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-Galactosylceramide
Demonstrates Its Immunopotentiating Effect by Inducing
Interleukin (IL)-12 Production by Dendritic Cells and
IL-12 Receptor Expression on NKT Cells
By
From the * Section of Genetic Engineering, Research Center for Genetic Engineering and Cell
Transplantation, and Department of Immunology, Tokai University School of Medicine, Isehara
259-1193, Japan; the § Division of Biological Sciences, Graduate School of Science, Hokkaido University,
Sapporo 060-0810, Japan; the Department of Immunology, Juntendo University School of Medicine,
Tokyo 113-0033, Japan; the ¶ Howard Hughes Medical Institute, Department of Microbiology and
Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and ** Core
Research for Evolutional Science and Technology (CREST) Project and Department of Molecular
Immunology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The natural killer T (NKT) cell ligand 
-galactosylceramide (-GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12-mediated antitumor activities. Because of these similarities between the activities of -GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by -GalCer. We first established, using
purified subsets of various lymphocyte populations, that -GalCer selectively activates NKT
cells for production of interferon (IFN)-
. Production of IFN- by NKT cells in response to
-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT
cells and DCs through CD40/CD40 ligand interactions. Moreover, -GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1
/ or
V14/ mice. This effect of -GalCer required the production of IFN- by NKT cells and
production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses
of -GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN- production. Collectively, these findings indicate an important role
for DC-produced IL-12 in the activation of NKT cells by -GalCer and suggest that NKT
cells may be able to condition DCs for subsequent immune responses. Our results also suggest
a novel approach for immunotherapy of cancer.
-galactosylceramide;
interleukin 12;
interleukin 12 receptor
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Natural killer T (NKT)1 cells represent a novel lymphoid lineage distinct from mainstream T cells, B
cells, and NK cells. NKT cells are characterized by the expression of an invariant TCR encoded by V14 and J281
gene segments and V
8, 7, or 2 gene segments (1, 2). It
was demonstrated recently that NKT cells are strongly
stimulated by the glycolipid -galactosylceramide (-GalCer), a potent inducer of antitumor immunity in mice
(3). Recognition of -GalCer by NKT cells appeared to
depend on the interaction of the invariant TCR of these
cells with -GalCer presented by the nonclassical MHC
molecule CD1d on APCs (6). Stimulation of NKT cells by
-GalCer resulted in the production of large amounts of
IFN- and some IL-4, and the development of a cytotoxic
phenotype (7).
The in vivo antitumor activity of -GalCer strongly resembles the antitumor activity mediated by the cytokine
IL-12 (8, 9). Moreover, both -GalCer and IL-12 are
strong inducers of NKT cell activity and exert their antitumor activities through activation of these cells (8, 9). Because of these striking similarities between -GalCer and
IL-12 for activation of NKT cells, we decided to investigate whether -GalCer activation of NKT cells involves
regulation by IL-12. First, we demonstrated that NKT cells
are the main, if not the only, target for activation by -GalCer in spleen cell populations of mice. Second, we showed that endogenous IL-12 produced by dendritic cells (DCs) is
critically important for the activation of NKT cells by
-GalCer and that the interaction between DCs and NKT
cells involves CD40 and its ligand. Third, -GalCer induced the expression of IL-12R on NKT cells, which required the production of IFN- by NKT cells. Fourth, -GalCer acted synergistically with IL-12 in the activation
of natural killing activity and IFN- production in vivo.
Collectively, these findings indicate that -GalCer exerts
its function through IL-12 and suggest a novel approach
for therapeutic intervention in cancer and other disease processes.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mice.
C57BL/6 mice were purchased from Charles River Japan. V14 NKT cell-deficient (J281/) and CD1d/ mice
were established by specific deletion of the J281 and CD1d gene segment, respectively (3, 10). All mice used in this study
were at 5-8 wk of age and were maintained in specific pathogen- free conditions.
-GalCer.
-GalCer [(2S,3S,4R)-1-O-(-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol] used for
this study was provided by Dr. Y. Koezuka (Kirin Brewery Co.,
Ltd., Gunma, Japan [4, 5]). The stock solution of -GalCer (220 µg/ml) was diluted in 0.5% polysorbate 20 (Nikko Chemical) in
0.9% NaCl solution. This stock solution was further diluted into
an appropriate concentration with saline and used for the experiments. A vehicle control solution was prepared from a solution of
0.5% polysorbate 20 in 0.9% NaCl solution. The vehicle control
was used in all experiments.
