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Molecular Medicine Program [M. C., M. G., K. H., K. O., J. T., R. G. V.] and Department of Immunology [M. C., R. G. V.], Mayo Clinic, Rochester, Minnesota 55905
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ABSTRACT |
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 Top ABSTRACT Overview SummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or CCL20 (Mip-3) in tumors. We show that expression of these chemokines in the colorectal tumor model CMT93 significantly decreases tumorigenesis. This decrease is associated with an increase in CD8 T cells, natural killer cells, and Class II DCs in the tumor within the first 24 h. Furthermore, studies in immunodeficient mice show that both natural killer cells and T cells are required for this decrease in immunogenicity. CCL3 and CCL20 expression alone did not significantly inhibit the development of the B16 melanoma tumor. However, coexpression of the Herpes Simplex Virus thymidine kinase gene (HSVtk) and CCL20, cured large established tumors where HSVtk expression alone was not sufficient. Finally, coexpression of HSVtk with either CCL3 or CCL20 was able to significantly increase protection against subsequent tumor rechallenge. |
Overview Summary |
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TopABSTRACTOverview SummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or CCL20 (Mip-3) alone, or in tandem, with the Herpes Simplex Virus thymidine kinase gene, HSVtk, in tumors. Coexpression of HSVtk and CCL20 cured large established B16 tumors where chemokine or HSVtk expression alone was not sufficient. In addition, coexpression of HSVtk with either CCL3 or CCL20 was able to significantly increase protection against subsequent tumor rechallenge. Our data demonstrate that as well as provision of signals to recruit and activate DCs, a source of antigen release is also required to generate optimal antitumor responses. |
INTRODUCTION |
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TopABSTRACTOverview SummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of the cytokine GM-CSF in irradiated tumor vaccines is an effective way to generate an antitumor immune response (7)
. One observation in GM-CSF therapy is the observed infiltrate of DCs into the tumor site, and it is this influx of DCs that has been postulated to be responsible for the long-term T-cell mediated immunity (8, 9, 10)
. However, GM-CSF is not a chemotactic factor for DCs, and GM-CSF has multiple effects on other immune cells, including macrophages and endothelial cells, which may be the intermediate effectors in the recruitment of DCs (11)
. Vaccination with irradiated GM-CSF-expressing tumors results in an increased expression of the chemokine Mip-1 at the tumor site that may be the cause of DC recruitment (12)
. Additionally, GM-CSF may have confounding effects attributable to its role in hematopoiesis, which includes profound leukocytosis and fatality in mice receiving tumors expressing GM-CSF (7)
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The evolving field of chemokine biology has identified a surprising specificity in the matching of chemokine expression patterns in immune processes, with patterns of receptor expression by cells and cell subsets, that appear to play key roles in orchestrating immune responsiveness through location (13, 14, 15, 16, 17) . Chemokines may, therefore, represent an ideal option to recruit a particular repertoire of immune cells characteristic of a specific T cell-mediated response, including DC, macrophages, NK cells, CTL, and Th1 cells, while excluding the broader effects of cytokine secretion alone.
To determine the role of DC recruitment to tumors in activation of antigen-specific antitumor immune responses, we hypothesized that we could use chemokines to selectively recruit immature DCs to the tumor site. Although DC recruitment is an important step in the generation of an immune response, this is only part of the story. The release of antigen at appropriate levels and in appropriate form is also required for T-cell priming (9 , 18, 19, 20) . Therefore, we have used a model that distinguishes between the roles of immune cell recruitment and/or antigen release in the generation of an antitumor immune response. On the basis of in vitro data, we show that murine bone marrow-derived DCs express the chemokine receptors CCR1, CCR5, and CCR6. Therefore, we chose mCCL3 and mCCL20, the ligands for these receptors, to recruit immune cells into tumors. We also coexpressed these chemokines with the HSVtk, a viral enzyme that converts gancyclovir from a prodrug to a toxic agent that kills the cells and is associated with a bystander effect (21) . Using an IRES, we coexpressed HSVtk with a chemokine in order to induce cytotoxicity with concomitant release of tumor antigen, thus combining recruitment of DCs and inflammatory cells with antigen release. Our data demonstrate that coexpression of HSVtk and chemokine is able to increase tumor immunity in animal models where chemokine or HSVtk expression alone is not sufficient.
