Materials and Methods

Ethics Statement
All experiments were approved and carried out according to the Guide for the Care and Use of Animals [Animal Care Committee of Chungbuk National University, Korea (CBNUA-045-0902-01)].

Animals
The CCR5 wild type (CCR5+/+) and CCR5 knockout (CCR5−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, Maine 04609 USA). The mice were housed and bred under specific pathogen free conditions at the Laboratory Animal Research Center of Chungbuk National University, Korea. The mice (n = 4/cage) were maintained in a room with a constant temperature of 22±1°C, relative humidity of 55±10%, and 12-h light/dark cycle, and fed standard rodent chow (Samyang, Korea) and purified tap water ad libitum. The CCR5+/+ mice (n = 20) and CCR5−/− mice (n = 20), that were used, had matched ages (10–11 weeks old) and weights (16–19 g). Control mice (B6129PF2/J) were F2 hybrid mice from the C57BL/6J-AW-J and 129P3/J parental strains. CCR5 mice (B6, 129P2-Ccr5tm1Kuz/J) have the entire coding region of the CCR5 gene deleted from the parental strains, B6;129P2-Ccr5tm1kuz and B6;129P2-Cmkbr5tm1Kuz.

Tumor Inoculation and Tumor Monitoring
B16 melanoma cells were injected subcutaneously [5×105 tumor cells in 0.1 ml Phosphate buffered saline(PBS)/animal] using a 27 G needle into the right-lower flanks of the carrier mice as previously described [54]. The body weights and tumor volumes of the animals were monitored twice weekly. The tumor volumes were measured with Vernier calipers and calculated using the following formula: (A×B2)/2, where A is the larger and B is the smaller of the two dimensions. At the end of the experiment, the animals were sacrificed. The tumors were separated from the surrounding muscles and dermis.

Western Blotting
Tumor tissues were homogenized with protein extraction solution (PRO-PREPTM, Intron Biotechnology, Seoul, Korea), and lysed by 60 min incubation on ice. The tissue lysate centrifuged at 15,000 rpm for 15 min at 4°C. Equal amounts of proteins (40 µg) were separated on a SDS/12%-polyacrylamide gel, and then transferred to a polyvinylidene difluoride (PVDF) membrane (GE Water and Process technologies, Trevose, PA, USA). Blots were blocked for 1 h at room temperature with 5% (w/v) non-fat dried milk in Tris-Buffered Saline Tween-20 [TBST: 10 mM Tris (pH 8.0) and 150 mM NaCl solution containing 0.05% Tween-20]. After a short washing in TBST, the membranes were immunoblotted with the following primary antibodies: mouse monoclonal antibodies directed against p65 and p50 (1∶500 dilutions; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antibodies directed against Bax and PARP (1∶500 dilutions; Santa Cruz Biotechnology), and against caspase-3, caspase-9, Bcl-2 and c-IAP-1 (1∶1000 dilutions; Cell Signaling Technology, Beverly, MA). The blots were incubated with the respective horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1∶4000 dilutions; Santa Cruz Biotechnology). Immunoreactive proteins were detected with the ECL detection system.

Immunohistochemistry
All specimens were fixed in formalin and embedded in paraffin for examination. Sections (4 µm thick) were stained with H&E and analyzed by immunohistochemistry. The paraffin-embedded sections were deparaffinized and rehydrated, washed in distilled water, and then subjected to heat-mediated antigen retrieval. Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide in methanol for 15 min, followed by clearing in PBS for 5 min. The sections were blocked for 30 min with 3% normal horse serum diluted in PBS, blotted, and incubated with primary mouse anti-mouse proliferating cell nuclear antigen (PCNA, 1∶200 dilution) monoclonal antibodies in blocking serum for 4 h at room temperature. Thereafter, the slides were washed three times for 5 min each in PBS and incubated with biotinylated anti-mouse and anti-rabbit antibodies for 2 h. The slides were washed in PBS, followed by the addition of the avidin biotin peroxidase complex (ABC kit; Vector Laboratories, Burlingame, CA, USA). The slides were washed and the peroxidase reaction was developed with diaminobenzidine (DAB) and peroxide, followed by counterstaining with hematoxylin, mounting in aqua-mount, and evaluation under a light microscope (magnification×200; Olympus, Tokyo, Japan). A negative control was included in all experiments by omitting the primary antibody. For the detection of apoptotic cell death in the tumor tissues, the paraffin-embedded sections were incubated in a mixture of the labeling solution (540 µl) and enzyme solution (60 µl) for 1 h at 37°C, and then washed three times in 0.1 M PBS for 5 min each, according to the manufacturer’s instructions. Next, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at 37°C. Finally, the sections were rinsed, mounted on slides, and cover-slipped for fluorescence microscopy (DAS microscope).

Immunofluorescence
Sections were treated with 10% bovine serum albumin in PBS for 1 h at room temperature, incubated overnight at 4°C with p50 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA), p65 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA), Bax (1∶200 dilution), cleaved caspase-3 (1∶100), CD8 (1∶10), CD57 (1∶50) and IL-1Ra (1∶500) antibodies, followed by incubation in anti-mouse IgG conjugated with Alexa 488 (1∶100 dilution, Molecular Probes, Eugene, OR, USA) and anti-rabbit IgG conjugated with Alexa 568 (1∶100 dilution, Molecular Probes) for 40 min at room temperature. Finally, the sections were rinsed, mounted on slides, and cover-slipped for fluorescence microscopy and photography using ApoTome microscopy (Carl Zeiss, Thornwood, NY, USA).

Gel Electromobility Shift Assay
A gel electromobility shift assay (EMSA) was performed according to the manufacturer’s recommendations (Promega, Madison, WI). The tumor tissues were briefly homogenized in 200 µl of solution A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonylfluoride), vortexed vigorously, incubated on ice for 10 min, and then centrifuged at 15000 rpm for 15 min. The pelleted nuclei were resuspended in solution C (solution A supplemented with 420 mM NaCl and 20% glycerol), and incubated on ice for 20 min. The resuspended pellet was centrifuged at 15000 rpm for 15 min, and the resulting nuclear extracts supernatant were collected in a chilled Eppendorf tube. Consensus oligonucleotides were end-labeled using T4 polynucleotide kinase and [P32]-ATP for 10 min at 37°C. The gel shift reactions were assembled and incubated at room temperature. Subsequently, 1 ml of gel loading buffer was added to each reaction and loaded onto a 6% non-denaturating gel. The gel was subjected to electrophoresis until the dye was four-fifths of the way down the gel. The gel was dried for 1 h at 80°C and exposed to film overnight at -70°C.

Flow Cytometric Analysis
Lymphocytes were obtained from tumor and spleen tissues for phenotype analysis. Spleen and tumor lymphocytes were isolated from fresh spleen and tumor biopsies obtained from three mice per group. Spleen and tumor biopsies were briefly homogenized mechanically in PBS, filtered (100 µm cell strainer; BD PharMingen) and then placed in lysis buffer (ACK lysing buffer, LONZA) to remove red blood cells. One million splenocytes were washed once in PBS containing 1% bovine serum albumin (BSA) and re-suspended in 100 µl of PBS/BSA buffer. Meanwhile, one million cells obtained from tumor tissues were placed in RPMI medium, incubated to obtain suspension cell for 1hour at 37°C, centrifuged (1200 rpm, 3 min) and then washed in PBS. Next, the lymphocytes were incubated with various conjugated monoclonal antibodies, including CD4-APC, CD8-FITC and CD19-PE for 20min at 4°C, washed twice in PBS, and re-suspended in 400 µl of PBS. A flow cytometric analysis was performed on a FACSAria flow cytometry (BD Biosciences), and the data were analyzed using the WinMDI statistical software (Scripps, La Jolla, CA, USA). Forward and side scatter parameters were used to gate on lymphocytes.

Protein Immuno-arrays
The tumor and spleen tissues were excised and homogenized in PBS with protease inhibitor cocktail (Sigma-Aldrich, USA) and Triton X-100 (final concentration 1%). The samples were frozen at −70°C, thawed, and centrifuged at 10,000×g for 5 min to remove cellular debris. The tissue lysates were performed protein assay in the same way as for western blotting. 4.5 mg of proteins, collected from three samples per group, were used for Mouse cytokine array as the protocol provided by supplier (Proteome Profiler™, #ARY006, R&D Systems, USA).

Statistical Analysis
The data were analyzed using the GraphPad Prism 4 ver. 4.03 software (GraphPad Software, La Jolla, CA). Data are presented as mean ± SD. Difference between CCR5−/− mice and CCR5+/+ mice were compared using t test. A value of p<0.05 was considered to be statistically significant. Survival data were presented by Kaplan-Meier survival estimates and compared and calculated by Log-rank (Mantel-Cox) Test in GraphPad Prism.