Mice and cell lines
C57Bl/6 and C57Bl/6 IL-10
/J #002251) mice  were maintained in standard mouse facility at the Faculdade de Medicina, Universidade de São Paulo (Ribeirão Preto, SP, Brazil) with irradiated food and autoclaved water, with cycles of light and dark of 12 hours.
Alternatively, C57Bl/6 mice used for the neutralizing antibodies assays were maintained at the Ludwig Institute for Cancer Research mouse facility in spf (specific pathogen free) conditions. All mouse protocols were approved by the Ethics Committee for Animal Experimentation of Fundação Antônio Prudente, São Paulo, Brazil, protocol 05/05.
TC-1 cell line was kindly donated by Dr. TC Wu (John Hopkins, Baltimore, MD). This cell line is derived of lung epithelium transformed with HPV16 E6 and E7 oncogenes and Ha-ras . These cells were cultivated in RPMI supplemented with 10% bovine fetal serum and 400 μg/ml Neomicin (Invitrogen, Carlsbad, CA) in incubators with atmosphere of 5% CO2.
TC-1 cells were suspended in Phosphate Buffered Saline, PBS++ (1 mM CaCl2, 0.5 mM MgCl2) at a concentration of 105cells/100 μl of suspension. Each mouse was injected with 105 TC-1 cells subcutaneously in the right dorsal flank. Tumor formation and size was measured with a caliper until a maximum diameter of 1.5 cm. Mice were observed and measured with intervals of 2 or 3 days from the day when they were injected. Tumor volume was calculated using the formula V = D*d2/2, where V is the tumor volume, D is the largest tumor measured diameter and d is the smallest measured tumor diameter.
Neutralizing antibody treatment
Mice received intraperitoneal doses of 10 mg of anti-IL-10 antibody (mAb417 R&D Systems, Minneapolis, MN) alternated with intraperitoneal doses of 10 μg of anti-IL-10R antibody (AF-474-NA, R&D Systems, Minneapolis, MN) spaced by 4 days. The first dose was given in the same day as the TC-1 cells injection and it was a double dose of anti-IL-10 and anti-IL-10R. Alternatively, mice were treated with 500 μg of protein G purified antiIL10R clone 1B13a. As controls, mice were treated with equal amounts of rat IgG, catalog number 6-001-A (R&D Systems, Minneapolis, MN) or catalog number I4131 (Sigma-Aldrich, St Louis, MO).
All cell preparations were performed in 1× Hanks' buffered saline solution supplemented with 5% fetal bovine serum and 0.5 U/ml DNase.
For tumor preparations, these were harvested after mouse euthanasia, a fraction was frozen in Tissue-Tek OCT (Sakura Finetek Europe, NL) for immunofluorescence studies, the rest of the tumor was minced and digested in 100 μg/ml Collagenase under agitation at 37°C for 1 to 2 hours. The cell suspension was filtered through a 70 μm mesh and washed twice before use. Spleen and lymph node suspensions were made by mechanical dissociation of the tissue and filtration through nylon mesh. Splenocytes were submitted to hypotonic cell lyses to eliminate red cells. Peritoneal macrophages were harvested after mouse euthanasia; by injecting 5 ml ice cold PBS into the peritoneal cavity and aspiration of the solution containing the resident cells. After centrifugation, cells were ressuspended in Hanks' buffer, counted and used for macrophage sorting.
Single cell suspensions were incubated with the antibodies indicated in each figure. Tumor and spleen suspensions were blocked with Fc ligand (CD16/CD32, clone 2.4G2) (BD Biosciences, San Diego, CA) before incubation with the specific antibodies. The antibodies used in this work were anti-CD4 (cloneGK1.5), anti-CD8 (clone 53-6.7), antiGr1 (clone RB6-8C5), anti-CD11b (clone M1/70), anti-CD19 (clone 1D3), anti-CD45 (clone 30-F11), anti-IL-10 (clone JES5-16E3) purchased from BD Biosciences (San Diego, CA), PE-Cy5.5 conjugated F4/80 (clone BM8) and APC conjugated anti-Perforin were purchased from eBiosciences (San Diego, CA). MHC-I tetramers containing the HPV16 E749-57 epitope or an irrelevant peptide were a kind donation from Dr. Immanuel Luescher and Dr. Philippe Guillaume (Ludwig Institute for Cancer Research, Lausanne, Switzerland). The lymphocyte populations were analyzed after gating on the CD45+F4/80- compartment. For intracellular staining, cells were incubated with antibodies against surface markers first, then fixed and permeabilized with Cytofix/Cytoperm kit from BD Biosciences (San Diego, CA). Cells were analyzed in a FACSCalibur (BD Biosciences, San Diego, CA). The number of acquired events is indicated in the figure legends. For cell cycle analyzes, TC-1 cells were harvested by tripsinization, fixed in 4% buffered formaldehyde and incubated in a buffer containing 0.1% (v/v) Triton X-100, 0.2 mg/ml RNase (DNase free) (Sigma, St Louis, MO) and 10 mg/ml propidium iodide (BD Biosciences, San Diego, CA) for 15 min at 37°C with gentle agitation. Cells were subsequently washed and analyzed in a FACSCalibur (BD Biosciences, San Diego, CA).