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MATERIALS AND METHODS |
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TopABSTRACTOverview SummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
The murine melanoma B16 and B16tk lines and the colorectal tumor CMT93 used in this study have been described previously (20)
. The murine macrophage cell line IC-21 was obtained from the American Type Culture Collection and grown according to American Type Culture Collection guidelines. All cell lines were monitored routinely and found to be free of Mycoplasma infection. For cell killing in vitro, medium was supplemented with GCV (Cytovene; Roche, Indianapolis, IN) to a final concentration of 5 µg/ml. B16neo, B16CCL3, B16CCL20, B16tkIRESCCL3, and B16tkIRESCCL20 are stable clones generated by transfection of B16 cells with pCR3.1 alone or the appropriate expression vector followed by selection in 5 mg/ml G418 (Life Technologies, Inc.). CMT93neo, CMT93CCL3, CMT93CCL20, CMT93tkIRESCCL3, and CMT93tkIRESCCL20 are stable clones generated by transfection of CMT93 cells with pCR3.1 alone or the appropriate expression vector followed by selection in 1 mg/ml G418.
DC Culture.
DCs were cultured from the bone marrow of C57BL/6 mice as described previously (22)
. Briefly, bone marrow was isolated from the tibia and femur, and after RBC lysis, the resulting cell suspension was incubated in 1 ml of RPMI 1640 containing 10 µg/ml antibodies to MHC class II (I-Ab), Mac 3, CD8a (HO2.2), B220, CD3e, and Gr-1 (all from PharMingen, San Diego, CA) on ice for 20 min. The cells were washed and suspended in baby rabbit complement (Accurate Chemical and Scientific Corp., Westbury, NY) at a concentration of 107 cells/ml (1:15, complement: medium) at 37°C for 60 min. Cells were washed and plated at a concentration of 106 cells/ml in RPMI 1640 supplemented with 10 ng/ml GM-CSF for IDCs and 20 ng/ml GM-CSF plus 1 ng/ml IL-4 for MDCs. Cultures were washed at 3 days to remove nonadherent cells. IDCs were collected on day 7, whereas MDCs were supplemented with LPS on day 7 and cells collected on day 9.
Detection of Chemokine Production from Cell Lines.
Cell line chemokine production was detected by specific ELISA. Antibodies and recombinant protein were purchased from R&D Systems (Minneapolis, MN). Five µg/ml antimouse CCL3 (AF-450-NA) or CCL20 (AF-760) capture antibody were coated on treated 96-well ELISA plates (Rainin). Recombinant murine CCL3 (450-MA) or CCL20 (760-M3) were used as standards alongside cell supernatants. One µg/ml biotinylated antimouse CCL3 (BAF450) or CCL20 (BAF760) was used for detection followed by 0.2 µg/ml peroxidase-conjugated Strepavidin (016–030-084; Jackson ImmunoResearch Laboratories, West Grove, PA). Finally, TMB substrate reagent (PharMingen) was added and stopped after 20 min using 100 µl of 2N H2SO4.
RPA.
For analysis of DC chemokine receptor expression, a custom multiprobe template set was assembled to order by PharMingen, with templates including mCCR1, mCCR5, mCCR6, mCCR7, and the housekeeping control genes L32 and GAPDH. RNA probes were synthesized from the template mix using a T7 MAXIscript in vitro transcription kit (Amnion, Austin, TX) incorporating radiolabelled [32P] UTP (NEN) according to manufacturer’s instructions. DNA template sequences were digested for 30 min with DNaseI, and RNA templates were purified from unincorporated nucleotides using a Chromaspin-30 column (Clontech, Palo Alto, CA). RNA hybridization was performed using an RPAIII kit (Ambion). Briefly, 5 µg of total RNA prepared using RNeasy kit (Qiagen) was combined with 4 x 104 cpm labeled probe, denatured at 95°C for 3 min, and then incubated overnight at 56°C. Single-stranded RNA was digested with an RNaseA/T1 mix for 45 min at 45°C; then samples were precipitated and run on a 5% acrylamide sequencing gel until resolved. For analysis, gels were transferred to filter paper and exposed to X-ray film overnight at -70°C. For quantitation, films were scanned, and band intensities were calculated using the public domain NIH Image program.
Calcium Mobilization Assay.
Changes in levels of intracellular Ca2+ in response to chemokine were analyzed using calcium-responsive dye fluorescence by flow cytometry. IDCs were washed and resuspended at a concentration of 107/ml in HBSS containing 10 mM HEPES and combined with an equal volume of 20 µM Indo-1AM (Molecular Probes, Eugene, OR) in HBSS for 30 min at 37°C. The cells were washed and resuspended in HBSS containing 10 mM HEPES 0.05% BSA. The sample was analyzed with a FACStar+ flow cytometer (Becton Dickinson, San Jose, CA) using a UV laser for excitation with violet (390 nm) and blue (500 nm) fluorescence emissions recorded. Recombinant chemokine (R&D Systems) or cell supernatant was added after a 60-s baseline reading. Data plots were generated using the FlowJo software program (Tree Star, Palo Alto, CA).
In Vivo Injection of Chemokine-expressing Tumor Cells.
C57BL/6, beige, or SCID mice were age and sex matched for individual experiments. For tumorigenicity studies, 1 x 106 CMT93 or 5 x 105 B16 cells or clones were injected s.c. (100 µl) into the flank region. Animals were examined daily until the tumor became palpable; thereafter, the diameter, in two dimensions, was measured three times weekly using calipers. For tumor free data, animals were scored as tumor free if they failed to develop tumors > 0.3 cm in diameter. For survival data, animals were euthanized when tumor size reached 1 cm in any one perpendicular direction. Statistical analysis of survival curves was done using the Log-rank test. A P of <0.05 was considered significant.
In Vivo Injection of HSVtk-expressing Tumor Cells.
Tumors were established by injection of 5 x 105 s.c. (100 µl). Once tumors reached a size of 0.3 cm in one diameter, appropriate groups received daily i.p. injection of GCV (70 mg/kg) in 100-µl volumes for seven consecutive days. If primary GCV treatment failed, when tumors reached a size of 0.5 x 0.5 cm, mice received surgery to remove tumors. For rechallenge experiments, mice received an s.c. injection of 1 x 105 parental B16 cells on the opposite flank 10 days after the last surgical removal of tumor in any of the cohorts, along with a control group of naive mice. Animals were euthanized when tumor size reached 1 x 1 cm in two perpendicular directions. Statistical analysis of survival curves was done using the Log-rank test. A P of <0.05 was considered significant.
Analysis of Immune Cell Infiltrate in Vivo.
To study immune infiltrate, randomly selected untreated tumors were removed when they reached a size of 
0.5 x 0.5 cm. Half of the tumors were fixed, and sections were H&E stained. The remaining half was FACS analyzed for immune infiltrate. Tumors were dissociated and three color stained with directly conjugated antibodies as follows: CD45 PerCP; CD8 PE; CD4 PerCP; CD3e FITC; NK1.1 PE; CD11c PE; Mac3 PE; I-ab PE; CD11c FITC; Ly-6G PE; I-ab FITC; and CD14 FITC (BD PharMingen). To study immune infiltrate in CMT93 tumors where chemokine expression prevented tumor development, we used a gelfoam matrix to isolate cells infiltrating the inoculation site based on a modification of a protocol described previously (38)
. Briefly, sterile gelfoam sponges (Pharmacia and Upjohn, Kalamazoo, MI) were cut to a size of 1.5 x 1.5 x 1 cm and rehydrated in sterile HBSS. The sponge was implanted s.c. on the back of the mice, and the incision site was closed with wound clips. Three days after implantation, 1 x 106 CMT93 cells (100 µl) were injected into the sponge, and 24 h later, the sponge was removed. Sponges were finely chopped and then incubated in 15 ml of collagenase enzyme cocktail (20 mg/ml BSA and 400 units/ml collagenase in Saline G, 1.1 grams/liter glucose, 8 grams/liter NaCl, 0.4 gram/liter KCl, 0.29 gram/liter Na2HPO4.7H2O, 0.15 gram/liter KH2PO4, 0.15 gram/liter MgSO4.7H2O, and 0.016 gram/liter CaCl2.H2O in endotoxin free water) for 3 h at 37°C with agitation. The suspension was passed through a 100-µm filter, washed with HBSS, and analyzed for specific antibody staining by FACS as above. Infiltrating cells from three mice per group were pooled, and total cells were counted by trypan blue exclusion on a hemacytometer and phenotypically characterized by FACscan analysis.
