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NK- and CD8⁺ T Cell-Mediated Eradication of Established Tumors by Peritumoral Injection of CpG-Containing Oligodeoxynucleotides¹

Division of Molecular Immunology, Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany

	Abstract

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Unmethylated cytosine-phosphorothioate-guanine (CpG) containing oligodeoxynucleotides (CpG-ODN) are known to act as adjuvants and powerful activators of the innate immune system. We investigated the therapeutic effect of CpG-ODN on a variety of established mouse tumors including AG104A, IE7 fibrosarcoma, B16 melanoma, and 3LL lung carcinoma. These tumors are only weakly immunogenic and notoriously difficult to treat. Repeated peritumoral injection of CpG-ODN resulted in complete rejection or strong inhibition of tumor growth, whereas systemic application had only partial effects. The CpG-ODN-induced tumor rejection was found to be mediated by both NK and tumor-specific CD8⁺ T cells. Comparison of parental tumors and variants rendered more antigenic by transfection with tumor Ags suggested that the efficiency of the CpG-ODN therapy correlated with the antigenicity of the tumors. Peritumoral CpG-ODN treatment was even effective in a situation where the immune system was tolerant for the tumor Ag, as shown by breakage of tolerance and tumor elimination. These results suggest that peritumoral application of CpG-ODN acts locally by inducing NK cells, and also leads to efficient presentation of tumor Ags and stimulation of CD8⁺ effector and memory T cells, thus providing a powerful antitumor therapy that can be also applied without knowledge of the tumor Ag.

	Introduction

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Although many tumors are known to be antigenic in syngeneic hosts, they fail to induce an effective immune response. Diverse reasons could account for this phenomenon, e.g., lack of costimulatory molecules that are required for efficient T cell activation (1, 2, 3, 4); secretion of inhibitory factors such as VEGF, IL-10, TGF- and PGE₂ (5, 6, 7); or ignorance, inferring that the tumor is not seen by the immune system (8). In other situations, tumors induce a T cell response that can be identified as tumor-infiltrating lymphocytes (TILs)³ or, in the periphery, by staining with HLA tetramers, but this response is often not effective, possibly due to induction of peripheral tolerance or anergy (9, 10). Therefore, immunization strategies are required that not only expand tumor-specific lymphocytes but also sustain their activity. Recent progress in the development of tumor vaccines is based on the identification of tumor-associated rejection Ags, or peptides thereof, that are presented by MHC molecules to T lymphocytes (11). In other approaches dendritic cells (DC) were pulsed with whole tumor extracts or tumor lysates (12, 13). Hybrids generated by fusion of tumor cells with APCs (14, 15) and tumor-derived heat-shock proteins that carry tumor-specific peptides have also been used as tumor vaccines (16, 17, 18).

Despite the fact that most of these vaccines are powerful activators of tumor-specific T lymphocytes their clinical efficacy is, so far, limited. Immune evasion of solid tumors may be related to an intrinsic resistance for lymphocytic infiltration (19, 20), to tolerance, or to a failure to sustain T cell activity (8). Thus, additional or alternative strategies are required for the eradication of solid tumors. NK cells and macrophages have been shown to be critical antitumor effector cells (21). NK cells are especially efficient against tumors with low MHC class I expression (22), which are likely to evade T cell mediated immune responses. Among various cytokines produced by NK cells, IL-12 mediates cytotoxicity, whereas IFN- is a strong promoter of Th1 immune responses and, in addition, activates macrophages. Ideally, immunological tumor therapy should take advantage of both the innate and the adaptive immune system.

Bacterial unmethylated cytosine-phosphorothioate-guanine (CpG)-rich oligodeoxynucleotides (CpG-ODN) are potent activators of innate immunity and induce cellular response mediated by Toll-like receptor 9 (23) Thus, CpG-ODN are strong inducers of NK cells (24), probably through induction of IL-12 (25, 26). In addition, CpG-ODN have been shown to induce maturation of DC (27, 28, 29) and to act as efficient adjuvants for priming of CD8⁺ T cells when coinjected with Ag (30). In this study, we investigated the effect of peritumoral injection of CpG-ODN on induction of tumor immunity. A panel of murine transplantation tumors was selected which differ in their antigenicity, but grow progressively in the host and, therefore, represent a particular challenge for immunotherapy. Our data show that peritumoral application of CpG-ODN leads to local activation of NK cells and priming of tumor-specific CD8⁺ cells resulting in growth suppression or rejection of established tumors.

