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Transfection of Macrophage Inflammatory Protein 1 into B16 F10 Melanoma Cells Inhibits Growth of Pulmonary Metastases But Not Subcutaneous Tumors¹

Hendrik W. van Deventer^2,*,, Jonathon S. Serody^*,, Karen P. McKinnon, Casey Clements, W. June Brickey^, and Jenny P.-Y. Ting^,

^* Division of Hematology/Oncology, Department of Medicine, Lineberger Comprehensive Cancer Center, and Department of Immunology and Microbiology, University of North Carolina, and University of North Carolina School of Medicine, Chapel Hill, NC 27599

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Macrophage inflammatory protein 1 (MIP-1), a CC chemokine, is a chemoattractant for T cells and immature dendritic cells. Plasmacytoma cells expressing MIP-1 generate a cytotoxic T cell response without affecting tumor growth. To understand this discrepancy, we compared a local tumor model with a metastatic one using MIP-1-transfected B16 F10 melanoma cells. Clonal idiosyncrasies were controlled by selecting three lipotransfected tumor clones and two pcDNA vector transfected control clones with equivalent in vitro proliferative capacities. No significant differences were seen between the MIP-1-producing and control melanoma cells after s.c. injection in the hind leg. All animals had a leg diameter of 10 cm in 18.5–21.5 days. However, after i.v. injection the number of pulmonary foci was significantly reduced in the MIP-1-producing clones. Injection of 10⁶ control transfected cells resulted in a median of 98.5 tumor foci in 2 wk, whereas the injection of the MIP-1-producing clones resulted in 89.5, 26.5, and 0 foci. The number of metastatic foci was inversely proportional to the amount of MIP-1 produced by the clone in vitro. Flow cytometry showed a significant increase in CD8⁺ cells in lungs of mice with MIP-1-transfected tumors 3 days after injection. This increase was not maintained 10 days later despite continued production of MIP-1. The protection offered by transfection with MIP-1 was significantly impaired in ₂-microglobulin^-/- mice. Our findings suggest that MIP-1 is effective in preventing the initiation of metastasis, but not at sustaining an effective antitumor response.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Researchers have attempted to increase the effectiveness of antitumor vaccines by introducing vectors that deliver cytokines and chemokines to the tumor microenvironment at more relevant concentrations than could be given systemically. Using this technique, antitumor immunity was first established in a mouse model using GM-CSF-transfected B16 F10 melanoma cells (1). Subsequent human vaccine trials in melanoma were able to demonstrate cytotoxic T cell responses and even tumor destruction (2), although results in other cancers have not been as promising (3). Current efforts have focused on combining GM-CSF with other cytokines (reviewed in Ref. 4).

Chemokines are small molecules that serve as signals for cellular activation and migration (reviewed in Refs. 5 and 6). They are classified by the position of the cysteine residue in the N terminus (CXC, CC, C, or CX₃C), and all but fractalkine are secreted. Chemokines are highly basic, which is favorable to the formation of stable associations with sulfated proteins and proteoglycans. In sites of inflammation, chemokines recruit granulocytes, monocytes, immature dendritic cells (DC),³ and activated T cells. A different set of chemokines attracts lymphocytes and mature DCs to secondary lymphoid organs (5).

Several properties of the CC chemokine macrophage inflammatory protein 1 (MIP-1) make it a suitable candidate for transfection into a tumor-based vaccine. Although it is a chemoattractant for a wide variety of immune cells (reviewed in Ref. 7), it has its greatest effect on CD8⁺ T cells (8) and immature DCs (9). MIP-1 also facilitates the induction of a Th1 immune response (10), even independent of IL-12 (11). Finally, MIP-1 has been shown to improve CTL activity when combined with a plasmid DNA vaccine for HIV (12) and when used as an exogenous adjuvant in a hepatitis B vaccine (13).

Maric and Liu (14) have demonstrated that transfection of MIP-1 into a plasmacytoma model leads to a strong CTL response, but not to noticeable differences in tumor growth. Tumor growth in these experiments was assessed by measuring the diameter of the leg following s.c. inoculation of tumor cells. Why did MIP-1 not have a greater effect in this model?

