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Oral Administration of High Molecular Mass Poly--Glutamate Induces NK Cell-Mediated Antitumor Immunity¹

Tae Woo Kim^*, Tae Young Lee, Hyun Cheol Bae^*, Jeong Ho Hahm, Yang Hyun Kim, Chung Park, Tae Heung Kang^*, Chul Joong Kim, Moon Hee Sung^,¶ and Haryoung Poo^2,

^* Laboratory of Infection and Immunology, Graduate School of Medicine, Korea University, Gyeonggi-Do, Korea; Mucosal Immunology Laboratory, System Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea; Bioleaders, Daejeon, Korea; College of Veterinary Medicine, Chungnam National University, Daejeon, Korea; and ^¶ Department of Bio and Nanochemistry, Kookmin University, Seoul, Korea

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
We analyzed the in vivo tumor regression activity of high molecular mass poly--glutamate (-PGA) from Bacillus subtilis sups. chungkookjang. C57BL/6 mice were orally administered 10-, 100-, or 2000-kDa -PGA or -glucan (positive control), and antitumor immunity was examined. Our results revealed higher levels of NK cell-mediated cytotoxicity and IFN- secretion in mice treated with higher molecular mass -PGA (2000 kDa) vs those treated with lower molecular mass -PGA (10 or 100 kDa) or -glucan. We then examined the effect of oral administration of 10- or 2000-kDa -PGA on protection against B16 tumor challenge in C57BL/6 mice. Mice receiving high molecular mass -PGA (2000 kDa) showed significantly smaller tumor sizes following challenge with the MHC class I-down-regulated tumor cell lines, B16 and TC-1 P3 (A15), but not with TC-1 cells, which have normal MHC class I expression. Lastly, we found that -PGA-induced antitumor effect was decreased by in vivo depletion of NK cells using mAb PK136 or anti-asialo GM1 Ab, and that was completely blocked in NK cell-deficient B6 beige mice or IFN- knockout mice. Taken together, we demonstrated that oral administration of high molecular mass -PGA (2000 kDa) generated significant NK cell-mediated antitumor activity in mice bearing MHC class I-deficient tumors.

	Introduction

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Poly--glutamate (-PGA)³ is a safe and edible biomaterial that is naturally synthesized by Bacillus subtilis and is composed of D- or L-glutamic acid polymerized via -amide linkages (1). Recently, Bacillus subtilis sups. chungkookjang, isolated from chungkookjang, a traditional Korean fermented soybean food, was reported to produce -PGA, a copolymer of D- and L-glutamate that has a higher molecular mass (>1000 kDa) than -PGA (10–10,000 kDa) produced from B. subtilis (natto) (2). Currently, we studied the antitumor effects of the high molecular mass -PGA isolated from B. subtilis (chungkookjang) to determine whether this might be a source for an effective oral antitumoral agent and the basis underlying these effects.

Effective tumor surveillance and elimination require the activation of complex host immune systems (3), which include a large number of molecular and cellular components, such as NK cells and CTLs. Specifically, NK cells mediate the natural process of cytotoxicity, and are the major components of the immune surveillance mechanism responsible for recognizing and killing transformed cells. MHC class I-down-regulated tumors, which have malignant and metastatic capacities, are not destroyed by cytotoxic CD8⁺ T cells, but may be killed by NK cells in vitro and in vivo, making NK cells an important component of tumor surveillance (4, 5, 6). Another major mediator of innate and adaptive immune responses, IFN-, is believed to oversee the host-protective response against the growth of transplanted tumors and the formation of chemically induced and spontaneous tumors (7, 8, 9, 10, 11, 12, 13, 14).

To assess the antitumor activity of -PGA isolated from B. subtilis (chungkoojang), we performed a tumor challenge assay by orally dosing C57BL/6 mice with PBS, various molecular mass versions of -PGA, or -glucan (curdlan), an immunomodulating antitumor agent known to activate NK cells (15, 16). We then s.c. injected these mice with tumor cells, examining NK cell-mediated antitumor effects in these mice. To our knowledge, this is the first report showing that -PGA has antitumor activity via activation of NK cells.