	Materials and Methods

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Mice

Female C3H/HeN (C3H), C57BL/6, (C3H/HeN x C57BL6)F₁ mice were purchased from Charles River Wiga (Sulzfeld, Germany). Alb.K^bxDes.TCR (H-2^kxd) mice, expressing K^b under the control of the albumin promoter exclusively in the liver and the K^b-specific TCR (Des.TCR), display peripheral tolerance toward the K^b molecule, as described previously (31, 32). All mice were kept under specific pathogen-free conditions at the central animal facilities of the German Cancer Research Center (Heidelberg, Germany).

Tumor cell lines

AG104A, a spontaneous fibrosarcoma of C3H (H-2^k) mice and the L^d transfectant, AG104A-L^d, were kindly provided by Dr. H. Schreiber (University of Chicago, Chicago, IL) (19). P815.K^b is the K^b transfectant of P815 mastocytoma cells derived from DBA/2 (H-2^d) mice (32). These cells were cultured in DMEM (Life Technologies, Eggenstein, Germany) with 5% heat-inactivated FCS (Biochrom, Berlin, Germany), 10 mM HEPES (Gerbu, Gaibeg, Germany), 2 mM L-glutamine (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 100 U/ml penicillin/streptomycin (Life Technologies), and 0.05 mM 2-ME (Merck, Darmstadt, Germany) at 37°C and 5% CO₂.

IE7 is a metastatic clone from the methylcholanthrene-induced T10 sarcoma of H-2^b x H-2^k mice, and IE7K^k is a H-2K^k transfectant (33). B16 is a melanoma from C57BL/6 mice and MO4 is an OVA transfectant of B16 cells (34), kindly provided by S. Schnell (Memorial Sloan-Kettering Cancer Center, New York, NY). D122 is a highly metastatic variant of the 3LL spontaneous lung carcinoma of C57BL/6 mice (35). These tumor cells were cultured in RPMI 1640 medium (Life Technologies), supplemented with 10% heat-inactivated FCS, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin/streptomycin, and 0.05 mM 2-ME. For s.c. injection, adherent cells were trypsinized (0.25% trypsin, 2.5 mM EDTA) and resuspended in Dulbecco’s PBS (Life Technologies).

Reagents

Phosphothioate-stabilized CpG ODN 1668 (5'-TCCATGACGTTCCTGATGCT-3') was synthesized by the central oligonucleotide facility of the German Cancer Research Center.

Animal experiments

AG104A and AG104A-L^d (2 x 10⁵), IE7 and IE7K^k (2 x 10⁵), B16 and MO4 (2 x 10⁵), 3LL (5 x 10⁵), and P815.K^b (5 x 10⁵) cells were inoculated s.c. or into the hind foot pad (IE7 and IE7K^k) of mice. CpG-ODN 1668 (20 µg) was injected peritumorally at various intervals starting day 5 (B16 and MO4) or day 7 (AG104A, AG104A-L^d, IE7, IE7K^k, 3LL, and P815.K^b). Tumor volumes were measured with a caliper and the formula (volume = 0.5 x length x (width)²) was applied to determine tumor growth kinetics. For each experiment, groups of 6–10 mice were used.

For in vivo depletion studies, 1 mg of the anti-CD8 mAb YTS 105.18.10 (36) was injected i.p. on days 6, 13, 20, and 27 (for AG104A-L^d) or days 5, 12, and 19 (for MO4) after tumor inoculation. Depletion of CD8⁺ cells was 90–100% effective as judged by staining of peripheral blood lymphocytes for CD8. NK cells were depleted in vivo by i.p. injection of 0.5 mg of the anti-IL-2 receptor -chain mAb TM-1 (37) on days 6 and 20 (for AG104A-L^d) or days 5 and 19 (for MO4). After treatment with mAb TM-1, poly(I:C) treatment did not result in induction of NK cell activity. TM-1 treatment does not affect allogeneic cytotoxic T cell induction (37).