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RESULTS |
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TopABSTRACTOverview SummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Immature DCs expressed RNA for all four receptors, but on maturation with LPS, RNA expression was lost for mCCR6 and mCCR5, remained constant for mCCR1, and up-regulated for mCCR7 (Fig. 1A)
. The response of IDC to mCLL3 and mCLL20 was analyzed using a calcium mobilization assay. Bone marrow-derived IDCs mobilized calcium in response to both CCL3 (40 ng/ml) and CCL20 (200 ng/ml; Fig. 1B
) but not to a similarly formulated CCL24/eotaxin 2 negative control (40 ng/ml; data not shown). Thus, these chemokines were shown to be able to activate specific chemokine receptors expressed by bone marrow-derived immature DCs in mice. On the basis of these data, we decided to express CCL3 and CCL20 in tumor cells to recruit immature DCs into the tumor.
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. To confirm the biological activity of the secreted CCL3 or CCL20, calcium mobilization assays were performed on IDCs using supernatants from B16 clones expressing either CCL3 or CCL20. Supernatants from both B16 CCL3 and B16 CCL20 induced calcium mobilization in IDCs, but no calcium flux was seen using supernatants from parental B16 cells (Fig. 2)
. All HSVtk-coexpressing cell clones showed 100% cytotoxicity when grown in 5 µg/ml GCV (data not shown). Additionally, the in vitro bystander cytotoxicity for clones expressing HSVtk alone or HSVtk plus chemokines to non-HSVtk-expressing parental cells was similar (data not shown), indicating similar levels of gene expression.
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in a chemokine dose-dependent fashion. Thus, for chemokine tumors mixed with CMT93 neo tumors an increasing number of mice developed tumors (Table 2)
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. However, the failure of CMT93 expressing CCL3 and CCL20 to develop tumors made it difficult to analyze in vivo immune infiltrate by histology. For this reason, we used a gelfoam matrix to isolate cells from the site of tumor inoculation. Both CCL3- and CCL20-transfected CMT93 tumors showed an overall increase in leukocytes (CD45+) and, more specifically, an increase in CD8+, NK1.1, and Class II+/CD11c+ cells but no difference in Ly6G+/CD14- or Class II+/CD11c- cells (Fig. 3, A and B)
. Thus, within 24 h of implantation, chemokine expression attracted cells that phenotypically resembled DCs consistent with our earlier in vitro studies on bone marrow-derived DCs (Fig. 1B)
. In addition, we observed infiltrates of NK and T cells.
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and SCID mice that lack functional T and B cells (Fig. 4B)
were challenged with CMT93 tumors expressing CCL3, CCL20, or neor. All tumors developed at a similar rate in beige mice. Thus, no significant difference in survival between chemokine-expressing and parental tumors was observed in mice lacking NK cells. These data suggest that NK cells are necessary for the inhibition of tumor development in chemokine-expressing CMT93 cells. Similar results were observed in SCID mice (Fig. 4B)
, indicating a similar requirement for functional T and B cells.
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Neither Expression of CCL3 nor CCL20 from B16 Tumors Results in a Decrease in Tumorgenicity Despite Modifying the Immune Infiltrate into Tumors.
