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[Cancer Research 64, 8754-8760, December 1, 2004]
© 2004 American Association for Cancer Research

Immunology

Liposome-Encapsulated CpG Oligodeoxynucleotides as a Potent Adjuvant for Inducing Type 1 Innate Immunity

Yoshinori Suzuki1,2, Daiko Wakita1, Kenji Chamoto1, Yoshinori Narita1, Takemasa Tsuji1, Tsuguhide Takeshima1, Hiroshi Gyobu1,2, You Kawarada2, Satoshi Kondo2, Shizuo Akira3, Hiroyuki Katoh2, Hiroaki Ikeda1 and Takashi Nishimura1

1 Division of Immunoregulation, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; 2 Surgical Oncology, Cancer Medicine, Division of Cancer Medicine, Hokkaido University School of Medicine, Sapporo, Japan; and 3 Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Unmethylated cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs) exhibit potent immunostimulating activity by binding with Toll-like receptor 9 (TLR9) expressed on antigen-presenting cells. Here, we show that CpG-ODN encapsulated in cationic liposomes (CpG-liposomes) improves its incorporation into CD11c+ dendritic cells (DCs) and induces enhanced serum interleukin (IL)-12 levels compared with unmodified CpG-ODN. CpG-liposome potently activated natural killer (NK) cells (84.3%) and NKT cells (48.3%) to produce interferon-{gamma} (IFN-{gamma}), whereas the same dose of unmodified CpG-ODN induced only low numbers of IFN-{gamma}–producing NK cells (12.7%) and NKT cells (1.6%) to produce IFN-{gamma}. In contrast with the NKT cell agonist {alpha}-galactosylceramide, which induces both IFN-{gamma} and IL-4 production by NKT cells, CpG-liposome only induced IFN-{gamma} production by NKT cells. Such potent adjuvant activities of CpG-liposome were absent in TLR9-deficient mice, indicating that CpG-liposome was as effective as CpG-ODN in stimulating type 1 innate immunity through TLR9. In addition to TLR9, at least two other factors, IL-12 production by DCs and direct contact between DCs and NK or NKT cells, were essential for inducing type 1 innate immunity by CpG-liposome. Furthermore, ligation of TLR9 by CpG-liposome coencapsulated with ovalbumin (OVA) caused the induction of OVA-specific CTLs, which exhibited potent cytotoxicity against OVA-expressing tumor cells. These results indicate that CpG-liposome alone or combined with tumor antigen protein provides a promising approach for the prevention or therapy of tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It has been reported that antigen-presenting cells (APCs), especially dendritic cells (DCs), play a pivotal role in bridging innate and acquired immunity. These cellular interactions are essential for the induction of antigen-specific protective immunity against infectious diseases and malignant tumors (1 , 2) . It has been shown that Th1-dependent acquired immunity, via production of Th1 cytokines such as interferon-{gamma} (IFN-{gamma}) and interleukin (IL)-2, is important for the generation of CTLs. However, recent work has shown that innate effector cells, such as DCs, natural killer (NK) cells, and NKT cells, by producing IL-12 and/or IFN-{gamma}, also are involved in controlling type 1 immunity (3, 4, 5) .

DCs interact with pathogens using Toll-like receptors (TLRs). Engagement of TLRs induces the production of IL-12, IL-18, IFN-{gamma}, and/or IL-10, which influences the activation of NK cells, NKT cells, and conventional T cells (6 , 7) . IL-12 produced by DCs is the most important cytokine that induces IFN-{gamma}–producing NK, NKT, Th1, and Tc1 cells (8 , 9) . It also has been reported that IFN-{gamma}–producing CD8{alpha}+ DCs and type 1 IFN-producing plasmacytoid DCs play a critical role in inducing Th1-dominant immunity, whereas IL-10–producing regulatory DCs suppress cellular immune responses (10, 11, 12) . Thus, DCs can function as type 1 and type 2 innate effector cells by producing Th1-inducing or Th2-inducing cytokines, respectively. In contrast, NK cells invariably promote Th1 immunity by producing IFN-{gamma} in response to IL-12 derived from pathogen-stimulated DCs (13) . Although IL-12–activated NKT cells usually function as IFN-{gamma}–producing type 1 effector cells, {alpha}-galactosylceramide ({alpha}-GalCer)–activated NKT cells function as type 2 innate effector cells to suppress Th1-dependent immunity by producing IL-4 and IFN-{gamma} (14 , 15) .

In some circumstances, the early production of IL-4 and IL-13 by NKT cells suppressed antitumor immunity against intradermally injected tumor cells, despite the fact that NKT cells themselves exhibit antimetastatic activities (5 , 16 , 17) . Moreover, immunization with {alpha}-GalCer–pulsed DCs, which preferentially activate IFN-{gamma}–producing NKT cells rather than IL-4–producing NKT cells, potentiated antitumor activity in vivo (18) . These results indicate that selective activation of IL-12–producing DCs and IFN-{gamma}–producing NK and NKT cells (type 1 innate immunity) may be beneficial for developing a vaccine directed at inducing Th1-dependent cellular immunity. Although IL-12 is useful for the selective induction of IFN-{gamma}–producing NKT cells (19) , its application to the clinical setting is limited because of adverse in vivo activities. Therefore, it is important to develop new adjuvants that can selectively activate type 1 innate immunity.