Our investigation into this question began by comparing the effects of MIP-1 on B16 F10 melanoma cells inoculated s.c. with those administered i.v. B16 F10 melanoma is a poorly immunogenic tumor with no expression of MHC class II or costimulatory molecules. Subcutaneous injection of tumor cells models primary tumorigenesis by establishing a focus of these cells near the skin, the site of origin for melanoma. In contrast, tail vein injection models tumor metastasis by introducing multiple tumor foci throughout the lungs. Differences in tumor progression between the two models are dependent on both the number of tumor cells present and their respective environments.

Our hypothesis is that MIP-1 is more effective in inhibiting tumor growth in a metastasis model than in the s.c. model. This premise is based on the known activity of MIP-1 in the lung in a variety of biological processes. Our results support this hypothesis. We found that, like the plasmacytoma model (14), expression of MIP-1 by melanoma had no effect on s.c. tumor growth. However, pulmonary metastasis was significantly inhibited in the presence of MIP-1. Furthermore, this inhibition appears to be mediated by CD8⁺ cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Wild-type C57BL/6 and B6.129P2-B2 m^tm1Unc females between the ages of 8 and 12 wk were obtained from The Jackson Laboratory (Bar Harbor, ME). All animals were housed at University of North Carolina (Chapel Hill, NC) under specific pathogen-free conditions. All animal protocols were approved by University of North Carolina’s institutional animal care and use committee.

Tumor cells and in vitro transfection with MIP-1

B16 F10 melanoma cells were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in complete DMEM supplemented with 7.5% (v/v) FCS and 100 µg/ml streptomycin sulfate (Life Technologies, Grand Island, NY) and were cultured at 37°C in 5% CO₂. Following transfection, cells were maintained in a selection medium of complete DMEM plus 800 µg/ml Genetacin (Life Technologies).

Plasmid MIP-1 cDNA (GenBank accession no. NM-011337) (4) cloned into a pCLXSN retroviral expression vector was obtained from Imgenex (San Diego, CA). Transfection with MIP-1 and pcDNA3 (vector control) was performed with FuGENE6 (Roche, Indianapolis, IN). When nontransfected cells were no longer viable in the presence of Genetacin, the resultant polyclonal cells were expanded and frozen for later use. Stable clones were generated by two successive rounds of serial dilution and expansion. Twelve MIP-1-transfected clones and eight pcDNA3 clones were generated and screened for proliferative capacity and MIP-1 production. Three MIP-1-producing clones (P1B, P1C, and P1D) and two control clones (CTRL1 and CTRL6) were chosen for in vivo experimentation.

Characterization of transfected cells

MIP-1 production by transfected tumor cells was measured by collecting supernatants of 3 x 10⁵ cells/ml after 24 h of culture. The amount of MIP-1 was determined using an ELISA with commercial Abs (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The sensitivity of the assay was 0.004 ng/ml. This was determined by adding 2 SD of the mean absorbance to the value of the zero standard replicates and calculating the corresponding concentration.

In vitro proliferation of tumor cells was measured by thymidine uptake and calculation of doubling time. Cells in log phase growth (1 x 10⁴) were pulsed with 1 µCi [³H]thymidine/well. The cells were harvested 24 h later, and the incorporation of [³H]thymidine was quantified using a liquid scintillation counter. Doubling time was measured by plating 1 x 10⁴ cells, harvesting at 24, 36, 48, and 60 h and counting viable cells using trypan blue and a hemocytometer. Each clone was assessed four times.