Green fluorescent CellTracker (Molecular Probes, Leiden, The Netherlands) was used to monitor CpG-ODN-induced migration of skin DC into the draining lymph nodes as previously described (38). Briefly, 20 µg CpG-ODN were injected s.c. into the abdominal flanks of C3H mice and 400 µl CellTracker (1/20 in ethanol) applied on the skin at the site of CpG-ODN injection. Twenty-four hours later, cell suspensions from draining axillary and nondraining mesenteric lymph nodes were prepared by treatment in RPMI 1640 supplemented with 5% FCS and 100 µg/ml collagenase D for 30 min. The resulting cell suspension was stained with PE-labeled anti-mouse CD11c (DC), CD5 (T lymphocytes), or CD40 for FACS analysis. CD11c⁺ DC analysis was performed on large granular cells based on forward and side scatter.

CTL assay

Forty-five days after tumor inoculation, spleen cells were removed from mice which had rejected the AG104A-L^d tumor after peritumoral treatment with CpG-ODN. Splenocytes were cocultured with irradiated (100 Gy) Ag104A-L^d cells (5 x 10⁵/ml) in 6-well plates for 6 days in a humidified 7% CO₂ incubator at 37°C. For determination of cytotoxicity, target cells were labeled with 100 µCi of Na⁵¹CrO₄ (NEN Life Science, Zaventem, Belgium) for 90 min at 37°C and incubated with effector cells in 96-well round-bottom plates for 4 h of incubation at 37°C in 6% CO₂. Specific lysis was calculated according to the formula: the percentage of specific lysis = [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100.

	Results

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Peritumoral injection of CpG-ODN induces rejection of established tumors

AG104A is a fibrosarcoma of C3H (H-2^k) origin (39). Transfection with the MHC class I alloantigen L^d (AG104A-L^d) fails to prevent growth in C3H or even in 2C TCR transgenic mice carrying a TCR against L^d. Moreover, simultaneous injection of tumor cells and transplantation with L^d-positive skin grafts results only in the rejection of the graft, but not in rejection of the L^d-expressing tumor (19). This observation demonstrates that an ongoing immune response against a tumor-associated Ag does not guarantee tumor rejection.

To investigate a potential antitumoral effect of CpG-ODN, C3H mice were inoculated s.c. with 2 x 10⁵ AG104A fibrosarcoma cells or with the L^d transfectant (AG104A-L^d). 7 days later, when the tumors had grown to 50 mm³, 20 µg of CpG-ODN 1668 was injected peritumorally twice a week for a period of 4 wk. Complete tumor rejection was observed for AG104A wild-type and AG104A-L^d tumors after eight injections of CpG-ODN (Fig. 1, a and b). Two injections of CpG-ODN inhibited tumor growth only initially, but 3 wk later the tumors grew progressively (Fig. 1c). The therapeutic effect of CpG-ODN was also tested for two other pairs of tumors that differ with regard to their antigenicity. IE7 is a methylcholanthrene-induced metastatic fibrosarcoma of (C3HxC57 BL/6)F₁ mice which expresses D^b and D^k molecules, but lacks K^b and K^k. Restoration of K^k expression by transfection (IE7.K^k) renders the tumor immunogenic and prevents formation of metastasis but the primary tumor continues to grow despite the presence of a tumor-specific T cell response (33). Fig. 2, a and b, shows that peritumoral CpG-ODN injection inhibits the growth of IE7K^k and of the less antigenic parental IE7 tumors, albeit to a lower extent. Similar results were obtained with the B16 melanoma and the more antigenic OVA transfectant MO4 (34) (Fig. 2, c and d). Again, in the presence of a strong tumor Ag, OVA, CpG-ODN treatment was more efficient. Finally, the effect of CpG-ODN on the spontaneous, C57BL6-derived 3LL.D122 Lewis lung carcinoma was tested. This tumor cell line expresses only low levels of the K^b MHC class I molecule and, therefore, fails to induce a protective immune response when transplanted s.c. (35). Peritumoral application of CpG-ODN also resulted in suppression of the weakly antigenic 3LL.D122 tumor (Fig. 2e). Together, these results indicate that CpG-ODN treatment can suppress not only growth of immunogenic but also of nonimmunogenic tumors.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Inhibitory effect of peritumoral CpG-ODN treatment on growth of the AG104A tumor and its Ld transfectant. a and b, A total of 2 x 105 AG104A cells or AG104A-Ld fibrosarcoma cells were inoculated s.c. into the right flank of C3H female mice on day 0. Twenty micrograms of CpG-ODN 1668 or PBS were injected peritumorally twice a week starting on day 7. A total of 8 injections were applied as indicated by arrows. C, AG104A-Ld tumor-bearing mice were treated peritumorally two or eight times with 20 µg of CpG-ODN. Each group represents six mice. Results are presented as mean ± SE. The experiment was repeated several times with comparable results.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Inhibitory effect of peritumoral CpG-ODN treatment on IE7, B16, and 3LL tumors. a and b, A total of 2 x 105 of IE7 fibrosarcoma cells or the Kk transfectant, IE7Kk, were injected into the hind footpad of (C57BL6xC3H)F1 female mice. Seven days later the mice were peritumorally treated with 20 µg of CpG-ODN 1668 as indicated by arrows. c and d, A total of 2 x 105 B16 melanoma cells and the corresponding OVA transfectant, MO4, were inoculated s.c. into the right flank of C57BL/6 female mice. Five days later, the mice were injected peritumorally with 20 µg of CpG-ODN 1668 as indicated by arrows. E, A total of 5 x 105 of the spontaneous lung carcinoma cells 3LL.D122 were inoculated s.c. into the right flank of C57BL/6 female mice and the mice were treated with 20 µg of CpG-ODN 1668 as indicated. Each group represents six mice. Results are presented as mean ± SE.