Unmethylated cytosine-phosphorothioate-guanine (CpG) containing oligodeoxynucleotides (CpG-ODNs) now are well known to promote a Th1-type immune response by binding with TLR9 on APCs (6 , 7 , 20 , 21) . It has been reported that administration of CpG-ODNs induces a systemic Th1 response and enhances NK activity (22 , 23) . However, the cytokine profile produced by NKT cells in response to stimulation with CpG-ODN–activated DCs remains unclear.

It recently has been shown that TLR9, the receptor for CpG motifs (24 , 25) , localizes to endosomal/vacuolar/vesicular compartments but not to the cell surface (20) . Internalization of CpG-ODN is a prerequisite to activate TLR9 for initiation of signaling (26) . These results, together with the finding that lipofection of CpG-ODN into spleen cells enhances its immune stimulatory effects (27) , suggest that CpG-ODN encapsulated in liposomes (CpG-liposome) may increase its uptake by TLR9-expressing DCs to produce enhanced levels of IL-12 and downstream responses. To test this hypothesis, we examined the effect of in vivo administered CpG-liposome on the activation of type 1 innate immunity.

Our results show that (a) compared with unmodified CpG, CpG-liposomes exhibit higher incorporation into DCs and augmented IL-12 production by DCs; (b) CpG-liposomes induce IFN-{gamma} but not IL-4 production by NKT cells; (c) DC-derived IL-12 and a contact between DCs and NK (or NKT) cells are essential to induce type 1 innate immunity by CpG-liposomes; and (d) ligation of TLR9 by CpG-liposome coencapsulated with ovalbumin (OVA) induces Th1-dependent OVA-specific CTLs with potent cytotoxicity against OVA-expressing tumor cells. Collectively, our findings indicate that CpG-liposome, as compared with unmodified CpG-ODN, has superior activity to induce type 1 immunity.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
C57BL/6 mice were purchased from Charles River Japan (Yokohama, Japan). S. Akira (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) provided TLR9 knockout mice on the C57BL/6 background (6 , 7) . All of the mice used in this study were female, 5 to 8 weeks of age, and maintained in specific pathogen-free conditions.

CpG-ODN, {alpha}-GalCer, and Liposome.
Phosphothioate-stabilized CpG-ODN1668 (5'-TCCATGACGTTCCTGATGCT-3') was synthesized by Hokkaido System Science (Sapporo, Japan). {alpha}-GalCer [(2S,3S,4R)-1-O-({alpha}-D-galactopyranosyl)-2-(N-hexacosanoylamin)-1,3,4-octadecanetriol] used in this study was provided by Dr. Y. Koezuka (Kirin Brewery, Gunma, Japan; ref. 29 ). Cationic liposomes were purchased from NOF Corporation (Tokyo, Japan).

Flow Cytometric Staining and Analysis.
For analysis of cell surface markers, the samples were stained with phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated antibodies directed against the following markers: CD4-PE, CD8-PE, NK1.1-PE, CD11b-PE, CD11c-PE, and CD69-FITC (all from BD Pharmingen, San Diego, CA). Data were acquired on a Becton Dickinson FACSCalibur (Becton Dickinson, Franklin Lakes, NJ). Data were analyzed using CellQuest software (Becton Dickinson). For the detection of cytoplasmic cytokine expression, the samples first were stained with NK1.1-biotin, streptavidine-allophycocyanin, CD3-PerCP (all from Pharmingen), and CD69-FITC, then fixed with 4% paraformaldehyde, and treated with permeabilizing solution [50 mmol/L NaCl, 5 mmol/L EDTA, 0.02% NaN3, and 0.5% Triton X-100 (pH 7.5)], and the fixed cells then were stained with PE-conjugated anti–IFN-{gamma} monoclonal antibody (mAb) or PE-conjugated anti–IL-4 mAb (all from Pharmingen) for 45 minutes on ice. The percentage of cells expressing cytoplasmic IFN-{gamma} or IL-4 was determined by flow cytometry (FACSCalibur).

Monoclonal Antibodies.
Anti–IL-12 mAbs (C15.1 and C15.6) were gifts from Dr. G. Trinchieri (Schering-Plough Research Institute, Dardilly, France; ref. 8 ). Anti-biotin mAb-conjugated microbeads for the MACS system were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Rat-IgG, anti-CD154 (CD40L), was purchased from Pharmingen. IL-12 was donated by Genetics Institute (Cambridge, MA).

