The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baxevanis, C. N.
Right arrow Articles by Papamichail, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baxevanis, C. N.
Right arrow Articles by Papamichail, M.
Right arrowPubmed/NCBI databases
*Substance via MeSH
The Journal of Immunology, 2003, 171: 2953-2959.
Copyright © 2003 by The American Association of Immunologists

In Vivo Antitumor Activity of NKT Cells Activated by the Combination of IL-12 and IL-18 1

Constantin N. Baxevanis2, Angelos D. Gritzapis and Michael Papamichail

Cancer Immunology and Immunotherapy Center, St. Savas Hospital, Athens, Greece


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interleukin-12 and IL-18 have been demonstrated to potentiate innate immunity in a variety of experimental tumor models, but the functional roles of NK and/or NKT cells and their mechanism of action in these models have not been fully addressed. Through adoptive transfer of NKT cells activated in vitro with a combination of IL-12 plus IL-18 (IL-12/IL-18 NKT) into syngeneic animals, we demonstrate in this study that IL-12/IL-18 NKT cells are essential and collaborate with the host’s own NK cells in natural host immunity against the growth of ALC and MC57X syngeneic tumors. The relative roles of the adoptively transferred IL-12/IL-18 NKT cells and endogenous NK cells in host protection were first shown in normal C57BL/6 (B6) mice treated with anti-asialo GM1 Ab that selectively depletes NK cells; second, in B6.TCRJ{alpha}281-/- mice specifically deficient for NKT cells; and third, in B6.scid mice that also lack NKT cells. Furthermore, by injecting normal B6 mice with anti-IL-2 and/or anti-IFN-{gamma} mAb, we could demonstrate that effective innate immunity against both types of syngeneic tumors was dependent on the production of IL-2 and IFN-{gamma} by the adoptively transferred NKT cells. In vitro studies confirmed both the secretion of IL-2 and IFN-{gamma} by the IL-12/IL-18-activated NKT cells and their collaborative role with NK cells for lysis of ALC and MC57X syngeneic tumor targets. This is the first description of an antitumor function of IL-12/IL-18 NKT cells adoptively transferred into syngeneic hosts that provides the basis for a new modality in the cellular immunotherapy of cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human NK cells are members of the innate immune system and are thought to play a significant role both as regulatory and effector cells in defense mechanisms against tumor cells (for review, see Ref.1). Functionally, NK cells display spontaneous MHC-unrestricted cytolytic responses and produce cytokines, such as IFN-{gamma} and TNF-{alpha}, which further enhance their functional program (2, 3). NK cells also express NK receptors that recognize MHC class I ligands and regulate (inhibit or activate) the functional response to target cells (for review, see Ref.4). In addition, resting or unstimulated NK cells constitutively express a number of cytokine receptors and may be activated for increased cytotoxicity or cytokine production following stimulation by numerous cytokines (e.g., TNF-{alpha}, IL-1, IL-2, IL-12, IL-15, IL-18, or IL-21) (3, 4, 5, 6, 7, 8, 9, 10). NKT cells constitute a lymphocyte subpopulation that expresses TCR and NK markers (NK1 or NKR-P1A) and have been implicated in immunoregulatory roles, as well as in controlling tumor growth (for review, see Ref.11). One population of NKT cells expresses rearranged TCRs that are limited in heterogeneity (12, 13), whereas another category of NK 1.1+ TCR+ lymphocytes includes cells that have diverse TCRs (14, 15). NKT cells can be activated by a TCR-dependent manner recognizing {alpha}-galactosylceramide bound on CD1d (16) or peptides presented to them in the context of MHC (17, 18). In addition, NKT cells are also activated in a TCR-independent fashion through the action of several cytokines such as IL-2, IL-12, IL-15, or IL-18 (4, 6, 16, 19). Once activated, NKT cells can act as potent killers against tumor cells (16) or as regulatory cells by activating or suppressing cytotoxic responses (20, 21).

Recently, great interest has been focused on the role of IL-12 and/or IL-18 on the activation of NK or NKT cells. IL-12 is a 70-kDa heterodimeric cytokine composed of two covalently linked p35 and p40 subunits, both of which are required for biologic activity. IL-12 exerts multiple biologic activities that, besides NK cell activation, also include activation of CD8+ CTLs, differentiation of CD4+ T lymphocytes, induction of NO production by macrophages, and induction of type 1 responses by Th lymphocytes (reviewed in Refs.22 and 23). IL-12 has also been shown to possess potent antitumor activity in a wide variety of murine tumor models (24, 25, 26, 27, 28). Recent reports have demonstrated that the in vivo antitumor capacity of IL-12 is mediated via NK and/or NKT cells (13, 29).

IL-18 is a cytokine that was identified as a factor inducing IFN-{gamma} production by T and NK cells (28). Besides its IFN-{gamma}-inducing activity, IL-18 also augments both NK and NKT cell activity (29) and CTL development in vitro (30). In vivo, the antitumor effects of IL-18 have been studied in a number of tumor-bearing mouse models (31, 32, 33, 34, 35, 36). In these models, systemic administration of IL-18 (31, 32, 33, 34) or in vivo IL-18 gene transfer (35, 36) inhibited tumor growth and prolonged the survival of tumor-bearing mice by mechanisms not precisely defined; nevertheless, at least in some models, these included involvement of NK and T cells (33, 36). In a recent report, dendritic cells genetically modified to secrete IL-18 were found to promote antitumor activity in a mouse sarcoma model by inducing Th1-type immunity (37).

Both IL-12 and IL-18 have been demonstrated to act synergistically to enhance in vitro cytotoxicity against tumor targets and IFN-{gamma} production mediated by either NKT or NK cells (7, 38, 39). Moreover, in vivo administration of IL-18 has been shown to potentiate the antitumor effects mediated by locally secreted IL-12 (40). In this latter model, the synergistic effect of these cytokines was primarily mediated by NK cells and indirectly by CTL. B16 melanoma cells cotransfected with IL-12 and IL-18 genes were readily rejected when transplantated in syngeneic animals by mechanisms including activation of NK and CTL cytotoxicities (41), and combined systemic administration of IL-18 and IL-12 has been demonstrated to stimulate the antitumor activity of dendritic cells pulsed with tumor cDNA (42). Finally, in a most recent work, Hashimoto et al. (43) using NK-depleted mice or V{alpha} NKT cell-deficient animals indicated that conventional NK, but not NKT cells are major effectors in IL-18-induced innate immunity.