Isolation of Lymphoid Cell Subsets by FACS®.
Spleen cells were incubated on nylon wool columns for 45 min, and the nonadherent cells were used for the isolation of NKT cells, NK cells, CD4+ T cells, and CD8+ T cells by cell sorting using a FACS VantageTM instrument (Becton Dickinson). All mAbs used in these experiments (mAbs against NK1.1, CD4, CD8, and TCR-/) were purchased from PharMingen. Unless noted otherwise,
NK1.1+TCR-/+ cells were used as purified NKT cells. The
stained cells were isolated using the FACS VantageTM. The purity
of the sorted cells was >98%. The details of the staining and sorting have been described previously (11).
Coculture of DCs and NKT Cells.
DCs were prepared according to the method of Steinman et al. (12) with some modifications. In brief, spleen cells were incubated in 10-cm plastic dishes (Falcon; Becton Dickinson) for 2 h, and the nonadherent cells were removed from the culture. The adherent cells were further incubated overnight and the nonadherent cells were harvested. Then, CD11c+B220CD4CD8 cells were isolated from the
nonadherent populations by cell sorting and used as the source
of DCs. Generally, DCs (105) were cocultured with purified
NK1.1+TCR-/+ NKT cells (2 × 105) in the presence of 50 ng/ml of -GalCer in 96-well U-bottomed plates (Costar
Corp.). After incubation for 36 h, the culture supernatants were
harvested to detect cytokine levels.
Detection of Cytokine Activity.
IL-4 or IFN- activity in culture supernatants was determined using the BiotracTM mouse IL-4
or BiotracTM mouse IFN- ELISA system (Nycomed Amersham
plc). Serum samples were obtained from C57BL/6 mice 24 h after injection of -GalCer (200 ng/mouse) and/or IL-12 (200 U/mouse; donated by Genetics Institute, Inc., Cambridge, MA),
and cytokine levels were measured using ELISA kits (Nycomed Amersham plc). IL-12 p70 activity in culture supernatants was measured using Intertest-12XTM ELISA kits (Genzyme Corp.).
Cytotoxicity Assay.
The natural killing activity of spleen cells was determined by 4-h 51Cr-release assays using YAC-1 cells as target. 1 lytic unit (LU) was defined as the number of effector cells required to cause 25% lysis of 2,500 target cells as described previously (13).Measurement of the Synergistic Effect of -GalCer and IL-12 In Vivo.
-GalCer (200 ng/mouse i.v.), and 6 h later mice were injected with recombinant IL-12 (200 U/mouse i.p.) or saline. 1 d after treatment with IL-12, IFN- production in the serum and NK activity of spleen cells were determined. Control mice were treated with vehicle only.
Quantitative Reverse Transcription PCR Assay for IL-12R mRNA Measurement.
C57BL/6, CD1d/, and V14 NKT cell-deficient mice were injected with -GalCer (2 µg/mouse i.v.) or vehicle. At different time points (0-6 h) after treatment, mice were
killed and spleen cells were isolated. TaqManTM real-time quantitative reverse transcription (RT)-PCR assay was carried out for the
detection of IL-12R mRNA expression by these cells according
to published methods (14). In brief, total RNA extracts from the
cells were added to the master mixture. To detect the amount of
the IL-12R mRNA RT-PCR amplificon, target (IL-12R1,
IL-12R2) and control (glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) hybridization probes were mixed with target and
control PCR primers, respectively. This mixture was transferred
to a set of thermocycler tubes and transcribed at 42°C for 30 min,
followed by 40 cycles of amplification at 95°C for 15 s and 60°C
for 1.5 min, and analyzed using an ABI PRISM 7700 sequence
detector (Applied Biosystems). IL-12R1 and IL-12R2 mRNA
expression were estimated from the ratio of fluorescence intensity
to GAPDH. IL-12R expression induced by -GalCer is indicated in the figures as induction index, calculated as follows: induction index = IL-12R expression of -GalCer-stimulated
sample/IL-12R expression of unstimulated sample.
TaqManTM probes used for these analyses are as follows:
IL-12R1 mRNA-605T, 5'-CGGATGCCCACAACGAATTGGA-3'; IL-12R2 mRNA-551T, 5'-AGCCACCTCAAAACATATCATGTGTCCAGG-3'; GAPDH-542T, 5'-CCTGGCCAAGGTCATCCATGACAACTTT-3'.