Bioactivity of MIP-1

The bioactivity of MIP-1 expressed by the transfected B16 F10 cells was assessed by measuring the chemotaxis of immature DCs. DCs were prepared from bone marrow precursors using the method described by Lutz and Shuler (15). In brief, bone marrow cells were separated from erythrocytes using a Cappel LSM gradient (ICN Biomedicals, Aurora, OH). The cells were then resuspended at a concentration of 2 x 10⁵ cells/ml in RPMI 1640 supplemented with 10% FCS, 25 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate (Life Technologies). Ten milliliters of the cell suspension with 20 ng/ml murine GM-CSF (PeproTech, Rocky Hill, NJ) were then added to 100-mm bacterial culture plates (Fisher Scientific, Pittsburgh, PA). Ten milliliters of complete RPMI with 10 ng/ml GM-CSF were added on day 3. Loosely adherent immature DCs were harvested on day 6. These cells have low MHC class II surface expression and chemokine receptor expression consistent with immature DCs and will generate mature DCs with 2 days of treatment with LPS, TNF-, or CD40 ligand (data not shown).

In vitro chemotaxis was assessed using the 5-µm ChemoTx System (NeuroProbe, Gaithersburg, MD) according to the manufacturer’s protocol. Immature DCs were resuspended in complete DMEM at 80,000 cells/microplate well. Chemoattractants consisted of either medium from MIP-1-transfected cells or medium from control transfected cells with the addition of varying amounts of purified MIP-1. Chemokinesis was ruled out by resuspending a portion of the cells in medium with a chemoattractant. The plates were incubated for 3 h before counting. Results are reported as migration indexes, which are the average number of cells that migrate under experimental conditions divided by the number of cells that migrate to medium from control transfected cells. All conditions were tested in triplicate.

Tissue sections for immunohistochemistry were prepared by snap-freezing in OCT compound (Sakura, Torrance, CA) and liquid nitrogen. Sections were cut to 7 µm, mounted onto SuperFrost Plus slides (VWR Scientific, West Chester, PA) and stored at -70°C. On the day of staining, the slides were air-dried for 30 min, fixed with 2% paraformaldehyde/PBS for 30 min, and permeabilized with 1% saponin/HBSS. After blocking endogenous biotin/avidin activity (Vector Laboratories, Burlingame, CA), nonspecific binding was blocked with 1 µg/ml Fc block in 2% rabbit serum/HBSS for 30 min. Biotinylated polyclonal goat anti-mouse MIP-1 Ab (R&D Systems) was added for 30 min at 37°C. After washing three times with 0.1% saponin/HBSS, biotinylated rabbit anti-goat IgG (Vector Laboratories) was added for 30 min at 37°C. After three additional washes, avidin conjugated to FITC (Vector Laboratories) was added for 30 min at room temperature. The slides were washed with HBSS, and coverslips were mounted with Vectashield plus DAPI (Vector Laboratories). The tissue was examined using an Olympus BX40 microscope (New Hyde Park, NY). Images were captured and analyzed using Adobe Photoshop software (Adobe Systems, San Jose, CA).

In vivo levels of MIP-1

Lung tissue was harvested and homogenized in a 1 ml of a buffer consisting of 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 1 mM PMSF (Sigma-Aldrich, St. Louis, MO) in PBS. The samples were incubated for 30 min at 4^oC before centrifugation at 20,000 rpm for 20 min. The supernatants were diluted to 1/20, 1/100, and 1/500 before measuring MIP-1 levels by ELISA. The amount of MIP-1 was calculated based on the dilution that generated an absorbance in the linear portion of the standard curve.

Animal models

For local tumor growth, 15,000 tumor cells in 20 µl PBS were injected s.c. into the hind leg. Leg diameter was measured three times per week, and the average of the three measurements was recorded. The animals were euthanized when the leg was >10 cm in diameter. The length of time to reach 10 cm was interpolated from the last leg diameter that was <10 cm and the first measurement that was >10 cm.

To establish lung metastases, 1 x 10⁶ tumor cells were resuspended in 100 µl PBS and injected via the tail vein. On day 14 the mice were euthanized, and the lungs were perfused with Feketes solution via the trachea and removed en bloc. Lung metastases were counted under a dissection microscope by an investigator who was blinded to the treatment.

Flow cytometry

To form a single-cell suspension, anesthetized mice were first perfused with PBS via the right ventricle. The lungs were harvested, minced, and incubated for 45 min at 37^oC in 1 mg/ml collagenase A (Roche, Mannheim, Germany) and RPMI 1640 with 5% FBS (Life Technologies). After pelleting the cells and discarding the supernatant, the erythrocytes were lysed with buffered ammonium chloride. The cells were washed, resuspended in 10 ml medium and passed through a 70-µm pore size cell strainer. Ten milliliters of 40% (v/v) Percoll (Sigma-Aldrich) in medium was added, and the resultant suspension was centrifuged at 3000 rpm for 15 min. The pellet was washed twice and resuspended at 1 x 10⁶ cells/ml in blocking solution of 2% normal rat serum/PBS.

Surface marker phenotype was determined by single-color flow cytometry. After 20 min in the blocking solution, the cells were incubated for 1 h using the following fluorochrome-labeled Abs and appropriate isotype controls: CD4 (L3T4) CD8 (Ly-2), Gr-1 (RB6-8C3), Pan NK (DX5), CD11c (HL3), B220 (RA3-6B2; all from BD PharMingen, San Diego, CA), and F480 (C1:A3-1; Cedarlane Laboratories, Hornby, Ontario, Canada). The cells were then washed three times in PBS with 0.5% BSA. Propidium iodide was added 10 min before evaluation with the FACScan flow cytometer. Analysis of FACS results was performed using WinMDI version 2.8 (Scripps Research Institute, La Jolla, CA). Histogram markers were set to include 5% of the isotype control. The number of cells for each subpopulation was calculated by multiplying the percentage of cells, as determined by FACS, by the total number of cells for each mouse.

Statistics

Unless otherwise stated, data are presented as the mean value for at least three separate experiments, with error bars representing 1 SEM. Values of p 0.05 were considered significant using a homoscedastic t test assuming equal means.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transfected B16 F10 melanoma cells produce biologically active MIP-1

B16 F10 melanoma cells were transfected with either a plasmid containing MIP-1 or a control vector. Before clonal selection, polyclonal MIP1--transfected cells produced 0.52 ± 0.28 ng/ml/10⁵ cells of the chemokine (Fig. 1A). From these cells, several stably transfected clones were selected, ranging in MIP-1 production from 1.54 ± 0.2 ng/ml/10⁵ cells (clone P1B) to 0.01 ± 0.0001 ng/ml/10⁵ cells (clone P1D).

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Transfection of B16 F10 melanoma cells with a plasmid expressing MIP-1 leads to production of a biologically active chemokine. A MIP-1 construct was introduced into B16 F10 melanoma cells using lipotransfection. A, Supernatants were collected from polyclonal and stable transfectants, P1B, P1C, and P1D after 24 h of culture in fresh medium. MIP-1 levels were measured by ELISA. B, The biological activity of MIP-1 produced by transfected B16 F10 cells was verified by measuring the migration of immature DCs in a Transwell chemotaxis assay. The chemoattraction of medium plus increasing concentrations of purified MIP-1 was compared with that of the supernatants of the clones shown in A. The results were normalized to medium alone, which was set at 1.0

MIP-1 produced by the transfected melanoma cells induced chemotaxis comparable to that seen when purified MIP-1 was added to the supernatant. Clone P1B prompted a greater degree of chemotaxis than clone P1C. The amount of MIP-1 produced by P1D was below the threshold needed to induce chemotaxis. These relationships correlate with the amount of MIP-1 measured by ELISA.

MIP-1- and pcDNA vector-transfected subclones were selected with similar rates of proliferation in vitro

The proliferative capacity of the stable clones was assessed before their use in the in vivo experiments (Fig. 2, A and B). The selected MIP-1-producing clones (P1B, P1C, and P1D) and the two control clones (CTRL1 and CTRL6) had approximately equivalent thymidine uptake (1.7 x 10⁵ ± 0.19 x 10⁵ cpm over 20 h) and doubling times (19.9 ± 3.1 h).