Detailed analysis of the in vivo cellular mechanisms induced by IL-18 alone or in combination with IL-12, resulting in inhibition of tumor growth, has been hampered by the fact that these cytokines were administered exogenously or were secreted locally by gene-modified tumor cells, thereby influencing several immune pathways of which only few were analyzed (i.e., those concerning cytotoxic responses). In the present study, we activated in vitro highly purified NK or NKT lymphocytes by a mixture of IL-12 and IL-18, which were subsequently transferred into B6 normal, NKT-deficient (NKT-/-), or SCID mice inoculated with syngeneic tumors. By performing such experiments, we were able to demonstrate that NKT cells when activated in vitro with a combination of IL-12 and IL-18 are sufficient, via an IL-2- and IFN-{gamma}-dependent collaboration with the host’s own NK cells, to inhibit tumor progression in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Inbred (B6) mice were purchased from the animal breeding facility of the Hellenic Pasteur Institute (Athens, Greece), and B6.J{alpha}281-/- mice from M. Taniguchi (Chiba University School of Medicine, Chiba, Japan). B6.CB17-Prkdcscid (B6.scid) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice aged 6–8 wk old were used in all experiments, which were performed according to animal experimental ethics committee guidelines.

Cell lines

The two tumors used in this study are of B6 origin. MC57X is a methylcholanthrene-induced fibrosarcoma, and ALC is a T cell lymphoma induced by radiation leukemia virus D-RadLV. Both cell lines were provided by R. Kiessling (Department of Oncology and Pathology, Karolinska Hospital, Stockholm, Sweden). ALC was propagated by i.p. transfer in B6 mice, and it grew as an ascites tumor. MC57X was propagated by s.c. injection of cell suspensions prepared by passing the tumor tissue through a wire mesh in RPMI 1640 medium (Life Technologies, Grand Island, NY). In the experiments described below, ALC or MC57X cells were inoculated s.c. in the right flank and grew as solid tumors at the injection site.

Cell preparation

Mononuclear cells were obtained from spleens and liver of mice by purification over Histopaque-1083 (Sigma-Aldrich, St. Louis, MO). DX-5+ (NK.1.1+) cells were purified from murine mononuclear cells by positive selection using anti-DX-5 microbeads, according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). We targeted the DX-5 Ag for isolating total NK cells because this is much less mouse strain specific than the NK cell marker NK1.1. DX-5+ NK cells isolated by the same procedure were successfully used to study NK cell-mediated cytotoxicity in normal and immunodeficient mice in vitro (44) or by adoptive transfer (45). NK1.1+ CD3+ (NKT) and NK 1.1+ CD3- (NK) were further separated from total DX-5+ cells by cell sorting on an ALTRA cell sorter (Coulter, Hialeah, FL) using FITC-conjugated anti-CD3 mAb (clone 145-2C11) and PE-conjugated anti-NK 1.1 mAb (clone PK 136), both purchased from BD PharMingen (San Diego, CA). After sorting, NKT cells were >98% NK 1.1+ and >98% CD3+, whereas NK cells were >98% NK1.1+ and <1% CD3+. By applying this method, we have been able to isolate 2–2.5 x 106 NKT cells and 4–5 x 106 NK cells per mouse.

Cell cultures

Highly purified NKT or NK cells (1 x 106 cells/ml) were cultured in 14-ml conical tubes (Costar, Cambridge, MA) in RPMI 1640 culture medium supplemented with 10% FCS (Life Technologies), 2 mM L-glutamine (Sigma-Aldrich), 5 x 10-5 M 2-ME (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich) (complete medium). Cytokines were added at the following final concentrations: murine rIL-18 at 100 ng/ml (Peprotech EC, London, U.K.) and murine rIL-12 at 10 ng/ml (R&D Systems, Abington, U.K.). The doses of each of the cytokines used in this study have been assayed for optimal stimulation of NKT cells in preliminary experiments (data not shown). One day later, supernatants were removed for cytokine (IL-2 and IFN-{gamma}) determination using specific ELISA kits (R&D Systems). The cultured NKT cells were then washed once in RPMI 1640, resuspended in fresh complete medium, placed in 50-µl aliquots into wells of 96-well U-bottom plates (Costar), and incubated in CO2 incubators for 48 h with 50 µl containing equal number of freshly isolated syngeneic NK cells or NKT cells (as a control). In some experiments, NK cells were added in the IL-12/IL-18 NKT cells with mixtures of anti-IL-2 plus anti-IFN-{gamma} mAb, each at 10 µg/ml final concentration (included in the 50-µl aliquots). The same mAb were used for in vivo IL-2 and IFN-{gamma} neutralization (see below).

At the end of the incubation, 100-µl aliquots containing 5 x 103 51Cr-labeled tumor targets were added to each culture at the indicated E:T ratios, and incubation was prolonged for another 12 h. Labeling of tumor targets and estimation of percentage of cytotoxicity have been described in detail elsewhere (46).

Tumor rejection models

Mice were inoculated s.c. with 2 x 104 MC57X or ALC tumor cells, each in 0.5 ml PBS. Injections with IL-12/IL-18 NKT cells, which have been precultured for 24 h with IL-12/IL-18, were administered i.p. simultaneously with tumor cells on day 0 and on days 1 and 2 thereafter (0.5 x 106 cells/injection/mouse). Tumor growth in mice receiving only tumor cells was first observed 3 wk after tumor inoculation (palpable tumor), and mice were monitored for another 5 wk. The observation was terminated with the euthanasia of mice when the tumor mass grew up to 1.5 cm in diameter (usually between 60 and 70 days after tumor inoculation). Mice treated with cytokine-derived NKT effector cells were considered free of tumor if there was no palpable tumor at the site of injection during the time period of 8 wk. In vivo depletion of NK cells only was performed by injections of anti-asialo GM1 Ab (Cedarlane Laboratories, Ontario, Canada) (20–30 µl per injection) on days 1 and 0 (day of tumor inoculation) and weekly thereafter (47). Assessment of cell depletion was performed following preparation of cell suspensions from spleen and liver, as described (14, 47). This protocol was shown to maintain significant depletion of the NK population: in a total of seven mice tested, the percentage of NK cells was reduced from 3.5–5.0% in the spleens of untreated mice to 0.2–0.4%, and from 6–8% in their livers to 0.5–1.0% after treatment, whereas the levels of NKT cells remained unaffected (0.8–1.3% in the spleen and 20–30% in the liver). For in vivo IL-2 and/or IFN-{gamma} neutralization, mice received per injection 100 µg anti-mouse IL-2 mAb (clone JES6-1A12, IgG1) and/or 100 µg anti-mouse IFN-{gamma} mAb (clone 37895/11, IgG2{alpha}), both purchased from R&D Systems. Injections were given i.v. in 0.2 ml PBS on day 0 and weekly thereafter. Rat IgG1 (clone R3-34) and rat IgG2{alpha} (clone R35-95), both from BD PharMingen, were administered as isotype-matched controls for anti-IL-2 and anti-IFN-{gamma} mAb, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NK1.1+CD3+ (NKT) cells activated in vitro with IL-12 plus IL-18 and adoptively transferred into syngeneic mice induce protection against the growth of ALC and MC57X tumors