	Materials and Methods

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Cells

The HPV-16 E7-expressing murine tumor model, TC-1, has been described previously (17). In brief, HPV-16 E6, E7, and the ras oncogene were used to transform primary C57BL/6 mouse lung epithelial cells to generate TC-1 cells. TC-1 P3 (A15), a clone showing marked down-regulation of MHC class I expression, was isolated and expanded, as previously described (18, 19). Both TC-1 and TC-1 P3 (A15) cells were grown in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FBS (HyClone), 1% penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 2 mM nonessential amino acids, and 0.4 mg/ml G418 (all Invitrogen Life Technologies). B16 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, L-glutamine, sodium pyruvate, nonessential amino acids, and penicillin-streptomycin. YAC-1 cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, L-glutamine, sodium pyruvate, nonessential amino acids, and penicillin-streptomycin.

Preparation of -PGA

-PGA molecules of various molecular masses isolated from B. subtilis (chungkookjang) were provided by Bioleaders, and were dissolved in a neutral pH buffer. Briefly, the culture broth of B. subtilis sups. chungkookjang was collected and mixed with 3 vol of ethanol. The precipitate was lyophilized, dissolved in 10 mM Tris-HCl buffer (pH 7.5), treated with proteinase K, and then dialyzed against distilled water. -PGA was purified by anion-exchange chromatography, as follows. The dialyzed -PGA sample was subjected to a Sep-Pak Plus Waters Accell Plus QMA cartridge (Millipore) equilibrated with distilled water. After washing with distilled water, the cartridge column charged with -PGA was stepwise developed with NaCl solutions from 0.1 to 1.0 M. By estimating the concentration of glutamate in hydrolyzed -PGA using the amino acid analyzer, the content of -PGA was calculated by the following equation: content of -PGA (%) = (amount of glutamic acid/amount of sample) x (A/B) x 100. A = 129 (molecular mass of -glutamyl residue in -PGA); B = 147 (molecular mass of glutamic acid).

The number and weight-average molecular masses (Mn and Mw, respectively) along with the polydispersity (Mw/Mn) of the -PGA were measured by gel permeation chromatography using a GMPWXL column (Viscotek) and LR125 Laser Refractometer (Viscotek). Polyacrylamide (American Polymer Standard) standards were used to construct a calibration curve. The content of -PGA (10, 100, 2000 kDa) was increased >99%, and polydispersity was decreased after anion-exchange chromatography.

-PGA treatment and splenocyte isolation

Female 6- to 8-wk-old C57BL/6 mice were purchased from Folas International. NK cell-deficient C57BL/6J-Lyst^bg-J/J mice or IFN--deficient B6.129S7-Ifng^tm1Ts/J mice were provided by Y. Chul Sung at POSTEC (Pohang, Korea), and were as previously described (18, 20). Mice were housed in the specific pathogen-free animal facility at the Korea Research Institute Bioscience and Biotechnology. For experiments, 400 µg of -PGA or -glucan was suspended in 150 µl of PBS and given orally to the appropriate groups of mice on days 0, 1, 2, 3, and 4, and days 7, 8, 9, 10, and 11. Seven days after the final administration of -PGA, mice were anesthetized by diethyl ether inhalation. Spleens were removed and homogenized in HBSS, and samples were set on ice in 50-ml Falcon tubes to allow the debris to settle. Each supernatant was transferred to a fresh tube and centrifuged at 18,000 rpm for 10 min at 10°C. The resulting cell pellet was resuspended in 15 ml of Tris ammonium chloride buffer (8.3 g/L ammonium chloride and 10 mM Tris-HCl (pH 7.4)) and incubated for 10 min in a water bath at 37°C for lysis of RBC. Each tube was then filled with up to 50 ml of HBSS and centrifuged, and the resulting cell pellets were resuspended with RPMI 1640 supplemented with 10% FBS (HyClone).

NK cell cytolytic activity

Splenocytes were prepared from each mouse, as described above. For determination of NK cell activity, a ⁵¹Cr release assay was performed, as described previously (21). In brief, YAC-1 cells (used as the target cells; 2 x 10⁶ cells) were incubated in 200 µl of ⁵¹Cr-sodium (⁵¹Cr 1 mCi/ml) at 37°C for 1 h. The labeled cells were washed twice with complete medium and resuspended in complete medium at a final concentration of 5 x 10⁴ cells/ml. The effector cells (mouse lymphocytes) were washed and suspended in RPMI 1640 complete medium to obtain an E:T ratio of 100:1 when 100 µl of target cells and 100 µl of effector cells were combined in the wells of a 96-well plate. Triplicate control wells contained 100 µl of target cells (5 x 10⁴ cells/ml) either alone for spontaneous release, or with 100 µl of Triton X-100 (Sigma-Aldrich) for maximum release. The plates were incubated for 4 h at 37°C, and then 50 µl of supernatant was transferred to a tube and radioactivity was measured in a gamma counter. Cytotoxicity was calculated as follows: percentage of specific cytotoxicity = ((experimental release – spontaneous release)/(maximum release – spontaneous release)) x 100.

