Cutting Edge: Inhibition of Experimental Tumor Metastasis by Dendritic Cells Pulsed with α-

This information is current as Isao Toura, Tetsu Kawano, Yasunori Akutsu, Toshinori of October 1, 2021. Nakayama, Takenori Ochiai and Masaru Taniguchi J Immunol 1999; 163:2387-2391; ; http://www.jimmunol.org/content/163/5/2387 Downloaded from References This article cites 34 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/163/5/2387.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ●

Cutting Edge: Inhibition of Experimental Tumor Metastasis by Dendritic Cells Pulsed with ␣-Galactosylceramide1

Isao Toura,*† Tetsu Kawano,* Yasunori Akutsu,*† Toshinori Nakayama,* Takenori Ochiai,† and Masaru Taniguchi2*

␣-GalCer inhibits tumor metastasis almost completely in the liver Downloaded from ␣ A unique lymphoid lineage, V 14 NKT cells, bearing an in- or lung (10), indicating that V␣14 NKT cells are activated in vivo ␣ ␣ variant Ag receptor encoded by V 14 and J 281 gene seg- by the ligand and that the activated V␣14 NKT cells serve as a ments, play crucial roles in various immune responses, includ- critical component of host immunity to tumors. ing protective immunity against malignant tumors. A specific MHC peptides recognized by tumor-specific CTL are being de- ligand of V␣14 NKT cells is determined to be ␣-galactosylce- fined in various mouse and human tumor cells, some of which ramide (␣-GalCer) which is presented by the CD1d molecule.

indeed induce CTLs in vitro and also inhibit tumor growth in vivo http://www.jimmunol.org/ Here, we report that dendritic cells (DCs) pulsed with ␣-Gal- (11–13). These results suggest that tumor-specific peptides are par- Cer effectively induce potent antitumor cytotoxic activity by ticularly effective for the induction of immune reactions against specific activation of V␣14 NKT cells, resulting in the inhibi- tumors. It is well established that CTL induction usually follows tion of tumor metastasis in vivo. Moreover, a complete inhibi- peptide presentation by MHC class I molecules on APC, and, tion of B16 melanoma metastasis in the liver was observed therefore, APCs pulsed with peptides in vitro can stimulate Ag- when ␣-GalCer-pulsed DCs were injected even 7 days after specific CTLs when administered in vivo. In fact, dendritic cells transfer of tumor cells to syngeneic mice where small but mul- (DCs) pulsed with a soluble Ag are able to induce protective an- tiple metastatic nodules were already formed. The potential titumor immunity accompanied by tumor-specific CTL induction

␣ ␣ by guest on October 1, 2021 utility of DCs pulsed with ␣-GalCer for tumor immunotherapy (14–17). Since -GalCer specifically activates V 14 NKT cells in ␣ is discussed. The Journal of Immunology, 1999, 163: 2387– vitro and in vivo, we address whether DCs pulsed with -GalCer ␣ 2391. are able to activate V 14 NKT cells in vivo and induce protective antitumor immunity in situ.

he V␣14 NKT cell has been recently defined as a novel Materials and Methods lymphocyte lineage, characterized by the expression of an Mice ␣ ␣ invariant Ag receptor encoded by V 14 and J 281 gene V␣14 NKT mice (RAG-1Ϫ/Ϫ V␣14 transgenic (tg) V␤8.2tg mice) with a T Ϫ Ϫ segments (1–3), and detected in most of the peripheral tissues, C57BL/6 (B6) background were established by mating with RAG-1 / including lung, liver, bone marrow, and placenta (4). A V␤8.2tg and RAG-1Ϫ/Ϫ V␣14tg mice as described (5). In V␣14 NKT Ag, ␣-galactosylceramide (␣-GalCer),3 has been found to be a mice, only transgenic TCR␣␤ (V␣14tg and V␤8.2tg) was expressed and thus resulted in preferential development of V␣14 NKT cells with unde- ligand for V␣14 NKT cells and specifically presented by a class Ib Ϫ Ϫ tectable T cells, NK cells, and B cells (5). RAG-1 / mice were kindly molecule, CD1d (5–7), which is well conserved through mamma- provided by Dr. Susumu Tonegawa, MIT, Boston, MA (18). Pathogen-free lian evolution and lacks allelic polymorphism (8, 9). Injection of B6 mice were purchased from Japan SLC (Shizuoka, Japan). All mice used in this study were 8–10 wk old and were maintained in our animal facility under specific pathogen-free conditions.