PCR primers used for these analyses are as follows: IL-121
mRNA, forward primer (-563F) 5'-AATGTGTCTGAAGAGGCCGGT-3' and reverse primer (-657R) 5'-GAGTTAACCTGAGGTCCGCAGT-3'; IL-12R2 mRNA, forward primer (-529F)
5'-ATCTCAGTTGGTGTTGCTCCA-3' and reverse primer (-602R) 5'-GCCACAGTTCCATTTTCTCCT-3'; GAPDH, forward primer (-368F) 5'-CTTCACCACCATGGAGAAGGC-3'
and reverse primer (-605R) 5'-GGCATGGACTGTGGTCATGAG-3'.
Blocking of IL-12R Induction by Anti-IFN- mAb.
mAb
(R4-6A2; PharMingen) or IL-12 (C15.1 and C15.6, donated by
Dr. G. Trinchieri, Wistar Institute of Anatomy and Biology, Philadelphia, PA) at 0 and 1 d before priming with -GalCer. As a
control, the same amount of rat IgG1 (PharMingen) was injected intraperitoneally into control mice before injection of -GalCer.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-GalCer Selectively Activates NK1.1+TCR-/+ NKT
Cells.
To provide direct evidence that NKT cells are the
only target cells for activation by -GalCer, various lymphoid subsets were isolated from mouse spleen cell suspensions by flow cytometry and cocultured with DCs in the
presence of -GalCer. After 36 h of culture, the supernatants were harvested and their IL-4 and IFN- contents
were measured by ELISA. Fig. 1 shows that purified NK1.1+ T cells produce higher levels of IL-4 and IFN-
than unfractionated spleen cells. The IFN- produced in
these cultures was not derived from classical NK cells, because enrichment of NK1.1+TCR-/ NK cells showed
no significant cytokine production. In contrast, NK1.1+
TCR-/+ cells, which represent the NKT cell population, revealed markedly high levels of IL-4 and IFN- production. Although CD4+ T cells produced higher levels of
cytokines compared with unfractionated spleen cells, this
appeared to be due to the presence of CD4+NK1.1+ NKT
cells, because CD4+NK1.1 cells produced neither IL-4
nor IFN- in response to -GalCer. Culture of NK1.1+
TCR-/+ NKT cells alone or with DCs in the absence
of -GalCer caused no significant production of IFN- or
IL-4, indicating that DCs are essential for the stimulation of
cytokine production by NKT cells.
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Fig. 2 A shows that coculture of
DCs and NKT cells in the presence of -GalCer results in
high levels of IFN- production. However, addition of
anti-IL-12 mAb into these cultures caused a marked inhibition of IFN- production. Such inhibition was not observed when control anti-CD8 rat IgG mAb was added.
Therefore, these results indicated that endogenously produced IL-12 by DCs was essential for the early activation of
NKT cells by -GalCer. The effect of mAbs against CD40
and CD40L on the activation of NKT cells by -GalCer
was also investigated (Fig. 2 B). Both anti-CD40 mAb and
anti-CD40L mAb greatly inhibited the production of IFN- by NKT cells in response to -GalCer. These findings suggested that direct contact between DCs and NKT cells
through CD40/CD40L interactions is critically important
for the activation of NKT cells by -GalCer. To study the
requirements for IL-12 production by DCs in these cultures in further detail, IL-12 p70 activity in culture supernatants was measured by ELISA. As shown in Fig. 2 C,
DCs produced IL-12 p70 when cultured with NKT cells
and -GalCer. However, DCs did not produce IL-12 p70
when cultured with -GalCer alone or when cultured
with -GalCer and NK (NK1.1+TCR-/) cells.
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-GalCer Induces IL-12R Expression on NKT Cells.
