- Email: [email protected]
Adjuvant activity mediated by iNKT cells
Seminars in Immunology 22 (2010) 97–102
Contents lists available at ScienceDirect
Seminars in Immunology journal homepage: www.elsevier.com/locate/ysmim
Review
Adjuvant activity mediated by iNKT cells Shinichiro Fujii a , Shinichiro Motohashi b,c , Kanako Shimizu d , Toshinori Nakayama b , Yohei Yoshiga e , Masaru Taniguchi e,∗ a
Research Unit for Cellular Immunotherapy, RIKEN Research Center for Allergy and Immunology, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Japan Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, Japan c Thoracic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, Japan d Therapeutic Model, RIKEN Research Center for Allergy and Immunology, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Japan e Laboratory of Immune Regulation, RIKEN Research Center for Allergy and Immunology, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Japan b
a r t i c l e
i n f o
Keywords: iNKT cells Adjuvant effects Dendritic cells ␣-Galactosylceramide Clinical trial
a b s t r a c t Invariant natural killer T (iNKT) cells have adjuvant activity due to their ability to produce large amounts of IFN-␥, which activates other cells in innate and acquired systems, and orchestrates protective immunity. Based on these adjuvant mechanisms, we developed iNKT cell-targeted adjuvant therapy and carried out a phase I/IIa trial on advanced lung cancer patients. The patient group with increased numbers of IFN␥-producing cells showed prolonged survival with a median survival time of 31.9 months. Sixty percent of the patients in this group survived for more than 2 years with only a primary treatment and without tumor progression and metastasis. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Invariant natural killer T (iNKT) cells are characterized by the expression of an invariant antigen receptor encoded by V␣14J␣18 in mice and V␣24J␣18 in humans and also by the rapid production of both Th1 and Th2 cytokines after stimulation with their ligands [1–3]. An exogenous glycolipid, ␣-galactosylceramide (␣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 ␣-GalCer or the iNKTcell receptor are well conserved among species such as mouse, rat, sheep, and human [4–7]. In addition, the first four amino acids (Asp94, Arg95, Gly96, and Ser97) in the J␣18 region of the iNKTcell receptor important for the binding with ␣-GalCer and the CD1d molecule are also conserved in mice and humans. Thus, ␣-GalCer identified can be used to activate both human and mouse iNKT cells. Although ␣-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.
∗ Corresponding author. Tel.: +81 45 503 7001; fax: +81 45 503 7006. E-mail address: [email protected] (M. Taniguchi). 1044-5323/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2009.10.002
Because of their apparent self-reactivity and ability to quickly release large quantities of cytokines such as interferon-␥ (IFN-␥), 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-␥ 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-␥ 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.
98
S. Fujii et al. / Seminars in Immunology 22 (2010) 97–102
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, ␣-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 nonself-ligands, such as ␣-GalCer. After activation by ␣-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 [4,8–11]. IFN-␥ 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-␥ even after direct injection of IL-12 and appear functionally impaired. IFN-␥ 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. 2. 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, ␣-GalCer presented on immature DCs activates iNKT cells to proceed to maturation step of DCs. Concerning maturation of DCs, both co-stimulatory moleculeand cytokine-mediated signals are involved. A single injection of free ␣-GalCer into mice induces a burst of IL-4 (at 2 h), IL-12 (at 6 h) and IFN-␥ production (at 16–24 h) that is robust enough to be detectable in the serum [16]. Phenotypically, up-regulation of costimulatory molecules (CD40, CD80, CD86, and B7-DC) and MHC class II on DCs and CD40L expression on iNKT cells are detectable within 2–6 h after ␣-GalCer administration [17]. This phenotypic maturation returns to the basal level 72 h later. The iNKT cells are necessary for DC maturation because this process does not occur in J␣18-deficient mice (Fig. 1). iNKT cell-mediated adjuvant effects on the maturation of DCs have been studied in terms of inflammatory cytokines and CD40–CD40L signaling [17]. With regard to the inflammatory cytokines during the early phase after immunization, TNF-␣ and IFN-␥ are initially secreted and detectable in the serum. When TNF-␣ and IFN-␥ signaling is blocked in immunized mice, the costimulatory molecules are not up-regulated on DCs, suggesting a requirement for these cytokines. On the other hand, in ␣-GalCertreated CD40−/− and CD40L−/− mice, expression of co-stimulatory molecules (CD80, CD86, and B7-DC) on DCs is up-regulated to the same level as in WT mice. The findings have also been confirmed by Matsuda et al., who showed that IFN-␥ production from iNKT cells in CD40−/− mice can be detected at 2 h after ␣-GalCer administration [18]. These findings indicate that CD40 signaling is not crucial for the initial activation of DCs, but instead functions as an augmenting factor.
Fig. 1. iNKT cell-mediated adjuvant effects on anti-tumor protective responses. The ␣-GalCer-loaded immature DCs activate iNKT cells to produce IFN-␥. The resulting augmented expression of CD40L on iNKT cells initiates DC maturation, including the enhanced production of IL-12 and elevated expression of co-stimulatory molecules, which can induce potent antigen-specific CD4+ and CD8+ T cell responses (adaptive immunity). The IFN-␥ and IL-12 also induce the activation of NK cells (innate immunity).
S. Fujii et al. / Seminars in Immunology 22 (2010) 97–102
During interaction between iNKT cells and DCs, IL-12 is produced by the DCs. Since IL-12R expression is significantly higher on iNKT cells compared to other cell types, such as NK cells, IL-12 initially acts on and enhances iNKT cells capable of IFN-␥ production [19,20]. Accordingly, production of both IL-12 and IL-18 from DCs and iNKT cell production of IFN-␥ augments NK cell-mediated cytolytic function and IFN-␥ production [8,19,21,22]. In summary, during the initial step of ␣-GalCer anti-tumor adjuvant activity, immature DCs mainly activate CD4− iNKT cells, rather than CD4+ iNKT cells because the CD4− iNKT cell-mediated antitumor immunity is more potent than that by CD4+ iNKT cells in mice [23]. In a second step, naïve DCs undergo maturation due to their interaction with iNKT cells stimulated with DC-presented ␣GalCer. During the maturation process, DCs acquire the potential to produce cytokines such as TNF-␣, IL-12 and IL-18. These inflammatory cytokines activate a second element of innate immunity such as NK cells. Therefore, activation of the iNKT cell-mediated cellular cascade should be helpful for acquisition of resistance to tumors [24].
