1 Division of Immunoregulation, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
2 Section of Genetic Engineering, Research Center for Genetic Engineering and Cell Transplantation, Tokai University School of Medicine, Bohseidai, Isehara 259-1193, Japan
3 Department of Immunology, Juntendo University School of Medicine, Tokyo 113-0033, Japan
4 Howard Hughes Medical Institute, Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
5 CREST (Core Research for Evolutional Science and Technology) Project and Department of Molecular Immunology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
Correspondence to: T. Nishimura, Division of Immunoregulation, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
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Abstract |
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Keywords: -galactosylceramide, CD40, CD40 ligand, cytotoxic T lymphocyte, dendritic cell, NKT cells
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Introduction |
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Cells of the innate immune system, including macrophages and dendritic cells (DC), take up foreign antigens through pattern recognition receptors, combine peptide fragments of these antigens with MHC class I and class II molecules, and stimulate naive CD8+ and CD4+ T cells respectively (46). In addition, professional antigen-presenting cells (APC) communicate with T cells using positive and negative feedback systems, by producing cytokines such as IL-12, IL-18 and IL-10 (79). These interactions will influence the differentiation of naive CD4+ T cells into Th1 or Th2 lymphocytes that mediate cellular and humoral immunity respectively (1012). In order to understand host immune defenses against pathogenic microorganisms it is therefore important to study the molecular and cellular mechanisms that bridge innate and acquired immunity.
Recently, a novel lymphoid lineage, V14 NKT cells, distinct from mainstream T cells, B cells and NK cells, has been identified (13,14). These cells are characterized by co-expression of NK cell receptors and semi-invariant TCR encoded by V
14 and J
281 gene segments. Upon activation, NKT cells produce high levels of IFN-
and IL-4, and can influence immune responses against autoantigens and tumors (15,16). These cells were therefore implicated as key effector cells in innate immune responses. However, the potential role of NKT cells in the development of adaptive immune responses remains unclear. Recently, it was demonstrated that NKT cells can be activated by the glycolipid
-galactosylceramide (
-GalCer) bound with the MHC class I-like molecule CD1d (17,18). In vivo administration of
-GalCer induced strong cytolytic activity and cytokine production in wild-type mice, but not CD1d- or NKT-deficient mice. Furthermore, using highly purified cell populations, we demonstrated that
-GalCer selectively activates NKT cells, and that this responses required CD11c+ DC and CD40CD40 ligand (CD40L) interactions (19). This chemical therefore allowed us to directly test whether the interaction between NKT cells and DC early in an immune response affects the subsequent development of an adaptive immune response.
In the present paper we demonstrate that in vivo administration of the NKT cell ligand -GalCer induces the expression for the early activation marker CD69 on conventional CD4+ and CD8+ T cells, B cells, macrophages and NKT cells. These effects of
-GalCer were absent in CD1d- and NKT cell-deficient mice. We further showed that activation of CD69 on CD8+ T cells resulted in the acquisition of a cytolytic phenotype and IFN-
production. Administration of anti-CD40L antibodies prior to treatment with
-GalCer blocked the activation of CD8+ T cells. These findings provide direct evidence that antigen presentation by CD1- and CD40-expressing DC to NKT cells is critically important for bridging innate and adaptive immunity.
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Methods |
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-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, Gunma, Japan) (18). The stock solution of
-GalCer (220 µg/ml) was diluted in 0.5% polysorbate 20 (Nikko Chemical, Tokyo, Japan) 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 non-adherent cells were used for the isolation of CD4+ T cells and CD8+ T cells by cell sorting using a FACS Vantage instrument (Becton Dickinson, Mountain View, CA). All mAb used in these experiments [mAb against NK1.1 (PK136), CD3 (145-2C11), CD4 (GK1.5), CD8a (53-6.7), CD11b/Mac-1 (M1/70), CD11c (HL3), CD45/B220 (RA3-6B2) and CD69 (H1.2F3)] were purchased from PharMingen (San Diego, CA). The stained cells were isolated using FACS vantage. The purity of the sorted cells was >98%. The details of the staining and sorting have been described earlier (21).