Cytotoxicity Assay.
The natural killing activity of spleen cells was determined by 51Cr-release assays using YAC-1 cells as target. Briefly, 1 x 104 (Na251CrO4)-labeled target cells suspended in 100 µL Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal calf serum (assay medium) were seeded into V-bottomed microtiter wells. Various numbers of effector cells suspended in 50 µL assay medium were added to the wells and incubated for 4 to 6 hours, and 100 µL supernatant were collected from each well. The percentage of specific lysis was calculated as follows: (cpm experimental release – cpm spontaneous release)/(cpm maximal release – cpm spontaneous release) x100.

Isolation of NK1.1+ Cells.
Spleen cells were loaded on nylon-wool columns for 45 minutes, and the nonadherent cells were used for the isolation of NK1.1+ cells. After incubation with anti–NK1.1-biotin, NK1.1+ cells were positively selected using magnetized mAbs for anti-biotin (Miltenyi Biotec). The resulting population was consistently >93% NK1.1+ cells, with <0.5% TCR+ NK1.1– cells.

Coculture of Dendritic Cells and NK1.1+ Cells.
DCs were positively selected using magnetized mAbs for anti-CD11c (Miltenyi Biotec). DCs (2 x 105) were cocultured with purified NK1.1+ cells (2 x 105) in 96-well U-bottomed plates. After incubation for 24 hours, the culture supernatants were harvested to detect cytokine levels, and cells were evaluated in the intracellular cytokine-staining assay.

Detection of Cytokine Production.
IFN-{gamma} in culture supernatants was determined using Biotec mouse IFN-{gamma} ELISA system (Amersham Biosciences, Piscataway, NJ). After 24 hours of culture, the supernatants were harvested, and ELISA (Pharmingen) was used to measure their IFN-{gamma} level.

Measurement of Antitumor Immunity.
C57BL/6 mice were immunized with CpG-liposome coencapsulated with OVA (CpG + OVA-liposome), CpG-liposome, OVA-liposome, or saline-liposome (control) twice at 2-week intervals. Five days after the last immunization, lymphocytes were prepared from popliteal lymph nodes. To examine the generation of Tc1 or Th1 cells, the cells were restimulated with 5 µg/mL of class I binding OVA257–264 peptide or class II binding OVA323–339 peptide for 4 days. The generation of Tc1 or Th1 cells was determined by cytoplasmic staining of IFN-{gamma} among CD8+ T cells or CD4+ T cells. OVA-specific CTLs were generated by stimulation of immunized cells with 50 µg/mL of OVA protein for 4 days. The generation of CTLs was confirmed by two methods: (a) the cultured cells were stained with PE-conjugated H-2b-OVA257–264 peptide tetramers (Beckman Coulter, Fullerton, CA); and (b) the cytotoxicity of cultured cells against OVA-expressing EG-7 or parental EL-4 cells was measured by 4-hour 51Cr release assay.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CpG-Liposome Potently Induces Type 1 Innate Immunity.
C57BL/6 mice were intravenously treated with saline, unmodified CpG-ODN, or CpG-liposome and examined for activation of innate effector cells, including DCs, NK cells, and NKT cells. To examine uptake of CpG-ODN into DCs in vivo, we prepared FITC-conjugated CpG. As shown in Fig. 1ACitation , uptake of FITC–CpG-ODN into CD11c+ DCs was greatly enhanced when mice were treated with CpG-liposome compared with unmodified CpG-ODN. In addition to enhanced uptake of CpG-ODN by DCs, CpG-liposome induced increased levels of serum IL-12 (Fig. 1C)Citation compared with unmodified CpG-ODN (Fig. 1B)Citation . Administration of liposome alone could not induce any IL-12 production in serum, showing that IL-12 production was induced by the action of CpG-ODN (data not shown). Flow cytometric analysis of IL-12 production in DC population indicated that ~15% of CD11c+ DCs expressed cytoplasmic IL-12 1 hour after stimulation with CpG-liposome (Fig. 1C)Citation . Among neutral, cationic, and anionic liposomes, cationic liposomes were most potent in enhancing uptake by DCs, which is consistent with a previous report (data not shown; ref. 29 ).



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  		Fig. 1. Efficient uptake of CpG-liposome by DCs induces potent IL-12 production. A. Mice were treated intravenously with saline (control), unmodified FITC-labeled CpG or FITC-labeled CpG encapsulated in liposome (CpG-liposome). One hour after treatment, spleen cells were stained with PE-conjugated anti-CD11c mAb, and the uptake of FITC-labeled CpG into CD11c+ DCs was determined by two-color flow cytometry. B, elevation of serum IL-12 levels in mice treated with saline (control), unmodified labeled CpG, or CpG encapsulated in liposome (CpG-liposome). One hour after treatment, serum was harvested from the mice, and serum IL-12 levels were measured using ELISA. C. Spleen cells were obtained from the mice 1 hour after treatment with CpG-liposome or saline (control). IL-12–producing ability of DCs then was determined by cytoplasmic staining with PE-conjugated anti–IL-12 mAb, followed by cell surface staining with FITC-conjugated anti-CD11c mAb. Similar results were obtained in three different experiments.