We (5) and others (45) have shown that both ALC lymphoma (H-2b) and MC57X fibrosarcoma (H-2b) grow progressively slowly when injected into syngeneic animals at relatively low numbers (i.e., 1 x 103 to 2 x 104 cells), developing large solid tumors (1.5–2.0 cm diameter) after 6–7 wk. Adaptive immunity against these tumors can be generated following active immunization with irradiated ALC or MC57X tumor cells, whereby C57BL/6 mice develop T cell-mediated antitumor immune responses resulting in tumor rejection (48, 49). However, a clear picture as to how innate immunity can control the growth of these tumors has been lacking. Considering the important role of NKT cells in immunosurveillance of sarcomas (50) as well as the potential of IL-12 plus IL-18 to activate NKT cells independently of TCR engagement (38), we sought to induce protection in B6 recipients inoculated with ALC or MC57X tumors by adoptive transfer of NKT cells, preincubated in vitro with a combination of IL-12 and IL-18. The vast majority of ALC-injected B6 mice and all B6 mice inoculated with MC57X syngeneic tumors demonstrated complete inhibition of tumor growth when treated with syngeneic NKT cells (Fig. 1). Single cytokine incubation (i.e., IL-12 or IL-18) did not induce significant protection (Fig. 1). In contrast to normal mice, B6 mice specifically depleted of NK cells using anti-asialo GM1 injections were not protected against ALC or MC57X tumor growth upon treatment with IL-12/IL-18 NKT cells (Fig. 2). This suggested that host NK cells in synergy with the adoptively transferred syngeneic IL-12/IL-18 NKT cells were indispensable for innate protection from ALC- and MC57X-induced tumor formation. To investigate the role of endogenous NKT cells in these tumor models, we have used B6.J{alpha} 281-/- and B6.scid mice, both of which lack NKT cells. As shown in Fig. 2, both types of strains were protected upon transfer of IL-12/IL-18 NKT cells. As with normal B6 mice, treatment with anti-asialo GM1 rendered B6.J{alpha} 281-/- and B6.scid mice susceptible to tumor growth. These data suggested that neither endogenous NKT nor T cells (which are also absent in B6.scid mice) played a significant role in tumor rejection.



View larger version (60K):
[in this window]
[in a new window]
  		FIGURE 1. Adoptive transfer of NKT cells cultured in vitro with IL-12 plus IL-18 (IL-12/IL-18 NKT) protects B6 mice against ALC and MC57X tumor growth. Groups of B6 mice were inoculated s.c. with 2 x 104 ALC or MC57X tumor cells (day 0). Subgroups of B6 mice received then purified NKT cells (0.5 x 106 cells/mouse) preincubated for 24 h either in plain medium (NKT), or with 10 ng/ml rIL-12 (IL-12/NKT), 100 ng/ml rIL-18 (IL-18/NKT), or 10 ng/ml IL-12 plus 100 ng/ml IL-18 (IL-12/IL-18 NKT) via i.v. adoptive transfer (i.e., three injections per mouse starting on day 0 and for the following 2 days). Tumor development was recorded at 8 wk as a percentage of the mice in each group (in parentheses). Significant difference from the PBS-treated groups (B6) was determined using a Fisher’s exact test. *, p < 0.01. Mean values (±SD) from two independently performed experiments are shown.

 


View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 2. Depletion of NK cells in vivo by anti-asialo GM1 (anti-GM1) treatment abrogates the protective effect of the adoptively transferred IL-12/IL-18 NKT cells. Wild-type mice (B6), B6.J{alpha}281-/- mice, or B6.scid mice inoculated with ALC or MC57X tumor cells received injections with anti-GM1 during and after treatment with IL-12/IL-18 NKT cells, as described in Materials and Methods. Significant difference from mice that received no anti-GM1 injections was determined using a Fisher’s exact test. *, p < 0.01. Mean values (±SD) from two independently performed experiments are shown.

 
IL-12/IL-18 NKT cells produce IFN-{gamma} and IL-2 to activate NK cells for antitumor activity

Recent reports have demonstrated the capacity of NKT cells to mediate antitumor cytotoxicity directly through interaction with the target cells or indirectly by producing IFN-{gamma} or IL-2 to activate NK cells (12, 13). To test the potential mechanisms regulating potent antitumor innate immunity mediated by IL-12/IL-18 NKT cells in our experimental tumor models, we injected tumor cell-inoculated normal B6 mice with IL-2- and/or IFN-{gamma}-neutralizing mAb during and after treatment with IL-12/IL-18 NKT cells. We have found (Fig. 3) that upon treatment with anti-IL-2 or anti-IFN-{gamma}, the percentage of B6 mice with ALC- or MC57X-induced tumors increased to almost 60%, whereas combined administration of anti-IL-2 and anti-IFN-{gamma} mAbs totally eliminated the IL-12/IL-18 NKT-mediated protective effect (i.e., 100% of the mice developed tumors).



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 3. Combined treatment with anti-IL-2 plus anti-IFN-{gamma} mAbs inhibits the protective effect of the adoptively transferred IL-12/IL-18 NKT cells. Injections with the cytokine-specific mAbs or their isotype-matched controls (IgG1 + IgG2{alpha}) were given, as described in Materials and Methods. Significant differences with B6 mice that were treated only with IL-12/IL-18 NKT cells were determined using a Fisher’s exact test. **, p <= 0.05; *, p < 0.01. Mean values (±SD) from two independently performed experiments are shown.

 
The impact of IL-12 plus IL-18 on IFN-{gamma} and IL-2 production by the activated NKT cells was confirmed by quantitating the levels of both cytokines in vitro. After 24 h of incubation in the presence of IL-12 plus IL-18, NKT cells produced high levels of both IL-2 and IFN-{gamma} (Fig. 4). Significantly lower levels of both IL-2 and IFN-{gamma} were produced when NKT cells were incubated with each of the cytokines alone (data not shown). The same cells when coincubated for another 48 h with freshly isolated syngeneic NK cells induced lysis of both ALC and MC57X tumor targets (Fig. 4) at fairly high levels (50–60% cytotoxicity against both tumor targets at an E:T = 40), which were drastically reduced when mixtures of anti-IL-2 plus anti-IFN-{gamma} mAb were present throughout the 48-h incubation (Fig. 3). In addition, only low levels of tumor cell lysis were observed when NKT cells were added to the IL-12/IL-18 NKT cells (25 and 19% cytotoxicity at an E:T = 40 against ALC and MC57X tumor targets, respectively). These data suggested that IL-12/IL-18 NKT cells mainly act as regulatory cells producing IL-2 and IFN-{gamma} to induce NK cell-mediated cytotoxicity, although they themselves may also act as effectors, by lysing their targets, however, at significantly lower levels.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 4. In vitro activation of highly purified NKT cells with IL-12 plus IL-18. IL-2 and IFN-{gamma} production was determined in culture supernatants (Sup) of NKT cells incubated for 24 h with medium (medium NKT) or with IL-12 + IL-18 (IL-12/IL-18 NKT). Both medium NKT and IL-12/IL-18 NKT were then cultured for another 48 h with syngeneic NK or NKT cells in the presence or absence of anti-IL-2 plus anti-IFN-{gamma} mAb. Upon addition of ALC or MC57X targets, incubation was prolonged for 12 h, at which time point percentage of cytotoxicity was estimated. For more details, see Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented in this work demonstrated for the first time immunotherapy of syngeneic tumors by adoptively transferred NKT cells activated in vitro by a combination of IL-12 plus IL-18. They also show that in vivo collaboration between the transferred IL-12/IL-18 NKT cells with the host’s own NK cells is required for effective host protection from tumor growth. Furthermore, we demonstrate that IL-2 and IFN-{gamma} produced by the IL-12/IL-18-activated NKT cells are critical components in natural antitumor immunity.