3. iNKT cell-mediated adjuvant effects on acquired immunity Similar to their effects on the innate immunity, iNKT cellmediated 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 ␣-GalCer. DC transfer experiments from mice given OVA plus ␣-GalCer into naïve mice confirmed that ␣-GalCer matured DCs mediated the T cell immunity. In addition, Tsuji et al. have demonstrated that ␣-GalCer could serve as an adjuvant for a CD8+ T cell-based malaria vaccine in mice co-administered with ␣-GalCer and irradiated mouse malaria (P. yoelii) sporozoites [26]. This treatment induced highly protective anti-malaria immunity in the mice. Thus, after ␣-GalCer stimulation, iNKT cells augment CD8 T cell responses. The activation of iNKT cells can shift the balance from unresponsiveness to immunity. It is also worth mentioning the critical importance of timing of ␣-GalCer administration to induce iNKT cell-mediated adjuvant activity, because DCs reduce their ability to uptake antigens after maturation [13]. To elicit T cell immunity, ␣-GalCer needs to be given at the same time or close to the OVA injection. Therefore, the timing of DC maturation and antigen delivery to DCs is crucial. TNF-␣ and IFN-␥ are important for DC maturation. However, these molecules play a different role in T cell activation, because T cell immunity in mice where TNF-␣ and IFN-␥ signaling has been blocked is decreased to one-third of that of WT mice immunized with antigen plus ␣-GalCer, but not completely inhibited. Moreover, in CD40−/− and CD40L−/− mice, very little CD4+ and CD8+ T cell immunity is detected [17]. This suggests that CD40 signaling is crucial for induction of T cell immunity in the context of iNKT cellmediated adjuvant effects, while inflammatory cytokines would be accessory factors. In fact, when DCs from CD40−/− mice given cellassociated OVA antigen plus ␣-GalCer are transferred into wild type mice, very limited T cell responses could be induced [17]. Therefore, the role of CD40 is pivotal in the adjuvant effects during iNKT cell-mediated T cell interactions [17,24]. Collectively, the linkage between innate and acquired immunity via DCs and iNKT cells has several components under distinct controls, i.e., antigen presentation in the steady state, increases in co-stimulatory molecules dependent on inflammatory cytokines,
99
and CD40/CD40L signals that activate functional CD4+ and CD8+ T lymphocytes. 4. iNKT cell-mediated anti-tumor adjuvant activity using ␣-GalCer-loaded DC For effective iNKT cell activation, ␣-GalCer-loaded DCs (␣GalCer-DCs) have distinct advantages over an injection of free ␣-GalCer [16,17]. A single injection of ␣-GalCer-DCs induces a burst of IFN-␥ production by iNKT cells that lasts for 2 weeks, whereas the response to soluble ␣-GalCer is rapid, but disappears by day 2. In keeping with their effective Th1 cytokine production, ␣-GalCerDCs induce significant expansion of iNKT cells in the tissues and inhibit in vivo tumor growth in a murine model of lung cancer and liver metastasis [27,28]. Co-administration of B16 melanoma cells i.v. with ␣-GalCer-DCs resulted in a significant reduction of lung metastases in comparison to treatment with free ␣-GalCer alone. This protective effect is mainly mediated by NK cells, because the number of metastases is increased when the mice are treated with anti-asialo-GM1 mAb to eliminate NK cells. Overall, the effector phase in the iNKT cell-mediated adjuvant anti-tumor response is dependent on downstream components, such as NK cells, rather than due to the direct effects of iNKT cells (Fig. 1). Therefore, ␣-GalCer has different functional effects when administered as a free glycolipid or in association with DCs. 5. Adjuvant iNKT cell therapy by co-administration of antigen with ␣-GalCer As discussed above, ␣-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 ␣-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 ␣-GalCer, vaccinated mice are protected against tumor cells where both innate cells and acquired CD4+ and CD8+ T cells contribute to the tumor resistance induced by DCs capturing dying tumor cells in vivo [29]. For protection against virus infection, ␣-GalCer in combination with an inactivated influenza A virus promotes optimal virus specific CD8+ T cell immunity as well as virus specific IgG via iNKT cell activation [30–33]. ␣-GalCer could also act as a mucosal adjuvant for induction of protective immunity against genital herpes. Intranasal immunization with HSV-2 glycoprotein D (gD) in combination with ␣-GalCer elicits strong systemic gD-specific IgGAb response as well as gD-specific T cell activation [34]. In addition, the use of antigen-specific DNA vaccines plus ␣-GalCer has been successful in Leishmania and HIV-1 models [35,36]. 6. iNKT cell-mediated anti-tumor adjuvant therapy using tumor cells loaded with ␣-GalCer The iNKT cell-mediated adjuvant effects induced by ␣-GalCerDCs 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 ␣-GalCer and peptide-pulsed DCs has long been considered. In fact, Stober et al. have reported that ␣GalCer-loaded DCs pulsed with MHC I binding peptides stimulate CD8+ T cells better than DCs pulsed with peptide only. However, the efficiency of induction of T cell immunity was quite low, because only 2% of antigen-specific CD8 T cells were detected after using a dose of peptide (40 M) to pulse the DCs that was 40 times higher
100
S. Fujii et al. / Seminars in Immunology 22 (2010) 97–102
than the usual dose (1 M) [37]. Moreover, Fujii et al. have been unable to enhance T cell priming by simultaneously loading the DCs with MHC class I binding OVA peptide and ␣-GalCer. This may be due to the elimination of ␣-GalCer-loaded and peptide-pulsed DCs by activated iNKT cells or antigen-specific CD8 T cells. To overcome this problem, the use of ␣-GalCer-loaded tumor cells, instead of ␣-GalCer-loaded DCs, has been tested, based on the idea that tumor-specific CD8 T cell immunity could efficiently be mounted by the released tumor antigens under the influence of iNKT cell-mediated adjuvant effects when the ␣-GalCer-pulsed target tumor cells are killed by activated iNKT cells or antigen-specific CD8 T cells. In fact, in the B16 melanoma models, administration of tumor cells loaded with ␣-GalCer (␣-GalCer-tumor) has successfully induced protection against lung metastases [14]. In addition, i.v. injection of mice with ␣-GalCer-tumor induced protection against subcutaneous challenge with tumor cells [24,38]. Moreover, after tumor regression, T cell-mediated anti-tumor responses can be detected [15]. The mechanism of augmentation of T cell specific responses by ␣-GalCer-tumor is due to cross-presentation by DCs, because the maturing antigen-capturing DCs effectively elicit strong in vivo T cell immunity in situ. Our findings suggest several possibilities for the development of future treatment strategies.
7. Phase I/II clinical trial of adjuvant iNKT cell-targeted therapy for advanced lung cancer The administration of ␣-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 ␣-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, immunological responses, and clinical outcomes [40]. A total of 23 patients with advanced or recurrent non-small cell lung cancer refractory to the standard treatments were enrolled and, among them, 17 patients completed the protocol. The patient’s peripheral blood mononuclear cells (PBMCs) were cultured with GM-CSF and IL-2 for 7–14 days and then pulsed with ␣-GalCer [41]. The cultured PBMCs, including DCs, were intravenously administered (1 × 109 cells/m2 /injection) into the original patients 4 times, with a 1 month interval between the second and third administration. In the 17 patients who completed the protocol, the treatment was well-tolerated and no severe adverse event related to the treatment was observed, confirming the results of the previous phase I study of ␣-GalCer-pulsed DCs administration [42]. Six out of 17 patients showed a remarkable increase in circulating iNKT cell number after ␣-GalCer-pulsed DC administration. An enzymelinked immunospot (ELISPOT) assay was used to detect functional iNKT cells in the patients [43]. The ␣-GalCer-reactive IFN-␥ spot forming cells appeared to include both iNKT cells and NK cells [43,44], consistent with the notion that ␣-GalCer-activated iNKT cells subsequently stimulate NK cells to produce IFN-␥ [8,45]. Using this method, significant increases in the number of IFN-␥producing PBMCs were detected in 10 out of 17 patients. The most intriguing finding was that the patient group with increased numbers of IFN-␥-producing cells also showed a significantly prolonged overall survival (31.9 months) in comparison to the survival of the group with no increase in these cells (9.7 months) (Fig. 2). Because of the good correlation between the increased numbers of IFN-␥-producing PBMCs and the prolonged survival, IFN-␥ may be a good biological marker for predicting the clinical course in response to ␣-GalCer-pulsed DC administration. Although this pre-
Fig. 2. Clinical effect of iNKT cell-targeted immunotherapy. (Upper panel) Overall survival curve of all patients (black), high IFN-␥ producers (red) and low IFN-␥ producers (blue) after ␣-GalCer-pulsed DCs treatment. High IFN-␥ producers (n = 10) showed a significantly prolonged overall survival rate in comparison to low IFN-␥ producers (n = 7). MST, median survival time. (Lower panel) Chest X-ray images of a patient who received ␣-GalCer-pulsed DCs treatment and was categorized as having stable disease. Note the tumor size was unchanged 14 months after the treatment.