 
Concomitant with the activation of DCs, NK cells and NKT cells also were activated by CpG-liposome (Fig. 2A, a–j)Citation . Intravenous administration of unmodified CpG caused significant induction of IFN-{gamma} production by NK cells (12.7%) and NKT cells (1.6%). However, treatment with CpG-liposome caused more profound IFN-{gamma} production by NK (84.3%) and NKT cells (48.3%). Although CpG-liposome was able to induce expression of the early activation molecule CD69 on conventional T cells (Fig. 2B)Citation , it induced negligible levels of IFN-{gamma} by T cells (Fig. 2A, k–o)Citation . Conversely, empty liposome induced neither IFN-{gamma} production nor CD69 expression (Fig. 2A and B)Citation . Thus, our results show that CpG-liposome is a potent adjuvant for inducing IFN-{gamma} production by NK and NKT cells. Moreover, we showed that the potent adjuvant activity of CpG-liposome for inducing IFN-{gamma}–producing NK and NKT cells was almost completely abolished in TLR9-deficient mice (Fig. 2A, e, j, and o)Citation . Therefore, the CpG-liposomes exhibit potent adjuvant activity for NK and NKT cells in a TLR9-dependent mechanism.



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  		Fig. 2. CpG liposome induces IFN-{gamma} production by NK and NKT cells and activates innate and acquired immunity. A, wild-type C57BL/6 mice (a–d, f–i, k–n) were intravenously treated with CpG-liposome, unmodified CpG, liposome, or saline (control). TLR9–/– mice (e, j, o) were intravenously treated with CpG-liposome. Four hours after treatment, spleen cells were prepared and analyzed by four-color flow cytometry to determine IFN-{gamma}–producing ability among NK1.1+ CD3– NK cells (a–e), NK1.1+ CD3+ NKT cells (f–j), or NK1.1– CD3+ T cells (k–o). B. C57BL/6 mice were intravenously treated with CpG-liposome, unmodified CpG, liposome, or saline (control). Four hours after treatment, spleen cells were harvested, and the activation status of various immunoregulatory cells (NK1.1+ NK and NKT cells, CD4+ T cells, CD8+ T cells, CD11b+ macrophages, and CD11c+ DCs) was determined by measuring expression of the early activation marker CD69 using FITC-antiCD69 mAb. Similar results were obtained in three different experiments.

 
It generally has been accepted that NKT cells play a pivotal role in innate immunity through production of IFN-{gamma} and IL-4 (9 , 30) . When {alpha}-GalCer, which is a CD1d-binding NKT cell ligand, was administered into mice, NKT cells produced both IFN-{gamma} and IL-4 (Fig. 3A and B)Citation . In striking contrast, CpG-liposome induced only IFN-{gamma} but not IL-4 production by NKT cells in vivo (Fig. 3)Citation . Thus, CpG-liposome is a potent and selective adjuvant for inducing IL-12–producing DCs and IFN-{gamma}–producing NK and NKT cells, which are critical cytokines involved in type 1 immunity.



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  		Fig. 3. CpG-liposome–activated NKT cells produce IFN-{gamma} but not IL-4. C57BL/6 mice were treated with CpG-liposome or {alpha}-GalCer. A. Two hours after treatment, IL-4 production by NK and NKT cells was determined by four-color staining analysis. B. IFN-{gamma} production by NK and NKT cells was measured by flow cytometry 4 hours after treatment with CpG-liposome or {alpha}-GalCer injection. Four-color staining was carried out using PE/anti–IL-4 mAb, FITC/anti-CD69 mAb, PerCP/anti-CD3 mAb, and biotinylated anti-NK1.1 mAb + avidin APC. Similar results were obtained in three different experiments.

 
IL-12 Production by CpG-Liposome–Activated DCs Is Critical for Inducing IFN-{gamma} Production by NK and NKT Cells.
As shown in Fig. 4Citation , the capacity of NK (54.8%) and NKT cells (23%) to produce IFN-{gamma} in CpG-liposome–treated mice was greatly reduced to 27.4% (NK) and 7.3% (NKT) when C57BL/6 mice were treated with anti–IL-12 mAb. Thus, IL-12 produced by CpG-liposome–activated TLR9+ DCs plays a major role in activating NK and NKT cells by CpG-liposome. To confirm this conclusion, we isolated CD11c+ DCs from spleen cells of the mice treated with or without CpG-liposome. As shown in Fig. 5ACitation , CpG-liposome–activated DCs supported the production of IFN-{gamma} by whole NK1.1+ (NK + NKT) cells. Analysis by cytoplasmic staining for IFN-{gamma} indicated that CpG-liposome–activated DCs supported the induction of IFN-{gamma}–producing NK and NKT cells (Fig. 5B)Citation . Such supporting effect on IFN-{gamma} production was not observed when NK1.1+ cells were cocultured with untreated DCs. Moreover, this effect was strongly inhibited by anti–IL-12 mAbs. Thus, IL-12 induced by CpG-liposome is a critical factor for the capacity of CpG-liposomes to induce IFN-{gamma} production by NK and NKT cells.