To date, most studies addressing the role of NKT cells in various experimental models of immune response have investigated pathways leading to their activation upon TCR engagement (16). However, only a restricted set of Ags has been found to stimulate NKT cells via TCR in a CD1-dependent manner (11, 12). In apparent contrast with the above studies, NKT cells have also been demonstrated to promote potent tumor rejection in response to exogenous factors in a TCR-independent manner. In a recently published work, Leite-de-Moraes et al. (38) demonstrated that NKT cells can be fully activated in the absence of TCR cross-linking, in response to IL-18 associated with IL-12. In the same report, it was found that NKT cells stimulated with IL-12 and IL-18 produced high levels of IFN-{gamma} and expressed increased cytotoxic potential against Fas-positive targets. No TCR engagement in NKT cells has also been demonstrated in IL-12-induced tumor rejection models (29, 50) in which NKT cells played an essential role. In these tumor models, IFN-{gamma} and/or perforin were required for IL-12-induced and NKT cell-mediated antitumor immunity. These results agree with the TCR-independent stimulation of NKT cells in our model and imply that several death-inducing ligands must be involved in the capacity of NKT cells to induce target cell apoptosis.

Although NKT cell activation in our system does not require treatment with an exogenous CD1d-binding ligand, we still cannot exclude the possibility that the adoptively transferred IL-12/IL-18 NKT cells may recognize tumor-derived material presented to them in the context of CD1d expressed either by the tumor cell themselves or by host APCs. Crowe et al. (50) have most recently shown that NKT cells in the absence of exogenous stimulatory factors play a significant role in immunosurveillance of 3-methylcholanthrene (MCA) 3-induced sarcomas, and that this effect depends on CD1d recognition and requires additional involvement of NK cells. Although NK cells are known to be activated following stimulation of NKT cells by {alpha}-galactosylceramide ({alpha}-GalCer) (51, 52, 53), no evidence for activation of NKT cells by {alpha}-GalCer was provided in the above-mentioned study (50). Thus, it seems that in addition to activation with {alpha}-GalCer or IL-12, NKT cells can be stimulated to exert potent antitumor effectors by a variety of TCR-dependent or TCR-independent stimuli that may not be mutually exclusive, that is, NKT cells can be activated both through recognition of CD1d-bound ligands and through the action of a single cytokine or cytokine combination. With respect to this, several studies suggest that dendritic cells upon contact with NKT cells via TCR-CD1d interaction are stimulated to produce IL-12 that further activates NKT cells to lyse tumor targets (54, 55). Further work will be required to precisely determine the dynamics between dendritic cells and NK as well as NKT cells in vivo in our tumor model.

Certainly, the data described in this report do not necessarily ascribe a direct in vivo antitumor effect to the transferred IL-12/IL-18 NKT cells, because tumor growth inhibition was not observed when mice were depleted of endogenous NK cells. In addition, our in vitro data clearly demonstrate efficient tumor cell lysis only when the IL-12/IL-18 NKT cells are coincubated with syngeneic NK cells. Thus, we could assume that the cytokine-induced NKT cells mediate antitumor cytotoxicity indirectly by producing IFN-{gamma} and IL-2 to activate NK cells. A dual function of NKT cells was recently reported by Metelitsa et al. (12), who found that NKT cells recognizing CD1d-bound ligands on tumor targets are stimulated to perform cytotoxicity and also to produce IL-2 that is required for inducing cytotoxic responses in NK cells. In the same report, it was shown that IFN-{gamma} synergistically acted with IL-2 to enhance NK cell cytotoxicity. The likelihood that IFN-{gamma} produced by NKT cells may also lead to NK cell activation was shown in earlier studies in which in vivo injection of {alpha}-GalCer in wild-type mice resulted in IFN-{gamma} production and CD69 induction by NK cells. This, however, was not the case when {alpha}-GalCer was injected in RAG- or CD1-deficient mice or in wild-type mice pretreated with anti-IFN-{gamma} Abs (56), which suggested that NK cell activation was a secondary event that depended on IFN-{gamma} release by NKT cells. Given the fact that upon a 24-h incubation with IL-12/IL-18 our NKT cells produce both IL-2 and IFN-{gamma}, we suggest that these may have a putative regulatory role by activating syngeneic NK cells. Of course, we cannot rule out the possibility that the critical contribution of IL-12/IL-18 NKT cells in vivo may be direct tumor cell lysis. Our in vitro data demonstrated low cytotoxicity levels against both ALC and MC57X tumor targets mediated by the IL-12/IL-18 NKT effectors. However, the in vitro data do not necessarily reflect the functional status of effector cells in vivo. For example, cytokine-induced CD3+CD56+ killer cells were demonstrated to mediate superior antitumor effects in vivo, although their in vitro cytotoxicity levels against the same tumor cells were somehow lower compared with those mediated by lymphokine-activated killer cells (57). This is not to say that the IL-12/IL-18 NKT cells protect mice from tumor development by direct lysis, as our data indicate that most of the antitumor cytotoxicity is conducted by NK cells. However, direct tumor cell lysis in vivo by the cytokine-induced NKT cells through feedback stimulation by the already activated NK cells or through TCR-CD1d interactions cannot be excluded. In this respect, it would also be important to examine the in vivo antitumor activity of IL-12/IL-18 NK cells in relation to trans activation of NKT cells. Work is now in progress in our laboratory to address these points.