Detection of cytokine activity
IL-4 or IFN- activity in serum samples or culture supernatants was determined using the Biotrac mouse IL-4 or Biotrac mouse IFN-
ELISA system (Amersham, Little Chalfont, UK). Serum samples were obtained from C57BL/6 mice 24 h after injection of
-GalCer (2 µg/mouse) and cytokine levels were measured using ELISA kits (Amersham).
Cytotoxicity assay
The NK activity of spleen cells was determined by 4 h 51Cr-release assays using YAC-1 cells as target. Reverse antibody-dependent cellular cytotoxicity (R-ADCC) CTL activity was measured using Fc receptor-positive P815 mastocytoma cells in the presence of anti-CD3 mAb. Tumor antigen-specific CTL activity was detected using MBL-2 T lymphoma cells. One lytic unit was defined as the number of effector cells required to cause 25% lysis of 2500 target cells. The cytotoxicity (%) was calculated as described previously (13,22).
Quantitative RT-PCR assay for IL-12 receptor (IL-12R) mRNA measurement
C57BL/6, CD1d/ and V14 NKT cell-deficient mice were injected i.v. with
-GalCer (2 µg/mouse) or vehicle. At 4 h after treatment, mice were sacrificed and spleen cells were isolated. TaqMan real-time quantitative RT-PCR assay was carried out for the detection of IL-12R mRNA expression by these cells according to the method previously described (19). IL-12Rß1 and IL-12Rß2 mRNA expression was estimated from the ratio of fluorescence intensity as compared with GAPDH. IL-12R expression induced by
-GalCer was indicated in Table 1
as an induction index, which was calculated by the following formula: induction index = (IL-12R expression of
-GalCer-stimulated sample/IL-12R expression of unstimulated sample) x100.
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PCR primers used for these analyses are as follows: IL-12ß1 mRNA: forward primer (563F) 5'-AATGTGTCTGAAGAGGCCGGT-3' and reverse primer (657R) 5'-GAGTTAACCTGAGGTCCGCAGT-3'; IL-12Rß2 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'.
Immunopotentiating effect of -GalCer in vivo
C57BL/6 mice were i.v. injected with -GalCer (2 µg/mouse) and the mice were sacrificed 46 h after the treatment to examine serum cytokine levels, NK activity, IL-12R induction and acquisition of the early activation marker CD69 in spleen cell subpopulations. The generation of CD8+ CTL was investigated using the purified CD8+CD69+ T cells or CD8+CD69 T cells from the spleen of
-GalCer-injected mice. The acquisition of non-specific CTL activity was determined by measuring R-ADCC as described above. The ability of
-GalCer-activated CD4+ T cells and CD8+ T cells to produce IFN-
was determined after culture with IL-2 (20 U/ml; kind gift from Shionogi Pharmaceutical Institute, Osaka, Japan) plus IL-12 (20 U/ml; kindly donated by Genetics Institute, Cambridge, MA) for 2 days. IFN-
levels in culture supernatants were measured by ELISA.
The generation of tumor antigen-specific CTL by -GalCer administration in vivo
C57BL/6 mice were hyperimmunized with mitomycin C-treated syngeneic MBL-2 T lymphoma cells twice with a 2 week interval. This protocol is sufficient to induce antitumor memory T cells, as described previously (23). Then, the immunized mice were i.v. injected with 2 µg/mouse of -GalCer twice in 2 day intervals. Six hours after the final boosting with
-GalCer, CD8+ T cells, CD8+CD69 T cells and CD8+CD69+ T cells were isolated from the spleen by flow cytometry, and their ability to differentiate into tumor antigen-specific CTL was assessed after culture with IL-2 plus IL-12 for 2 days. The generation of tumor-antigen-specific CTL was determined by measuring their cytotoxicity against MBL-2 T lymphoma cells. As control target cells, syngeneic B16 melanoma cells were used.
Blocking of IL-12R induction by anti-CD40L mAb
Wild-type C57BL/6 mice were injected i.p. with 100 µg anti-CD154 (CD40L) mAb (MR1; PharMingen) at 0 and 1 days before priming with -GalCer. As a control, the same amount of hamster IgG (PharMingen) was injected i.p. into control mice prior to injection of
-GalCer.
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Results |
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-GalCer activates immunoregulatory cells involved in both innate and acquired immunity
To assess whether -GalCer, in addition to its effects on NKT cells, also influences the function of other immunoregulatory cells, we examined the effect of this agent on the acquisition of the early activation marker CD69 on NKT, macrophages, T and B cells. As shown in Fig. 1
(A and F), CD69 antigen expression on NK1.1+ cells was greatly enhanced 4 h after the injection of
-GalCer in vivo. Further analysis by three-color flow cytometry demonstrated that >98% of NK1.1+TCR
ß+ NKT cells expressed CD69 antigen (data not shown). Surprisingly, we also found that
-GalCer stimulated the expression of CD69 antigen on CD4+ T cells, CD8+ T cells, B cells and macrophages within 4 h after
-GalCer treatment (Fig. 1BJ
). To evaluate whether CD69 up-regulation on macrophages, T and B cells was directly induced by
-GalCer or indirectly induced via the activation of NKT cells, we examined the effect of
-GalCer administration on CD69 antigen expression in CD1d/ and NKT-deficient mice. As illustrated in Fig. 2
, neither CD1d/ nor NKT-deficient mice revealed significant up-regulation of CD69 antigen expression on spleen cells. These data demonstrated that the acquisition of CD69 antigen on T cells, B cells and macrophages was not induced by direct stimulation with
-GalCer but was secondary to the activation of NKT cells.
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Discussion |
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To evaluate this issue, we tested whether the NKT cell ligand -GalCer could stimulate T cells and B cells involved in acquired immunity. Recently, Koezuka et al. (17,18) described a novel NKT cell-specific ligand,
-GalCer, which is a natural product isolated from marine sponges.
-GalCer can stimulate NK activity and cytokine production by NKT cells and exhibits potent antitumor activity in vivo (15). The finding that wild-type mice but not CD1d/ or NKT-deficient mice respond to
-GalCer suggests that both NKT cells and CD1+ DC are important for the activation of immune responses to
-GalCer (17). Indeed, we and others clearly demonstrated that
-GalCer selectively stimulated CD4+NK1.1+ NKT cells to induce NK and cytokine production in the presence of CD1d+ DC in vitro (19,24). We also demonstrated that the immunostimulating effect of
-GalCer was initiated by CD40CD40L-mediated NKTDC interactions (19). Consistent with these findings,
-GalCer showed a variety of immunoregulatory functions in vivo and these effects were absent in both CD1d/ and NKT-deficient mice (Table 1
). Using the newly defined NKT-specific glycolipid antigen,
-GalCer, it became possible to investigate the role of NKT cells in triggering acquired immunity. Our results showed that NKT cells influence immune responses mediated by conventional CD4+ T cells, CD8+ T cells and B cells.
To test the activation status of lymphoid cells early after -GalCer injection, we measured the expression of early-activation antigen CD69 on various lymphoid cells by flow cytometry. CD69 antigen is a type II integral membrane protein with a C-type lectin-binding domain and a member of the NK cell gene complex family of cell-surface receptors (28). CD69 has been demonstrated to be rapidly induced on the surface of activated lymphocytes as early as 34 h following stimulation and its expression is closely correlated with various immunological functions (29). As shown in Fig. 1
,
-GalCer induced up-regulation of CD69 expression on conventional CD4+ T cells, CD8+ T cells and B cells in addition to NKT cells within 4 h after
-GalCer treatment. Two possibilities are considered to explain why T cells and B cells are stimulated by NKT cell-specific ligand. First,
-GalCer by itself may stimulate the expression of CD69 antigen on T cells and B cells in addition to NKT cells. Second,
-GalCer-activated NKT cells in the presence of CD1+ DC may act as immunoregulating cells to bridge innate and acquired immunity.
To address this issue, we investigated whether -GalCer-induced up-regulation of CD69 antigen expression on T and B cells occurs in the absence of CD1+ DC or NKT cells using CD1d/ or NKT-deficient mice. As shown in Fig. 2
, neither CD1d/ nor NKT-deficient mice revealed induction of CD69 antigen expression on T and B cells. Therefore,
-GalCer by itself cannot stimulate CD69 antigen expression on T and B cells directly. These data strongly suggested that NKTDC interactions during the early phase of immune responses is critically important in the sequential activation of effector cells involved in acquired immunity.
As shown in Fig. 3, CD69+CD8+ T cells induced in vivo in response to
-GalCer-activated NKT cells showed strong R-ADCC CTL activity and higher levels of IFN-
production after culture with IL-2 and IL-12. However, CD69CD8+ T cells showed only low levels of cytokine production and R-ADCC CTL activity after culture with IL-2 and IL-12. Moreover,
-GalCer administration into mice hyperimmunized with syngeneic MBL-2 tumor cells induced the generation of antigen-specific CTL from CD69+CD8+ T cells, but not from CD69CD8+ T cells in the presence of IL-2 plus IL-12 (Fig. 4
). Therefore,
-GalCer-induced acquisition of CD69 antigen directly correlated with the functional maturation of CD8+ CTL. These data provided direct evidence that
-GalCer-activated NKT cells can induce the development of CD69+CD8+ CTL.
Recently, it has been reported that conditioned DC are critically important for the interaction of naive CD4+ Th and CD8+ T cells (4,30). During these responses, CD40CD40L molecules play a central role in cellcell interactions which stimulate IL-12 production by DC and IFN- production by naive Th cells. Consistent with this, CD40CD40L appeared to be key molecules in bridging innate and acquired immunity by
-GalCer. Indeed, in vivo administration of anti-CD40L mAb greatly blocked the development of CD69+CD8+ T cells, which showed CTL activity and IFN-
-producing ability in response to
-GalCer (Fig. 5
). Although anti-CD40L mAb strongly blocked CTL generation, it showed slight inhibition against CD69 antigen expression on CD8+ T cells (data not shown). Moreover, anti-IFN-
mAb also revealed partial blocking on
-GalCer-induced CD69 expression on CD8+ T cells, though it strongly inhibited CTL generation by
-GalCer (data not shown). Therefore, IFN-
produced by NKT cells through CD40CD40L interaction with DC appeared to be a key factor for the final maturation of CD69+CD8+ T cells into CTL but not for initial CD69 expression on CD8+ T cells. Our preliminary experiment using anti-LFA-1 mAb indicated that the LFA-1/ICAM-dependent pathway may be important for initial activation of naive CD8+ T cells to express CD69 antigen by
-GalCer administration. During the preparation of this paper, Carnaud et al. also reported that
-GalCer administration induced CD69 expression on CD8+ T cells in addition to NKT, NK and B cells (31). Taken together, the present data and our previous in vitro analysis (19) suggest the following sequential immune responses early after
-GalCer administration: (i)
-GalCer binds with CD1d on DC; (ii) NKT cells interact with
-GalCer-bound DC; (iii) during NKTDC interactions, DC produce IL-12 in a CD40CD40L-dependent manner and NKT cells express IL-12R; (iv) NKT cells produce IFN-
in response to DC-derived IL-12; (v) during NKTDC interactions, naive CD8+ T cells also interact with conditioned DC and express CD69 early activation antigen in an LFA-1-dependent, but not CD40CD40L and IFN-
-dependent manner; (vi) IFN-
produced via NKT
-GalCer-bound DC interactions induces the final maturation of CD69+CD8+ T cells into functional CTL.
In this paper, we have emphasized the effect of NKT cell activation on CD8+ CTL development. However, -GalCer induced the production of both IFN-
and IL-4, and also activated B cells. Moreover, repeated administration of
-GalCer caused down-modulation of IFN-
production but not IL-4 production (3234). These studies suggest that
-GalCer may play an equally important role in bridging innate immunity mediated by NKT cells, and humoral immunity mediated by B cells and Th2 cells. A recent study has shown that NKT cells are involved in CD1d-restricted IgG formation to GPI-anchored antigens (25). Thus, our data presented here provided a new biological concept of NKT cells and suggests that immunodeviation through the control of NKT cell function may be a useful strategy for the modulation of acquired immunity involved in autoimmune diseases and cancer.
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Acknowledgments |
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Abbreviations |
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APC antigen-presenting cells |
CD40L CD40 ligand |
CTL cytotoxic T lymphocyte |
DC dendritic cell |
IL-12R IL-12 receptor |
NKT natural killer T |
R-ADCC reverse antibody-dependent cellular cytotoxicity |
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Notes |
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Received 14 December 1999, accepted 7 March 2000.
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References |
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