* CREST (Core Research for Evolutional Science and Technology) Project and De- ␣-Galactosylceramide partment of Molecular Immunology, Graduate School of Medicine, School of Med- † icine, and Second Department of Surgery, School of Medicine, Chiba University, ␣-Galactosylceramide ((2S,3S,4R)-1-O-(␣-D-galactopyranosyl)-N-hexaco- Chiba, Japan sanoyl-2-amino-1,3,4-octadecanetriol; ␣-GalCer) was provided by Kirin Received for publication May 25, 1999. Accepted for publication July 6, 1999. Brewery (Gunma, Japan) and prepared as previously described (5, 10). 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 Preparation of DCs pulsed with -GalCer with 18 U.S.C. Section 1734 solely to indicate this fact. DCs were prepared from spleen of B6 mice according to the methods of 1 This work was supported in part by a Grant-in-Aid for Scientific Research on Pri- Crowly et al. (5, 19). Briefly, spleens were treated with collagenase type III ority Areas (06282103) from the Ministry of Education, Culture, Sports, and Science, (400 U/ml; Worthington Biochemical, Freehold, NJ) for 20 min at 37°C in Japan, and by a grant from Taisho Pharmaceutical Company, Japan. 5% CO2 and then disrupted on a metal screen. Resulting single cells were 2 Address correspondence and reprint requests to Dr. Masaru Taniguchi, Depart- ment of Molecular Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: [email protected]. 3 Abbreviations used in this paper: ␣-GalCer, ␣-galactosylceramide; DC, dendritic chiba-u.ac.jp cell; LLC, Lewis lung cancer; tg, transgenic; B6, C57BL/6.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