Sequential production of IFN-{gamma} by {alpha}-GalCer- or IL-12-activated NKT cells and NK cells has to date been reported to be essential for the innate control of MCA-induced sarcoma (50). In this study, we provide additional information on how tumors can be innately controlled by both NKT and NK cells by demonstrating that upon IL-12/IL-18 activation, NKT cells produce both IL-2 and IFN-{gamma}, which synergistically act to induce NK cells to mediate antitumor cytotoxicity. At present, we have not determined whether IFN-{gamma} alone could have a direct effect, or, as in agreement with earlier reports (58, 59), only enhances NK cytotoxicity induced by IL-2. IL-2 is well known to directly activate NK cell proliferation and cytotoxicity (60, 61), and, combined with IL-2, IFN-{gamma} may also augment NK cell activation (62, 63). Moreover, IFN-{gamma} produced by NKT cells in vivo may indirectly stimulate NK cell mobilization from blood (60) and induce NK cells themselves to produce IFN-{gamma} (56). However, IFN-{gamma} is not the sole effector molecule produced by the activated NK cells to confer antitumor immunity. It has been reported that NKT cell-dependent activation of NK cells may also result in enhanced perforin and TNF-{alpha} production (4, 13, 64), both of which may, additionally to IFN-{gamma}, confer antitumor immunity (5, 13). Therefore, it will be important not only to determine the nature of signals that activate NKT cells, i.e., whether they are either tumor cell dependent (i.e., glycolipid ligands) or independent (e.g., cytokines), but also the signals that regulate the outcome of NKT function to be able to manipulate immune responses toward effective tumor destruction.

Cytokine-induced NKT cell activation has been performed to date in cultures supplemented with 10% FCS or 10% human AB serum (3, 38, 39), thus raising the possibility that serum factors might interfere in these activation pathways. To address this, we repeated our in vitro experimentations also in protein-free hybridoma medium. NKT cells grown in this medium in the presence of IL-12/IL-18 produced comparable high levels of IFN-{gamma} and IL-2 in vitro and were as effective in vivo (data not shown) as the IL-12/IL-18-activated NKT cells described in this work that were grown in RPMI 1640 supplemented with 10% FCS. These data suggested that the activation of our NKT cells was dependent solely on the synergistic action of these cytokines.

NK and NKT cells have been shown to contribute to IL-12-induced immunity against tumors. For example, metastasis of the EL4-S3 lymphoma was strictly controlled by NK cells following therapy with high-dose IL-12 (29). Intriguingly, in melanoma B16F10 and lung tumor RM-1 models, both NK and NKT cells contributed to natural protection from tumor metastasis (29). In those models, a lower dose of IL-12 or delayed administration of IL-12 dictated a greater relative role of NKT cells in immune protection from tumor metastasis. A similar situation apparently also applies for the IL-18-induced antitumor immunity. Thus, although NK, but not NKT, cells were demonstrated to play an essential role in the reduction of B16 pulmonary metastases (43), a preceding report suggested that perforin-dependent cytotoxicity of liver NKT cells is augmented in vitro by IL-18 stimulation (65). Thus, it appears that the relative roles of each population are tumor and therapy dependent. In addition, the activation of NK and NKT by IL-12 or IL-18 may follow different kinetics depending on the expression of the cytokine-specific receptors on either subset. To this end, naive NK cells were found to express at high levels the IL-18R, whereas NKT cells did so only after IL-12 stimulation (43). Thus, our in vitro model for NKT cell activation with the combination of IL-12 plus IL-18 is in line with those reports. At present, it is not known whether the observed collaboration between IL-12/IL-18-activated NKT cells with syngeneic NK cells would be as efficient in metastatic tumor models. Smyth et al. (29) recently reported that in the i.v. RM-1 lung tumor model, NKT (in addition to NK) cells appeared to also contribute to regression of metastatic lesions. We are currently working on the establishment of lung and/or liver metastatic tumor models via i.v. injections of ALC or MC57X tumor cells to investigate this point.

Our work provides the first evidence that NKT cells activated in vitro with a combination of IL-12 plus IL-18 may be critical in natural immune responses against the growth of the MCA-induced MC57X fibrosarcoma and the ALC lymphoma. These results demonstrate the significance of a novel therapeutic mechanism and encourage clinical investigation of the immunoregulatory and antitumor activities of IL-12/IL-18-activated NKT cells.


   Acknowledgments
 
We thank Joanna Doukoumopoulou for her excellent secretarial assistance.


   Footnotes
 
1 This work was supported by a grant from the Regional Operational Program Attika No. 20, MIS Code 59605GR, to M.P. Back

2 Address correspondence and reprint requests to Dr. Constantin N. Baxevanis, Cancer Immunology and Immunotherapy Center, St. Savas Hospital, 171 Alexandras Avenue, 11522 Athens, Greece. E-mail address: baxevani{at}ath.forthnet.gr Back