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Transfected B16 F10 subclones were chosen with similar proliferation rates. The proliferation rates of the transfected B16 F10 melanoma cells were measured by two separate assays. A, Known quantities of cells in log phase growth were plated, harvested at various time points, and then counted using a hemocytometer. Doubling times were then calculated. B, In the second assay, 12,500 cells/well were plated and thymidine uptake was measured after 24 h. C, MIP-1 does not inhibit the proliferation of wild-type B16 F10 melanoma cells. Cells (20,000/well) were plated with varying concentrations of MIP-1. Thymidine uptake was measured after 24 h of culture.

B16 F10 cells transfected with MIP-1 continue to produce protein in vivo

In vitro protein expression does not always predict in vivo protein expression. To verify that the production of MIP-1 persists and is associated with the localized tumor, tissue sections of the s.c. tumor were taken 7 days after injection of P1B, an MIP-1 transfected subclone. MIP-1 was detected in areas of melanin production corresponding to transfected tumor cells using a fluorescently labeled Ab (Fig. 3A).

View larger version (63K): [in this window] [in a new window]   FIGURE 3. Transfected B16 F10 tumor cells produce MIP-1 in vivo. A, MIP-1 transfected tumor cells (15,000) were injected s.c. into the legs of B6 mice. Seven days later tissue was harvested and stained for MIP-1. The x10 images for green fluorescence (left panel) and light microscopy (right panel) show detection of MIP-1 in areas positive for melanin (arrow). B, On day 14 s.c. implanted tumors were harvested, dissociated, and placed in culture. Once viability was established, fresh medium was added, and supernatants were collected after 24 h. MIP-1 production was measured by ELISA. C, Lungs from mice injected with either P1B or mock-transfected B16 F10 cells were harvested on days 3 and 10 after injection. Homogenates were generated, and MIP-1 levels were measured by ELISA.

To further demonstrate the production of MIP-1 in vivo, tissue homogenates of the lungs were generated 3 and 10 days following injection of MIP-1 and control transfected clones. Injection with P1B tumor cells resulted in MIP-1 levels of 19.5 and 12.2 ng/ml on days 3 and 10, respectively. These were higher than levels seen on the same days after injection with mock-transfected tumors (0.2 ng/ml (p = 0.08) and 1.4 ng/ml (p = 0.03); Fig. 3C).

Transfection of MIP-1 into B16 F10 melanoma cells does not prevent the development of s.c. tumors

To evaluate the effect of MIP-1 on primary tumors, mice were injected s.c. with 15,000 transfected or control B16 F10 cell. All mice injected with polyclonal MIP-1-transfected B16 melanoma cells developed palpable tumors. The rate of growth of these tumors was quantified by measuring the diameter of the injected hind leg 3 days/wk. Polyclonal MIP-1-transfected B16 cells reached a 10-cm leg diameter in 18.9 ± 1.02 days compared with 18.9 ± 1.21 days in controls (Fig. 4A). Similarly, the stable clones P1B and P1C reached this end point in 18.5 ± 0.27 and 21.5 ± 1.69 days, respectively. These differences were not statistically significant (p > 0.05).

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Transfection of B16 F10 melanoma cells with MIP-1 does not significantly improve survival time in a local tumor model. MIP-1-transfected or mock-transfected B16 F10 cells (15,000) were injected s.c. into the left hind leg of B6 mice. A, The diameter of the injected leg was measured 3 days/wk and recorded. B, Mice were euthanized when the tumor-bearing leg diameter was >10 cm, and survival time (time to 10 cm) was extrapolated based on the last two measurements. The above data represent averages from four experiments.

Transfection of MIP-1 into B16 F10 melanoma cells inhibits pulmonary metastases

To test the effects of transfection with MIP-1 on the establishment of pulmonary metastases, 1 x 10⁶ cells from each clone were injected via tail vein into C57BL/6 mice. Two weeks later the lungs were harvested, and metastatic colonies were counted. Transfection of tumor cells with MIP-1 resulted in a substantial decrease in the number of pulmonary metastases, but not in the maximum size of the colonies (Fig. 5A). The median number of metastatic colonies in the control group was 98.5 colonies compared with 89.5 for P1D, 26.5 colonies for clone P1C, and no colonies for clone P1B (Fig. 5B). The reduction in pulmonary metastases was inversely proportional to the in vitro production of MIP-1 (refer to Fig. 1).