● 2388 CUTTING EDGE

FIGURE 1. Activation of V␣14 NKT cells in vivo by administration of ␣-GalCer-pulsed DCs. A, B6 mice were i.v. administered 1 ϫ 106 vehicle- pulsed DCs (⅜), 2 ϫ 105 ␣-GalCer-pulsed DCs (Œ), or 1 ϫ 106 ␣-GalCer- pulsed DCs (ⅷ) of B6 mice. Twenty four hours after the administration, their cytotoxicity was examined on YAC-1 cells at indicated E:T ratios. B, ␣-GalCer-pulsed (ⅷ) or vehicle-pulsed (⅜) DCs (3 ϫ 105) from RAG-1Ϫ/Ϫ mice were transferred i.v. into V␣14 NKT mice. Their cyto- toxicity was assayed 24 h after DC transfer on B16 melanoma and Colon 26 cells. Downloaded from loaded on a dense BSA (Pentex Path-O-Cyte 4; Bayer, IL) and centrifuged at 1500 ϫ g for 30 min at 4°C. The low density fraction was further applied to plastic culture dishes (Falcon, Franklin Lakes, NJ) for 90 min at 37°C in ␣ 5% CO2. Adherent cells were pulsed with -GalCer (100 ng/ml in 0.025% polysolvate 20) or control vehicle (0.025% polysolvate 20) overnight at 37°C. After washing extensively, nonadherent cells were used as ␣-GalCer-pulsed DCs. http://www.jimmunol.org/ 51Cr release cytotoxicity assay The 51Cr release assay was conducted by a standard method as described (10). In brief, ␣-GalCer-pulsed DCs were injected i.v. into normal B6 or V␣14 NKT mice, and, 24 h later, the cytotoxic activity of their spleen cells was assessed on YAC-1 cells, B16 melanoma cells, or Colon 26 cells labeled with 100 ␮Ci of sodium chromate (51Cr; Amersham Pharmacia Biotech, Uppsala, Sweden) for1hat37°C. Effector cells (3 ϫ 105) were mixed with 1 ϫ 104 tumor cells at indicated E:T ratios in a 96-well round- bottom plate (Falcon, Lincoln Park, NJ) in 150 ␮l of complete media at 51 FIGURE 2. Inhibition of tumor metastasis by ␣-GalCer-pulsed DCs. by guest on October 1, 2021 37°C in 5% CO2. Four hours later, released Cr was measured. The per- centage cytotoxicity was calculated, and mean values were shown with The antitumor effect of ␣-GalCer-pulsed DCs was evaluated using two SDs as described (10). different metastasis models: liver metastasis of B16 melanoma (A) and lung metastasis of LLC (B). Tumor cells were inoculated into B6 mice on day Tumor metastasis model 0. One day after the inoculation, indicated numbers of ␣-GalCer-pulsed B16 melanoma cells (3 ϫ 106) or Lewis lung carcinoma (LLC) cells (4 ϫ DCs were transferred i.v. Representative photographic views of metasta- 105) were inoculated into B6 mice on day 0 via spleen for liver metastasis sized organs are shown. The quantitative analysis of tumor metastasis was or via tail vein for lung metastasis, respectively. In the case of liver me- performed by measuring the amount of melanoma Ag, GM3 in tastasis model, mice were sacrificed on day 14, and the metastasis of B16 the liver (A, right), or the number of metastatic nodules in the lung (B, melanoma cells in the liver was evaluated by measuring the amount of right). The levels were indicated as filled or open bars in ␣-GalCer-pulsed melanoma Ag (GM3 ganglioside) as described (20). For the lung metastasis DCs or control vehicle-DCs, respectively. Five mice were used in each model, mice were sacrificed on day 18, and the number of metastatic nod- group, and mean values with SDs are shown. ules of LLC was counted under a light microscope. Three to five mice were used for each group in every experiment. ␣-GalCer-pulsed DCs were transferred i.v. istered 1 ϫ 106 ␣-GalCer-pulsed DCs. A potent antitumor cyto- toxicity against B16 melanoma and Colon 26 cells was detected Results only when ␣-GalCer-, but not vehicle-, pulsed DCs were injected ␣ ␣ Activation of V 14 NKT cells by -GalCer-pulsed DCs in vivo (Fig. 1B). We have reported that murine and human NKT cells recognize ␣ ␣-GalCer in a CD1d-restricted fashion (5, 6, 21–23). Moreover, Inhibition of tumor metastasis by -GalCer-pulsed DCs the administration of ␣-GalCer activates V␣14 NKT cells in vivo To test whether the transfer of ␣-GalCer-pulsed DCs induces in- and induces cytotoxic activity against various tumor cells in an hibition of tumor metastasis as a consequence of in vivo activation NK-like fashion (5, 10). Thus, we expected efficient activation of of V␣14 NKT cells, ␣-GalCer- or vehicle-pulsed DCs were i.v. resting V␣14 NKT cells with ␣-GalCer-pulsed DCs in vivo. To transferred into normal B6 mice on the next day (day 1) after test this assumption, graded numbers of ␣-GalCer-pulsed DCs intrasplenic injection of tumor cells on day 0 (day 0). Hepatic were i.v. administered into normal B6 mice, and their cytotoxic metastasis of B16 melanoma cells was inhibited by transfer of activity in the spleen cells was evaluated by 51Cr release assay ␣-GalCer-pulsed DCs in a dose-dependent manner (Fig. 2A). No (Fig. 1A). A significant cytotoxicity against YAC-1 cells was in- metastatic nodules were detected in the liver with injection of duced by more than 2 ϫ 105 cells of ␣-GalCer-, but not vehicle-, ␣-GalCer-, but not vehicle-, pulsed DCs (3 ϫ 106 cells) macro- pulsed DCs. This suggests that ␣-GalCer-pulsed DCs indeed ac- scopically (Fig. 2A) and microscopically (data not shown). No sig- tivate V␣14 NKT cells in vivo. To confirm the data in Fig. 1A, nificant melanoma Ag was detected by quantitation of B16 mela- V␣14 NKT mice bearing only V␣14 NKT cells were i.v. admin- noma Ag, GM3 ganglioside, when 3 ϫ 106 ␣-GalCer-pulsed DCs The Journal of Immunology 2389

were transferred (Fig. 2A, right). The inhibitory effect of tumor metastasis was also observed in a lung metastatic model using LLC cells. A single i.v. injection of ␣-GalCer-pulsed DCs (3 ϫ 106), but not vehicle-pulsed DCs, induced a complete inhibition of lung metastasis of LLC (Fig. 2B).

Eradication of established metastatic foci by ␣-GalCer-pulsed DCs The next experiments were designed to know whether the ␣-Gal- Cer-pulsed DCs were also effective on established tumors. To ad- dress this question, ␣-GalCer-pulsed DCs were i.v. transferred into B6 mice bearing multiple metastatic tumor foci in the liver. First, the levels of metastasis of B16 melanoma cells in the liver were monitored, and representative results on days 5, 7, and 9 are shown in Fig. 3A. On day 5, no metastatic foci were observed macro- scopically, whereas numerous single melanoma cells were micro- scopically identified in a section of the liver (arrowheads). On day 7, however, numerous metastatic foci were detected both macro-

scopically and microscopically. Their sizes were increased, and Downloaded from some were fused with each other on day 9. The sizes of tumor foci on day 7 were in the range between 0.1 and 0.5 mm in diameter, and about 100 foci were detected macroscopically (Fig. 3B). Con- sequently, we started to transfer 3 ϫ 106 vehicle- or ␣-GalCer- pulsed DCs on day 7, on day 9, and on day 11 and continued every other day until day 13. We chose the dose of DCs (3 ϫ 106) that http://www.jimmunol.org/ showed significant curative effects, as shown in Fig. 2. As we anticipated, almost complete eradication of established metastatic foci was observed when the transfer of ␣-GalCer-pulsed DCs (3 ϫ 106) started on day 7 (Fig. 3C). Little effect was observed whether the transfer started on day 9 or day 11. These results strongly indicated that ␣-GalCer-pulsed DCs exerted a strong antitumor effect, resulting in the regression of the established metastatic nod- ules. Finally, to evaluate the efficacy of ␣-GalCer-pulsed DCs, we

treated the melanoma-bearing B6 mice with simple injection of by guest on October 1, 2021 ␣-GalCer (100 ␮g/kg) started on day 1, 3, 5, and 7 (Fig. 3D). In contrast to the treatment with ␣-GalCer-pulsed DCs, significant antitumor effect was observed only when the ␣-GalCer injection started on day 1 and on day 3, suggesting a significant advantage for the use of ␣-GalCer-pulsed DCs in the eradication of the es- tablished tumor metastasis.

Discussion We have recently reported that the injection of ␣-GalCer (10) or FIGURE 3. Regression of established metastatic tumors by ␣-GalCer- IL-12 (20) prevents liver or lung metastasis of tumors via activa- pulsed DCs. The macroscopic and microscopic views of tumor foci (A) and tion of V␣14 NKT cells in vivo in situ. The antitumor effect is also their sizes and numbers on day 7 and day 9 are shown (B). Arrows in A indicate micrometastasis of B16 melanoma cells. C, Effects of i.v. admin- observed in various malignant tumor cells, including LLC (20), istration of ␣-GalCer-pulsed DCs on established B16 melanoma foci were erythroleukemia (Friend virus-induced lymphoma cell, FBL-3), assessed. B6 DCs (3 ϫ 106) were pulsed with vehicle (open bar) or ␣-Gal- Colon 26, or a TAP-2-deficient mutant of Rauscher virus-induced Cer (filled bar) and then i.v. transferred into tumor-bearing B6 mice. The lymphoma (RMA-S) (10). Although the antitumor effects are dra- transfer of pulsed DCs started on day 7, day 9, or day 11. The same num- matic, it is not clear whether the effects are also observed on the bers of pulsed DCs were injected every other day until day 13. All mice established tumor nodules. In this paper, we extended our inves- were sacrificed on day 14. Representative photographic views are shown. tigation by using ␣-GalCer-pulsed DCs and trying to treat estab- Three mice are used in each group, and mean values with SDs are shown. lished metastatic tumor foci. Seven days after intrasplenic inocu- The results of two independent experiments were similar.D,Tumor-bear- lation of B16 melanoma cells, the number of metastasized nodules ing B6 mice were treated with i.v. injection of vehicle (open bar) or ␣-Gal- ␮ in the liver were more than 100, and their sizes were in the range Cer (100 g/kg; filled bar) in vivo, which started on days 1, 3, 5, and 7 and continued every other day until day 13. The amounts of melanoma Ag were between 0.1 to 0.5 mm in diameter. These tumor nodules were measured and are depicted as bars. almost completely eradicated when the mice were treated with 3 ϫ 106 ␣-GalCer-pulsed DCs on days 7, 9, 11, and 13 (Fig. 3C). Since it takes 24 h to activate V␣14 NKT cells by the administration of 106) of DCs injected were not enough to eliminate established ␣-GalCer-pulsed DCs (Fig. 1 and Ref. 10), even slightly bigger tumor cells with exponential proliferation or bigger tumor sizes. In tumor nodules than those observed on day 7 are thought to be any event, these results encourage the view of clinical applications, regressed. We did not observe significant antitumor effect when particularly those that focus on the treatment of micrometastasis the treatment started on day 9, probably because the numbers (3 ϫ after radical operations. 2390 CUTTING EDGE

␣-GalCer is presented by a monomorphic CD1d on DCs and a unique subset of major histocompatibility complex class I-specific CD4ϩ and Ϫ Ϫ recognized by the invariant V␣14 NKTCR (5, 6). Injection of CD4 8 T cells in mice and humans. J. Exp. Med. 180:1097. 3. Makino, Y., R. Kanno, T. Ito, K. Higashino, and M. Taniguchi. 1995. Predom- ␣-GalCer in vivo, only when started soon after tumor cell transfer, ϩ ϩ inant expression of invariant V␣14 TCR ␣ chain in NK1.1 T cell populations. induces antitumor activity against metastatic tumors macroscopi- Int. Immunol. 7:1157. cally (5) and microscopically (24). This indicates that it takes a 4. Makino, Y., H. Koseki, Y. Adachi, T. Akasaka, K. Tsuchida, and M. Taniguchi. longer time for DCs to present ␣-GalCer, to activate V␣14 NKT 1994. Extrathymic differentiation of a T cell bearing invariant V␣14J␣ 281 TCR. cells, and to induce antitumor activities in vivo. However, ␣-Gal- Int. Rev. Immunol. 11:31. 5. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno, Cer-pulsed DCs are more effective than the direct injection of R. Nakagawa, H. Sato, E. Kondo, H. Koseki, and M. Taniguchi. 1997. CD1d- ␣-GalCer, as evidenced by the results showing that established restricted and TCR-mediated activation of V␣14 NKT cells by glycosylceram- metastatic foci can be regressed by ␣-GalCer-pulsed DCs but not ides. Science 278:1626. by ␣-GalCer injection (Fig. 3, C and D). 6. Burdin, N., L. Brossay, Y. Koezuka, S. T. Smiley, M. J. Grusby, M. Gui, M. Taniguchi, K. Hayakawa, and M. Kronenberg. 1998. Selective ability of The manipulation of tumor immunity is totally dependent on the mouse CD1 to present : ␣-galactosylceramide specifically stimulates induction of tumor-specific cytotoxicity, which is known to be V␣14ϩ NK T lymphocytes. J. Immunol. 161:3271. mediated by CTL (14–16). Thus, many efforts have been made to 7. Brossay, L., N. Burdin, S. Tangri, and M. Kronenberg. 1998. Antigen-presenting identify tumor-specific peptides, including human malignant mel- function of mouse CD1: one molecule with two different kinds of antigenic li- anoma-related Ags, such as gp100 (25), melanoma antigen recog- gands. Immunol. Rev. 163:139. 8. Porcelli, S. A. 1995. The CD1 family: a third lineage of antigen-presenting mol- nized by T cells-1 (MART-1 (11)), tyrosinase (11), and melanoma- ecules. Adv. Immunol. 59:1. associated antigen-1 (MAGE-1) and MAGE-3 (26). Indeed, some 9. Porcelli, S. A., B. W. Segelke, M. Sugita, I. A. Wilson, and M. B. Brenner. 1998. of these peptides have shown a successful induction of antimela- The CD1 family of antigen-presenting molecules. Immunol. Today 19:362. Downloaded from noma immunity (12). Furthermore, various tumor-associated Ags 10. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, H. Sato, E. Kondo, or specific peptides, such as PSA (27), papilloma virus protein M. Harada, H. Koseki, T. Nakayama, Y. Tanaka, and M. Taniguchi. 1998. Nat- ural killer-like nonspecific tumor cell lysis mediated by specific ligand-activated (28), receptor for advanced glycosylation and products-1 (RAGE-1 V␣14 NKT cells. Proc. Natl. Acad. Sci. USA 95:5690. (29)), and tumor associated mucin 1 (MUC1) (30, 31), are iden- 11. Kawakami, Y., S. Eliyahu, K. Sakaguchi, P. F. Robbins, L. Rivoltini, tified, and their ability to generate CTL is elucidated. J. R. Yannelli, E. Appella, and S. A. Rosenberg. 1994. Identification of the im- However, there are several critical problems for the application munodominant peptides of the MART-1 human melanoma antigen recognized by

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