Cytokine ELISA

Splenocyte suspension was treated with 5 µg/ml Con A (Sigma-Aldrich) for 48 h at 37°C. After incubation, supernatants were collected and tested for presence of IFN- by ELISA. The supernatants from the splenocyte cultures were collected, and murine IFN- levels were measured in triplicate using the BD optEIA mouse IFN- ELISA kit (BD Pharmingen), according to the manufacturer’s instructions. Briefly, 96-well microplates were coated with capture Ab diluted in coating buffer overnight at 4°C. Plates were then washed and blocked with assay diluent (BD Pharmingen) for 1 h at room temperature. After the addition of culture supernatants, working detector (detection Ab plus avidin-HRP) was added to the wells and incubated for 1 h at room temperature. Following incubation, plates were washed and tetramethylbenzidine substrate reagent was added (BD Pharmingen) to the wells for 30 min at room temperature in the dark. At the end of the incubation, stop solution (1 M H₃PO₄) was added, and absorbance was read at 450 nm using a Spectramax microplate spectrophotometer 250 (Molecular Devices).

Tumor challenge assay

Tumor cells were washed three times with PBS and resuspended in PBS. Five or ten mice from each group (1 wk after the final oral administration of -PGA in the case of experimental mice) were s.c. injected with 1 x 10⁴ B16, TC-1, or TC-1 P3 (A15) cells in 100 µl of PBS in the left inguinal region. Tumor size was assessed three times per week, using caliper measurements, and these values were used to calculate the tumor volume for each individual mouse.

Ab-mediated depletion of NK, CD4⁺, and CD8⁺ cells

To study the subsets of lymphocytes needed for the observed antitumor effects, we performed in vivo Ab depletion essentially as described (18), followed by a tumor protection experiment performed as described above. In brief, mice received daily oral treatments with 400 of -PGA for 32 days. Five days after completion of treatment, anti-asialo GM1 (anti-ASGM1) Ab (Wako Pure Chemical) (22) or PK136 (23) was used for NK depletion, mAb GK1.5 (24) was used for CD4 depletion, and mAb2.43 (25) was used for CD8 depletion. The flow cytometry analysis revealed that >95% of NK1.1⁺ lymphocyte subset was depleted with a normal level of other subsets during Ab depletion. Five days after completion of treatment, Ab depletion was initiated. One week after completion of the -PGA treatment, the mice were challenged with 1 x 10⁴ of B16 tumor cells, as described above. Ab depletion was continued until the animals were sacrificed.

Flow cytometric analysis

For flow cytometric analysis, mouse splenocytes were resuspended in PBS supplemented with 0.02% w/v sodium azide and blocked with 0.5% BSA in PBST at 4°C for 30 min. The cells were then incubated on ice with saturating amounts (1 µg/10⁶ cells) of FITC-conjugated anti-NK-1.1 mAbs and PE-Cy5-conjugated anti-CD3e mAbs (BD Pharmingen). The stained cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences), and data were processed using the CellQuest Pro software (BD Biosciences).

Statistical analysis

Survival was analyzed using the log-rank test. Student’s t tests were used for between-group comparisons, and ANOVA was used for comparison of multiple groups. All data are presented as mean ± SD from triplicate experiments.

	Results

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Oral administration of high molecular mass -PGA induces NK cell activity and IFN- secretion in mouse splenocytes