diction cannot be made prior to DC administration, the monitoring of IFN-␥-producing PBMCs by ELISPOT would be still valuable for patients receiving this immunotherapy. Although none of the 17 patients with advanced or recurrent lung cancer showed a major tumor regression, median survival time of all 17 patients (18.6 months) was superior to that of patients treated with various types of chemotherapy or radiotherapy, where the prognosis is essentially very poor [46–49]. The progression of tumor lesions in the lung appeared to be inhibited in some patients, categorized as having stable disease (Fig. 2 lower panels). iNKT cells in peripheral blood were markedly increased, but only once after ␣-GalCer-pulsed DC treatment in all cases where there was an increase, and the expansion occurred after the first two injections in five out of six cases. At present, it remains unclear why the marked expansion of iNKT cells occurred only once. Moreover, there is no direct evidence indicating that iNKT cell anergy was induced in patients receiving this immunotherapy, since PBMCs produced normal amounts of IFN-␥ in response to ␣-GalCer stimulation even during or after the third/fourth injection of ␣-GalCer-pulsed DCs [40]. In addition, iNKT cells obtained by a second apheresis a week after two initial DC injections still maintained their in vitro proliferative capacity when cultured with ␣-GalCer and IL-2. Thus, iNKT cells do not appear to become anergic during the course of the cell therapy. In any event, from the prognostic point of view, the increased number of IFN-␥-producing cells but not the increased number of iNKT cells in PBMCs was found to correlate with prolonged median survival time. In summary, the adjuvant iNKT cell-targeted immunotherapy using ␣-GalCer-pulsed DCs, which induces the activation of endogenous iNKT cells and iNKT cell-mediated adjuvant effects on other cell types, was well-tolerated. The increase in numbers of IFN-␥-producing cells in PBMCs was associated with prolonged median survival time, and thus this study encourages further eval-
S. Fujii et al. / Seminars in Immunology 22 (2010) 97–102
uation of this immunotherapy for survival benefit. In general, the immunotherapy may be more effective in patients with low tumor burden, and thus the most feasible application of this DC therapy would be for lung cancer patients with small tumor foci, including remaining micrometastasis after radical surgery or after receiving the established first-line therapy. 8. 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. For this purpose, cloned murine iNKT-ES cells have been generated by direct transfer of mature iNKT cell nuclei into unfertilized eggs, with the idea that the pre-rearranged invariant V˛14J˛18 gene would provide advantages in obtaining large number of functional iNKT cells in vitro. During their developmental progression, iNKT-ES cells generated cells with iNKT cell potential and developed into ␣-GalCer/CD1d dimer+ iNKT cells on day 20 to day 25 in the presence of Notch signaling. Interestingly, two types of functional iNKT cells could be generated when the cells with iNKT potential were further cultured on either the OP9/control (switch culture) or on OP9/Dll-1 cells. iNKT cells developed in the OP9/Dll-1culture were Th2-like cells producing IL-4, but not IFN-␥. By contrast, the majority of iNKT cells generated in the switch cultures were Th1-like iNKT cells which had a phenotype similar to DN iNKT cells and produced mainly IFN-␥. When iNKT cell-deficient J˛18−/− mice were reconstituted with Th1-like iNKT cells followed by immunization with OVA and ␣GalCer, there was a substantial increase in the number of OVAspecific IFN-␥-producing CD8+ T cells, 100-fold above that in mice without cell transfer. Thus, in vitro generated iNKT cells are able to function in vivo. The ability to generate iNKT cells with desired functions by use of a simple in vitro culture system offers a powerful new approach for the establishment of optimal iNKT cell therapy. 9. 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 ␣-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-␥ 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 combination therapy which also efficiently induces anti-tumor CD8 T cell responses in vivo in situ by using tumor peptides together with ␣-GalCer-loaded DCs. Another powerful treatment option will be available when it becomes possible to manipulate iNKT cell function in vivo to shift to Th1 type iNKT cells producing mainly IFN-␥ for the anti-tumor therapy. The most likely way to accomplish this will be to develop neoglycolipids with strong adjuvant activity. For example, the ␣C-GalCer analog of ␣-GalCer has been shown to induce mainly Th1 type iNKT cells and efficiently eradicate tumors in the murine model. Unfortunately, ␣-C-GalCer fails to stimulate human iNKT cells. Thus, development of new glycolipids with strong adjuvant potential in humans will be an important step to manipulate Th1 type NKT cell generation in vivo. Combination therapies that stim-
101
ulate CD8 T, NK and iNKT cell populations will probably yield the greatest clinical benefit. Acknowledgment The authors are grateful to Dr. Peter Burrows for helpful comments and constructive criticisms in the preparation of the manuscript. References [1] Taniguchi M, Harada M, Kojo S, Nakayama T, Wakao H. The regulatory role of V␣14 NKT cells in innate and acquired immune response. Annu Rev Immunol 2003;21:483–513. [2] Godfrey DI, MacDonald HR, Kronenberg M, Smyth MJ, Van Kaer L. NKT cells: what’s in a name? Nat Rev Immunol 2004;4:231–7. [3] Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annu Rev Immunol 2007;25:297–336. [4] Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Motoki K, et al. CD1d-restricted and TCR-mediated activation of V␣14 NKT cells by glycosylceramides. Science 1997;278:1626–9. [5] Spada FM, Koezuka Y, Porcelli SA. CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells. J Exp Med 1998;188:1529–34. [6] Brossay L, Chioda M, Burdin N, Koezuka Y, Casorati G, Dellabona P, et al. CD1dmediated recognition of an ␣-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution. J Exp Med 1998;188:1521–8. [7] Brigl M, Brenner MB. CD1: antigen presentation and T cell function. Annu Rev Immunol 2004;22:817–90. [8] Carnaud C, Lee D, Donnars O, Park SH, Beavis A, Koezuka Y, et al. Cutting edge: cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. J Immunol 1999;163:4647–50. [9] Eberl G, MacDonald HR. Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells. Eur J Immunol 2000;30:985–92. [10] Gumperz JE, Miyake S, Yamamura T, Brenner MB. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J Exp Med 2002;195:625–36. [11] Hayakawa Y, Takeda K, Yagita H, Smyth MJ, Van Kaer L, Okumura K, et al. IFN␥-mediated inhibition of tumor angiogenesis by natural killer T-cell ligand, ␣-galactosylceramide. Blood 2002;100:1728–33. [12] Fujii S, Shimizu K, Smith C, Bonifaz L, Steinman RM. Activation of natural killer T cells by ␣-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 2003;198:267–79. [13] Hermans IF, Silk JD, Gileadi U, Salio M, Mathew B, Ritter G, et al. NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells. J Immunol 2003;171:5140–7. [14] Shimizu K, Goto A, Fukui M, Taniguchi M, Fujii S. Tumor cells loaded with ␣galactosylceramide induce innate NKT and NK cell-dependent resistance to tumor implantation in mice. J Immunol 2007;178:2853–61. [15] Fujii S. Exploiting dendritic cells and natural killer T cells in immunotherapy against malignancies. Trends Immunol 2008;29:242–9. [16] Fujii S, Shimizu K, Kronenberg M, Steinman RM. Prolonged interferon-g producing NKT response induced with ␣-galactosylceramide-loaded dendritic cells. Nat Immunol 2002;3:867–74. [17] Fujii S, Liu K, Smith C, Bonito AJ, Steinman RM. 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 2004;199:1607–18. [18] Matsuda JL, Gapin L, Baron JL, Sidobre S, Stetson DB, Mohrs M, et al. Mouse V␣14i natural killer T cells are resistant to cytokine polarization in vivo. Proc Natl Acad Sci USA 2003;100:8395–400. [19] Kitamura H, Iwakabe K, Yahata T, Nishimura S, Ohta A, Ohmi Y, et al. The natural killer T (NKT) cell ligand ␣-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 1999;189:1121–8. [20] Seino K, Fujii S, Harada M, Motohashi S, Nakayama T, Fujisawa T, et al. V␣14+ NKT cell-mediated anti-tumor responses and their clinical application. Springer Semin Immunopathol 2005;27:65–74. [21] Kawamura T, Takeda K, Mendiratta SK, Kawamura H, Van Kaer L, Yagita H, et al. Critical role of NK1+ T cells in IL-12-induced immune responses in vivo. J Immunol 1998;160:16–9. [22] Smyth MJ, Wallace ME, Nutt SL, Yagita H, Godfrey DI, Hayakawa Y. Sequential activation of NKT cells and NK cells provides effective innate immunotherapy of cancer. J Exp Med 2005;201:1973–85. [23] Crowe NY, Coquet JM, Berzins SP, Kyparissoudis K, Keating R, Pellicci DG, et al. Differential antitumor immunity mediated by NKT cell subsets in vivo. J Exp Med 2005;202:1279–88. [24] Fujii S, Shimizu K, Hemmi H, Steinman RM. Innate V␣14+ natural killer T cells mature dendritic cells, leading to strong adaptive immunity. Immunol Rev 2007;220:183–98. [25] Liu K, Iyoda T, Saternus M, Kimura K, Inaba K, Steinman RM. Immune tolerance after delivery of dying cells to dendritic cells in situ. J Exp Med 2002;196:1091–7.