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  		Fig. 4. In vivo administration of anti–IL-12 mAb blocks NK and NKT cell activation by CpG-liposome. C57BL/6 mice were intravenously treated with CpG-liposome or saline. Four hours after the treatment, spleen cells were harvested, and IFN-{gamma} production by NK1.1+ CD3– NK cells (A–C), NK1.1+ CD3+ NKT cells (D--F), or NK1.1– CD3+ T cells (G–I) was examined by four-color flow cytometry analysis. To examine the role of IL-12 for inducing IFN-{gamma}–producing cells, mice were treated with anti–IL-12 mAb at 0 and 12 hours before treatment with CpG-liposome (B, E, and H). Similar results were obtained in three different experiments.

 


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  		Fig. 5. IL-12 derived from DCs and direct contact between DCs and NK (or NKT) cells are critical for inducing IFN-{gamma}–producing NK and NKT cells by CpG-liposome. A. Spleen cells were prepared from mice 30 minutes after intravenous treatment with saline or CpG-liposome. CD11c+ DCs or whole NK1.1+ (NK + NKT) cells then were isolated from spleen of saline-treated mice (DC, NK1.1) or CpG-liposome–treated mice (DC*, NK1.1+*) by MACS. To induce IFN-{gamma} production, these cells were cocultured in various combinations. CpG-liposome–activated DCs (DC*), but not control DCs (DC), were able to support IFN-{gamma} production by CpG-liposome–activated NK1.1+ cells (NK1.1+*) and saline-treated NK1.1+ cells (NK1.1+). B. To determine the frequency of IFN-{gamma}–producing NK and NKT cells among NK1.1+ cell populations, cytoplasmic expression of IFN-{gamma} among NK1.1+ cells was further analyzed by four-color flow cytometry. C. CpG-liposome–activated DCs (DC*), saline-treated DCs (DC), or saline-treated NK1.1+ cells (NK1.1+) isolated as described previously were cocultured in various combinations to induce IFN-{gamma} production. To examine the role of direct contact, DC*s (or DCs) were separately cultured with NK1.1+ cells using a transwell culture system. DC* + NK1.1+ (transwell), CpG-activated DCs were separately cultured with NK1.1+ cells; DC + NK1.1+ + rIL-12 (transwell), saline-treated DCs were separately cultured with NK1.1+ cells in the presence of IL-12. Similar results were obtained in three different experiments.

 
CD40/CD40L-mediated DC/NKT cell-cell interaction has been reported to be critical for {alpha}-GalCer–induced activation of NKT cells (9) . However, anti-CD40L mAb did not block the induction of IFN-{gamma}–producing NK1.1+ cells mediated by CpG-liposome, suggesting an alternative mechanism of NKT cell activation (Fig. 5B)Citation .

Induction of IFN-{gamma} by NK and NKT Cells in Response to CpG-Liposome Requires Direct DC–NK/NKT Cell Contact.
DCs derived from untreated mice were able to support the activation of NK and NKT cells with presence of IL-12 (Fig. 5C)Citation , further supporting a critical role of IL-12 derived from CpG-liposome–activated DCs for activating NK and NKT cells. However, IL-12 was unable to reconstitute the activation of NK and NKT cells when DCs and NK or NKT cells were separated using a transwell system. Similarly, transwell culture of IL-12–producing CpG-liposome–activated DCs and NK1.1+ cells failed to activate NK and NKT cells. Collectively, these data (Fig. 5C)Citation indicate that (a) IL-12 produced by CpG-liposome–activated DCs is critical for the activation of NK and NKT cells; and (b) direct contact between CpG-liposome–activated DCs and NK (or NKT) cells is essential for IL-12–dependent NK and NKT cell activation. However, which molecule(s) is important for the direct contact between DCs and NK or NKT cells remains unclear.

