increase of the amounts of NK and NKT cells in peripheral blood, as well as the produced IFN- level. Those for whom the IFN- growth was more than two-fold compared to the baseline had an estimated median survival time of almost 32 months. The median survival time of seven patients who were non- or poor responders was only 10 months. The authors concluded that iNKT secretion elevated the IFN- level, which may be associated with enhancement of the bearing patients’ cancer survival [113, 114].
Uchida et al. [106] also picked the -GalCer-loaded APCs, but injected them into the nasal submucosa of nine patients with HNSCC. The use of this novel route of administration resulted in rapid migration of the APCs into ipsilateral regional lymph nodes and started a regional anti-tumour response. The number of circulating iNKT cells increased in four patients and slightly decreased among others. The quantity of NK cells also increased in most cases. For the first time, an obvious regression of tumour could be observed. Its diameter decreased for one patient from 22 to 7 mm. Another positive aspect is that in five patients the disease has remained stable [60, 106].
In continuation of this study [115], the peripheral blood mononuclear cells collected from patients with head and neck squamous cell carcinoma (here 17) were cultivated with GM-CSF and IL-2, followed by the load of -GalCer on the day before administration. The injection, however, was applied into the nasal and oral submucosa. Depending on the route of administration, the difference in migration sites could had been observed. The APCs from the nasal route migrated to the lateral neck lymph nodes, whereas those from oral route migrated to the submandibular lymph nodes. A greater increase of the amount of iNKT cells and IFN- was detected in patients after the nasal administration. The unique microenvironment of submandibular lymph nodes is rich in Langerhans-like DCs, which tend to induce suppressive cells such as regulatory T cells. This is the probable background of the observed immunological suppression in patients after oral injection [114, 115].
In a subsequent study conducted by Nagato et al. [116], the researchers focused on closer examination of tumour infiltration by the iNKT under the influence of -GalCer-loaded APCs. Four patients with operable advanced lung cancer formed a treatment group, whose outcomes were compared with those from a control group of six patients.-GalCer-loaded GM-CSF and IL-2-cultured APCs were administered intravenously a week before the surgery, after which tumour infiltrating cells (TILs) were collected from the resected lung tissue. Further analysis revealed a considerable increase of the iNKT cells, especially among TILs, as well as the IFN- production in comparison to the control group. The induced iNKT cells’ activation and infiltration to the tumour microenvironment contributed to a higher anti-tumour response. Proposed administered post-operative therapy could help to decrease severe toxic effects of chemotherapeutical agents, normally applied after a non-small cell cancer surgery [116].

Administration of activated invariant natural killer T-cells

Patients suffering from cancer have been reported to have not only low levels of iNKT cells, but also a reduced ability of those cells to develop properly. Since iNKT cells can expand after proper in vivo stimulation, an idea of creating a clinical trial based on these reports emerged. Motohashi et al. [117] published the results of such a study in 2006. In order to obtain the activated iNKT cells, peripheral blood mononuclear cells were stimulated with -GalCer and IL-2 for up to three weeks. In vitro expanded cell fractions enriched with the activated iNKT cells were injected intravenously in two doses. The number of iNKT cells in peripheral blood increased in two out of three cases after the second dose, such as the IFN- production. The safety of this therapy was estimated as high; no severe adverse events developed. All six patients completed the study; however, none of them met the criteria of either complete or partial response. After applying this therapy, four of the patients were classified with stable, and two of them with progressive state of a disease. The authors have been following these two patients for nine and 12 months beginning from the end of the trial and confirm that their stable state has been maintained. It is possible that studies performed on a larger scale or for a longer period of time could have provided more precise and conclusive information. As iNKT cells have a potent antitumor activity both in vivo and in vitro, the application of the above method of treatment in cancer patients seems to be promising [60, 117, 118].