102
S. Fujii et al. / Seminars in Immunology 22 (2010) 97–102
[26] Gonzalez-Aseguinolaza G, Van Kaer L, Bergmann CC, Wilson JM, Schmieg J, Kronenberg M, et al. Natural killer T cell ligand ␣-galactosylceramide enhances protective immunity induced by malaria vaccines. J Exp Med 2002;195:617–24. [27] Akutsu Y, Nakayama T, Harada M, Kawano T, Motohashi S, Shimizu E, et al. Expansion of lung V␣14 NKT cells by administration of ␣-galactosylceramidepulsed dendritic cells. Jpn J Cancer Res 2002;93:397–403. [28] Motohashi S, Kobayashi S, Ito T, Magara KK, Mikuni O, Kamada N, et al. Preserved IFN-␥ production of circulating V␣24 NKT cells in primary lung cancer patients. Int J Cancer 2002;102:159–65. [29] Liu K, Idoyaga J, Charalambous A, Fujii S, Bonito A, Mordoh J, et al. Innate NKT lymphocytes confer superior adaptive immunity via tumor-capturing dendritic cells. J Exp Med 2005;202:1507–16. [30] Youn HJ, Ko SY, Lee KA, Ko HJ, Lee YS, Fujihashi K, et al. A single intranasal immunization with inactivated influenza virus and ␣-galactosylceramide induces long-term protective immunity without redirecting antigen to the central nervous system. Vaccine 2007;25:5189–98. [31] Kamijuku H, Nagata Y, Jiang X, Ichinohe T, Tashiro T, Mori K, et al. Mechanism of NKT cell activation by intranasal coadministration of ␣-galactosylceramide, which can induce cross-protection against influenza viruses. Mucosal Immunol 2008;1:208–18. [32] Kopecky-Bromberg SA, Fraser KA, Pica N, Carnero E, Moran TM, Franck RW, et al. ␣-C-galactosylceramide as an adjuvant for a live attenuated influenza virus vaccine. Vaccine 2009;27:3766–74. [33] Guillonneau C, Mintern JD, Hubert FX, Hurt AC, Besra GS, Porcelli S, et al. Combined NKT cell activation and influenza virus vaccination boosts memory CTL generation and protective immunity. Proc Natl Acad Sci USA 2009;106:3330–5. [34] Lindqvist M, Persson J, Thorn K, Harandi AM. The mucosal adjuvant effect of ␣-galactosylceramide for induction of protective immunity to sexually transmitted viral infection. J Immunol 2009;182:6435–43. [35] Dondji B, Deak E, Goldsmith-Pestana K, Perez-Jimenez E, Esteban M, Miyake S, et al. Intradermal NKT cell activation during DNA priming in heterologous prime-boost vaccination enhances T cell responses and protection against Leishmania. Eur J Immunol 2008;38:706–19. [36] Huang Y, Chen A, Li X, Chen Z, Zhang W, Song Y, et al. Enhancement of HIV DNA vaccine immunogenicity by the NKT cell ligand, ␣-galactosylceramide. Vaccine 2008;26:1807–16. [37] Stober D, Jomantaite I, Schirmbeck R, Reimann J. NKT cells provide help for dendritic cell-dependent priming of MHC class I-restricted CD8+ T cells in vivo. J Immunol 2003;170:2540–8.
[38] Shimizu K, Kurosawa Y, Taniguchi M, Steinman RM, Fujii S. Cross-presentation of glycolipid from tumor cells loaded with ␣-galactosylceramide leads to potent and long-lived T cell mediated immunity via dendritic cells. J Exp Med 2007;204:2641–53. [39] Toura I, Kawano T, Akutsu Y, Nakayama T, Ochiai T, Taniguchi M. Inhibition of experimental tumor metastasis by dendritic cells pulsed with ␣-galactosylceramide. J Immunol 1999;163:2387–91. [40] Motohashi S, Nagato K, Kunii N, Yamamoto H, Yamasaki K, Okita K, et al. A phase I-II study of ␣-galactosylceramide-pulsed IL-2/GM-CSF-cultured peripheral blood mononuclear cells in patients with advanced and recurrent non-small cell lung cancer. J Immunol 2009;182:2492–501. [41] Ishikawa E, Motohashi S, Ishikawa A, Ito T, Uchida T, Kaneko T, et al. Dendritic cell maturation by CD11c-T cells and V␣24+ natural killer T-cell activation by ␣-galactosylceramide. Int J Cancer 2005;117:265–73. [42] Ishikawa A, Motohashi S, Ishikawa E, Fuchida H, Higashino K, Otsuji M, et al. 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;11:1910–7. [43] Motohashi S, Ishikawa A, Ishikawa E, Otsuji M, Iizasa T, Hanaoka H, et al. 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 2006;12:6079– 86. [44] Motohashi S, Nakayama T. Clinical applications of natural killer T cell-based immunotherapy for cancer. Cancer Sci 2008;99:638–45. [45] Smyth MJ, Crowe NY, Pellicci DG, Kyparissoudis K, Kelly JM, Takeda K, et al. Sequential production of interferon-␥ by NK1.1+ T cells and natural killer cells is essential for the antimetastatic effect of ␣-galactosylceramide. Blood 2002;99:1259–66. [46] Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest 1997;111:1710–7. [47] Socinski MA. The role of chemotherapy in the treatment of unresectable stage III and IV nonsmall cell lung cancer. Respir Care Clin N Am 2003;9:207–36. [48] Shepherd FA, Dancey J, Ramlau R, Mattson K, Gralla R, O’Rourke M, et al. Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinumbased chemotherapy. J Clin Oncol 2000;18:2095–103. [49] Huisman C, Smit EF, Giaccone G, Postmus PE. Second-line chemotherapy in relapsing or refractory non-small-cell lung cancer: a review. J Clin Oncol 2000;18:3722–30.
