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The Journal of Immunology, 2003, 170: 4201-4208.
Copyright © 2003 by The American Association of Immunologists

Host Absence of CCR5 Potentiates Dendritic Cell Vaccination1

Judith Ng-Cashin*,{dagger},{ddagger}, Jennifer J. Kuhns{dagger},{ddagger}, Susan E. Burkett{ddagger}, John D. Powderly*,{ddagger}, Robin R. Craven{dagger},{ddagger}, Hank W. van Deventer*,{ddagger}, Suzanne L. Kirby*,{ddagger},§ and Jonathan S. Serody2,*,{dagger},{ddagger}

Departments of * Medicine, {dagger} Microbiology and Immunology, {ddagger} Lineberger Comprehensive Cancer Center, and § Pathology, University of North Carolina School of Medicine, Chapel Hill, NC 27599


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous work has shown that dendritic cells (DCs) express specific chemokine receptors that allow for coordinated movement in vivo. To test the in vivo relevance of this, we used a murine melanoma system and knockout mice to investigate the function of the chemokine receptor CCR5 and its ligands, CCR ligand (CCL)3 and CCL5. We found that the lack of CCR5 in the host mouse resulted in delayed tumor growth, but this effect was overcome at a higher tumor load. With the administration of tumor charged DCs, CCR5-/- mice that had previously been injected with tumor were completely protected from tumor. This effect was dependent on the dose of tumor cells and the expression of CCR5 on the DC and its absence in the host. In contrast, the loss of the CCR5 ligand, CCL3, led to an early delay in tumor growth that did not persist, while the absence of the CCR5 ligand, CCL5, had no effect. Blocking the activity of CCR5 in the host may represent a new strategy for enhancing the activity of a therapeutic melanoma DC vaccine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The incidence of malignant melanoma is increasing at a rate that exceeds all other cancers in humans. Of the skin cancers, melanoma is the most lethal by far, responsible for six of every seven deaths caused by skin cancer (1, 2). When detected early, patients with melanoma can be cured by surgical excision (3). Although metastatic melanoma may respond to chemotherapy or radiation therapy, the long term outcome for these patients is poor (1, 4, 5, 6).

Because of the demonstrable host immune response to melanoma and the current understanding of tumor immunology, vaccine therapy is being investigated as a therapeutic strategy for melanoma (7, 8, 9, 10, 11, 12, 13). Dendritic cells (DCs)3 are the most potent APCs, highly specialized to prime T cell-dependent Ag-specific immune responses (14, 15, 16, 17). Unlike other APCs, DCs can present Ags to naive and memory T cells (18). These characteristics make the DC an obvious candidate for cellular vaccines that activate T cells toward specific tumor Ags (19).

DCs have distinct trafficking properties. The capacity of DCs to migrate first to the site of inflammation where Ags are taken up and processed by DCs and then to the draining lymph nodes where DCs stimulate Ag-specific T cells is dependent primarily on the expression of chemokine receptors (20, 21, 22, 23, 24, 25, 26). Chemokines, or chemotactic cytokines, predominantly are 8- to 10-kDa secreted proteins that regulate migration and activation of leukocytes (26, 27, 28, 29). Chemokines are classified into four groups based on the position and spacing of the first two conserved cysteines. Most chemokines fall into two subfamilies: the C-X-C and the C-C chemokine families. Chemokines also can be divided functionally into inflammatory and constitutive chemokines. Chemokine receptors span the plasma membrane seven times and are part of the G protein-coupled receptor superfamily. These receptors are identified by the chemokine family of their initially defined ligand (i.e., CXCRs and CCRs). The chemokine ligand-receptor interaction has significant redundancy, allowing chemokines to bind several different receptors and receptors to bind multiple chemokines (26, 27).

Others have shown that, in vitro, DCs express receptors for inflammatory chemokines such as CCR5, as well as CCR1, CCR2, and CXCR1, allowing them to follow chemokine gradients to sites of inflammation. Once maturation has been initiated, the levels of these inflammatory chemokine receptors decrease modestly, and constitutive chemokine receptors such as CCR7 and CXCR4 are expressed (20, 30). These receptors guide the mature DC toward the lymphatics and then into T cell-rich areas of the lymphoid organ.