CpG-Liposome Coencapsulated with a Model Tumor Antigen Potentiates the Generation of Tumor Antigen-Specific Th1 and Tc1 Responses.
Finally, we investigated whether CpG-liposome is applicable to tumor vaccine therapy. For this purpose, we prepared CpG-liposome coencapsulated with OVA, which was used as a model tumor antigen. C57BL/6 mice were immunized with saline, CpG-liposome, OVA-liposome, or CpG + OVA encapsulated in liposome (CpG + OVA-liposome) twice at a 2-week interval. Five days after the second immunization, spleen cells were restimulated with class I or class II binding OVA peptide or OVA protein for 4 days and examined for levels of IFN-{gamma} production and frequency of tetramer+-OVA–specific CTLs in the culture. As shown in Fig. 6ACitation , mice vaccinated with CpG-liposome, OVA-liposome, or liposome alone did not have a significant increase in CD4+ IFN-{gamma}–producing Th1 cells. However, mice vaccinated with CpG + OVA-liposome had increased frequency of CD4+ IFN-{gamma}–producing Th1 cells (8.1%; Fig. 6A, e–hCitation ). Similar results were obtained for CD8+ IFN-{gamma}–producing Tc1 cells (Fig. 6A, a–d)Citation . Thus, these findings indicate that CpG + OVA-liposome activates Th1 and Tc1 immunity concomitant with the elevation of IFN-{gamma} production. CpG + OVA-liposome successfully induced OVA-specific CTLs, which was confirmed by staining with PE-conjugated OVA257–264-binding H-2kb tetramer. As shown in Fig. 6BCitation , vaccination of mice with CpG + OVA-liposome induced increased frequency (8.0%) of tetramer+ CD8+ T cells compared with mice treated with CpG-liposome (0.5%), OVA-liposome (2.4%), or liposome alone (0.9%). CTLs of mice vaccinated with CpG + OVA-liposome exhibited strong cytotoxicity and specifically lysed OVA-expressing EG-7 but not parental EL-4 cells (Fig. 6C)Citation . These results show that CpG-liposome or CpG-liposome coencapsulated with model tumor antigen protein acts as a potent adjuvant for bridging type 1 innate immunity with type 1 adaptive immunity. We already have shown that such enhanced generation of antigen-specific CTLs was induced in mice bearing OVA-expressing EG-7 tumor (data not shown). Thus, CpG-liposome combined with tumor antigen protein may be applicable to the therapy of tumor.



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  		Fig. 6. CpG-liposome coencapsulated with OVA induces antigen-specific IFN-{gamma}–producing Tc1 and Th1 cells and antitumor effect against OVA-expressing tumors. A. C57BL/6 mice were immunized with CpG-liposome coencapsulated with OVA (CpG + OVA-liposome), CpG-liposome, OVA-liposome, or saline-liposome (control) twice at a 2-week interval. Five days after the second immunization, lymphocytes were prepared from popliteal lymph nodes and restimulated with class I binding OVA257–264 peptide (a–d) or class II binding OVA323–339 peptide (e–h) for 4 days. Cytoplasmic expression of IFN-{gamma} among CD8+ T cells (a–d) and CD4+ T cells (e–h) then was examined by two-color flow cytometry using PE/anti–IFN-{gamma} mAb, FITC/anti-CD8 mAb, or FITC/anti-CD4 mAb. B. C57BL/6 mice were immunized with CpG + OVA-liposome, CpG-liposome, OVA-liposome, or saline-liposome (control) twice at a 2-week interval. Five days after the second immunization, lymphocytes were prepared from popliteal lymph nodes and restimulated with OVA protein (50 µg/mL) for 4 days. B, The generation of tetramer+ CTLs was determined by flow cytometry using FITC/anti-CD8 mAb and PE-conjugated H-2kb–OVA257–264 peptide tetramer. C. After 4 days of in vitro culture, cells were harvested, and their cytotoxicity against OVA-expressing EG-7 tumor cells or parental EL-4 tumor cells was examined by 4-hour 51Cr release assay. Similar results were obtained in three different experiments.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this article, we have shown that CpG-liposome is a potent adjuvant that can selectively induce type 1 innate immunity through activation of IL-12–producing DCs and IFN-{gamma}–producing NK and NKT cells (Figs. 1Citation 2Citation 3Citation ). Although it generally has been accepted that CpG-ODNs induce type 1 immunity through binding to TLR9 on APCs (20, 21, 22, 23) , the detailed mechanisms underlying NK and NKT cell activation by CpG-ODN have remained unclear. It has been reported that human NK, NKT, T cells and macrophages (31) and murine NK, NKT, and T cells (32 , 33) express little TLR9 and are not directly activated by CpG-ODNs. Thus, other immunoregulatory cells such as DCs, macrophages, and B cells are considered to be critical for the subsequent activation of NK and NKT cells in the murine system. Here, we have shown that two factors, i.e., (a) IL-12 produced by CpG-activated DCs and (b) direct contact between CpG-activated DCs and NK or NKT cells, are essential to induce IFN-{gamma}–producing NK and NKT cells. We also showed that ligation of TLR9 with CpG-liposome coencapsulated with antigenic protein links type 1 innate and acquired immunity and induces antigen-specific CTLs.