Contents lists available at ScienceDirect
Seminars in Immunology journal homepage: www.elsevier.com/locate/ysmim
Review
Adjuvant activity mediated by iNKT cells Shinichiro Fujii a , Shinichiro Motohashi b,c , Kanako Shimizu d , Toshinori Nakayama b , Yohei Yoshiga e , Masaru Taniguchi e,∗ a
Research Unit for Cellular Immunotherapy, RIKEN Research Center for Allergy and Immunology, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Japan Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, Japan c Thoracic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, Japan d Therapeutic Model, RIKEN Research Center for Allergy and Immunology, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Japan e Laboratory of Immune Regulation, RIKEN Research Center for Allergy and Immunology, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Japan b
a r t i c l e
i n f o
Keywords: iNKT cells Adjuvant effects Dendritic cells ␣-Galactosylceramide Clinical trial
a b s t r a c t Invariant natural killer T (iNKT) cells have adjuvant activity due to their ability to produce large amounts of IFN-␥, which activates other cells in innate and acquired systems, and orchestrates protective immunity. Based on these adjuvant mechanisms, we developed iNKT cell-targeted adjuvant therapy and carried out a phase I/IIa trial on advanced lung cancer patients. The patient group with increased numbers of IFN␥-producing cells showed prolonged survival with a median survival time of 31.9 months. Sixty percent of the patients in this group survived for more than 2 years with only a primary treatment and without tumor progression and metastasis. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Invariant natural killer T (iNKT) cells are characterized by the expression of an invariant antigen receptor encoded by V␣14J␣18 in mice and V␣24J␣18 in humans and also by the rapid production of both Th1 and Th2 cytokines after stimulation with their ligands [1–3]. An exogenous glycolipid, ␣-galactosylceramide (␣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 ␣-GalCer or the iNKTcell receptor are well conserved among species such as mouse, rat, sheep, and human [4–7]. In addition, the first four amino acids (Asp94, Arg95, Gly96, and Ser97) in the J␣18 region of the iNKTcell receptor important for the binding with ␣-GalCer and the CD1d molecule are also conserved in mice and humans. Thus, ␣-GalCer identified can be used to activate both human and mouse iNKT cells. Although ␣-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.
∗ Corresponding author. Tel.: +81 45 503 7001; fax: +81 45 503 7006. E-mail address: [email protected] (M. Taniguchi). 1044-5323/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2009.10.002
Because of their apparent self-reactivity and ability to quickly release large quantities of cytokines such as interferon-␥ (IFN-␥), 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-␥ 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-␥ 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.
98
S. Fujii et al. / Seminars in Immunology 22 (2010) 97–102
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, ␣-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 nonself-ligands, such as ␣-GalCer. After activation by ␣-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 [4,8–11]. IFN-␥ 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-␥ even after direct injection of IL-12 and appear functionally impaired. IFN-␥ 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. 2. 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, ␣-GalCer presented on immature DCs activates iNKT cells to proceed to maturation step of DCs. Concerning maturation of DCs, both co-stimulatory moleculeand cytokine-mediated signals are involved. A single injection of free ␣-GalCer into mice induces a burst of IL-4 (at 2 h), IL-12 (at 6 h) and IFN-␥ production (at 16–24 h) that is robust enough to be detectable in the serum [16]. Phenotypically, up-regulation of costimulatory molecules (CD40, CD80, CD86, and B7-DC) and MHC class II on DCs and CD40L expression on iNKT cells are detectable within 2–6 h after ␣-GalCer administration [17]. This phenotypic maturation returns to the basal level 72 h later. The iNKT cells are necessary for DC maturation because this process does not occur in J␣18-deficient mice (Fig. 1). iNKT cell-mediated adjuvant effects on the maturation of DCs have been studied in terms of inflammatory cytokines and CD40–CD40L signaling [17]. With regard to the inflammatory cytokines during the early phase after immunization, TNF-␣ and IFN-␥ are initially secreted and detectable in the serum. When TNF-␣ and IFN-␥ signaling is blocked in immunized mice, the costimulatory molecules are not up-regulated on DCs, suggesting a requirement for these cytokines. On the other hand, in ␣-GalCertreated CD40−/− and CD40L−/− mice, expression of co-stimulatory molecules (CD80, CD86, and B7-DC) on DCs is up-regulated to the same level as in WT mice. The findings have also been confirmed by Matsuda et al., who showed that IFN-␥ production from iNKT cells in CD40−/− mice can be detected at 2 h after ␣-GalCer administration [18]. These findings indicate that CD40 signaling is not crucial for the initial activation of DCs, but instead functions as an augmenting factor.
Fig. 1. iNKT cell-mediated adjuvant effects on anti-tumor protective responses. The ␣-GalCer-loaded immature DCs activate iNKT cells to produce IFN-␥. The resulting augmented expression of CD40L on iNKT cells initiates DC maturation, including the enhanced production of IL-12 and elevated expression of co-stimulatory molecules, which can induce potent antigen-specific CD4+ and CD8+ T cell responses (adaptive immunity). The IFN-␥ and IL-12 also induce the activation of NK cells (innate immunity).
S. Fujii et al. / Seminars in Immunology 22 (2010) 97–102
During interaction between iNKT cells and DCs, IL-12 is produced by the DCs. Since IL-12R expression is significantly higher on iNKT cells compared to other cell types, such as NK cells, IL-12 initially acts on and enhances iNKT cells capable of IFN-␥ production [19,20]. Accordingly, production of both IL-12 and IL-18 from DCs and iNKT cell production of IFN-␥ augments NK cell-mediated cytolytic function and IFN-␥ production [8,19,21,22]. In summary, during the initial step of ␣-GalCer anti-tumor adjuvant activity, immature DCs mainly activate CD4− iNKT cells, rather than CD4+ iNKT cells because the CD4− iNKT cell-mediated antitumor immunity is more potent than that by CD4+ iNKT cells in mice [23]. In a second step, naïve DCs undergo maturation due to their interaction with iNKT cells stimulated with DC-presented ␣GalCer. During the maturation process, DCs acquire the potential to produce cytokines such as TNF-␣, IL-12 and IL-18. These inflammatory cytokines activate a second element of innate immunity such as NK cells. Therefore, activation of the iNKT cell-mediated cellular cascade should be helpful for acquisition of resistance to tumors [24].