3 Abbreviations used in this paper: MCA, 3-methylcholanthrene; {alpha}-GalCer, {alpha}-galactosylceramide. Back

Received for publication February 3, 2003. Accepted for publication July 16, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cerwenka, A., L. L. Lanier. 2001. Natural killer cells, viruses and cancer. Nat. Rev. Immunol. 1:41.[Medline]
  2. Chuang, S. S., P. R. Kumaresan, P. A. Mathew. 2001. 2B4 (CD244)-mediated activation of cytotoxicity and IFN-{gamma} release in human NK cells involves distinct pathways. J. Immunol. 167:6210.[Abstract/Free Full Text]
  3. Fehniger, T. A., M. H. Shah, M. J. Turner, J. B. Van Densen, S. P. Whitman, M. A. Cooper, K. Suzuki, M. Wechser, F. Goodsaid, M. A. Caligiuri. 1999. Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response. J. Immunol. 162:4511.[Abstract/Free Full Text]
  4. Smyth, M. J., Y. Hayakawa, K. Takeda, H. Yagita. 2002. New aspects of natural-killer-cell surveillance and therapy of cancer. Nat. Rev. Immunol. 2:850.
  5. Baxevanis, C. N., I. F. Voutsas, O. E. Tsitsilonis, M. L. Tsiatas, A. D. Gritzapis, M. Papamichail. 2000. Compromised antitumor responses in tumor necrosis factor-{alpha} knockout mice. Eur. J. Immunol. 30:1957.[Medline]
  6. Cooper, M. A., T. A. Fehniger, A. Ponnapan, V. Mehta, M. D. Wewers, M. A. Caligiuri. 2001. Interleukin-1{beta} costimulates interferon-{gamma} production by human natural killer cells. Eur. J. Immunol. 31:792.[Medline]
  7. Lauwerys, B. R., N. Garot, J.-C. Renauld, F. A. Houssian. 2000. Cytokine production and killer activity of NK/T-NK cells derived with IL-2, IL-15, or the combination of IL-12 and IL-18. J. Immunol. 165:1847.[Abstract/Free Full Text]
  8. Dunne, J., S. Lynch, C. O’ Farrelly, S. Todryk, J. E. Hegarty, C. Feighery, D. G. Doherty. 2001. Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15. J. Immunol. 167:3129.[Abstract/Free Full Text]
  9. Son, Y.-I., R. M. Dallal, R. B. Mailliard, S. Egawa, Z. L. Jonak, M. T. Lotze. 2001. Interleukin-18 (IL-18) synergizes with IL-2 to enhance cytotoxicity, interferon-{gamma} production, and expansion of natural killer cells. Cancer Res. 61:884.[Abstract/Free Full Text]
  10. Parrish-Novak, J., R. Dillon, A. Nelson, A. Hammond, C. Sprecher, J. A. Gross, J. Johnston, K. Madden, W. Xu, J. West, et al 2000. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature 408:57.[Medline]
  11. Kronenberg, M., L. Gapin. 2002. The unconventional lifestyle of NKT cells. Nat. Rev. Immunol. 2:557.[Medline]
  12. Metelitsa, L. S., O. V. Naidenko, A. Kant, H.-W. Wu, M. J. Loza, B. Perussia, M. Kronenberg, R. C. Seeger. 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.[Abstract/Free Full Text]
  13. Smyth, M. J., K. Y. Thia, S. E. Street, E. Cretney, J. A. Trapani, M. Taniguchi, T. Kawano, S. B. Pelikan, N. Y. Crowe, D. I. Godfrey. 2000. Differential tumor surveillance by natural killer (NK) and NKT cells. J. Exp. Med. 191:661.[Abstract/Free Full Text]
  14. Hammond, K. J. L., S. B. Pelikan, N. Y. Crowe, E. Randle-Barett, T. Nakayama, M. Taniguchi, M. J. Smyth, I. R. van Driel, R. Scollay, A. G. Baxter, D. I. Godfrey. 1999. NKT cells are phenotypically and functionally diverse. Eur. J. Immunol. 29:3768.[Medline]
  15. Slifka, M. K., R. R. Pagarigan, J. L. Whitton. 2000. NK markers are expressed on a high percentage of virus-specific CD8+ and CD4+ T cells. J. Immunol. 164:2009.[Abstract/Free Full Text]
  16. Soloski, M. J.. 2001. Recognition of tumor cells by the innate immune system. Curr. Opin. Immunol. 13:154.[Medline]
  17. Kambayashi, T., E. Assarsson, J. Michailsson, P. Berglund, A. D. Diehl, B. J. Chambers, H.-G. Ljunggren. 2000. Emergence of CD8+ T cells expressing NK cell receptors in influenza: a virus-infected mice. J. Immunol. 165:4964.[Abstract/Free Full Text]
  18. Baxevanis, C. N., A. D. Gritzapis, O. E. Tsitsilonis, H. L. Katsoulas, M. Papamichail. 2002. Her-2/neu-derived peptide epitopes are also recognized by cytotoxic CD3+CD56+ (natural killer T) lymphocytes. Int. J. Cancer 98:864.[Medline]
  19. Fehniger, T. A., M. A. Caligiuri. 2001. Interleukin 15: biology and relevance to human disease. Blood 97:14.[Free Full Text]
  20. Smyth, M. J., D. I. Godfrey, J. A. Trapani. 2001. A fresh look at tumor immunosurveillance and immunotherapy. Nat. Immun. 2:293.
  21. Nasaki, T., S. Matsui, N. Noben-Trauth, H. Chen, C. Watson, D. D. Donaldson, D. P. Carbone, W. E. Paul, J. A. Berzofsky. 2000. NKT cell-mediated regression of tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway. Nat. Immun. 1:515.[Medline]
  22. Trinchieri, G.. 1995. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adoptive immunity. Annu. Rev. Immunol. 13:251.[Medline]
  23. Trinchieri, G.. 1997. Cytokines acting on or secreted by macrophages during intracellular infection (IL-15, IL-12, IFN-{gamma}). Curr. Opin. Immunol. 9:17.[Medline]
  24. Brunda, M. J.. 1994. Interleukin-12. J. Leukocyte Biol. 55:280.[Abstract]
  25. Zou, J. P., N. Yamamoto, T. Fuki, H. Takenaka, M. Kobayashi, S. H. Herrmann, S. R. Wolf, H. Fujiwara, T. Hamaoka. 1995. Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-{gamma} production by antitumor T cells. Int. Immunol. 7:1135.[Abstract/Free Full Text]
  26. Brunda, M. J., L. Luistor, R. R. Warrier, R. B. Wright, B. R. Hubbard, M. Murphy, S. F. Wolf, M. K. Gately. 1993. Antitumor and antimetastatic activity of interleukin 12 against murine tumors. J. Exp. Med. 178:1223.[Abstract/Free Full Text]
  27. Robertson, M. J., J. Ritz. 1996. Interleukin 12: basic biology and potential applications in cancer treatment. Oncologist 1:88.[Abstract/Free Full Text]
  28. Okamura, H., H. Tsutsi, T. Komatsu, M. Yutsudo, A. Hakura, T. Taminoto, K. Torigoe, T. Okura, Y. Nukada, K. Hattori, et al 1995. Cloning of a new cytokine that induces IFN-{gamma} production by T cells. Nature 378:88.[Medline]
  29. Smyth, M. J., M. Taniguchi, S. E. Street. 2000. The antitumor activity of IL-12: mechanisms of innate immunity that are dose and model dependent. J. Immunol. 165:2665.[Abstract/Free Full Text]
  30. Okamoto, I., K. Kohno, T. Tanimoto, H. Ikegami, M. Kurimoto. 1999. Development of CD8+ effector T cells is differentially regulated by IL-18 and IL-12. J. Immunol. 162:3202.[Abstract/Free Full Text]