Because NK cells are a major component of innate immunity and play a major role in immune surveillance (4, 5), we speculated that these cells might be key players in -PGA-mediated antitumor effects. Thus, we used a ⁵¹Cr release assay against NK-sensitive YAC-1 cells to investigate whether the cytotoxic activity of NK cells in tumor-bearing mice increased following treatment with -PGA. Splenic NK cells from C57BL/6 mice given 10-, 100-, or 2000-kDa -PGA, -glucan, or PBS were allowed to interact with ⁵¹Cr-labeled YAC-1 cells, and cytotoxicity was measured. As shown in Fig. 1A, our results demonstrated that NK cell activity in mice treated with 2000-kDa -PGA (58.9 ± 6.5%) was significantly higher than that in mice treated with PBS (4.7 ± 1.7%), 10 (20.2 ± 10.5%)- or 100-kDa -PGA (34.3 ± 7.8%), or -glucan (12.9 ± 6.2%).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. High molecular mass -PGA treatment increases NK cell cytotoxicity and IFN- production in the spleens of B16-bearing mice. Various molecular mass -PGAs or -glucan (400 µg each) were orally administered on days 0–4 and 7–11. One week after the final administration of -PGA, mice were anesthetized by diethyl ether inhalation, and spleens were homogenized in HBSS. A, NK cell (effector) activity was determined by a 51Cr release assay against YAC-1 (target) cells, with cells diluted to an E:T cell ratio of 100:1. B, IFN- levels from spleen samples were measured in triplicate, using the BD optEIA mouse IFN- ELISA kit (BD Pharmingen). The data presented in this figure are representative of triplicate experiments. C, Intracellular cytokine staining and flow cytometry analysis of splenocytes to determine the percentage of IFN--producing lymphocyte subset from spleen samples.

Intracellular IFN- staining and flow cytometry analysis were performed to determine the percentage of IFN--producing lymphocyte subset from spleen of mice, treated with 2000-kDa -PGA or PBS. As shown in Fig. 1C, NK cell population was slightly increased in the splenocytes from the -PGA-treated animals. Moreover, significant increase in IFN--secreting NK cells was observed in spleen from mice treated with 2000-kDa -PGA (0.08 vs 0.20%). In contrast, there was no difference in percentage of IFN--secreting NKT cell and CD4⁺ and CD8⁺ T cell populations between -PGA- and PBS-treated groups. These results suggest that NK cell is a major subset of immune cells that secretes IFN- in response to -PGA treatment. Collectively, mice treated with 2000-kDa -PGA show antitumor activities, such as significantly augmented NK cell-mediated cytotoxicity and IFN- secretion.

High molecular mass -PGA has antitumor activity in a mouse B16 model

We then examined the antitumor effect of orally administered -PGA having various molecular masses, using a B16 mouse tumor model. Mice were orally treated with 400 µg of PBS, 10-kDa -PGA, 2000-kDa -PGA, or -glucan on days 0–4 and 7–11, and then s.c. inoculated with B16 melanoma cells 6 days after the initial treatment. As shown in Fig. 2A, the average tumor size in the 2000-kDa -PGA-treated group (380.0 mm², n = 10) was significantly smaller than that in the PBS control group (1174.5 mm², n = 10) or the 10-kDa -PGA-treated group (670.3 mm², n = 10) at 6 wk after tumor injection. Notably, the average tumor size in the 2000-kDa -PGA-treated group was also smaller than in the group receiving the well-known tumor-inhibition polypeptide, -glucan (564.4 mm², n = 10).

View larger version (15K): [in this window] [in a new window]   FIGURE 2. High molecular mass -PGA is more effective than low molecular mass -PGA as an antitumoral agent. Mice were given daily oral treatments with 400 µg of high (2000-kDa) or low (10-kDa) molecular mass -PGA, -glucan (positive control), or PBS (negative control) for 28 days, and were challenged with 1 x 104 of B16 tumor cells 1 wk after the initial treatment. A, Tumor volume was measured at 5 wk after tumor injection. B, Survival rate of B16 tumor-bearing mice was checked. The data presented in this figure are representative of triplicate experiments.

The antitumor effects of high molecular mass -PGA appear to require MHC class I down-regulation

Because NK cells are capable of spontaneously eradicating MHC class I-down-regulated tumors (4, 5), we next examined the effects of high molecular mass -PGA against mice inoculated with TC-1 cells having normal MHC class I expression, or TC-1 P3 (A15) cells having decreased MHC class I expression (18, 19). Our results revealed that treatment with 2000-kDa -PGA significantly decreased tumor volumes in TC-1 P3 (A15)-inoculated mice (Fig. 3B), but not in TC-1-inoculated mice (Fig. 3A), suggesting that high molecular mass -PGA is more effective against tumor cells having low MHC class I expression. In terms of survival rate, -PGA-treated TC-1 P3 A15-bearing mice showed significantly better survival than control mice (Fig. 3D) and -PGA-treated TC-1-bearing mice (Fig. 3C). Taken together, these results support the notion that the antitumor effects of -PGA are mediated by NK cells, which act on MHC class I-down-regulated tumors.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. High molecular mass -PGA inhibits MHC class I-down-regulated tumor growth in C57BL/6 mouse. A total of 400 µg of -PGA (2000 kDa) or -glucan was given orally to group mice on days 0–4 and 7–11. Volume for oral inoculation per head was 150 µl each time. One week after the final administration of -PGA, C57BL/6 mice were challenged with 1 x 104 of tumor cells in 100 µl of PBS into the left inguinal region by s.c. injection. The group of PBS-treated mice was used as a negative control. Tumor size was measured in A, TC-1 tumor cells, or B, TC-1 P3 (A15), a MHC class I-down-regulated clone from TC-1-bearing mice. Survival of C, TC-1- or D, TC-1 P3 (A15)-bearing mice was monitored for 77 days after tumor injection. The data presented in this figure are representative of triplicate experiments.