The in vivo relevance of the coordinated surface expression of chemokine receptors in DCs has not been explored in a tumor model. We used a murine melanoma system and knockout mice to investigate the function of the chemokine receptor CCR5 and two of its chemokine ligands, CCR ligand (CCL)3 (also known as macrophage inhibitory protein (MIP)-1{alpha}) and CCL5 (also known as RANTES. We found that the lack of CCR5 in the host resulted in delayed tumor growth, but this effect was overcome at a higher tumor load. We found that the protective effect found with the absence of CCR5 on host cells was enhanced by an Ag-loaded DC vaccine. This work may have significant impact on the use of clinical vaccines to treat tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

The C57BL/6 mouse strain (B6) and CCL3-/- were purchased from The Jackson Laboratory (Bar Harbor, ME). CCR5-/- mice were backcrossed onto a B6 background for eight generations (N. Maeda and W. Kuziel, unpublished data); CCL3-/- mice were backcrossed to B6 for 10 generations (31); and CCL5-/- mice were backcrossed to B6 for six generations (32). Pretreatment, mice were housed and bred under specific pathogen-free conditions at the University of North Carolina (Chapel Hill, NC). After tumor inoculation, mice were housed under conventional conditions. Donor mice were 10- to 16-wk-old. Recipient mice were 6- to 12-wk-old.

Cell lines

B16-F10 melanoma cells were purchased from American Type Culture Collection (Rockville, MD). These cells were grown in an adherent monolayer in DMEM with high glucose (Life Technologies, Grand Island, NY) supplemented with 10% bovine calf serum (Sigma-Aldrich, St. Louis, MO), 2 mM L-glutamine, and 0.025 mM 2-ME (D/10CS).

DC preparation and culture

DCs were cultured from mouse bone marrow cells. Bone marrow cells were harvested from donor mice by disarticulating the femur, removing the proximal and distal ends, and flushing the marrow out with 3 ml RPMI 1640 (Life Technologies) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine, and 0.025 mM 2-ME (R/10F). The cells were resuspended in R/10F with 20 ng/ml GM-CSF (PeproTech, Rocky Hill, NJ) using 2.25 ml/femur. The cells were plated at 1.5 ml/well in 6-well low cluster tissue culture plates (Costar, Corning, NJ) to minimize DC adherence. Cells were refed with cytokine on day 4. On days 6 and 8, cells were refed again with the addition of 10 ng/ml IL-4 (PeproTech). On day 11, the DCs were harvested and split 1:2; the cells were resuspended in R/10F with 5 ng/ml GM-CSF, 10 ng/ml IL-4, and 10 ng/ml TNF-{alpha} (PeproTech) and plated in double the number of previous wells at 1.5 ml/well in 6-well low cluster culture plates. On day 12, cells were refed with R/10F with 10 ng/ml TNF-{alpha}. On day 13, the DCs were harvested again and counted. They were resuspended at 1.3 x 106 cells/ml in R/10F with 5 ng/ml GM-CSF, 10 ng/ml IL-4, and 10 ng/ml TNF-{alpha} and plated at 1.5 ml/well (2 x 106 cells/well) in 6-well low cluster plates.

An equal number of B16-F10 melanoma cells were harvested from the cell culture and resuspended at 4 x 106 cells/ml in R/10F. The cell suspension was irradiated to 16,000 Gy. After irradiation, the melanoma cells were not viable in culture (data not shown). A tumor cell lysate was made by subjecting the cells to two freeze/thaw cycles from -70°C to 56°C, followed by two passes through a 25-gauge needle, then two passes through a 30-gauge needle. GM-CSF, IL-4, and TNF-{alpha} were added to the lysate at the same concentrations as above. The lysate was added to the plated DCs at 0.5 ml/well (2 x 106 cells/well). The DCs were pulsed with the lysate overnight at 37°C with 6% CO2. On day 14, the pulsed DCs were harvested and counted. They were resuspended at 1 x 107 cells/ml in sterile PBS. Human CD40 ligand (CD40L) (PeproTech) was added at 2 µg/ml to facilitate DC maturity. The cells were incubated at 37°C with 6% CO2 for 1 h.