  31. Osaki, T., J. M. Peron, Q. Cai, H. Okamura, P. D. Robbins, M. Kurimoto, S. Nagata, M. T. Lotze, H. Tahara. 1998. IFN-{gamma}-inducing factor/IL-18 administration mediates IFN-{gamma}- and IL-12-independent antitumor effects. J. Immunol. 160:1742.[Abstract/Free Full Text]
  32. Micalleh, M. J., K. Yoshida, S. Kawai, T. Hamaya, K. Kohno, S. Arai, T. Tanimoto, K. Torigoe, M. Fujii, M. Ikeda, M. Kurimoto. 1997. In vivo antitumor effects of murine interferon-{gamma}-inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites. Cancer Immunol. Immunother. 43:961.
  33. Takeda, K., H. Tsutsui, T. Yoshimoto, O. Adachi, N. Yoshida, T. Kishimoto, H. Okamura, K. Nakanishi, S. Akira. 1998. Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 8:383.[Medline]
  34. Akamatsu, S., N. Arai, T. Hanaya, S. Arai, T. Tanimoto, M. Fujii, K. Kohno, M. J. Micallef, M. Ikeda, M. Kurimoto. 2002. Antitumor activity of interleukin-18 against the murine T-cell leukemia/lymphoma EL-4 in syngeneic mice. J. Immunother. 25:(Suppl. 1):28.
  35. Wang, Q., H. Hu, D. W. Ju, L. He, J. P. Pan, D. J. Xia, L. H. Zhang, X. Cao. 2001. Intratumoral IL-18 gene transfer improves therapeutic efficacy of antibody-targeted superantigen in established murine melanoma. Gene Ther. 8:542.[Medline]
  36. Ju, D. W., Y. Yang, Q. Tao, W. G. Song, L. He, G. Chen, S. Gu, C. C. Ting, X. Cao. 2000. Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity. Gene Ther. 19:1672.
  37. Tatsumi, T., A. Gambotto, P. D. Robbins, W. J. Storkus. 2002. Interleukin 18 gene transfer expands the repertoire of antitumor Th1-type immunity elicited by dendritic cell-based vaccines in association with enhanced therapeutic efficacy. Cancer Res. 62:5853.[Abstract/Free Full Text]
  38. Leite-de-Moraes, M. C., A. Hameg, A. Arnould, F. Machavoine, Y. Koezuka, E. Schneider, A. Herbelin, M. Dy. 1999. A distinct IL-18-induced pathway to fully activate NKT lymphocytes independently from TCR engagement. J. Immunol. 163:5871.[Abstract/Free Full Text]
  39. Hyodo, Y., K. Matsui, N. Hayashi, N. T., S. I. Tsutsui, H. Kashiwamura, K. Yamauchi, K. Hiroishi, Y.-I. Takeda, Y. Twakura Tagawa, et al 1999. IL-18 up-regulates perforin-mediated NK activity without increasing perforin messenger RNA expression by binding to constitutively expressed IL-18 receptor. J. Immunol. 162:1662.[Abstract/Free Full Text]
  40. Nagai, H., I. Hara, T. Horikawa, M. Fujii, M. Kurimoto, S. Kamidono, M. Ichihashi. 2000. Antitumor effects on mouse melanoma elicited by local secretion of interleukin-12 and their enhancement by treatment with interleukin-18. Cancer Invest. 18:206.[Medline]
  41. Kishida, T., H. Asada, E. Satoh, M. Shinya, H. Hirai, M. Iwai, H. Tahara, J. Imanishi, O. Mazda. 2001. In vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice. Gene Ther. 8:1234.[Medline]
  42. Yamanaka, R., N. Yajia, N. Tsuchiya, J. Homna, R. Tanaka, J. Ramsey, M. Blaese, K. G. Xanthopoulos. 2002. Administration of interleukin-12 and -18 enhancing the antitumor immunity of genetically modified dendritic cells that had been pulsed with Semliki forest virus-mediated tumor complementary DNA. J. Neurosurg. 97:1184.[Medline]
  43. Hashimoto, W., F. Tanaka, P. D. Robbins, M. Taniguchi, H. Okamura, M. T. Lotze, H. Tahara. 2003. Natural killer, but not natural killer T cells play a necessary role in the promotion of an innate antitumor response induced by IL-18. Int. J. Cancer 103:508.[Medline]
  44. Tomasello, E., P. O. Desmoulins, K. Chemin, S. Gruia, H. Cremer, J. Ortaldo, P. Love, D. Kaiserlian, E. Vivier. 2000. Combined natural killer and dendritic functional deficiency in KARAP/DAP12 loss-of-function mutant mice. Immunity 13:355.[Medline]
  45. Shi, F.-D., K. Takeda, S. Akira, N. Sarvetnick, H.-G. Ljunggren. 2000. IL-18 directs autoreactive T cells and promotes autodestruction in the central nervous system via induction of IFN-{gamma} by NK cells. J. Immunol. 165:3099.[Abstract/Free Full Text]
  46. Baxevanis, C. N., I. F. Voutsas, O. E. Tsitsilonis, A. D. Gritzapis, R. Sotiriadou, M. Papamichail. 2000. Tumor-specific CD4+ T lymphocytes from cancer patients are required for optimal induction of cytotoxic T cells against the autologous tumor. J. Immunol. 164:3902.[Abstract/Free Full Text]
  47. Smyth, M. J., N. Y. Crowe, D. I. Godfrey. 2000. NK cells and NKT cells collaborate in host protection from methyl-cholanthrene-induced fibrosarcoma. Int. Immunol. 13:459.
  48. Wen, T., K. Kono, A. Shahinian, R. Kiessling, T. W. Mak, G. Klein. 1997. CD28 is not required for rejection of unmanipulated syngeneic and autologous tumors. Eur. J. Immunol. 27:1988.[Medline]
  49. Wen, T., L. Truemper, W. Fung-Leung, A. Rahemtulla, E. Klein, G. Klein, T. W. Mak. 1998. Requirement of the CD8+ or CD4+ T lymphocyte subsets for the rejection of lymphoma and fibrosarcoma grafts studied in gene knockout hosts. Immunol. Lett. 61:187.[Medline]
  50. Crowe, N. Y., M. J. Smyth, D. I. Godfrey. 2002. A critical role for natural killer T cells in immunosurveillance of methylcholanthrene-induced sarcomas. J. Exp. Med. 196:119.[Abstract/Free Full Text]
  51. Hayakawa, Y., K. Takeda, H. Yagita, S. Katuta, Y. Iwakura, L. Van Kaer, I. Saiki, K. Okumura. 2001. Critical contribution of IFN-{gamma} and NK cells, but not perforin-mediated cytotoxicity, to anti-metastatic effect of {alpha}-galactosylceramide. Eur. J. Immunol. 31:1720.[Medline]
  52. Nakagawa, R., I. Nagafune, Y. Tazunoki, H. Ehara, H. Tomura, R. Iijima, K. Motoki, M. Kamishohara, S. Seki. 2001. Mechanisms of the anti-metastatic effect in the liver and of the hepatocyte injury induced by {alpha}-galactosylceramide in mice. J. Immunol. 166:6578.[Abstract/Free Full Text]
  53. Smyth, M. K., N. Y. Crowe, D. G. Pellici, K. Kygarissondis, J. M. Kelly, K. Takeda, H. Yagita, D. I. Godfrey. 2002. Anti-metastatic effect of {alpha}-galactosylceramide is mediated by sequential production of IFN-{gamma} by NKT and NK cells. Blood 99:1259.[Abstract/Free Full Text]