3. iNKT cell-mediated adjuvant effects on acquired immunity Similar to their effects on the innate immunity, iNKT cellmediated 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 ␣-GalCer. DC transfer experiments from mice given OVA plus ␣-GalCer into naïve mice confirmed that ␣-GalCer matured DCs mediated the T cell immunity. In addition, Tsuji et al. have demonstrated that ␣-GalCer could serve as an adjuvant for a CD8+ T cell-based malaria vaccine in mice co-administered with ␣-GalCer and irradiated mouse malaria (P. yoelii) sporozoites [26]. This treatment induced highly protective anti-malaria immunity in the mice. Thus, after ␣-GalCer stimulation, iNKT cells augment CD8 T cell responses. The activation of iNKT cells can shift the balance from unresponsiveness to immunity. It is also worth mentioning the critical importance of timing of ␣-GalCer administration to induce iNKT cell-mediated adjuvant activity, because DCs reduce their ability to uptake antigens after maturation [13]. To elicit T cell immunity, ␣-GalCer needs to be given at the same time or close to the OVA injection. Therefore, the timing of DC maturation and antigen delivery to DCs is crucial. TNF-␣ and IFN-␥ are important for DC maturation. However, these molecules play a different role in T cell activation, because T cell immunity in mice where TNF-␣ and IFN-␥ signaling has been blocked is decreased to one-third of that of WT mice immunized with antigen plus ␣-GalCer, but not completely inhibited. Moreover, in CD40−/− and CD40L−/− mice, very little CD4+ and CD8+ T cell immunity is detected [17]. This suggests that CD40 signaling is crucial for induction of T cell immunity in the context of iNKT cellmediated adjuvant effects, while inflammatory cytokines would be accessory factors. In fact, when DCs from CD40−/− mice given cellassociated OVA antigen plus ␣-GalCer are transferred into wild type mice, very limited T cell responses could be induced [17]. Therefore, the role of CD40 is pivotal in the adjuvant effects during iNKT cell-mediated T cell interactions [17,24]. Collectively, the linkage between innate and acquired immunity via DCs and iNKT cells has several components under distinct controls, i.e., antigen presentation in the steady state, increases in co-stimulatory molecules dependent on inflammatory cytokines,
99
and CD40/CD40L signals that activate functional CD4+ and CD8+ T lymphocytes. 4. iNKT cell-mediated anti-tumor adjuvant activity using ␣-GalCer-loaded DC For effective iNKT cell activation, ␣-GalCer-loaded DCs (␣GalCer-DCs) have distinct advantages over an injection of free ␣-GalCer [16,17]. A single injection of ␣-GalCer-DCs induces a burst of IFN-␥ production by iNKT cells that lasts for 2 weeks, whereas the response to soluble ␣-GalCer is rapid, but disappears by day 2. In keeping with their effective Th1 cytokine production, ␣-GalCerDCs induce significant expansion of iNKT cells in the tissues and inhibit in vivo tumor growth in a murine model of lung cancer and liver metastasis [27,28]. Co-administration of B16 melanoma cells i.v. with ␣-GalCer-DCs resulted in a significant reduction of lung metastases in comparison to treatment with free ␣-GalCer alone. This protective effect is mainly mediated by NK cells, because the number of metastases is increased when the mice are treated with anti-asialo-GM1 mAb to eliminate NK cells. Overall, the effector phase in the iNKT cell-mediated adjuvant anti-tumor response is dependent on downstream components, such as NK cells, rather than due to the direct effects of iNKT cells (Fig. 1). Therefore, ␣-GalCer has different functional effects when administered as a free glycolipid or in association with DCs. 5. Adjuvant iNKT cell therapy by co-administration of antigen with ␣-GalCer As discussed above, ␣-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 ␣-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 ␣-GalCer, vaccinated mice are protected against tumor cells where both innate cells and acquired CD4+ and CD8+ T cells contribute to the tumor resistance induced by DCs capturing dying tumor cells in vivo [29]. For protection against virus infection, ␣-GalCer in combination with an inactivated influenza A virus promotes optimal virus specific CD8+ T cell immunity as well as virus specific IgG via iNKT cell activation [30–33]. ␣-GalCer could also act as a mucosal adjuvant for induction of protective immunity against genital herpes. Intranasal immunization with HSV-2 glycoprotein D (gD) in combination with ␣-GalCer elicits strong systemic gD-specific IgGAb response as well as gD-specific T cell activation [34]. In addition, the use of antigen-specific DNA vaccines plus ␣-GalCer has been successful in Leishmania and HIV-1 models [35,36]. 6. iNKT cell-mediated anti-tumor adjuvant therapy using tumor cells loaded with ␣-GalCer The iNKT cell-mediated adjuvant effects induced by ␣-GalCerDCs 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 ␣-GalCer and peptide-pulsed DCs has long been considered. In fact, Stober et al. have reported that ␣GalCer-loaded DCs pulsed with MHC I binding peptides stimulate CD8+ T cells better than DCs pulsed with peptide only. However, the efficiency of induction of T cell immunity was quite low, because only 2% of antigen-specific CD8 T cells were detected after using a dose of peptide (40 M) to pulse the DCs that was 40 times higher
100
S. Fujii et al. / Seminars in Immunology 22 (2010) 97–102
than the usual dose (1 M) [37]. Moreover, Fujii et al. have been unable to enhance T cell priming by simultaneously loading the DCs with MHC class I binding OVA peptide and ␣-GalCer. This may be due to the elimination of ␣-GalCer-loaded and peptide-pulsed DCs by activated iNKT cells or antigen-specific CD8 T cells. To overcome this problem, the use of ␣-GalCer-loaded tumor cells, instead of ␣-GalCer-loaded DCs, has been tested, based on the idea that tumor-specific CD8 T cell immunity could efficiently be mounted by the released tumor antigens under the influence of iNKT cell-mediated adjuvant effects when the ␣-GalCer-pulsed target tumor cells are killed by activated iNKT cells or antigen-specific CD8 T cells. In fact, in the B16 melanoma models, administration of tumor cells loaded with ␣-GalCer (␣-GalCer-tumor) has successfully induced protection against lung metastases [14]. In addition, i.v. injection of mice with ␣-GalCer-tumor induced protection against subcutaneous challenge with tumor cells [24,38]. Moreover, after tumor regression, T cell-mediated anti-tumor responses can be detected [15]. The mechanism of augmentation of T cell specific responses by ␣-GalCer-tumor is due to cross-presentation by DCs, because the maturing antigen-capturing DCs effectively elicit strong in vivo T cell immunity in situ. Our findings suggest several possibilities for the development of future treatment strategies.
7. Phase I/II clinical trial of adjuvant iNKT cell-targeted therapy for advanced lung cancer The administration of ␣-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 ␣-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, immunological responses, and clinical outcomes [40]. A total of 23 patients with advanced or recurrent non-small cell lung cancer refractory to the standard treatments were enrolled and, among them, 17 patients completed the protocol. The patient’s peripheral blood mononuclear cells (PBMCs) were cultured with GM-CSF and IL-2 for 7–14 days and then pulsed with ␣-GalCer [41]. The cultured PBMCs, including DCs, were intravenously administered (1 × 109 cells/m2 /injection) into the original patients 4 times, with a 1 month interval between the second and third administration. In the 17 patients who completed the protocol, the treatment was well-tolerated and no severe adverse event related to the treatment was observed, confirming the results of the previous phase I study of ␣-GalCer-pulsed DCs administration [42]. Six out of 17 patients showed a remarkable increase in circulating iNKT cell number after ␣-GalCer-pulsed DC administration. An enzymelinked immunospot (ELISPOT) assay was used to detect functional iNKT cells in the patients [43]. The ␣-GalCer-reactive IFN-␥ spot forming cells appeared to include both iNKT cells and NK cells [43,44], consistent with the notion that ␣-GalCer-activated iNKT cells subsequently stimulate NK cells to produce IFN-␥ [8,45]. Using this method, significant increases in the number of IFN-␥producing PBMCs were detected in 10 out of 17 patients. The most intriguing finding was that the patient group with increased numbers of IFN-␥-producing cells also showed a significantly prolonged overall survival (31.9 months) in comparison to the survival of the group with no increase in these cells (9.7 months) (Fig. 2). Because of the good correlation between the increased numbers of IFN-␥-producing PBMCs and the prolonged survival, IFN-␥ may be a good biological marker for predicting the clinical course in response to ␣-GalCer-pulsed DC administration. Although this pre-
Fig. 2. Clinical effect of iNKT cell-targeted immunotherapy. (Upper panel) Overall survival curve of all patients (black), high IFN-␥ producers (red) and low IFN-␥ producers (blue) after ␣-GalCer-pulsed DCs treatment. High IFN-␥ producers (n = 10) showed a significantly prolonged overall survival rate in comparison to low IFN-␥ producers (n = 7). MST, median survival time. (Lower panel) Chest X-ray images of a patient who received ␣-GalCer-pulsed DCs treatment and was categorized as having stable disease. Note the tumor size was unchanged 14 months after the treatment.