Flow cytometric analysis

Single-cell suspensions of DCs were stained in 1.2-ml microtubes. Cells were suspended in PBS with 2% FBS, then preincubated with mouse IgG (Sigma-Aldrich) for 20 min at 4°C. Then, cells were stained with specific mAbs or their isotype controls conjugated to FITC or PE for 20 min at 4°C. After washing, cells were fixed in 1% paraformaldehyde in PBS. The following staining reagents were used: CD11b-FITC (clone M1/70, IgG2b, {kappa}), CD11c-PE (clone HL3, IgG1, {lambda}), CD80-FITC, and -PE (clone 16-10A1, IgG2, {kappa}), CD86-FITC (clone GL1, IgG2a, {kappa}), and A-Ib-FITC (clone AF6-120.1, IgG2a, {kappa}) (BD PharMingen, San Jose, CA). FACS analysis was performed using a FACScan flow cytometer (BD Biosciences, San Jose, CA) and CellQuest software.

Mixed lymphocyte reaction

After maturation with CD40L, cultured DCs were resuspended in R/10F and seeded at 1 x 104 cells/well in a 96-well plate. T cells were isolated from spleens from C57BL/6 (H-2b) and BALB/c (H-2d) mice (Jackson ImmunoResearch Laboratories, West Grove, PA). The spleens were harvested, and RBCs were lysed using RBC lysis buffer. A single-cell suspension was prepared from the remaining splenocytes in PBS with 2 mM EDTA and 0.5% BSA at 1 x 108 cells/ml. T cells were positively selected using monoclonal rat anti-mouse CD90 (Thy1.2) Abs conjugated to colloidal superparamagnetic microbeads per the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). The selected T cells were resuspended in R/10F and plated in triplicate with the DCs at 1 x 105 or 1 x 106 cells/well. After 2 days in culture, the reaction was pulsed with 1 µCi/well of [3H]thymidine. Eighteen hours after incubation, the [3H]thymidine incorporation was measured.

Chemotaxis assay

After maturation with CD40L, cultured DCs were resuspended in R/10F at 5 x 105 cells/ml. MIP-1{beta}, a CCR5-specific chemokine, or eotaxin, a CCR5-irrelevant chemokine, was added to each bottom well of 96-well ChemoTx plates (NeuroProbe, Cabin John, MD). The chemokines were added using 29 µl of varying concentrations (100, 10, 1, or 0 nM) of chemokine in triplicate. After a filter was placed on top of the well plate, 1 x 104 DCs in 20 µl were pipetted onto the upper surface of each well. The plate was incubated at 37°C with 6% CO2 for 3 h to allow chemotaxis across the filter. After incubation, the cells remaining on top of the filter were aspirated. A total of 20 µl of 1 mM EDTA in PBS was added to each well, and the plate was incubated for 20 min at 4°C. After removing the EDTA and PBS, and after one wash with PBS, the plate was centrifuged at 1500 rpm for 5 min. The filter was removed, and the cells that had migrated to the bottom of the wells were harvested and counted using a hemocytometer.

Ribonuclease protection assay

RNA was extracted from DCs by TRIzol (Life Technologies) according to the manufacturer’s protocol. The RNA probe for CCR5 (BD PharMingen) was synthesized using the mCR-5 template set according to the manufacturer’s instructions. A total of 40 µg of DC-derived RNA was used per sample according to the manufacturer’s protocol. RNA was separated on a 20 by 1.5 cm 5% acrylamide per 8 M urea gel, then transferred to a Hybond membrane (Amersham Pharmacia Biotech, Arlington Heights, IL) using a vertical submarine transfer unit (C.B.S. Scientific, Del Mar, CA). The membrane was dried in an oven at 80°C for 2 h. The membrane was washed twice with blocking buffer (0.2% I-Block Reagent; Tropix, Bedford, MA), 1x PBS and 0.5% SDS (Sigma-Aldrich) and then incubated in avidin-alkaline phosphatase conjugate (Tropix) for 5 min before exposure onto chemiluminescent hyperfilm (Amersham Pharmacia Biotech). The CCR5 expression was normalized by analyzing the ratio of GAPDH to CCR5 using a densitometer with the NIH Image software program (National Institutes of Health, Bethesda, MD).