View larger version (68K): [in this window] [in a new window]   FIGURE 5. Transfection of B16 F10 melanoma cells with MIP-1 leads to a significant decrease in the number of pulmonary metastases. B16 F10 melanoma cells (1 x 106) transfected with MIP-1 or a control vector were injected into the tail vein of B6 mice. The lungs were harvested 2 wk later and perfused with Fekety’s solution. A, Representative lungs after injection of clone P1C and CTRL1 are shown. The thick white lines show equivalent distances demonstrating equivalent size of the largest colony. B, Metastatic colonies were counted using a dissecting scope by an investigator who was blinded to treatment group. These data are a compilation of five experiments.

Inhibition of pulmonary metastases by MIP-1 is mediated through CD8⁺ cells

To evaluate the cells recruited into the lungs after administration of MIP-1 B16 cells, single-cell suspensions of lungs from injected mice were analyzed with flow cytometry. Three days after injection there was a significant increase in the total number of cells from the lungs with MIP-1-producing tumors compared with control tumors (92 x 10⁶ vs 15.3 x 10⁶; p < 0.05). The population of cells that showed the most significant difference was CD8⁺ cells (20.2 ± 6.5 vs 1.5 ± 1.2). There was a trend to increased number of CD4⁺ cells (4.6 ± 0.7 vs 1.6 ± 0.8; Fig. 6A). There were no detectable differences in other immune cells, including granulocytes, NK cells, B cells, DCs, and macrophages.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. Inhibition of pulmonary metastasis by MIP-1-producing B16 F10 melanoma cells is due to CD8+ T cells. A, B16 F10 melanoma cells (1 x 106) transfected with either MIP-1 or a control vector were injected into the tail veins of B6 mice. On days 3 and 10 a single-cell suspension of the lung was formed and analyzed using flow cytometry. B, 2m-/- mice were i.v. injected with either 2.5 x 105 P1B cells or mock-transfected B16 F10 melanoma cells. At 2 wk the lungs were harvested and perfused with Fekete’s solution, and the individual colonies were counted.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study we have shown that transfection of B16 F10 melanoma cells with MIP-1 can inhibit the growth of pulmonary metastases. These results are the first demonstration of improvement in a clinical end point using transfection with MIP-1. However, we did not see any improvement when the same tumors were implanted s.c., suggesting that the site of the tumor may make a difference in such therapy.

To establish the validity of this result, we developed a range of stable MIP-1-producing clones and control vector clones. We then chose three MIP-1-producing clones and two control clones with similar proliferative properties based on two different assays. We observed that the number of pulmonary metastases was inversely related to the level of MIP-1 production in vitro, suggesting that MIP-1 was responsible for this improvement. Furthermore, this inverse correlation was noted in three MIP-1-transfected subclones and two control subclones, making it unlikely that the introduction of vector led to other unintended cellular differences.

The decrease in pulmonary metastases appears to be predominantly mediated by CD8⁺ T cells. Our FACS data establish that CD8⁺ cells are the most prevalent inflammatory cell type in the lungs 3 days after injection of an MIP-1-producing B16 cell. We did not see a similar increase in our control tumor cells. Furthermore, this protection was significantly reduced in the ₂m^-/- mice, which have few CD8⁺ cytotoxic T cells.

The influx of T cells is probably due to MIP-1 from the modified tumor cells. This hypothesis is consistent with MIP-1’s known chemotactic effect on T cells (8). In vitro, MIP-1 has a greater effect on CD8⁺ T cells than on CD4⁺ T cells, a relationship that we have now demonstrated in vivo. Maric et al. (20) have demonstrated recruitment of CD8 cells by MIP-1 from activated T cells and from plasmacytoma cells modified to express MIP-1 (14). MIP-1 from the tumor cells may have also led to an increase in immature DCs and thus indirectly caused an increase in T cell infiltration. As DCs mature, they produce additional chemokines that attract T cells. Differences in DC populations between treated and untreated tumors may have gone undetected because of their small number compared with the total number of cells in the lung.