diction cannot be made prior to DC administration, the monitoring of IFN-␥-producing PBMCs by ELISPOT would be still valuable for patients receiving this immunotherapy. Although none of the 17 patients with advanced or recurrent lung cancer showed a major tumor regression, median survival time of all 17 patients (18.6 months) was superior to that of patients treated with various types of chemotherapy or radiotherapy, where the prognosis is essentially very poor [46–49]. The progression of tumor lesions in the lung appeared to be inhibited in some patients, categorized as having stable disease (Fig. 2 lower panels). iNKT cells in peripheral blood were markedly increased, but only once after ␣-GalCer-pulsed DC treatment in all cases where there was an increase, and the expansion occurred after the first two injections in five out of six cases. At present, it remains unclear why the marked expansion of iNKT cells occurred only once. Moreover, there is no direct evidence indicating that iNKT cell anergy was induced in patients receiving this immunotherapy, since PBMCs produced normal amounts of IFN-␥ in response to ␣-GalCer stimulation even during or after the third/fourth injection of ␣-GalCer-pulsed DCs [40]. In addition, iNKT cells obtained by a second apheresis a week after two initial DC injections still maintained their in vitro proliferative capacity when cultured with ␣-GalCer and IL-2. Thus, iNKT cells do not appear to become anergic during the course of the cell therapy. In any event, from the prognostic point of view, the increased number of IFN-␥-producing cells but not the increased number of iNKT cells in PBMCs was found to correlate with prolonged median survival time. In summary, the adjuvant iNKT cell-targeted immunotherapy using ␣-GalCer-pulsed DCs, which induces the activation of endogenous iNKT cells and iNKT cell-mediated adjuvant effects on other cell types, was well-tolerated. The increase in numbers of IFN-␥-producing cells in PBMCs was associated with prolonged median survival time, and thus this study encourages further eval-
S. Fujii et al. / Seminars in Immunology 22 (2010) 97–102
uation of this immunotherapy for survival benefit. In general, the immunotherapy may be more effective in patients with low tumor burden, and thus the most feasible application of this DC therapy would be for lung cancer patients with small tumor foci, including remaining micrometastasis after radical surgery or after receiving the established first-line therapy. 8. 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. For this purpose, cloned murine iNKT-ES cells have been generated by direct transfer of mature iNKT cell nuclei into unfertilized eggs, with the idea that the pre-rearranged invariant V˛14J˛18 gene would provide advantages in obtaining large number of functional iNKT cells in vitro. During their developmental progression, iNKT-ES cells generated cells with iNKT cell potential and developed into ␣-GalCer/CD1d dimer+ iNKT cells on day 20 to day 25 in the presence of Notch signaling. Interestingly, two types of functional iNKT cells could be generated when the cells with iNKT potential were further cultured on either the OP9/control (switch culture) or on OP9/Dll-1 cells. iNKT cells developed in the OP9/Dll-1culture were Th2-like cells producing IL-4, but not IFN-␥. By contrast, the majority of iNKT cells generated in the switch cultures were Th1-like iNKT cells which had a phenotype similar to DN iNKT cells and produced mainly IFN-␥. When iNKT cell-deficient J˛18−/− mice were reconstituted with Th1-like iNKT cells followed by immunization with OVA and ␣GalCer, there was a substantial increase in the number of OVAspecific IFN-␥-producing CD8+ T cells, 100-fold above that in mice without cell transfer. Thus, in vitro generated iNKT cells are able to function in vivo. The ability to generate iNKT cells with desired functions by use of a simple in vitro culture system offers a powerful new approach for the establishment of optimal iNKT cell therapy. 9. 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 ␣-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-␥ 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 combination therapy which also efficiently induces anti-tumor CD8 T cell responses in vivo in situ by using tumor peptides together with ␣-GalCer-loaded DCs. Another powerful treatment option will be available when it becomes possible to manipulate iNKT cell function in vivo to shift to Th1 type iNKT cells producing mainly IFN-␥ for the anti-tumor therapy. The most likely way to accomplish this will be to develop neoglycolipids with strong adjuvant activity. For example, the ␣C-GalCer analog of ␣-GalCer has been shown to induce mainly Th1 type iNKT cells and efficiently eradicate tumors in the murine model. Unfortunately, ␣-C-GalCer fails to stimulate human iNKT cells. Thus, development of new glycolipids with strong adjuvant potential in humans will be an important step to manipulate Th1 type NKT cell generation in vivo. Combination therapies that stim-
101
ulate CD8 T, NK and iNKT cell populations will probably yield the greatest clinical benefit. Acknowledgment The authors are grateful to Dr. Peter Burrows for helpful comments and constructive criticisms in the preparation of the manuscript. References [1] Taniguchi M, Harada M, Kojo S, Nakayama T, Wakao H. The regulatory role of V␣14 NKT cells in innate and acquired immune response. Annu Rev Immunol 2003;21:483–513. [2] Godfrey DI, MacDonald HR, Kronenberg M, Smyth MJ, Van Kaer L. NKT cells: what’s in a name? Nat Rev Immunol 2004;4:231–7. [3] Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annu Rev Immunol 2007;25:297–336. [4] Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Motoki K, et al. CD1d-restricted and TCR-mediated activation of V␣14 NKT cells by glycosylceramides. Science 1997;278:1626–9. [5] Spada FM, Koezuka Y, Porcelli SA. CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells. J Exp Med 1998;188:1529–34. [6] Brossay L, Chioda M, Burdin N, Koezuka Y, Casorati G, Dellabona P, et al. CD1dmediated recognition of an ␣-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution. J Exp Med 1998;188:1521–8. [7] Brigl M, Brenner MB. CD1: antigen presentation and T cell function. Annu Rev Immunol 2004;22:817–90. [8] Carnaud C, Lee D, Donnars O, Park SH, Beavis A, Koezuka Y, et al. Cutting edge: cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. J Immunol 1999;163:4647–50. [9] Eberl G, MacDonald HR. Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells. Eur J Immunol 2000;30:985–92. [10] Gumperz JE, Miyake S, Yamamura T, Brenner MB. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J Exp Med 2002;195:625–36. [11] Hayakawa Y, Takeda K, Yagita H, Smyth MJ, Van Kaer L, Okumura K, et al. IFN␥-mediated inhibition of tumor angiogenesis by natural killer T-cell ligand, ␣-galactosylceramide. Blood 2002;100:1728–33. [12] Fujii S, Shimizu K, Smith C, Bonifaz L, Steinman RM. Activation of natural killer T cells by ␣-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 2003;198:267–79. [13] Hermans IF, Silk JD, Gileadi U, Salio M, Mathew B, Ritter G, et al. NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells. J Immunol 2003;171:5140–7. [14] Shimizu K, Goto A, Fukui M, Taniguchi M, Fujii S. Tumor cells loaded with ␣galactosylceramide induce innate NKT and NK cell-dependent resistance to tumor implantation in mice. J Immunol 2007;178:2853–61. [15] Fujii S. Exploiting dendritic cells and natural killer T cells in immunotherapy against malignancies. Trends Immunol 2008;29:242–9. [16] Fujii S, Shimizu K, Kronenberg M, Steinman RM. Prolonged interferon-g producing NKT response induced with ␣-galactosylceramide-loaded dendritic cells. Nat Immunol 2002;3:867–74. [17] Fujii S, Liu K, Smith C, Bonito AJ, Steinman RM. 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 2004;199:1607–18. [18] Matsuda JL, Gapin L, Baron JL, Sidobre S, Stetson DB, Mohrs M, et al. Mouse V␣14i natural killer T cells are resistant to cytokine polarization in vivo. Proc Natl Acad Sci USA 2003;100:8395–400. [19] Kitamura H, Iwakabe K, Yahata T, Nishimura S, Ohta A, Ohmi Y, et al. The natural killer T (NKT) cell ligand ␣-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 1999;189:1121–8. [20] Seino K, Fujii S, Harada M, Motohashi S, Nakayama T, Fujisawa T, et al. V␣14+ NKT cell-mediated anti-tumor responses and their clinical application. Springer Semin Immunopathol 2005;27:65–74. [21] Kawamura T, Takeda K, Mendiratta SK, Kawamura H, Van Kaer L, Yagita H, et al. Critical role of NK1+ T cells in IL-12-induced immune responses in vivo. J Immunol 1998;160:16–9. [22] Smyth MJ, Wallace ME, Nutt SL, Yagita H, Godfrey DI, Hayakawa Y. Sequential activation of NKT cells and NK cells provides effective innate immunotherapy of cancer. J Exp Med 2005;201:1973–85. [23] Crowe NY, Coquet JM, Berzins SP, Kyparissoudis K, Keating R, Pellicci DG, et al. Differential antitumor immunity mediated by NKT cell subsets in vivo. J Exp Med 2005;202:1279–88. [24] Fujii S, Shimizu K, Hemmi H, Steinman RM. Innate V␣14+ natural killer T cells mature dendritic cells, leading to strong adaptive immunity. Immunol Rev 2007;220:183–98. [25] Liu K, Iyoda T, Saternus M, Kimura K, Inaba K, Steinman RM. Immune tolerance after delivery of dying cells to dendritic cells in situ. J Exp Med 2002;196:1091–7.