The
effect of -GalCer on the induction of IL-12R mRNA expression in spleen cells was examined by RT-PCR. As
shown in Fig. 3, intravenous injection of -GalCer into
C57BL/6 mice caused the induction of mRNA for both
IL-12R1 and IL-12R2 in spleen cells within 4 h. This
upregulation of IL-12R was strongly blocked by administration of anti-IL-12 mAb or anti-IFN- mAb before injection of -GalCer (Fig. 4). Moreover, the IL-12R induction by -GalCer was almost completely abolished in both
CD1d/ and V14 NKT cell-deficient mice (Fig. 5, A
and B). Thus, these results suggested that CD1d-dependent
-GalCer-induced IFN- production by NKT cells may
be critically important for the upregulation of IL-12R on
NKT cells. To provide direct evidence for this hypothesis, we measured the expression of IL-12R on purified NKT
cells that were previously activated in the presence of DCs
and -GalCer, either in vitro or in vivo. Fig. 5 C shows
that in vitro activation of spleen cells by DCs plus -GalCer strongly induced the expression of IL-12R on NKT
cells. Similar findings were made when mice were injected
in vivo with -GalCer (Fig. 5 D).
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-GalCer Synergistically Acts with Exogenously Administered
IL-12 in the Activation of Natural Killing Activity and IFN-
Production In Vivo.
C57BL/6 mice were injected intravenously with -GalCer, and their splenic natural killing activity against YAC-1 cells was determined 24 h later. As
shown in Fig. 6 A, a suboptimal dose of neither -GalCer
nor IL-12 was able to activate natural killing activity in
vivo. However, combined administration of -GalCer and
IL-12 at a suboptimal dose caused a marked augmentation
of natural killing.
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A similar synergistic effect of -GalCer and IL-12 was
demonstrated for the elevation of serum IFN- production. As shown in Fig. 6 B, the administration of -GalCer
plus IL-12 resulted in a strong enhancement of serum IFN-
levels in C57BL/6 mice compared with mice treated with
-GalCer or IL-12 only.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The finding that NKT cells recognize -GalCer presented by DCs in a CD1d-dependent manner represents a
novel recognition mechanism in the immune system (15).
NKT cells, which can produce both IFN- and IL-4 (16,
17), play an important role in immunoregulation and have
been considered to play a central role as innate effector cells
involved in both the protection and the onset of immune diseases (18). The NKT cell ligand -GalCer has a strong
immunopotentiating effect in vivo, and this chemical mediates strong antitumor activity (3, 9). Therefore, it is
important to dissect the mechanism by which -GalCer
activates NKT cells.
The previous finding (3) that NKT-deficient mice did
not respond to -GalCer strongly suggested that NKT cells
may be the primary target cells to -GalCer. However, it
still remained unclear whether only NKT cells responded
to -GalCer. To answer this question, we used highly purified splenic NK cells, NKT cells, CD4+ T cells, and CD8+
T cells and determined their responsiveness to -GalCer in
the presence of DCs. The data illustrated in Fig. 1 clearly
demonstrate that NKT cells are the only cells that respond
to -GalCer (3). It is surprising that neither classical NK
cells nor mainstream CD4+ T cells or CD8+ T cells revealed a significant response to -GalCer even in the presence of DCs. Together with previous findings (3), the
present data indicate that -GalCer selectively stimulates
NKT cells in the presence of DCs.
Recently, the mechanisms of activation of naive CD4+
T cells through interaction with DCs have been examined
(12, 19). Cell-cell adhesion between CD4+ T cells and
DCs through CD40/CD40L and B7.1/CD28 resulted in the activation of both DCs and T cells, which triggered the
production of IL-12 by DCs and IFN- by Th1 cells (12,
19, 20, 23). Such conditioned DCs were able to prime
cytotoxic T cells (22, 26, 27). This recognition system has
resemblance to that discussed here. As shown in Fig. 2,
IL-12 production by DCs appears to be essential for NKT
cell activation by -GalCer, because neutralization of endogenously produced IL-12 by anti-IL-12 mAb caused a
strong inhibition of IFN- production by NKT cells. The
important role of CD40/CD40L for the production of
IFN- in the cocultures of DCs and NKT cells with
-GalCer is also apparent from these experiments (Fig. 2
B). As demonstrated in Fig. 2 C, DCs produce IL-12 only when they are cultured with -GalCer in the presence of
NKT cells, indicating that direct contact between -GalCer-bound DCs and NKT cells may be essential for IL-12
production by DCs. This interaction may be required for
the production of IFN- by IL-12-activated NKT cells,
because mAbs directed against CD40/CD40L greatly inhibited IFN- production by NKT cells (Fig. 2). These
findings indicate that the interaction of NKT cells with
DCs may be very similar to the interaction of helper T cells
with DCs (22, 26, 27). Since the interactions between DCs
and NKT cells occur very quickly after administration of
-GalCer, NKT cells may be able to condition DCs very
early in an immune response, and affect subsequent adaptive responses.
In this paper, we also demonstrate that -GalCer upregulates IL-12R expression in vivo (Fig. 3). IL-12R upregulation is blocked by mAbs against IL-12 or IFN- and is
absent in CD1d/ and NKT-deficient mice (Figs. 4 and
5). Moreover, activation of NKT cells in vitro and in vivo
results in a strong induction of IL-12R1 and IL-12R2
on these cells (Fig. 5, C and D). Therefore, we speculate
that the following series of events is induced upon culture
of -GalCer with DCs and NKT cells: (a) -GalCer first binds to CD1d molecules on DCs; (b) NKT cells recognize
-GalCer-bound DCs via their TCRs and also interact
with DCs via CD40/CD40L; (c) during this interaction,
DCs produce IL-12; (d) the endogenously produced IL-12
stimulates IFN- production by NKT cells; and (e) IFN-
produced by NKT cells upregulates IL-12R on NKT cells
in an autocrine manner. The dramatic synergistic effect of
suboptimal -GalCer and exogenously administered IL-12
indicates that expression of IL-12R1 and 2, detected by
quantitative RT-PCR, is functionally upregulated in vivo.
Moreover, since this synergistic effect of -GalCer and IL-12 was not demonstrated in NKT-deficient mice, we conclude that in wild-type mice coadministration of -GalCer and IL-12 leads to upregulation of IL-12R on CD1-dependent NKT cells.
Both -GalCer and IL-12 have been demonstrated to
exhibit potent antitumor activity in vivo. IL-12 has multiple effects on the immune system that are beneficial for the
induction of antitumor immunity in vivo (28). However, the unexpected severe side effects of IL-12 have made
it difficult to use this cytokine in clinical trials (31). We
demonstrated that -GalCer synergistically acts with small
doses of IL-12 in vivo to activate NKT cells and to induce
IFN- production (Fig. 6). These findings suggest that coadministration of -GalCer with IL-12 could be used as
a new approach for tumor immunotherapy.
Recent studies have demonstrated that Th1 immunity regulated by IL-12 and IFN- plays a critical role in the induction of protective immunity against tumors and infectious
agents (32, 33). Although NKT cells are involved in both
Th1 and Th2 immunity through IFN- or IL-4 production,
the immunomodulating protocol using -GalCer and IL-12
preferentially induces NKT cells that produce large amounts
of IFN- (34). These NKT cells may facilitate the development of Th1-dominant cellular immunity essential for the induction of protective immunity against tumors and some infectious agents. Recently, it was demonstrated that -GalCer
can stimulate human NKT cells in a CD1d-dependent manner (35, 36), indicating that our proposed immunotherapy
protocol using -GalCer and IL-12 will be useful for the application to human immune diseases, including cancer.
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Footnotes |
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Address correspondence to Takashi Nishimura, Section of Genetic Engineering, Research Center for Genetic Engineering and Cell Transplantation, Department of Immunology, Tokai University School of Medicine, Bohseidai, Isehara 259-1193, Japan. Phone: 81-463-93-1121; Fax: 81-463-96-5438; E-mail: tak24{at}is.icc.u-tokai.ac.jp
Received for publication 9 November 1998 and in revised form 21 January 1999.
We would like to thank Dr. S.H. Herrmann and Dr. M. Kobayashi (Genetics Institute, Inc.) for their kind
gift of IL-12. We also thank Dr. G. Trinchieri for his kind gift of anti-IL-12 mAbs, and Dr. Y. Koezuka for
providing -GalCer.
This work was supported in part by a Grant-in-Aid from The Science Frontier Program and a Grant-in-Aid for Scientific Research on Priority Areas, both from the Ministry of Education, Science, Sports and Culture, a Grant-in-Aid from the Ministry of Health and Welfare for Cancer Control, and a Grant-in-Aid for the IL-12 Project of Tokai University School of Medicine.
Abbreviations used in this paper
-GalCer, -galactosylceramide;
DC, dendritic cell;
GAPDH, glyceraldehyde 3-phosphate dehydrogenase;
NKT, natural killer T;
RT, reverse transcription.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| TABLE OF CONTENTS |
|
) and IFN-
)
and IL-12R
) mRNA was measured by quantitative RT-PCR. The
IL-12R mRNA levels are represented as an induction index, as described
in Materials and Methods. The bars represent mean ± SE of triplicate
samples.