  54. Kitamura, H., K. Iwakabe, T. Yahata, S.-I. Nishimura, A. Ohta, Y. Ohmi, M. Sato, K. Takeda, K. Okumura, L. van Kaer, 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.[Abstract/Free Full Text]
  55. Fernadez, N. C., A. Lozier, C. Flament, P. Riccardi-Castagnoli, D. Belleti, M. Suter, M. Perricuadet, T. Tursz, E. Maraskovsky, L. Zitvogel. 1999. Dendritic cells directly trigger NK cell functions: cross-talk relevant on innate antitumor immune responses in vivo. Nat. Med. 5:405.[Medline]
  56. Carnand, C., D. Lee, O. Donnars, S.-H. Park, A. Beavis, Y. Koezuka, A. Bendelac. 1999. Cutting edge: cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. J. Immunol. 163:4647.[Abstract/Free Full Text]
  57. Lu, P.-H., R. S. Negrin. 1994. A novel population of expanded human CD3+CD56+ cells derived from T cells with potent in vivo antitumor activity in mice with severe combined immunodeficiency. J. Immunol. 153:1687.[Abstract]
  58. Sayers, T. J., L. H. Mason, T. A. Wiltrout. 1990. Trafficking and activation of murine natural killer cells: differing roles for IFN-{gamma} and IL-2. Cell. Immunol. 127:311.[Medline]
  59. Sayers, T. J., A. T. Mason, J. R. Ortaldo. 1986. Regulation of human natural killer cell activity by interferon-{gamma}: lack of a role in interleukin-2-mediated augmentation. J. Immunol. 136:2176.[Abstract]
  60. Lotze, M. T., E. A. Grimm, A. Mazumder, J. L. Strausser, S. A. Rosenberg. 1981. Lysis of fresh and cultured autologous tumor by human lymphocytes cultured in T-cell growth factor. Cancer Res. 41:4420.[Abstract/Free Full Text]
  61. Rosenstein, M., I. Yron, Y Kaufmann, S. A. Rosenberg. 1984. Lymphokine-activated killer cells: lysis of fresh syngeneic natural killer-resistant murine tumor cells by lymphocytes cultured in interleukin 2. Cancer Res. 44:1946.[Abstract/Free Full Text]
  62. Frey, J. R., M. Kamber, R. Peck. 1987. Recombinant interferons or interleukin-2 increase cytotoxicity by human monocytes and NK cells. Lymphokine Res. 6:215.[Medline]
  63. Ochoa, A. C., G. Gromo, B. J. Alter, P. M. Sondel, B. H. Bach. 1987. Long-term growth of lymphokine-activated killer (LAK) cells: role of anti-CD3, {beta}-IL-1, interferon-{gamma} and -{beta}. J. Immunol. 138:2728.[Abstract]
  64. Smyth, M. J., J. M. Kelly, A. G. Baxter, H. Korner, J. D. Sedgwick. 1998. An essential role for tumor necrosis factor in natural killer cell-mediated tumor rejection in the peritoneum. J. Exp. Med. 108:1611.
  65. Dao, T., W. Z. Mehal, I. N. Crispe. 1998. IL-18 augments perforin-dependent cytotoxicity of liver NK-T cells. J. Immunol. 161:2217.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
S. Bessoles, F. Fouret, S. Dudal, G. S. Besra, F. Sanchez, and V. Lafont
IL-2 triggers specific signaling pathways in human NKT cells leading to the production of pro- and anti-inflammatory cytokines
J. Leukoc. Biol., July 1, 2008; 84(1): 224 - 233.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. Hyka-Nouspikel, L. Lucian, E. Murphy, T. McClanahan, and J. H. Phillips
DAP10 Deficiency Breaks the Immune Tolerance against Transplantable Syngeneic Melanoma
J. Immunol., September 15, 2007; 179(6): 3763 - 3771.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Cheng, A. Ueno, S. Cho, J. S. Im, S. Golby, S. Hou, S. A. Porcelli, and Y. Yang
Efficient Activation of V{alpha}14 Invariant NKT Cells by Foreign Lipid Antigen Is Associated with Concurrent Dendritic Cell-Specific Self Recognition
J. Immunol., March 1, 2007; 178(5): 2755 - 2762.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. M. Coquet, K. Kyparissoudis, D. G. Pellicci, G. Besra, S. P. Berzins, M. J. Smyth, and D. I. Godfrey
IL-21 Is Produced by NKT Cells and Modulates NKT Cell Activation and Cytokine Production
J. Immunol., March 1, 2007; 178(5): 2827 - 2834.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
D. S. Dace, P. W. Chen, H. Alizadeh, and J. Y. Niederkorn
Ocular immune privilege is circumvented by CD4+ T cells, leading to the rejection of intraocular tumors in an IFN-{gamma}-dependent manner
J. Leukoc. Biol., February 1, 2007; 81(2): 421 - 429.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. J. Subleski, V. L. Hall, T. C. Back, J. R. Ortaldo, and R. H. Wiltrout
Enhanced Antitumor Response by Divergent Modulation of Natural Killer and Natural Killer T Cells in the Liver.
Cancer Res., November 15, 2006; 66(22): 11005 - 11012.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. D. Gritzapis, L. G. Mahaira, S. A. Perez, N. T. Cacoullos, M. Papamichail, and C. N. Baxevanis
Vaccination with Human HER-2/neu (435-443) CTL Peptide Induces Effective Antitumor Immunity against HER-2/neu-Expressing Tumor Cells In vivo.
Cancer Res., May 15, 2006; 66(10): 5452 - 5460.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. N. MacGregor, Q. Li, A. E. Chang, T. M. Braun, D. P.M. Hughes, and K. T. McDonagh
Ex vivo Culture with Interleukin (IL)-12 Improves CD8+ T-Cell Adoptive Immunotherapy for Murine Leukemia Independent of IL-18 or IFN-{gamma} but Requires Perforin.
Cancer Res., May 1, 2006; 66(9): 4913 - 4921.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
B. Acres, M. Gantzer, C. Remy, N. Futin, N. Accart, O. Chaloin, J. Hoebeke, J.-M. Balloul, and S. Paul
Fusokine Interleukin-2/Interleukin-18, a Novel Potent Innate and Adaptive Immune Stimulator with Decreased Toxicity
Cancer Res., October 15, 2005; 65(20): 9536 - 9546.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. D. Wesley, S. H. Robbins, S. Sidobre, M. Kronenberg, S. Terrizzi, and L. Brossay
Cutting Edge: IFN-{gamma} Signaling to Macrophages Is Required for Optimal V{alpha}14i NK T/NK Cell Cross-Talk
J. Immunol., April 1, 2005; 174(7): 3864 - 3868.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Q. Li, A. L. Carr, E. J. Donald, J. J. Skitzki, R. Okuyama, L. M. Stoolman, and A. E. Chang
Synergistic Effects of IL-12 and IL-18 in Skewing Tumor-Reactive T-Cell Responses Towards a Type 1 Pattern
Cancer Res., February 1, 2005; 65(3): 1063 - 1070.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Baxevanis, C. N.
Right arrow Articles by Papamichail, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Baxevanis, C. N.
Right arrow Articles by Papamichail, M.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS