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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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
V
14
/
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.
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Introduction |
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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 J
281
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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Mice.
C57BL/6 mice were purchased from Charles River Japan. V-GalCer.
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-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+B220Detection of Cytokine Activity.
IL-4 or IFN-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.
Quantitative Reverse Transcription PCR Assay for IL-12R mRNA Measurement.
C57BL/6, CD1dBlocking of IL-12R Induction by Anti-IFN- mAb.
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Results |
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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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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-12R
1 and IL-12R
2 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 V
14 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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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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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-12R
1 and IL-12R
2
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-12R
1 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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