Injection and tumor monitoring protocol

Before all injections, recipient mice were sedated with 2.5% Avertin (2, 2, 2-tribromoethanol (Sigma-Aldrich) in tertiary amyl alcohol) injected i.p. All cellular injections were in a volume of 0.1 ml sterile PBS/dose and were injected s.c. into the right leg or flank using a 22-gauge needle. On day 0, B16-F10 melanoma cells were harvested and resuspended in sterile PBS at 2.5 x 104 or 1 x 105 cells/ml. Mice were shaved on the right flank and leg and then injected s.c. with 2.5 x 103 or 1 x 104 cells. Mice that received 2.5 x 103 tumor cells were vaccinated with 1 x 106 syngeneic, tumor lysate pulsed, matured DCs on days 7 and 14. Mice that received 1 x 104 tumor cells were vaccinated on days 3 and 10. Tumor growth was monitored two times a week by measuring perpendicular diameters. Mice were killed when the tumors displayed severe ulceration, when they limited ambulation, or when the tumor size was >600 mm2.

Tumor rechallenge

The CCR5-/- mice that showed no tumor growth after receiving melanoma cells followed by two vaccinations of tumor lysate pulsed DCs were rechallenged with tumor cells. On day 70, B16-F10 melanoma cells were harvested and resuspended in sterile PBS at 2.5 x 104. Mice were sedated with 2.5% Avertin injected i.p. and then were shaved on the right flank and leg. A total of 2.5 x 103 melanoma cells in 0.1 ml of sterile PBS were injected s.c. with a 22-gauge needle. Tumor growth was monitored as mentioned above.

Histology

On day 17, mice that received 1 x 104 melanoma cells followed by two vaccinations of 1 x 106 tumor lysate pulsed DCs were sacrificed for histology. Mice were injected i.p. with a lethal dose of 2.5% Avertin. The skin, s.c. tissue, and surrounding fascia and muscle around the tumor or tumor injection site were harvested. The tissues were paraffin embedded and sectioned for staining. The tissue slides were reviewed for evidence of microscopic tumor by one of the authors who was blinded to the treatment given.

Statistics

Differences in tumor growth were compared using Student’s t test. Differences in survival were compared using Fisher’s Exact test. All tests were two tailed. The p values <=0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro generation of DCs

We produced DCs from mouse bone marrow similarly to methods previously described (33). By day 8, the cells appeared large and homogeneous. By day 13, the cells expressed high levels of CD11c and increased levels of CD86 and MHC II (data not shown). On day 14, after incubation with CD40L, the cells expressed very high levels of CD11b, CD11c, and CD80, and moderate but significant levels of CD86, demonstrating a DC phenotype (Fig. 1A). Two-color staining was performed, and it confirmed simultaneous expression of CD11c and I-Ab, CD80, and CD86, respectively (Fig. 1B). These cells also demonstrated a DC functional phenotype. In an MLR, these cells induced CD8+ T cell proliferation. Allogeneic BALB/c CD8+ T cells demonstrated a stimulation index of 5.3 when compared with syngeneic B6 CD8+ T cells (data not shown). In a chemotaxis assay, these cells demonstrated a 3-fold increase in chemotaxis to a CCR5-specific chemokine, CCL4 (also known as MIP-1{beta}), compared with control cells (data not shown). Ribonuclease protection assay (RPA) confirmed both the absence of CCR5 in the DCs derived from the CCR5-/- mice and the expression of CCR5 in the DCs derived from the wild-type mice (Fig. 2).



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  		FIGURE 1. Characterization of bone marrow derived DCs after maturation with TNF-{alpha} and CD40L. Murine bone marrow cells were harvested from femurs and cultured ex vivo in medium containing, at various times, GM-CSF, IL-4, and TNF-{alpha}. On day 14 of culture, the cells were harvested and incubated with CD40L for 1 h. The cells were centrifuged, washed, and stained with mAbs for DC surface markers as indicated in the text. A, Representative histograms demonstrating the expression of CD11b (a), CD80 (b), CD11c (c), and CD86 (d). The percentage of positive cells is given for each histogram. B, Representative dot plots demonstrating control (a), unstained DCs, and the simultaneous expression of CD11c and I-Ab (b), CD11c and CD80 (c), and CD11c and CD86 (d). The percentage of double-positive cells is given for each plot.

 


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  		FIGURE 2. Absence of CCR5 in the CCR5-/- mice confirmed by RPA. On day 14 of culture, bone marrow-derived DCs from C57BL/6 wild-type and CCR5-/- mice were harvested. A total of 5 x 106 DCs were lysed in TRIzol reagent, RNA was extracted, and 40 µg of RNA was used per sample. RPA was performed as described in Materials and Methods. Results of the RPA are shown for CCR5, CCR1, and control, GAPDH. Left lane: C57BL/6 DCs; right lane: CCR5-/- DCs.

 
The native absence of CCR5 results in delayed tumor growth

Host absence of CCR5 may result in slower tumor growth kinetics. To investigate the impact of host inability to produce CCR5 on tumor growth, we injected 2.5 x 103 B16-F10 melanoma cells s.c. in C57BL/6 and CCR5-/- mice. Previous work by our group has demonstrated that this dose of tumor cells produces grossly visible s.c. tumor (seen at sacrifice) in 50% of wild-type mice at 3 days and 100% of mice in 7 days (data not shown). The CCR5-/- mice demonstrated a statistically significant delay in tumor growth through day 26 (p = 0.03) (Fig. 3A). We investigated this result further by injecting a higher tumor cell inoculum, 1 x 104 melanoma cells, in C57BL/6 and CCR5-/- mice. At the higher tumor dose, the melanoma growth kinetics did not differ between the different strains of mice (Fig. 3B). Therefore, the lack of CCR5 in the host caused a delay in melanoma growth. This effect was overcome by the injection of a larger number of tumor cells.



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  		FIGURE 3. B16-F10 melanoma tumor growth in CCR5-/- and wild-type mice. C57BL/6 and CCR5-/- mice were injected s.c. with two different doses of B16-F10 melanoma cells. A, A total of 2.5 x 103 tumor cells (seven mice/strain). The CCR5-/- mice demonstrated a statistically significant delay in tumor growth through day 26 (p = 0.03). B, A total of 1 x 104 tumor cells (seven mice/strain). There was no significant difference in tumor growth kinetics comparing the different strains of mice. Statistics by Student’s t test.

 
Vaccination of CCR5-/- mice with a wild-type DC vaccine enhances the delay in tumor growth

We hypothesized that the protective effect found in the CCR5-/- mice could be enhanced by improving the antitumor immune response. To investigate this, we used a DC vaccine to induce a specific antimelanoma immune response in our mouse model. We injected C57BL/6 and CCR5-/- mice with 2.5 x 103 B16-F10 melanoma cells s.c. followed by two injections of tumor lysate-pulsed wild-type DCs. We found that tumor growth was completely eliminated in the CCR5 knockout mice. The vaccinated CCR5-/- mice did not demonstrate any measurable tumor growth through day 70 (p = 0.01) (Fig. 4A) with a statistically significant survival rate (p = 0.03) (Fig. 4C).



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  		FIGURE 4. B16-F10 melanoma tumor growth in CCR5-/- and wild-type mice receiving wild-type DC vaccine. C57BL/6 and CCR5-/- mice were injected s.c. with two different doses of B16-F10 melanoma cells followed by two s.c. vaccinations with 1 x 106 tumor lysate-pulsed matured DCs from wild-type mice. A, A total of 2.5 x 103 tumor cells followed by DC vaccine on days 7 and 14 (four mice/strain). The vaccinated CCR5-/- mice did not develop measurable tumor growth through day 70 (p = 0.01, by Student’s t test). B, A total of 1 x 104 tumor cells followed by DC vaccine on days 3 and 10 (six mice/strain). One-half of the vaccinated CCR5-/- mice did not develop measurable tumor growth through day 70. This delayed tumor growth was statistically significant through day 29 (p = 0.03) and approached significance through day 53 (p = 0.06, p values by Student’s t test). C, With 2.5 x 103 tumor cells, the CCR5-/- mice demonstrated a statistically significant survival rate compared with wild-type mice (p = 0.03, by Fisher’s Exact test). With 1 x 104 tumor cells, the CCR5-/- mice showed a trend toward improved survival compared with the wild-type controls.

 
To evaluate the effect of the DC vaccine at the higher tumor dose that initially did not result in delayed tumor growth kinetics in the CCR5 knockout mice, we repeated this experiment with 1 x 104 B16 F10 melanoma cells. We found that tumor growth was delayed in the CCR5-/- mice. This delay was statistically significant through day 29 (p = 0.03) and approached significance through day 53 (p = 0.06) (Fig. 4B). In fact, one-half of the CCR5 knockout mice did not develop a measurable tumor through day 70, while all the wild-type mice did (data not shown). However, this did not translate into a statistically significant difference in survival (Fig. 4C). Thus, the combination of a wild-type DC vaccine and the absence of CCR5 in the host led to enhanced antitumor responses at both tumor doses evaluated. At low inoculum, tumor lysate pulsed DCs were 100% effective at eliminating tumor from CCR5-/- mice. At a higher inoculum, a delay in early tumor growth was found with 50% of the animals protected from tumor.

Vaccination of CCR5-/- mice with a CCR5-/- DC vaccine does not affect tumor growth

To examine the interaction of CCR5 in the DC vaccine with CCR5 in the host, we vaccinated CCR5-/- and wild-type mice that had received 2.5 x 103 melanoma cells with Ag-loaded DCs from CCR5-/- mice. There was a trend toward a delay in tumor growth in the CCR5-/- mice, but this delay was not statistically significant (p = 0.11 at day 24) (Fig. 5A). There was no significant difference in survival comparing CCR5-/- DCs in wild-type and CCR5-/- mice (Fig. 5B). This suggests that the optimal benefit of DC vaccination in the CCR5-/- mice is dependent on the expression of CCR5 on the DC.



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  		FIGURE 5. B16-F10 melanoma tumor growth in CCR5-/- and wild-type mice receiving CCR5-/- DC vaccine. C57BL/6 and CCR5-/- mice were injected s.c. with 2.5 x 103 B16-F10 melanoma cells. On days 7 and 14, the mice were vaccinated s.c. with 1 x 106 tumor lysate-pulsed matured DCs from CCR5-/- mice (four mice/strain). A, There was no significant difference in tumor growth between the wild-type and the CCR5-/- mice (by Student’s t test). B, There was no significant difference in survival between wild-type and CCR5-/- mice (by Fisher’s Exact test).

 
Vaccination of CCR5-/- mice with a wild-type DC vaccine generates protective antitumor immunity

Vaccinated CCR5-/- mice that had not developed tumors through 70 days after initial melanoma cell injection were rechallenged with tumor to confirm that the lack of tumor in these CCR5-/- mice was immune mediated. On day 70, the CCR5-/- mice were rechallenged with a s.c. injection of 2.5 x 103 B16-F10 melanoma cells and not vaccinated. These mice remained tumor-free through 60 days after rechallenge, indicating that the DC vaccine imparted antitumor immunity in vivo in the CCR5-/- mice.

Histology

We hypothesized that the protective effect of the wild-type DC vaccine in CCR5-/- mice was caused by diminished recruitment of a regulatory or suppressive population to the tumor injection site. To test this, we sacrificed and examined C57BL/6 and CCR5-/- mice that had received 1 x 104 B16-F10 melanoma cells followed by two injections of tumor lysate-pulsed DCs. At day 17 after tumor injection, we found no evidence of gross tumor growth in the CCR5-/- mice and tumor in the wild-type mice. Interestingly, microscopic examination of the tumor injection site revealed a cellular infiltrate around the tumor in the wild-type mice (Fig. 6, A and B). There was no microscopic evidence of tumor or cellular infiltration at the tumor injection site in the CCR5-/- mice (Fig. 6C).



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  		FIGURE 6. Absence of tumor in CCR5-/- mice receiving wild-type DC vaccine at day 17. C57BL/6 and CCR5-/- mice were injected s.c. with 1.0 x 104 B16-F10 melanoma cells. On days 3 and 10, the mice were vaccinated s.c. with 1 x 106 tumor lysate-pulsed matured DCs from wild-type mice (four mice/strain). Representative mice from both groups were sacrificed at day 17, and the tumor injection sites were fixed for histologic examination. A, Cellular infiltrate encompassing tumor cell aggregate at the tumor injection site in a wild-type mouse, low power. B, High power view of wild-type mouse with cellular infiltrate with areas of tumor cell aggregate and tumor cell necrosis. C, Absence of both identifiable tumor cells and active infiltrate at the tumor injection site in a CCR5-/- mouse.

 
Tumor growth kinetics and vaccination in CCL3-/- and CCL5-/- mice

To investigate the role of CCR5 chemokine ligands in this model, we performed analogous experiments using CCL3-/- and CCL5-/- mice. After injection with B16-F10 melanoma cells, tumor growth kinetics did not differ between the CCL3-/- or CCL5-/- mice and wild-type mice (data not shown). We injected C57BL/6, CCL3-/-, and CCL5-/- mice with 2.5 x 103 B16-F10 melanoma cells s.c. followed by two injections of tumor lysate-pulsed wild-type DCs. There was not a significant difference in the growth of tumors or survival in the vaccinated CCL5-/- mice compared with the wild-type controls. In the CCL3-/- mice, initially there was a significant delay in tumor growth compared with the wild-type mice (Fig. 7A). This difference approached statistical significance at day 34 (p = 0.06), but the difference was not significant by day 41 (p = 0.26). There was no significant difference in survival (Fig. 7B). To test the function of CCL3 production by DCs in the setting of a host without CCL3, we transferred tumor lysate pulsed CCL3-/- DCs into C57BL/6 and CCL3-/- mice previously injected with tumor. We did not find that injecting tumor lysate pulsed CCL3-/- DCs resulted in a significant difference in tumor growth between C57BL/6 and CCL3-/- mice or survival (data not shown). These results are similar but attenuated compared with what we found using CCR5-/- mice.



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  		FIGURE 7. B16-F10 melanoma tumor growth in CCL3-/- and wild-type mice receiving CCL3-/- DC vaccine. C57BL/6 and CCL3-/- mice were injected s.c. with 2.5 x 103 B16-F10 melanoma cells. On days 7 and 14, the mice were vaccinated s.c. with 1 x 106 tumor lysate-pulsed matured DCs from wild-type mice (four mice/strain). A, There was an initial delay in tumor growth in the CCL3-/- mice that approached statistical significance at day 34 (p = 0.06) that was extinguished by day 41 (p = 0.26; both by Student’s t test). B, There was no significant difference in survival between wild-type and CCL3-/- mice (by Fisher’s Exact test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CCR5 and its chemokine ligands may be important for both tumor virulence and induction of antitumor immune response. DCs are critical to tumor immunity and require chemokine ligand-receptor binding to migrate to the tumor site and then, once loaded with Ag, into the lymph nodes. We have investigated the role of CCR5 and two of its chemokine ligands, CCL3 and CCL5, in tumor growth kinetics, DC function, host immune response, and the interaction between them. Unexpectedly, we found that the absence of CCR5 led to a decrease in early tumor growth and, more importantly, that DC vaccination in CCR5-/- mice previously injected with tumor led to complete regression. This process required the expression of CCR5 in the DCs and the absence of CCR5 expression in the mouse. Mice that rejected tumors maintained memory, as injection of tumor 70 days after the initial tumor was rejected did not result in tumor growth in any of the animals. This strongly suggests that the mechanism(s) involved were immune mediated.

At this time, the mechanism by which the loss of CCR5 on both the DC and in the host markedly affects the function of a DC vaccine is not entirely clear. Previous work has stressed the expression of CCR7 on mature DCs. However, we have shown that the DCs used in this study, while expressing less CCR5 than immature DCs, still express significant quantities of this receptor. Interestingly, we found that the expression of CCR5 by DCs is critical in the enhanced response to DC vaccination found in CCR5-/- mice. It is possible that the presence of CCR5 on the DC allows for migration of the DC to the site of tumor and endogenous loading of the DC with tumor Ags. This may induce a broader immune response compared with that induced by ex vivo loading of DCs with tumor lysate. Additional experiments examining tumor site histology at time points earlier than day 17 should improve our understanding of this mechanism.

There are several hypotheses that can explain the mechanism by which the absence of CCR5 in the host mouse distinctly influences the function of a DC vaccine. Endogenous cells capable of blocking the induction of an immune response might also express CCR5 and be capable of interacting with DCs in the tumor bed to either inhibit their function or induce apoptosis. T cells as well as NK cells, monocytes, and immature DCs express CCR5. Recently, a number of investigators have characterized the function of CD4+CD25+ T regulatory (Treg) cells that are capable of blocking T cell proliferation in vitro and in vivo (34, 35, 36, 37). Regulatory or suppressor T cells that express CCR5 could be important in the process observed in this study. Our group and others have found that these Treg cells express CCR5 and migrate in response to chemokines that bind to it, such as CCL4 (38). Thus, it is possible that the absence of CCR5 in the host mouse impairs the migration of these Treg cells and leads to enhanced T cell activity after vaccination. The presence of a cellular infiltrate around tumor in the vaccinated wild-type mice and its absence in the CCR5-/- mice supports this. Alternatively, immature DCs could be recruited to the site of tumor via the interaction of CCR5 with its ligands, and the loading of tumor proteins onto these cells in the absence of an inflammatory response could result in the induction of anergy. Currently, we are conducting experiments to evaluate these hypotheses.

Whereas DCs can generate proinflammatory chemokines after interaction with LPS or tumor lysate, the production of the proteins CCL3 and CCL5 was not found to be critical to the function of these DCs. This suggests either that the production of these chemokines plays no role in DC function or, more likely, that redundant mechanisms compensate for the loss of their production. This could include the production of the CCR5 ligand, CCL4, or other chemokine ligands.

The host absence of local production of the proinflammatory chemokine CCL3 improved the activity of tumor lysate loaded DCs, but not to the degree found using CCR5-/- mice. Again, this suggests that proinflammatory chemokines may be able to recruit a CCR5-expressing cell to the site of the tumor that suppresses the function of lysate loaded DCs. The reason for the incomplete protection found using CCL3-/- mice would be caused by redundant activity of the ligands. Further work is needed to evaluate the mechanisms underlying these findings.

One important aspect of this work is the clinical implications involved for DC vaccine therapy. Modified chemokines such as methionylated-RANTES and aminooxypentane-RANTES that block the function of CCR5 are currently being evaluated clinically. A combination of these proteins with an Ag-specific or tumor-lysate loaded DC vaccine may hold significant promise for the treatment of locally advanced or metastatic disease. Importantly, our findings that DC vaccination can result in the regression of established tumors in the absence of CCR5 are directly applicable to the treatment of individuals with locally advanced or metastatic melanoma. Currently, our group is testing these proteins in combination with tumor lysate-pulsed DCs in animal models.


   Footnotes
 
1 This work was supported by the National Cancer Institute Grants CA58223 and CA89961 (to J.S.S.). J.N. was supported by National Institute of Allergy and Infectious Disease Training Grant 5T32 AI07273-17. Back

2 Address correspondence and reprint requests to Dr. Jonathan S. Serody, Lineberger Comprehensive Cancer Center, Campus Box 7295, University of North Carolina, Chapel Hill, NC 27599-7295. E-mail address: Jonathan_Serody{at}med.unc.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; CCL, CCR ligand; MIP, macrophage inhibitory protein; CD40L, CD40 ligand; RPA, ribonuclease protection assay; Treg, T regulatory. Back

Received for publication April 16, 2002. Accepted for publication February 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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