The antitumor effects of -PGA are dependent on NK cell and IFN-

To determine the contribution various lymphocyte subsets play an important role in -PGA-induced antitumor effects, infiltrated subsets from tumor of the mice treated with -PGA or PBS were analyzed at 2 wk after tumor challenge. The percentages of infiltrated CD4⁺ and CD8⁺ cells as well as NK1.1⁺/CD3^– NK cells on the gated lymphocyte population were dramatically increased in tumor of mice treated with -PGA, compared with the PBS-treated group (Fig. 4A).

View larger version (34K): [in this window] [in a new window]   FIGURE 4. NK 1.1+ cells were important for high molecular mass -PGA-mediated antitumor effects. Mice were given daily oral treatments with 400 µg of high (2000-kDa) molecular mass -PGA or PBS (negative control) and were challenged with 1 x 104 of B16 tumor cells 1 wk after the initial treatment. A, Infiltrated subsets from tumor of the mice treated with -PGA or PBS were analyzed by flow cytometry. Ab depletion was performed to attain lymphocyte subset depletion 5 days after -PGA treatment. mAb GK1.5 was used for CD4 depletion, mAb2.43 was used for CD8 depletion, and PK136 and anti-ASGM1 Ab were used for NK depletion. Flow cytometry analysis revealed that >95% of the appropriate lymphocyte subset was depleted with a normal level of other subsets. One week after -PGA treatment, mice were challenged with 1 x 104 of B16 (B) or TC-1 P3 (A15) tumor cells (C). Tumor size was measured up to 24 days after tumor injection. The data presented in this figure are representative of triplicate experiments.

We also performed in vivo Ab depletion experiments in TC-1 P3 (A15) bearing mice using anti-NK1.1 mAb (PK136) to further determine whether NK cells play an important role in -PGA-induced antitumor effects against MHC class I-down-regulated tumor cells. Mice were inoculated with TC-1 P3 (A15) cells, with or without oral prior administration of 2000-kDa -PGA. Our results revealed that the NK cell-depleted mice by anti-NK1.1 Ab developed TC-1 P3 (A15) tumors of similar size (p < 0.17), whereas the tumors were significantly smaller in -PGA-treated mice not subjected to NK cell depletion (Fig. 4C), indicating NK cells were essential for the antitumor effect generated by -PGA against various MHC class I-down-regulated tumor cell lines, including TC-1 P3 (A15) as well as B16.

To further confirm whether NK cells and IFN- are required for -PGA-induced antitumor effects, tumor sizes were monitored following injection of B16 tumor cells into normal C57BL/6 mice or the previously reported NK cell-deficient C57BL/6 mice (26) or IFN--deficient C57BL/6 mice with and without prior administration of 2000-kDa -PGA. Our results revealed that tumor sizes were consistently smaller in normal C57BL/6 mice treated with -PGA (p < 0.01), whereas treatment with 2000-kDa -PGA had no effect on tumor size in NK cell-deficient C57BL/6 mice and IFN--deficient C57BL/6 mice (Fig. 5). These findings collectively suggest that NK cells and IFN- are important for the observed antitumor effects of high molecular mass -PGA.

View larger version (13K): [in this window] [in a new window]   FIGURE 5. NK cells and IFN- production are mediated in high molecular mass -PGA-induced antitumor activity. C57BL/6 wild-type mice, C57BL/6 NK cell-deficient beige mice, and tumor size was measured at 24 days. C57BL/6 IFN--deficient mice were treated with daily oral treatment of 400 µg of -PGA (2000 kDa), and were challenged with 1 x 104 B16 tumor cells 1 wk after the initial -PGA treatment. The data presented in this figure are representative of triplicate experiments.

	Discussion

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The increased NK cell-mediated cytotoxicity and IFN- secretion were observed from splenocytes of mice treated with high molecular mass -PGA (2000 kDa), but not lower molecular mass -PGA (10 kDa) or PBS (Fig. 1). In vitro treatment of -PGA (2000 kDa) did not increase the NK cell-mediated cytotoxicity (data not shown) in splenocytes of C57BL/6 mice. When peritoneal macrophage and bone marrow dendritic cells (DCs) isolated from C57BL/6 mice were treated in vitro with -PGA (1 µg/ml, 2000 kDa), TNF- and IL-12 were produced, respectively (data not shown). Thus, NK cell activation and IFN- secretion may be induced by the activation of immune cells such as macrophages and DCs in high molecular mass -PGA-fed mice. The effect of high molecular mass -PGA on the immune cells may be mediated by TLR(s). Further studies are required to understand the molecular mechanism of the antitumor effect induced by oral administration of high molecular mass -PGA.

In accordance with this observation, in vivo depletion of NK cells by mAb PK136 and anti-ASGM1 Ab completely abrogated anti-B16 tumor activity in 2000-kDa -PGA-treated mice, and the high molecular mass -PGA showed no anti-B16 tumoral effects in NK cell-deficient C57BL/6 mice (Figs. 4 and 5). Notably, -PGA-induced antitumor activity was observed in a TC-1 P3 (A15) cell tumor model, but not in a TC-1 tumor model. Previous in vivo Ab depletion experiments showed that CD8⁺ T cells were essential for antitumor immunity against TC-1 tumor cells, whereas NK cells were essential for the antitumor immunity against TC-1 P3 (A15) cells (18). Because TC-1 tumor cells show normal MHC class I expression, whereas TC-1 P3 (A15) cells (18, 19) and B16 cells (27) show marked down-regulation of MHC class I expression, this finding suggests that MHC class I status is important for the antitumoral effects of -PGA. This provides further support for the involvement of NK cells in this antitumor effect, because it is widely accepted that MHC class I-negative tumor cells are susceptible to NK cells, whereas MHC class I-positive tumor cells are not. Recognition of class I MHC molecules delivers inhibitory signals to NK cells, forming the basis for the specific NK cell response to class I MHC-deficient cells (4, 5). Most mouse NK cells express MHC class I-inhibitory receptors such as Ly49 receptors, which contain tyrosine residues capable of binding to the Src homology 2 domains of Src homology region 2 domain-containing phosphatase 1 upon phosphorylation, and counteracting the kinase activity by recruiting a tyrosine phosphatase, Src homology region 2 domain-containing phosphatase 1. Thus, our present findings strongly suggest that high molecular mass -PGA (2000 kDa) might generate antitumor effects against tumors with down-regulated MHC class I expression via NK cell activation.

NK cells are widely believed to play a crucial role in bridging innate and adaptive immunity through modulation of the cytokine network (28). NK cells influence adaptive immunity via altering the activity of APCs. For instance, activated NK cells induce DCs to mature toward type 1 polarized DCs (DC1), in an IFN-- and TNF--dependent fashion, leading to induce cellular T cell response. As shown in Fig. 4, the percentage of infiltrated CD8⁺ T cells and CD4⁺ T cells as well as NK cells was significantly increased in tumor in mice treated with -PGA. Because there was a significant increase of tumor size in the treated mice after CD8⁺ T cell depletion, CD8⁺ T cells may also play a part in antitumor effect induced by -PGA. These results suggest that high molecular mass -PGA could be used as an adjuvant in vaccines leading to induction of a synergistic antitumor effect against MHC class I-down-regulated malignant cancers.

In sum, in this study, we report for the first time that oral administration of high molecular mass -PGA (2000 kDa) generated significant antitumor activity in mice bearing MHC class I-deficient tumors. These novel findings indicate that high molecular mass -PGA mediates antitumor immunity via the activation of NK cells, suggesting that high molecular mass -PGA could be a good candidate for development as a novel adjuvant for cancer immunotherapy.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
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.

¹ This work was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A050562), and a grant from Korea Research Institute Bioscience and Biotechnology Initiative Program to H.P.

² Address correspondence and reprint requests to Dr. Haryoung Poo, Mucosal Immunology Laboratory, Korea Research Institute of Bioscience and Biotechnology, Daejon 305-600, Korea. E-mail address: haryoung{at}kribb.re.kr

³ Abbreviations used in this paper: -PGA, poly--glutamate; anti-ASGM1, anti-asialo GM1; DC, dendritic cell.

Received for publication November 6, 2006. Accepted for publication April 24, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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