In the present article, it was shown that encapsulation of CpG in liposome dramatically enhances the immunostimulatory effect of CpG-ODN. The precise mechanism for this enhancement remains unclear. One possible mechanism for this enhancement is that encapsulation in liposome enhanced the uptake of FITC-labeled CpG by DCs (Fig. 1A)Citation . Compared with soluble CpG, intradermally injected CpG-liposome exhibited no higher immunopotentiating effect in terms of NK-cell activation in contrast to intravenously injected CpG-liposome (data not shown). Therefore, intravenously injected CpG-liposome efficiently induced NK-cell activation because of its superior trafficking property into NK-rich lymphoid organs, such as spleen and liver. However, vaccination of tumor-bearing mice with intradermal injection of CpG-liposome + OVA caused higher induction of therapeutic antitumor activity compared with intradermal injection of soluble CpG + OVA (data not shown). This may be because of the long-term duration of adjuvant effect by CpG-liposome compared with soluble CpG. Therefore, CpG-liposome appears to exhibit immunopotentiating effect by intravenous and intradermal injection routes. Since the identification of the gene(s) encoding the tumor rejection antigen (TRA), many investigators have tried to identify MHC class I or class II binding TRA peptides for development of a tumor-specific vaccine (34, 35, 36) . However, clinical studies have indicated that although vaccination with TRA peptides can elicit antigen-specific CTLs in vivo, this treatment is unable to eliminate tumors in vivo (37) . This low effectiveness of CTLs may be because (a) CTLs fail to react with tumor cells because of lack or down-regulation of MHC and/or costimulatory molecules essential to induce full activation of T cells (38 , 39) ; and (b) CTLs may fail to be fully activated in the tumor-bearing host because of strong immunosuppression and defective helper T-cell function (40 , 41) .

Type 1 innate and acquired immunity plays a pivotal role to overcome strong immunosuppression in tumor-bearing animals (3 , 8) . We also have proposed that Th1-dominant immunity is critical to induce CTL-mediated tumor eradication in vivo (42, 43, 44) . As such, it may be possible to induce tumor-specific protective immunity with a vaccine protocol that selectively activates type 1 innate and acquired immunity, as described here.

Recent results have shown that innate effector cells, such as DCs and NK and NKT cells, play a critical role for controlling acquired immunity via cytokine production and/or cell-cell contact (1, 2, 3, 4 , 45) . For example, activation of DCs with the CD1d-binding NKT cell ligand {alpha}-GalCer induced IL-12 production by DCs and IL-12 receptor expression on NKT cells through CD40/CD40L-mediated cell-cell interactions (9) . In response to {alpha}-GalCer, NKT cells produced both IFN-{gamma} and IL-4. The cytokines produced by innate effector cells following {alpha}-GalCer administration also influenced T and B cell–mediated acquired immunity (46 , 47) . Production of IL-10, transforming growth factor ß, and IL-4 by innate effector cells polarizes acquired immunity toward type 2, whereas IL-12, IL-18, and IFN-{gamma} production promote type 1 immunity (48) . Production of Th2 cytokines, such as IL-13 and IL-4, by NKT cells in vivo exhibits potent immunoregulatory activities by suppressing Th1-dependent immunity (14 , 15) . {alpha}-GalCer treatment of solid tumor-bearing mice suppressed antitumor immunity via IL-13 produced by NKT cells (16) . These results suggest that the development of a vaccine protocol for selective activation of IFN-{gamma}–producing NKT cells concomitantly with IL-12–producing DCs and IFN-{gamma}–producing NK cells may be beneficial for inducing type 1 acquired immunity. Fujii et al. (18) recently showed that vaccination with {alpha}-GalCer–bound DCs, which induce prolonged activation of IFN-{gamma}–producing NKT cells, is suitable to induce antitumor activity in vivo.

As shown in Fig. 1Citation , we have shown that CpG encapsulated in cationic liposome is efficiently taken up by DCs and induces enhanced IL-12 production by DCs compared with unmodified CpG-ODNs. Moreover, in contrast to {alpha}-GalCer, CpG-liposome induced IFN-{gamma} but not IL-4 production by NKT cells (Figs. 2ACitation and 3Citation ). Thus, these results show that CpG-liposome is a superior adjuvant that selectively activates type 1 innate immunity. Although the precise mechanisms underlying activation of type 1 innate immunity by CpG-liposome remain unclear, IL-12 production by DCs appeared to be essential for induction of IFN-{gamma} from NK and NKT cells (Figs. 4Citation and 5Citation ). Because blocking of IL-12 by mAb could not completely inhibit IFN-{gamma} production from NK and NKT cells cocultured with CpG-activated DCs, other cytokines secreted from CpG-activated DCs may be involved in remaining IFN-{gamma} production. It has been shown that IL-12 is the most important DC-derived cytokine for the activation of NK cells by in vivo CpG administration, consistent with our results (32) . They also indicated that CpG-induced DC-derived cytokines, such as TNF-{alpha} and type 1 interferons, also play a partial role in CpG-induced NK cell activation. Therefore, it is possible to suppose that type 1 interferon and TNF-{alpha} may be partially involved in our NK cell activation system by CpG-liposome injection. It was shown that IL-18 production from DCs was induced by the injection with CpG-liposome but not with CpG alone (49) . Therefore, IL-18 may not be involved in NK-cell activation induced by the administration with CpG alone.

We show that separation of CpG-liposome–activated DCs and NK (or NKT) cells by transwell culture failed to induce IFN-{gamma} production (Fig. 5)Citation . This indicates that direct contact between DCs and NK or NKT cells is required for the induction of IFN-{gamma}. It recently has been reported that direct contact between DCs and NKT cells via CD40/CD40L is essential for {alpha}-GalCer–mediated activation of NKT cells (29 , 45) . CD40/CD40L and CD28/B7 interactions also were reported to be required for activation of human NK cells by DCs (50) . Blocking experiments using mAbs against CD40L (Fig. 5)Citation and other molecules, such as LFA-1 and B7 (data not shown), did not show significant effects on CpG-liposome–mediated activation of NK and NKT cells. CD1d molecules expressed by DCs are critical for DC/T-cell interaction (51) . However, CD1d is not involved in CpG-liposome–mediated activation of DCs and NK cells because similar activation of type 1 innate immunity was shown in CD1d-deficient mice (data not shown). Interactions of TNF/TNF receptor superfamily member molecules, such as 4–1BB/4–1BBL and OX40/OX40L, were reported to induce activation of T cells (52 , 53) . However, from the blocking experiment using mAbs, none of these molecules appeared to be involved in the activation of NK and NKT cells by DCs (data not shown). We currently are investigating the molecules essential for DC/NK (or NKT) cell interaction during activation with CpG-liposome.

We further showed that type 1 innate immunity induced by CpG-liposome is beneficial for activation of antigen-specific type 1 acquired immunity (Fig. 6)Citation . We showed that CpG-liposome coencapsulated with model tumor antigen induces tumor-specific tetramer+-CTLs, which specifically lyse antigen-expressing tumor cells. It has been reported that CpG-ODNs induce protective immunity against tumors when CpG vaccination is carried out before tumor inoculation (54 , 55) . The therapeutic effect of CpG-ODN plus tumor peptide also was shown using mice bearing a small tumor mass (<3 mm; ref. 56 ). Because we showed the strong vaccination activity of CpG + antigen-liposome in untreated mice (Fig. 6)Citation , CpG + antigen-liposome is expected to be a prophylactic reagent against virus or tumor. However, in the preliminary experiments, we recently have found that CpG-liposome encapsulated with model tumor antigen cures mice bearing a large tumor mass (9 to 10 mm; data not shown). Thus, selective activation of DC-based type 1 innate immunity by CpG-liposome effectively induces type 1 acquired immunity.

Antitumor immunity in normal or small tumor-bearing mice can be induced by vaccination with tumor antigen plus immunostimulating adjuvant (54, 55, 56) . However, the generation of antitumor immunity in late tumor-bearing mice is considered difficult because of strong immunosuppression mechanisms. Recent findings have indicated that (a) the suppressive activity of T-regulatory cells is abolished by CpG-activated type 1 immunity (57) ; and (b) Th1 cells are essential for the generation of fully activated CTLs and long-term maintenance of CTL memory (58, 59, 60) . Collectively, the present results indicate that CpG-liposomes are an excellent tool to induce type 1 innate and acquired immunity. We currently are investigating the therapeutic activity of CpG + antigen-liposome in mice bearing a large tumor or spontaneous tumor to develop an effective vaccination protocol applicable for human cancer patients. It recently has been reported that repeated injection or intravenous injection of CpG-ODN has adverse side effects in mice (61 , 62) . However, no serious adverse events have been attributed to the use of CpG in >500 subjects studied in >12 clinical trials. These clinical trials showed that CpG exhibits attractive immunomodulating activities for therapeutic intervention in allergic reactions and cancer (63) . Therefore, CpG-liposome alone or combined with tumor antigen protein provides a promising approach for the prevention or therapy of tumors.


   ACKNOWLEDGMENTS
 
We thank Dr. Luc Van Kaer (Vanderbilt University School of Medicine, Nashville, TN) for reviewing this article. We also thank Dr. Michiko Kobayashi (Genetics Institute, Cambridge, MA) and Takuko Sawada (Shionogi Pharmaceutical Institute Co., Osaka, Japan) for their donations of IL-12 and IL-2, respectively.


   FOOTNOTES
 
Grant support: Grant-in-aid for Science Research on Priority Areas and Millennium Project from the Ministry of Education, Culture, Sports, Science, and Technology.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Y. Suzuki and D. Wakita contributed equally to this work.

Requests for reprints: Takashi Nishimura, Division of Immunoregulation, Institute for Genetic Medicine, Hokkaido University, N-15 W-7, Kita-ku, Sapporo 060-0815, Japan. Phone and Fax: 81-11-706-6835; E-mail: tak24{at}igm.hokudai.ac.jp

Received 5/13/04. Revised 8/27/04. Accepted 9/28/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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