The effect of MIP-1 on CD8⁺ cells is transitory. By day 10 there are no differences in T cell population or number despite persistent production of MIP-1. This may be due to the lack of a costimulatory signal, effects of suppressive cytokines, or desensitization to MIP-1. Maric et al. (20) generated a sustained accumulation of CD8⁺ T cells in a tumor by expressing B7-1 on the tumor surface. Transfection of tumor cells with GM-CSF also leads to an accumulation of CD8⁺ cells by increasing the number of DCs at the tumor site (21). DCs in this setting both secrete MIP-1 and provide the requisite costimulatory signal. Costimulatory molecules are not present on B16 F10 cells.

Alternatively, the chemoattractant capacity of MIP-1 may be down-regulated by cytokines such as IL-10. This cytokine can be detected soon after inoculation with B16 F10 tumor (22), and it may down-regulate CCR5 on T cells (23), although others have disputed this result (24). Down-regulation of CCR5 would make the T cells less responsive to MIP-1.

MIP-1 may be a chemoattractant at low doses and may inhibit chemotaxis at higher doses. Such a biphasic response has been demonstrated for T cells in vitro (17). The overall level of MIP-1 in the lung may remain relatively constant; however, the distribution of the chemokine may become quite patchy. Areas near tumor foci may have levels high enough to inhibit chemotaxis. With s.c. injection, inhibitory levels of MIP-1 may be obtained more rapidly, which could also explain why the clinical course in this model was worse.

Our findings have several possible implications for the use of chemokines in cancer immunotherapy. First, the effectiveness of such therapy may vary with tissue type. In our experiments MIP-1 was effective in the lung, but not in the s.c. tissue. This may be a manifestation of a greater biological effect of MIP-1 in the lung. MIP-1 is active in a variety of pulmonary processes, including influenza pneumonitis (25), allergic airway disease (26), and bone marrow transplant-induced injury (27). Rolling in the pulmonary capillary bed is negligible, because the diameter of the vessels is smaller than most (28). This difference may enhance the recruitment of T cells to the lung compared with the s.c. tissue. Regardless of the explanation, the implication is that immunotherapy using chemokines will need to be tailored to the target tissue.

Our findings also suggest that chemokine-directed therapy may be more effective in preventing the establishment of metastases than in treating primary tumors. Transfection with MIP-1 produced a significant difference in the number of metastatic foci, but not noticeable differences in the size of these foci. This observation suggests that once the tumor cell is able to establish itself, it is unencumbered in its growth. In this model, the CD8⁺ cell eliminates the initiation of a metastatic focus, but is unable to control an established tumor. This model suggests that the lack of clinical efficacy associated with MIP-1 transfection of an s.c. tumor may be due to the large initial inoculum of cells.

Despite significant improvement following transfection of tumor cells with MIP-1, much work is still required to make this a reasonable clinical strategy. Transient recruitment of a large number of T cells to foci of metastatic tumor is beneficial to reducing the number of such foci, but does not guarantee their elimination. Further progress may come from additional activation of the T cells and/or sustained T cell recruitment. Designing such strategies depends on understanding the state and fate of these cells. This is currently an area of active investigation in our laboratory.

	Footnotes

 
¹ This work was supported by National Cancer Institute Grants CA89217 (to H.W.v.D.) and CA58223 (to J.S.S.), and National Institutes of Health Grants AI41580, AI29564, and AI41751 (to J.P.-Y.T.).

² Address correspondence and reprint requests to Dr. Hendrik W. van Deventer, Division of Hematology/Oncology, University of North Carolina, Room 3009, Old Clinic Building, Chapel Hill, NC 27599-7305. E-mail address: hvand{at}med.unc.edu

³ Abbreviations used in this paper: DC, dendritic cell; MIP-1, macrophage inflammatory protein 1; ₂m^-/-, ₂-microglobulin knockout; CTRL, pcDNA control vector transfected B16 F10 cell; P1, MIP-1-transfected B16 F10 cell.

Received for publication November 21, 2001. Accepted for publication May 28, 2002.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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