CMT93 tumors have a higher level of immunogenicity when compared with the murine melanoma B16. Specifically, vaccination with irradiated CMT93 tumors protects against subsequent rechallenge in the majority of cases. By contrast, vaccination with irradiated B16 does not lead to protection (data not shown). We therefore wanted to see whether chemokine expression in a less immunogenic tumor model would lead to a similar decrease in tumorgenicity. However, expression of CCL3 and CCL20 in poorly immunogenic B16 tumors showed no decrease in tumorigenicity when compared with tumors expressing neor alone (Fig. 5A)
. It was possible that B16 was secreting a factor that blocked the effects of the chemokines in vivo. However, based on our earlier data, we had shown in vitro that supernatants from chemokine-expressing B16 cells resulted in calcium mobilization within IDCs (Fig. 2)
. The outgrowth of tumors was not attributable to loss of chemokine expression. Reverse transcription-PCR of RNA prepared from growing tumors demonstrated the presence of sustained chemokine gene expression in chemokine-transfected tumors but not in controls (data not shown). Furthermore, we went on to look at the immune infiltrate in chemokine-expressing tumors by FACS. An overall increase in leukocytes (CD45+) was observed in both CCL3- and CCL20-expressing tumors (Fig. 6A)
. Specifically, increased numbers of CD4+, CD8+, and NK1.1+ (Fig. 6B)
lymphocytes were detected, along with increased numbers of Class II+, Mac3+, and CD11c+ APCs (Fig. 6C)
. These data are consistent with those seen with gelfoam sponges in CMT93 tumors described above (Fig. 4, A and B)
. Examination of H&E-stained histological sections of tumors expressing chemokine indicated an increase in necrosis in tumors where chemokine was expressed (Fig. 7, A–C)
. Additionally, a perivascular cuff of mononuclear cells (lymphocytes plus macrophages) was observed within the CCL20 tumor, indicating recruitment of immune cells into the tumor from the vasculature (Fig. 7D)
. This perivascular cuffing was never observed in B16 tumors. Thus, chemokine expression alone causes an increased infiltrate of specific immune cells into B16 tumors that was associated with an increase in necrosis but was unable to affect tumor growth.
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). Mice bearing tumors expressing HSVtk IRES CCL3 or HSVtk alone showed significant delay in tumor growth when compared with parental B16 tumors (P < 0.0001; Fig. 5B
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To test for the development of effective immune responses not observable during primary tumor development, mice in all groups receiving late GCV therapy underwent surgery to remove any remaining tumors. Ten days after the last surgery, all four groups plus a naive group were challenged with parental B16 tumors on the opposite flank. In both the naive group and that in which mice were initially challenged with B16, all mice succumbed to tumor rechallenge. In mice initially challenged with HSVtk-expressing B16 cells, the majority of mice developed tumor on rechallenge, whereas both CCL3- and CCL20-expressing tumors showed a significant increase in protection against subsequent tumor rechallenge (P < 0.0001; Fig. 5C
). So despite the fact that chemokine expression from B16 cells is sufficient to modify the infiltrate, additional provision of tumor antigen in the appropriate context, through HSVtk/GCV-mediated cell death, was needed to generate a sufficient immune response to clear residual tumor cells. This was true for CCL20-expressing tumors but not for CCL3 tumors. However, coexpression of either CCL20 or CCL3 with HSVtk was able to generate protection against subsequent tumor rechallenge.
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DISCUSSION |
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TopABSTRACTOverview SummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have shown that, although in some models, chemokine expression alone is sufficient to prevent tumor development, this is not the case for all tumors. There may be several reasons why this is true. Although chemokine expression will attract a repertoire of immune cells into tumors, other environmental factors within the tumor may influence the ability of these cells to initiate or perpetuate an immune response, e.g., certain tumors aggressively suppress immune cell activation through the release of anti-inflammatory molecules, such as IL-10, transforming growth factor-ß, and vascular endothelial growth factor (27, 28, 29) . Other factors that may influence the immune response within tumors include the relative susceptibility of tumor cells to innate immune recognition by cells such as macrophages and NK cells.
Another consideration is that tumors may release varying amounts of antigen as they develop. The context in which this antigen is presented to the immune cells is important, in some cases leading to tolerance and in other instances leading to activation (30) . Therefore, although DCs may be recruited into tumors, without antigen release in the appropriate context, they will be either unable to cross-prime naïve T cells in draining lymph nodes, or this cross-priming will lead to T-cell suppression. Expression of the cytotoxic gene HSVtk with the chemokine CCL20 was able to eliminate large B16 tumors. We have described previously that B16 tumors killed by HSVtk undergo a nonapoptotic cell death (20) . Furthermore, we have shown that nonapoptotic cell death is associated with increased immunogenicity in vivo (26) . The enhanced effect of HSVtk when coexpressed with chemokines that recruit DCs may in part be caused by this release of antigen in the appropriate context for DCs to phagocytose and cross-present. Certainly, both CCL3 and CCL20 were able to enhance the immunogenicity of B16 HSVtk tumors.
Other studies have examined the combinations of cytokines with cytotoxic gene expression. Although some cytokines, such as GM-CSF, in combination with HSVtk have been shown to enhance protection against subsequent tumor rechallenge, they showed no enhancement in the regression of established primary tumors. In contrast, although IL-2 coexpression with HSVtk has been shown to enhance the regression of primary tumor, this did not extend to protection against subsequent tumor rechallenge (31) . Significantly, here we have shown that CCL20 was capable of doing both. This may reflect the importance in choosing an appropriate chemokine that will recruit DCs to prime a naive T cell as well as effector cells, such as NK and T cells.
The different results of different chemokines, despite some overlap in responsive cells, emphasize additional influences within the immune response. It will be necessary to further understand the cell types that contribute to the success or failure of protective immunity. In addition, it will be necessary to determine the relative contribution of chemokine expression in initiation of de novo immune responses and in attracting effector cells for tumor clearance once immune responses have begun.
Studies investigating the expression effects of chemokines have shown inhibition of tumor development to varying degrees. HCCL3 delayed tumor progression in a murine adenocarcinoma model after foot pad inoculation but was unable to completely eliminate tumors in the majority of cases (32) . Adenoviral delivery of hCCL20 into murine tumors inhibited tumor progression in a CD8 T cell-dependent fashion. In these studies, the effectiveness of the chemokine may have been enhanced as a result of damage and antigen release during delivery (33) . In our B16 model, we were unable to show that chemokine expression alone was sufficient to prevent tumor growth if it was expressed over the course of the development of tumor. Therefore, we conclude that cell death along with antigen release in an immunogenic context is required in the B16 model.
The present studies indicate that chemokine expression and immune cell recruitment alone are sufficient in some tumor models to inhibit tumor development, but that in other tumors, additional factors may be required. Consistent with this, chemokine expression, including CCL20, has been shown within human tumors and is clearly not sufficient to inhibit tumor development (34, 35, 36) . Furthermore, some chemokines, including RANTES, have been shown to enhance formation of human tumors in nude mice (37) . We show here that cytotoxic gene expression resulting in release of antigen from tumor cells for uptake and cross-presentation by infiltrating DCs significantly enhances the generation of tumor immunity when compared with chemokine expression alone. We would propose that future approaches to tumor therapy focus on both recruitment of inflammatory cells into tumors and provision of antigen through appropriate cell death that allows DC to pick up antigen, mature, and migrate to draining lymph nodes. We have shown in previous studies that the mode of cell death associated with HSVtk expression in B16 tumors is proinflammatory. Signals released from B16 tumors as they die, such as Hsp-70, activate macrophages that can provide signals for DC maturation (26) . Therefore, provision of signals to recruit and activate DCs will all be required to generate optimal antitumor responses.
These data emphasize local expression of chemotactic factors within established tumors to generate immune-mediated tumor clearance. Clinical translation of these data would be most compatible with gene therapy approaches targeting accessible tumor deposits, e.g., in an adjuvant setting. Additional work is underway to generate suitable gene therapy vectors to test this hypothesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grant R01 CA94180-01 and the Mayo Foundation. 
2 To whom requests for reprints should be addressed, at Molecular Medicine Program, Guggenheim 18, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Phone: (507) 284-9941; Fax: (507) 266-2122; E-mail: vile.richard{at}mayo.edu
3 Present address: Chester Beatty Laboratories, Fulham Road, London, United Kingdom.
4 The abbreviations used are: DC, dendritic cell; IDC, immature dendritic cell; GM-CSF, granulocyte macrophage colony-stimulating factor; APC, antigen presenting cell; IRES, internal ribosomal entry site; HSVtk, herpes simplex virus thymidine kinase; IL, interleukin; GCV, ganciclovir; RPA, ribonuclease protection assay; LPS, lipopolysaccharide; NK, natural killer; MDC, mature dendritic cell; FACS, fluorescence-activated cell sorter.
Received 3/17/03. Revised 5/22/03. Accepted 5/28/03.
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REFERENCES |
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), CMT93 CCL3 (
), and CMT93 CCL20 (
).
), B16 HSVtk IRES CCL3 (10 mice, 
]), and B16 HSVtk IRES CCL3 (7 mice, 