Because local application of CpG-ODN greatly enhanced the antitumor response, we investigated whether CpG-ODN would also induce systemic antitumor immunity. In one set of experiments, AG104A or AG104A-L^d cells were injected s.c., followed by CpG-ODN injection in the opposite, tumor-free flank. In the more immunogenic L^d-transfectant, contratumoral CpG-ODN treatment retarded tumor growth (Fig. 3a), however, the parental cell line grew progressively (Fig. 3b). In another approach, AG104A-L^d cells were inoculated into both flanks of C3H mice, but only one tumor was peritumorally injected with CpG-ODN. In this experiment, the treated tumor was completely rejected in five of six mice and tumor growth was strongly inhibited in the remaining animal. Three of six mice rejected the distal tumor on the other flank and growth suppression was observed in the other three mice (Fig. 3c). These results suggest that s.c. CpG-ODN application not only stimulates a local, but also a systemic, antitumor response implying a role for T cells.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Effect of local vs distal application of CpG-ODN. a and b, A total of 2 x 105 AG104A-Ld or AG104A wild-type cells were inoculated into the right flank of C3H female mice. Mice were injected with PBS or 20 µg CpG-ODN starting at day 7 as indicated by arrows, either peritumorally or into the opposite tumor free flank. c, A total of 2 x 105 AG104A-Ld cells were inoculated into both flanks of C3H female mice. Seven days later the tumor on the right flank was treated peritumorally with 20 µg of CpG-ODN or PBS. CpG-ODN inhibited growth of directly treated tumors in six of six mice, whereas the tumor on the distal flank was rejected in three of six mice. Results are presented as mean ± SE.

To investigate whether memory immune responses were induced by CpG-ODN treatment of tumors, C3H mice that had rejected the AG104A-L^d tumor were rechallenged 10–13 wk later with the AG104A wild-type tumor or the AG104A-L^d variant, without further CpG-ODN application. Only 4 of 14 mice developed tumors after a second challenge with AG104A-L^d cells, whereas the parental AG104A tumor grew in 7 of 8 (Table I). These results show that CpG-ODN treatment induces a tumor-specific memory response (in this case L^d-specific), the strength of which appears to correlate with the tumor’s antigenicity. To demonstrate the presence of primed L^d-specific CD8⁺ T cells, splenocytes from mice that had rejected the AG104A-L^d tumor following CpG-ODN treatment were restimulated in vitro with AG104A-L^d cells. Fig. 4 shows the resulting CTLs which specifically lysed AG104A-L^d target cells but not unrelated B16 tumor cells and only weakly wild-type AG104A cells, indicating that the majority of CTL were L^d specific.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Peritumoral CpG-ODN application induces tumor-specific CTL. A total of 2 x 105 AG104A-Ld cells were inoculated s.c. into C3H mice and treated eight times with 20 µg of CpG-ODN 1668, resulting in tumor rejection. On day 45 after primary tumor inoculation, splenocytes were restimulated in vitro for 6 days with AG104A-Ld cells. Targets were 51Cr-labeled AG104A, AG104A-Ld, and B16 tumor cells.

CpG-ODN is known to trigger innate immune responses and to enhance, thereby, T cell-mediated immunity. To analyze the cellular components of the immune system that were responsible for tumor rejection after local treatment with CpG-ODN, we focused on two likely candidates and depleted CD8⁺ and NK cells in vivo with specific Abs. The growth of PBS-treated AG104A-L^d tumors was not altered by depletion of CD8⁺ or NK cells. In contrast, depletion of each cell population alone significantly reduces the antitumor effects seen after CpG-ODN treatment (Fig. 5, a and b). Simultaneous depletion of CD8⁺ and NK cells completely abrogates the therapeutic effect of CpG-ODN treatment (Fig. 5c). Comparable results were obtained in the MO4 tumor system (Fig. 5d). These data demonstrate that both CD8⁺ and NK cells are crucial mediators of CpG-ODN-induced tumor rejection, and that they act synergistically in the eradication of AG104A-L^d and MO4 tumors.

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Both NK cells and CD8+ T cells synergistically mediate CpG-ODN-induced tumor rejection. a–c, C3H mice were inoculated s.c. with 2 x 105 AG104A-Ld cells. NK cells (a), CD8+ (b), or both NK and CD8+ (c) cells were depleted and 20 µg of CpG-ODN 1668 or PBS were injected peritumorally as indicated by arrows. d, C57BL/6 mice were inoculated s.c. with 2 x 105 MO4 cells. NK cells or CD8 cells were depleted and tumors were treated nine times with 20 µg of CpG-ODN 1668 or PBS. On day 21 tumor volume were measured. The volume of the PBS treated tumor was set to 100%. Six mice per group were analyzed. Results are presented as mean ± SE.

View larger version (45K): [in this window] [in a new window]   FIGURE 6. CpG triggers migration of skin DC to the draining lymph nodes. CellTracker was applied on the skin at the site of CpG-ODN injection (20 µg s.c. in the abdominal flanks). Twenty-four hours later, draining axillary lymph node cells were analyzed for presence of migrating (CellTracker-positive) cells. DC were identified as large granular CD11c+ cells. Irrelevant isotype-matched Abs and cells from mice not treated with CellTracker were used to set gates. The upper left panel shows that 25% of DC in draining lymph node migrated from the skin. A negligible amount of DC migrated to the draining lymph in the absence of CpG (upper middle panel). No migrating DC were detected in the distal mesenteric lymph nodes (not shown). T cells (bottom panel), B cells, or granulocytes (not shown) in draining lymph nodes were not CellTracker positive.

There is increasing evidence that tumors can induce specific peripheral tolerance or anergy (9, 10). Therefore, we investigated whether CpG-ODN treatment could break tolerance in a scenario where the hosts CD8⁺ T cells are tolerant toward the tumor Ag. For this purpose we used Alb.K^bxDes.TCR mice, which express K^b exclusively on hepatocytes and a K^b-specific TCR. These mice display peripheral tolerance to the K^b Ag and fail to reject K^b-positive tumor cells (32). Alb.K^bxDes.TCR mice were inoculated s.c. with P815.K^b tumor cells which are known to be NK cell resistant (40). Peritumoral treatment with CpG-ODN resulted in reduced growth in some animals (summarized as group A in Fig. 7) or complete rejection in others (group B) despite the tolerant status of the mice. To demonstrate that tolerance had been broken, all mice were challenged again with P815.K^b cells on day 35. Fig. 7 shows that all mice which had rejected the first tumor were protected against a further challenge (group B), whereas mice of group A developed a second tumor. These results demonstrate that operational tolerance to K^b can be broken by CpG-ODN resulting in tumor rejection and protection in 50% of the mice.

View larger version (35K): [in this window] [in a new window]   FIGURE 7. Peritumoral injection of CpG-ODN breaks tolerance. A total of 5 x 105 P815.Kb tumor cells were inoculated s.c. into the right flank of Kb tolerant Alb.KbxDes.TCR mice. Seven days later the resulting tumors were treated with 20 µg of CpG-ODN 1668. This treatment resulted in reduced growth in five of nine mice (summarized as group A) or complete rejection in four of nine mice (group B). On day 35 all of the mice received a second challenge of P815.Kb tumor cells, and tumor growth was evaluated 10 days later. All rejector mice (group B) were protected, whereas mice in group A developed tumors. The growth rate could not be determined because these mice had to be sacrificed due to the load of the primary tumors. Results are presented as mean ± SE.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In previous studies, injection of protein Ags or peptides together with CpG-ODN as an adjuvant was shown to result in priming of cytolytic CD8 T cells (30, 43). CpG-ODN does not directly activate T lymphocytes, but exerts its effect indirectly via induction of maturation and migration of DC (27- 29, 44, 45), thereby enhancing their ability to stimulate Ag-reactive T cells (46). Thus, peritumoral treatment with CpG-ODN is likely to activate Ag-presenting cells that have taken up tumor-derived material, and to enhance their maturation and migration to local lymph nodes where tumor-specific T cells are primed. Our observation that s.c. application of CpG-ODN results in activation and migration of CD11c⁺ DC into the draining lymph node supports this concept. CpG-ODN-induced emigration of Langerhans’ cells from skin has also been found by Ban et al. (45), but these authors did not follow trafficking into the draining lymph node. Another important factor for effective antitumor immunity appears to be sustained T cell activity (8).⁴ It is likely that repetitive CpG-ODN treatment leads to a continuous priming of T cells by APCs. Finally, once activated, T effector cells have to return from the lymph node to the tumor site. In recent studies we have observed that activated T cells fail to attack the tumor or an organ expressing a respective target Ag because of poor cellular infiltration. However, a local inflammatory response leads to strong infiltration and autoimmunity or tumor destruction (32, 47). Thus, a further beneficial effect of peritumoral CpG-ODN application may be a change in the tumor microenvironment that improves accessibility for effector T cells. As expected, histological examination showed that local CpG-ODN treatment increased cellular infiltration into the tumor (data not shown). The local effects of CpG-ODN, such as NK cell priming and APC activation in the presence of tumor derived Ag, may explain why peritumoral treatment with CpG-ODN is superior to application at a site distal to the tumor. As tumors are known to induce tolerance (9, 32), it was of interest to see whether CpG-ODN treatment was also effective in such a situation. Indeed, peritumoral CpG-ODN application was able to break tolerance, possibly through the induction of cytokines such as IL-2 (32), resulting in tumor rejection and protection in a fraction of animals. It is possible that in the remaining mice tolerance was not sufficiently broken.

Taken together, the results indicate that CpG-ODN is a potent inducer of antitumor immunity for a variety of transplantation tumors when applied locally. This approach may also be useful in cases where the tumor Ag/peptide has not been identified. However, the limited efficacy of systemic treatment indicates that further modifications are needed for clinical application when local treatment is not possible. An improved strategy would directly target CpG-ODN or substances with CpG-ODN-like activity to a primary tumor or its metastasis.

	Acknowledgments

 
We thank Dr. Hans Schreiber (University of Chicago) for kindly providing the AG104A and AG104-L^d fibrosarcoma cells and 2C TCR mice, Dr. Toshiyuki Tanaka (Biomedical Research Center, Osaka University, Osaka, Japan) and Dr. Daniela Männel (University of Regensburg, Regensburg, Germany) for TM-1 Abs, Dr. Stefan Schnell (Memorial Sloan-Kettering Cancer Center) for MO4 cells, and Dr. Hermann-Josef Gröne (German Cancer Research Center, Heidelberg, Germany) for histological examination.

	Footnotes

 
¹ This study was supported by Deutsche Forschungemeinschaft (SFB 405) and European Union (Qol-CT-1999-00202).

² Address correspondence and reprint requests to Dr. Günter J. Hämmerling, Division of Molecular Immunology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. E-mail address: hammerling{at}dkfz-heidelberg.de

³ Abbreviations used in this paper: TIL, tumor-infiltrating lymphocytes; CpG, cytosine-phosphorothioate-guanine; CpG-ODN, CpG-rich oligodeoxynucleotide.

⁴ Y. Kawarada, R. Ganss, N. Garbi, T. Sacher, B. Arnold, and G. J. Hämmerling. NK^- and CD8⁺ T cell-mediated eradication of established tumors by peritumoral injection of CpG-containing oligodeoxynucleotides. Submitted for publication.

Received for publication April 23, 2001. Accepted for publication August 29, 2001.

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