102
S. Fujii et al. / Seminars in Immunology 22 (2010) 97–102
[26] Gonzalez-Aseguinolaza G, Van Kaer L, Bergmann CC, Wilson JM, Schmieg J, Kronenberg M, et al. Natural killer T cell ligand ␣-galactosylceramide enhances protective immunity induced by malaria vaccines. J Exp Med 2002;195:617–24. [27] Akutsu Y, Nakayama T, Harada M, Kawano T, Motohashi S, Shimizu E, et al. Expansion of lung V␣14 NKT cells by administration of ␣-galactosylceramidepulsed dendritic cells. Jpn J Cancer Res 2002;93:397–403. [28] Motohashi S, Kobayashi S, Ito T, Magara KK, Mikuni O, Kamada N, et al. Preserved IFN-␥ production of circulating V␣24 NKT cells in primary lung cancer patients. Int J Cancer 2002;102:159–65. [29] Liu K, Idoyaga J, Charalambous A, Fujii S, Bonito A, Mordoh J, et al. Innate NKT lymphocytes confer superior adaptive immunity via tumor-capturing dendritic cells. J Exp Med 2005;202:1507–16. [30] Youn HJ, Ko SY, Lee KA, Ko HJ, Lee YS, Fujihashi K, et al. A single intranasal immunization with inactivated influenza virus and ␣-galactosylceramide induces long-term protective immunity without redirecting antigen to the central nervous system. Vaccine 2007;25:5189–98. [31] Kamijuku H, Nagata Y, Jiang X, Ichinohe T, Tashiro T, Mori K, et al. Mechanism of NKT cell activation by intranasal coadministration of ␣-galactosylceramide, which can induce cross-protection against influenza viruses. Mucosal Immunol 2008;1:208–18. [32] Kopecky-Bromberg SA, Fraser KA, Pica N, Carnero E, Moran TM, Franck RW, et al. ␣-C-galactosylceramide as an adjuvant for a live attenuated influenza virus vaccine. Vaccine 2009;27:3766–74. [33] Guillonneau C, Mintern JD, Hubert FX, Hurt AC, Besra GS, Porcelli S, et al. Combined NKT cell activation and influenza virus vaccination boosts memory CTL generation and protective immunity. Proc Natl Acad Sci USA 2009;106:3330–5. [34] Lindqvist M, Persson J, Thorn K, Harandi AM. The mucosal adjuvant effect of ␣-galactosylceramide for induction of protective immunity to sexually transmitted viral infection. J Immunol 2009;182:6435–43. [35] Dondji B, Deak E, Goldsmith-Pestana K, Perez-Jimenez E, Esteban M, Miyake S, et al. Intradermal NKT cell activation during DNA priming in heterologous prime-boost vaccination enhances T cell responses and protection against Leishmania. Eur J Immunol 2008;38:706–19. [36] Huang Y, Chen A, Li X, Chen Z, Zhang W, Song Y, et al. Enhancement of HIV DNA vaccine immunogenicity by the NKT cell ligand, ␣-galactosylceramide. Vaccine 2008;26:1807–16. [37] Stober D, Jomantaite I, Schirmbeck R, Reimann J. NKT cells provide help for dendritic cell-dependent priming of MHC class I-restricted CD8+ T cells in vivo. J Immunol 2003;170:2540–8.
[38] Shimizu K, Kurosawa Y, Taniguchi M, Steinman RM, Fujii S. Cross-presentation of glycolipid from tumor cells loaded with ␣-galactosylceramide leads to potent and long-lived T cell mediated immunity via dendritic cells. J Exp Med 2007;204:2641–53. [39] Toura I, Kawano T, Akutsu Y, Nakayama T, Ochiai T, Taniguchi M. Inhibition of experimental tumor metastasis by dendritic cells pulsed with ␣-galactosylceramide. J Immunol 1999;163:2387–91. [40] Motohashi S, Nagato K, Kunii N, Yamamoto H, Yamasaki K, Okita K, et al. A phase I-II study of ␣-galactosylceramide-pulsed IL-2/GM-CSF-cultured peripheral blood mononuclear cells in patients with advanced and recurrent non-small cell lung cancer. J Immunol 2009;182:2492–501. [41] Ishikawa E, Motohashi S, Ishikawa A, Ito T, Uchida T, Kaneko T, et al. Dendritic cell maturation by CD11c-T cells and V␣24+ natural killer T-cell activation by ␣-galactosylceramide. Int J Cancer 2005;117:265–73. [42] Ishikawa A, Motohashi S, Ishikawa E, Fuchida H, Higashino K, Otsuji M, et al. 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;11:1910–7. [43] Motohashi S, Ishikawa A, Ishikawa E, Otsuji M, Iizasa T, Hanaoka H, et al. 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 2006;12:6079– 86. [44] Motohashi S, Nakayama T. Clinical applications of natural killer T cell-based immunotherapy for cancer. Cancer Sci 2008;99:638–45. [45] Smyth MJ, Crowe NY, Pellicci DG, Kyparissoudis K, Kelly JM, Takeda K, et al. Sequential production of interferon-␥ by NK1.1+ T cells and natural killer cells is essential for the antimetastatic effect of ␣-galactosylceramide. Blood 2002;99:1259–66. [46] Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest 1997;111:1710–7. [47] Socinski MA. The role of chemotherapy in the treatment of unresectable stage III and IV nonsmall cell lung cancer. Respir Care Clin N Am 2003;9:207–36. [48] Shepherd FA, Dancey J, Ramlau R, Mattson K, Gralla R, O’Rourke M, et al. Prospective randomized trial of docetaxel versus best supportive care in patients with non-small-cell lung cancer previously treated with platinumbased chemotherapy. J Clin Oncol 2000;18:2095–103. [49] Huisman C, Smit EF, Giaccone G, Postmus PE. Second-line chemotherapy in relapsing or refractory non-small-cell lung cancer: a review. J Clin Oncol 2000;18:3722–30.