Combination strategy

On the basis of encouraging results of previous clinical trials, the idea of combination immunotherapy appeared. Such phase I study was performed on eight participants who suffered from HNSCC. The protocol assumed an infusion of -GalCer-pulsed APCs into the nasal submucosa (twice), followed by the administration of in vitro expanded iNKT cells into the tumour-feeding artery. In seven out of eight cases the results showed a significant increase in the number of iNKT cells and IFN--producing cells [119]. Similar positive changes in the level of iNKTs have previously been reported after -GalCer-loaded APC monotherapy [106]. The benefits of extension by adding the activated iNKT cells included a higher level of IFN- producing cells, and hence significant antitumor activity. This approach was well tolerated by all the participants, three of whom revealed strong partial response, and four were classified with stable disease [114, 119].
The combination therapy was also applied in a designed phase II study with participation of 10 patients suffering from HNSCC. The observed changes in the amount of the iNKT cells, especially within the tumour-infiltrating lymphocytes, led to measurable clinical effects: five out of 10 patients demonstrated tumour regression. Such good results encourage further research in this direction [120].

Conclusions

Studies conducted in recent years have revealed the existence of a certain regularity among many patients suffering from different cancer types: a low amount of iNKT cells. Moreover, surgical tumour removal or radiotherapeutic approach did not restore the iNKT cell levels [121]. Research has often linked this observation with poor clinical outcomes in patients with malignancies [18], noted for instance in cases of HNSCC [92]. A low level of iNKT cells infiltrating the tumour tissue of colorectal cancer or neuroblastoma has also been associated with worse survival capabilities [94]. Therefore, the current approaches focus much attention on deepening our knowledge of the iNKT biology and methods of rejuvenating the proper amount and functional condition of the iNKT cells in tumour-bearing patients. Identification of the best possible agonists that could successfully activate iNKT cells without inducing anergy (a state when cells do not respond on activation) has also become a very important issue [54]. For now, only -GalCer is used in clinical trials. Under its influence, the activated iNKT cells mediate in the anti-tumour response with other elements of the immune system, e.g. NK cells. In a paper published in Clinical Cancer Research, Giaccone et al. [99], for the first time in history, have applied -GalCer in humans. The intravenous injection temporarily reduced the number of circulating iNKT cells for at least one week and increased the GM-CSF and TNF-levels, but only in patients with a relatively high pre-treatment iNKT level [99]. Although this method did not result in any significant clinical effect, it has laid the foundation for others to emerge and follow in this direction. Researchers have cultured different types of APCs, loaded them with -GalCer, administered, and examined during clinical trials. DCs transpired to have a superior ability to present antigens and express costimulatory molecules on their surface, which enables antitumor properties of the used ligand [54]. The matured forms of DCs provided higher expansion of IL-12 and IFN- levels in the serum [105]. Other undertaken courses of action have been focused on combination therapy or different routes of administration: intra-arterial, submucosal, and sub-nasal [106, 115, 117, 119, 120]. The outcomes indicate an increased level of the iNKT, as well as IFN--producing cells in blood, compared to the -GalCer APCs level in monotherapy [106]. However, there remains much to be determined: the optimal internal route of administration and the interval between the doses or the appropriate agonists, which would induce iNKT activation without driving them into an anergy state. Efforts should also be made to approach the iNKTs with type II NKTs and learn more about their possible interactions during immunosurveillance.
So far, all immunotherapeutic approaches based on -GalCer usage in patients suffering from different tumours have appeared to be safe and well tolerated by the patients. No severe adverse events have been reported. It is now clear that iNKT cells possess valuable anti-tumour activity against many types of cancer and are able to induce a total or at least partial clinical response, even for aggressive tumour types. This is a substantial promise of changes in tumour-fighting therapies that might be implicated in the near future. Immunotherapy affecting the iNKT cells could have been used after radical surgery in non-small cell lung cancer patients in order to suppress growth of irremovable micro-metastases [104]. Unfortunately, the clinical responses observed at present in many trials have not been consistent and predictable. Further studies are required for the realisation of a widespread clinical administration dream.

This work was supported by research grant of the Medical University of Lublin DS 458.
The authors declare that there are no conflict of interests regarding the publication of this paper.

References

1. Berzins SP, Cochrane AD, Pelliccic DG, et al. (2005): Limited correlation between human thymus and blood NKT cell content revealed by an ontogeny study of paired tissue samples. Eur J Immunol 35: 1399-1407.
2. Lee PT, Putnam A, Benlagha K, et al. (2002): Testing the NKT cell hypothesis of human IDDM pathogenesis. J Clin Invest 110: 793-800.
3. Berzins SP, Smyth MJ, Baxter AG (2011): Presumed guilty: natural killer T cell defects and human disease. Nat Rev Immunol 11: 131-142.
4. Slauenwhite D, Johnston B (2015): Regulation of NKT Cell Localization in Homeostasis and Infection. Front Immunol 6: 255.
5. Ito H, Seishima M (2010): Regulation of the induction and function of cytotoxic T lymphocytes by natural killer T cell. J Biomed Biotechnol 2010: 1-8.
6. Lee PT, Benlagha K, Teyton L, Bendelac A (2002): Distinct functional lineages of human V24 natural killer T cells. J Exp Med 95: 637-641.
7. Godfrey DI, Stankovic S, Baxter AG (2010): Raising the NKT cell family. Nat Immunol 11: 197-206.
8. Terabe M, Berzofsky JA (2008): The role of NKT cells in tumor immunity. Adv Cancer Res 101: 277-348.
9. Bendelac A, Rivera MN, Park S, Roark JH (1997): Mouse CD1- specific NK1 T cells: development, specificity, and function. Annu Rev Immunol 15: 535-562.
10. Bendelac A, Savage PB, Teyton L (2007): The biology of NKT cells. Annu Rev Immunol 25: 297-336.
11. Van Kaer L (2007): NKT cells: T lymphocytes with innate effector functions. Curr Opin Immunol 19: 354-364.
12. Van den Heuvel MJ, Garg N, Van Kaer L, Haeryfar SM (2011): NKT cell costimulation: experimental progress and therapeutic promise. Trends Mol Med 17: 65-77.
13. McNab FW, Pellicci DG, Field K, et al. (2007): Peripheral NK1.1 NKT cells are mature and functionally distinct from their thymic counterparts. J Immunol 179: 6630-6637.
14. Assarsson E, Kambayashi T, Sandberg JK, et al. (2000): CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo. J Immunol 165: 3673-3679.
15. Exley M, Garcia J, Balk SP, Porcelli S (1997): Requirements for CD1d recognition by human invariant V24+ CD4CD8 T cells. J Exp Med 186: 109-120.
16. Kinjo Y, Tupin E, Wu D, et al. (2006) Natural killer T cells recognize diacylglycerol antigens from pathogenic bacteria. Nat Immunol 7: 978-986.
17. Neparidze N, Dhodapkar MV (2009): Harnessing CD1d- restricted T cells towards anti-tumor immunity in humans. Ann N Y Acad Sci 1174: 61-67.
18. Roark JH, Park SH, Jayawardena J, et al. (1998): CD1.1 expression by mouse antigen-presenting cells and marginal zone B cells. J Immunol 160: 3121-3127.
19. Matsuda JL, Naidenko OV, Laurent G, et al. (2000): Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J Exp Med 192: 741-754.
20. Porcelli S, Yockey CE, Brenner MB, Balk SP (1993): Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD48 alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain. J Exp Med 178: 1-16.
21. Lantz O, Bendelac A (1994): An invariant T cell receptor alpha chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD48 T cells in mice and humans. J Exp Med 180: 1097-1006.
22. Fujii S (2008): Exploiting dendritic cells and natural killer T cells in immunotherapy against malignancies. Trends Immunol 29: 242-249.
23. Gumperz JE, Miyake S, Yamamura T, Brenner MB (2002): Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J Exp Med 195: 625-636.
24. MacDonald HR (1995): NK1.1+ T cell receptor-alpha/beta+ cells: new clues to their origin, specificity, and function. J Exp Med 182: 633-638.
25. Crowe NY, Coquet JM, Berzins SP, et al. (2005): Differential antitumor immunity mediated by NKT cell subsets in vivo. J Exp Med 202: 1279-1288.
26. Kronenberg M (2005): Toward an understanding of NKT cell biology: progress and paradoxes. Annu Rev Immunol 23: 877-900.
27. Kim PJ, Pai SY, Brigl M, et al. (2006): GATA-3 regulates the development and function of invariant NKT cells. J Immunol 177: 6650-6659.
28. Lee YJ, Holzapfel KL, Zhu J, et al. (2013): Steady state production of IL-4 modulates immunity in different strains and is determined by lineage diversity of iNKT cells. Nat Immunol 14: 10.1038/ni.2731.
29. Michel ML, Keller AC, Paget C. et al. (2007): Identifcation of an IL-17-producing NK1.1- iNKT cell population involved in airway neutrophilia. J Exp Med 204: 995-1001.
30. Watarai H, Sekine-Kondo E, Shigeura T et al (2012): Development and function of invariant natural killer T cells producing T(h)2- and T(h)17-cytokines. PLoS Biology 10: e1001255.
31. Sag D, Krause P, Hedrick CC et al (2014): IL-10-producing NKT10 cells are a distinct regulatory invariant NKT cell subset. J Clin Invest 124: 3725-3740.
32. Terabe M, Berzofsky JA (2007): NKT cells in immunoregulation of tumor immunity: a new immunoregulatory axis. Trends Immunol 28: 491-496.
33. Cardell S, Tangri S, Chan S, et al. (1995): CD1-restricted CD4+ T cells in major histocompatibility complex class II-deficient mice. J Exp Med 182: 993-1004.
34. Macho-Fernandez E, Brigl M (2015): The extended Family of CD1d-restricted NKT cells: sifting through a mixed bag of TCRs, antigens, and functions. Front Immunol 6: 362.
35. Farra AR, Wub W, Choia B, et al. (2014): CD1d-unrestricted NKT cells are endowed with a hybrid function far superior than that of iNKT cells. PNAS 111: 12841-12846.
36. Peralbo E, Alonso C, Solana R (2007): Invariant NKT and NKT-like lymphocytes: two different T cell subsets that are differentially affected by ageing. Exp Gerontol 42: 703-708.
37. Zdrazilova­Dubska L, Valik D, Budinska E, et al. (2012): NKT-like Cells are Expanded in Solid Tumour Patients. Klin Onkol 25 Suppl 2: 2s21-2s25.
38. Ortaldo JR, Winkler-Pickett RT, Yagita H, et al. (1991): Comparative studies of CD3- and CD3+ CD56+ cells: examination of morphology, functions, T cell receptor rearrangement, and pore-forming protein expression. Cell Immunol 136: 486-495.
39. Hoyle C, Bangs CD, Chang P, et al. (1998): Expansion of Philadelphia chromosome-negative CD3(+)CD56(+) cytotoxic cells from chronic myeloid leukemia patients: in vitro and in vivo efficacy in severe combined immunodeficiency disease mice. Blood 92: 3318-3327.
40. Pievani A, Borleri G, Pende D, et al. (2011): Dual-functional capability of CD3+CD56+ CIK cells, a T-cell subset that acquires NK function and retains TCR-mediated specific cytotoxicity. Blood 118: 3301-3310.
41. Guilmot A, Carlier Y, Truyens C (2014): Differential IFN- production by adult and neonatal blood CD56+ natural killer (NK) and NK-like-T cells in response to Trypanosoma cruzi and IL-15. Parasite Immunology 36: 43-52.
42. Linn YC, Lau SK, Liu BH, et al. (2009): Characterization of the recognition and functional heterogeneity exhibited by cytokine-induced killer cell subsets against acute myeloid leukaemia target cell. Immunology 126: 423-435.
43. Jadidi-Niaragh F, Jeddi-Tehrani M, Ansaripour B, et al. (2012): Reduced frequency of NKT-like cells in patients with progressive chronic lymphocytic leukemia. Med Oncol 29: 3561-3569.
44. Linn YC, Hui KM (2010): Cytokine-Induced NK-Like T Cells: From Bench to Bedside. J Biomed Biotechnol 2010: 435745.
45. Renukaradhya GI, Sriram V, Du W, et al. (2006): Inhibition of antitumor immunity by invariant natural killer T cells in a T-cell lymphoma model in vivo. Int J Cancer 118: 3045-3053.
46. Metelitsa LS, Weinberg KI, Emanuel PD, Seeger RC (2003): Expression of CD1d by myelomonocytic leukemias provides a target for cytotoxic NKT cells. Leukemia 17: 1068-1077.
47. Spada FM, Borriello F, Sugita M, et al. (2000): Low expression level but potent antigen presenting function of CD1d on monocyte lineage cells. Eur J Immunol 30: 3468-3477.
48. Canchis PW, Bhan AK, Landau SB, et al. (1993): Tissue distribution of the non-polymorphic major histocompatibility complex class I-molecule CD1d. Immunology 80: 561-565.
49. Exley M, Garcia J, Wilson SB, et al. (2000): CD1d structure and regulation on human thymocytes peripheral blood T cells, B cells and monocytes. Immunology 100: 37-47.
50. Racke FK, Clare-Salzer M, Wilson SB (2002): Control of myeloid dendritic cell differentiation and function by CD1d-restricted (NK) T cells. Front Biosci 7: d978-d985.
51. Metelitsa LS (2011): Anti-tumor potential of type-I NKT cells against CD1d-positive and CD1d-negative tumors in humans. Clin Immunol 140: 119-129.
52. Renukaradhya GJ, Khan MA, Vieira M, et al. (2008): Type I NKT cells protect (and type II NKT cells suppress) the host’s innate antitumor immune response to a B-cell lymphoma. Blood 111: 5637-5645.
53. Song L, Asgharzadeh S, Salo J, et al. (2009): V24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages. J Clin Invest 119: 1524-1536.
54. Vivier E, Ugolini S, Blaise D, et al. (2012): Targeting natural killer cells and natural killer T cells in cancer. Nat Rev Immunol 12: 239-252.
55. Wu DY, Segal NH, Sidobre S, et al. (2003): Cross-presentation of Disialoganglioside GD3 to Natural Killer T cells. J Exp Med 198: 173-181.
56. Smyth MJ, Godfrey DI (2000): NKT cells and tumor immunity – a double-edged sword. Nat Immunol 1: 459-460.
57. Smyth MJ, Thia KY, Street SE, et al. (2000): Differential tumor surveillance by natural killer (NK) and NKT cells. J Exp Med 2000: 661-668.
58. Crowe NY, Smyth MJ, Godfrey DI (2002): A Critical Role for Natural Killer T Cells in Immunosurveillance of Methylcholanthrene-induced Sarcomas. J Exp Med 196: 119-127.
59. Swann JB, Uldrich AP, van Dommelen S, et al. (2009): Type I natural killer T cells suppress tumors caused by p53 loss in mice. Blood 113: 6382-6385.
60. Motohashi S, Nakayama T (2008): Clinical applications of natural killer T cell-based immunotherapy for cancer. Cancer Sci 99: 638-645.
61. Weinkove R, Brooks CR, Carter JM, et al. (2013): Functional invariant natural killer T-cell and CD1d axis in chronic lymphocytic leukemia: implications for immunotherapy. Haematologica 98: 376-384.
62. Matsuda JL, Mallevaey T, Scott-Browne J, Gapin L (2008): CD1d-restricted iNKT cells, the ‘Swiss-Army knife’ of the immune system. Curr Opin Immunol 20: 358-368.
63. Matsuda JL, Gapin L, Baron JL, et al. (2003): Mouse V alpha 14i natural killer T cells are resistant to cytokine polarization in vivo. Proc Natl Acad Sci U S A 100: 8395-8400.
64. Stetson DB, Mohrs M, Reinhardt RL, et al. (2003): Constitutive cytokine mRNAs mark natural killer (NK) and NK T cells poised for rapid effector function. J Exp Med 198: 1069-1076.
65. Coquet JM, Kyparissoudis K, Pellicci DG, et al. (2007): IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production. J Immunol 178: 2827-2834.
66. Paget C, Ivanov S, Fontaine J, et al. (2012): Interleukin-22 is produced by invariant natural killer T lymphocytes during influenza A virus infection: potential role in protection against lung epithelial damages. J Biol Chem 287: 8816-8829.
67. Kitamura H, Iwakabe K, Yahata T, et al. (1999): The natural killer T (NKT) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells. J Exp Med 189: 1121-1128.
68. Uemura Y, Liu TY, Narita Y, et al. (2009): Cytokine-dependent modification of IL-12p70 and IL-23 balance in dendritic cells by ligand activation of Valpha24 invariant NKT cells. J Immunol 183: 201-208.
69. Germanov E, Veinotte L, Cullen R, et al. (2008): Critical role for the chemokine receptor CXCR6 in homeostasis and activation of CD1d-restricted NKT cells. J Immunol 181: 81-91.
70. Caux C, Massacrier C, Vanbervliet B, et al. (1994): Activation of human dendritic cells through CD40 cross-linking. J Exp Med 1994; 180: 1263-1272.
71. Fujii S, Liu K, Smith C, Bonito AJ, et al. (2004): 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 199: 1607-1618.
72. Smyth MJ, Crowe NY, Pellicci DG, et al. (2002): Sequential production of interferon-gamma by NK1.1(+) T cells and natural killer cells is essential for the antimetastatic effect of alpha-galactosylceramide. Blood 99: 1259-1266.
73. Hermans IF, Silk JD, Gileadi U, et al. (2003): NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells. J Immunol 171: 5140-5147.
74. Gottschalk C, Elisabeth Mettke E, Kurts C (2015): The Role of Invariant Natural Killer T Cells in Dendritic Cell Licensing, Cross-Priming, and Memory CD8+ T Cell Generation Front Immunol 2015: 379.
75. Gonzalez-Aseguinolaza G, Van Kaer L, Bergmann CC, et al. (2002): Natural killer T cell ligand alpha-galactosylceramide enhances protective immunity induced by malaria vaccines. J Exp Med 1995: 617-624.
76. Tan JQ, Xiao W, Wang L, He YL (2010): Type 1 natural killer T cells: naturally born for fighting. Acta Pharmacol Sin 31: 1123-1132.
77. Haraguchi K, Takahashi T, Nakahara F, et al. (2006): CD1d expression level in tumor cells is an important determinant for anti-tumor immunity by natural killer T cells. Leuk Lymphoma 47: 2218-2223.
78. Metelitsa LS, Naidenko OV, Kant A, et al. (2001): Human NKT cells mediate antitumor cytotoxicity directly by recognizing target cell CD1d with bound ligand or indirectly by producing IL-2 to activate NK cells. J Immunol 167: 3114-3122.
79. Introna M, Mantovani A (1983): Natural killer cells in human solid tumors. Cancer Metastasis Rev 2: 337-350.
80. Facchetti P, Prigione I, Ghiotto F, et al. (1996): Functional and molecular characterization of tumour-infiltrating lymphocytes and clones thereof from a major-histocompatibility-complex-negative human tumour: neuroblastoma. Cancer Immunol Immunother 42: 170-178.
81. Lin EY, Li JF, Gnatovskiy L, et al. (2006): Macrophages regulate the angiogenic switch in a mouse model of breast cancer. Cancer Res 66: 11238-11246.
82. Zheng Y, Cai Z, Wang S, et al. (2009): Macrophages are an abundant component of myeloma microenvironment and protect myeloma cells from chemotherapy drug-induced apoptosis. Blood 114: 3625-3628.
83. Qian B, Deng Y, Im JH, et al. (2009): A distinct macrophage population mediates metastatic breast cancer cell extravasation, establishment and growth. PLoS One 4: e6562.
84. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V (2012): Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol 12: 253-268.
85. Sica A, Bronte V (2007): Altered macrophage differentiation and immune dysfunction in tumor development. J Clin Invest 117: 1155-1166.
86. Almand B, Clark JI, Nikitina E, et al. (2001): Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer. J Immunol 166: 678-689.
87. Serafini P, Borrello I, Bronte V (2006): Myeloid suppressor cells in cancer: recruitment, phenotype, properties, and mechanisms of immune suppression. Semin Cancer Biol 16: 53-65.
88. Ko HJ, Lee JM, Kim YJ (2009): Immunosuppressive myeloid-derived suppressor cells can be converted into immunogenic APCs with the help of activated NKT cells: an alternative cell-based antitumor vaccine. J Immunol 182: 1818-1828.
89. Lee JM, Seo JH, Kim YJ, et al. (2012): The restoration of myeloid-derived suppressor cells as functional antigen-presenting cells by NKT cell help and all-trans-retinoic acid treatment. Int J Cancer 131: 741-751.
90. Gebremeskel S, Clattenburg DR, Slauenwhite D, et al. (2015): Natural killer T cell activation overcomes immunosuppression to enhance clearance of postsurgical breast cancer metastasis in mice. Oncoimmunology 4: e995562.
91. Manjili MH, Payne KK (2012): Cancer immunotherapy: Re-programming cells of the innate and adaptive immune systems. Oncoimmunology 1: 201-204.
92. Molling JW, Langius JAE, Langendijk JA, et al. (2007): Low levels of circulating invariant natural killer t cells predict poor clinical outcome in patients with head and neck squamous cell carcinoma. J Clin Oncol 25: 862-868.
93. Najera Chuc AE, Cervantes LA, Retiguin FP, et al. (2012): Low number of invariant NKT cells is associated with poor survival in acute myeloid leukemia. J Cancer Res Clin Oncol 138: 1427-1432.
94. Tachibana T, Onodera H, Tsuruyama T, et al. (2015): Increased intratumor v24-positive natural killer T cells: a prognostic factor for primary colorectal carcinomas. Clin Cancer Res 11: 7322-7327.
95. Coca S, Perez-Piqueras J, Martinez D, et al. (1997): The prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma. Cancer 79: 2320-2328.
96. Burdin N, Brossay L, Koezuka Y, et al. (1998): Selective ability of mouse CD1 to present glycolipids: -galactosylceramide specifically stimulates V14+ NK T lymphocytes. J Immunol 161: 3271-3281.
97. Toura I, Kawano T, Akutsu Y, et al. (1999): Cutting edge: inhibition of experimental tumor metastasis by dendritic cells pulsed with -galactosylceramide. J Immunol 163: 2387-2391.
98. Hayakawa Y, Rovero S, Forni G, Smyth MJ (2003): Alpha-galactosylceramide (KRN7000) suppression of chemical- and oncogene-dependent carcinogenesis. Proc Natl Acad Sci U S A 100: 9464-9469.
99. Giaccone G, Punt CJ, Ando Y, et al. (2002): A phase I study of the natural killer T-cell ligand alpha-galactosylceramide (KRN7000) in patients with solid tumors. Clin Cancer Res 8: 3702-3709.
100. Crowe NY, Uldrich AP, Kyparissoudis K, et al. (2003): Glycolipid antigen drives rapid expansion and sustained cytokine production by NKT cells. J Immunol 171: 4020-4027.
101. Parekh VV, Lalani S, Kim S, et al. (2009): PD-1:PD-L blockade prevents anergy induction and enhances the anti-tumor activities of glycolipid-activated iNKT cells. J Immunol 182: 2816-2826.
102. Fujii S, Shimizu K, Kronenberg M, Steinman RM (2002): Prolonged IFN--producing NKT response induced with -galactosylceramide-loaded DCs. Nat Immunol 3: 867-874.
103. Nieda M, Okai M, Tazbirkova A, et al. (2004): Therapeutic activation of Valpha24+Vbeta11+ NKT cells in human subjects results in highly coordinated secondary activation of acquired and innate immunity. Blood 103: 383-389.
104. Ishikawa A, Motohashi S, Ishikawa E, et al. (2005): A phase I study of -galactosylceramide (KRN7000)-pulsed dendritic cells in patients with advanced and recurrent non-small cell lung cancer. Clin Cancer Res 2005; 11: 1910-1917.
105. Chang DH, Osman K, Connolly J, et al. (2005): Sustained expansion of NKT cells and antigen-specific T cells after injection of alpha-galactosyl-ceramide loaded mature dendritic cells in cancer patients. J Exp Med 201: 1503-1517.
106. Uchida T, Horiguchi S, Tanaka Y, et al. (2008): Phase I study of -galactosylceramide-pulsed antigen presenting cells administration to the nasal submucosa in unresectable or recurrent head and neck cancer. Cancer Immunol Immunother 57: 337-345.
107. Nicol AJ, Tazbirkova A, Nieda M (2011): Comparison of clinical and immunological effects of intravenous and intradermal administration of -galactosylceramide (KRN7000)-pulsed dendritic cells. Clin Cancer Res 17: 5140-5151.
108. Fujii S, Shimizu K, Steinman RM, Dhodapkar MV (2003): Detection and activation of human V24+ natural killer T cells using -galactosyl ceramide-pulsed dendritic cells. J Immunol Methods 272: 147-159.
109. Ikarashi Y, Mikami R, Bendelac A, et al. (2001): Dendritic cell maturation overrules H-2D-mediated natural killer T (NKT) cell inhibition: critical role for B7 in CD1d-dependent NKT cell interferon gamma production. J Exp Med 194: 1179-1186.
110. Richter J, Neparidze N, Zhang L, et al. (2013): Clinical regressions and broad immune activation following combination therapy targeting human NKT cells in myeloma. Blood 121: 423-430.
111. Haslett PA, Corral LG, Albert M, Kaplan G (1998): Thalidomide costimulates primary human T lymphocytes, preferentially inducing proliferation, cytokine production, and cytotoxic responses in the CD8+ subset. J Exp Med 187: 1885-1892.
112. Motohashi S, Kobayashi S, Ito T, et al. (2002): Preserved IFN- production of circulating V24 NKT cells in primary lung cancer patients. Int J Cancer 102: 159-165.
113. Motohashi S, Nagato K, Kunii N, et al. (2009): A phase I-II study of alpha-galactosylceramide-pulsed IL-2/GM-CSF-cultured peripheral blood mononuclear cells in patients with advanced and recurrent non-small cell lung cancer. J Immunol 182: 2492-2501.
114. Salio M, Silk JD, Jones EY, Cerundolo V (2014): Biology of CD1-and MR1-restricted T cells. Annu Rev Immunol 32: 323-366.
115. Kurosaki M, Horiguchi S, Yamasaki K, et al. (2011): Migration and immunological reaction after the administration of GalCer-pulsed antigen-presenting cells into the submucosa of patients with head and neck cancer. Cancer Immunol Immunother 60: 207-215.
116. Nagato K, Motohashi S, Ishibashi F, et al. (2012): Accumulation of activated invariant natural killer T cells in the tumor microenvironment after -galactosylceramide-pulsed antigen presenting cells. J Clin Immunol 32: 1071-1081.
117. Motohashi S, Ishikawa A, Ishikawa E, et al. (2006): A phase I study of in vitro expanded natural killer T cells in patients with advanced and recurrent non-small cell lung cancer. Clin Cancer Res 12: 6079-6086.
118. Motohashi S, Okamoto Y, Nakayama T (2012): Clinical trials of invariant natural killer T cell-based immunotherapy for cancer. In: Terabe M, Berzofsky JA, eds. Natural killer T cells. Balancing the regulation of tumor immunity. Springer Science + Business Media, LLC; 185-198.
119. Kunii N, Horiguchi S, Motohashi S, et al. (2009): Combination therapy of in vitro-expanded natural killer T cells and -galactosylceramide-pulsed antigen-presenting cells in patients with recurrent head and neck carcinoma. Cancer Sci 100: 1092-1098.
120. Yamasaki K, Horiguchi S, Kurosaki M, et al. (2011): Induction of NKT cell-specific immune responses in cancer tissues after NKT cell-targeted adoptive immunotherapy. Clin Immunol 138: 255-265.
121. Schneiders FL, Scheper RJ, Bontkes HJ, et al. (2012): Clinical Trials with a-Galactosylceramide (KRN7000) in Advanced Cancer. In: Terabe M, Berzofsky JA, eds. Natural killer T cells. Balancing the regulation of tumor immunity. Springer Science + Business Media, LLC; 172-183.
122. Gao B, Radaeva S, Park O (2009): Liver natural killer and natural killer T cells: immunobiology and emerging roles in liver diseases. J Leukoc Biol 86: 513-528.
123. Tsuji M (2006): Glycolipids and phospholipids as natural CD1d-binding NKT cell ligands. Cell Mol Life Sci 63: 1889-1898.