CD45+ cells were sorted from total tumor suspensions by positive selection after incubation with biotin conjugated anti-CD45 and streptavidin conjugated magnetic beads (Miltenyi Biotec, Germany) and loading in columns exposed to magnetic field (MACS LS+ Separation Columns) (Miltenyi Biotec, Germany). The negative fraction was used as CD45- population for RNA expression analyzes. In general, we obtained 95% pure cells with at least 90% viability.
Peritoneal macrophages were sorted from the peritoneal resident cells by positive selection with purified anti-F4/80 antibody, followed by anti-rat PE and and isolation using the EasySep anti-PE magnetic beads (Stem Cell Technologies, Vancouver, CA).
For protein expression experiments, tumors were digested as described before, and single cell suspensions were blocked with Fc blocking reagent and stained with FITC conjugated anti-CD11b, PE conjugated anti-Gr1, PECy5.5 conjugated anti-F4/80 and APC conjugated anti-CD45. Populations CD45+CD11b+F4/80+ or CD45+CD11+Gr1+ were sorted in a FACSCalibur (BD Biosciences, San Diego, CA). These sortings resulted in populations with 90-95% viability, approximately 90% purity for the F4/80+ population, which is the most abundant in the tumors, and approximately 80% purity for the Gr1+ population.
Lymphocyte ex-vivo cultures
Total cell suspensions from peripheral lymph nodes were seeded in 24 well culture dishes, 1 × 106 cells/well in 10% fetal bovine serum and stimulated with 15 μg/ml each HPV16 E749-57 peptide and HPV16 E6. After 5 days of incubation, cells were harvested, stained with anti-CD4 (cloneGK1.5), anti-CD8 (clone 53-6.7), fixed and stained with anti-Foxp3 (clone FJK-16S) using the FITC anti-rat/mouse Foxp3 staining set (eBioscience, San Diego, CA). Cells were analyzed by flow cytometry, when 100.000 events were acquired for posterior analyzes.
Cytokine expression analyses were performed by RT-PCR using RNA obtained from tumors collected 20 days post injection of TC-1 cells. Total RNA was extracted from infiltrating leukocytes (CD45+) and tumor cells (CD45-) using the RNeasy mini kit (Qiagen, Germany) according to the manufacture's recommended protocol. cDNA synthesis was performed using M-MLV reverse transcriptase (Invitrogen, Calrsbad, CA), oligo-dT and random primers, according to the manufacturer recommendation. We used 50 ng of cDNA in each amplification reaction. Expression of the constitutive gene HPRT was used as internal control. Amplified fragments were resolved in 1% agarose gels stained with 0.5 μg/ml ethidium bromide (Sigma-Aldrich, St Louis, MO).
Quantitative Real-time PCR analyses for IL-10 expression were done in CD45+ and CD45- cells isolated from TC-1 tumors. The reaction was performed in an ABI 7300 detection system (Applied Biosystems, Foster City, CA) with SYBR Green Mastermix (Applied Biosystems, UK) and specific primers for IL-10 (Forward CCCTGGGTGAGAAGCTGAAG and Reverse CACTGCCTTGCTCTTATTTTCACA) and GAPDH (Forward GGGCTGGCATTGCTCTCA and Reverse TGCTGTAGCCGTATTCATTGT). The relative quantification of IL-10 in comparison to a reference gene (GAPDH) was determined as described . The relative expression ratio was calculated based on Real-time PCR efficiency and the crossing points for the IL-10 and GAPDH transcripts. The following formula was applied Ratio = EIL-10(Ctcontrol-Ctsample)/EGAPDH(Ctcontrol-Ctsample), where E is the efficiency, Ctcontrol and Ctsample are the cycle numbers where GADPH and IL-10 amplification is detected above the threshold, respectively.
In order to analyze a panel of genes involved in the inflammatory response in our tumor model, Mouse Inflammatory Cytokines and Receptors RT2 Profiler PCR Array System (PAMM-011A) were used (SABiosciences Corporation, Frederick, MD). RT2 Profiler PCR Array System analysis was performed using cDNA obtained from total RNA of CD45+ and CD45-sorted cells. Samples of cDNA were synthesized with M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA), oligo-dT primer (IDT) and random primers. The reactions were performed on an ABI Prism 7300 sequence detection system (Applied Biosystems, Foster City, CA) using SYBR Green Mastermix (Applied Biosystems, UK). The mRNA expression levels were calculated as the ratio of the Ct averages of the housekeeping genes (HKG) and the Ct averages of the target gene (TG), where Ct (Cycle threshold) is the number of amplification cycles required for detection of fluorescence in a given cDNA sample. The amplification efficiencies of the targets (TG) and references (HKG) displayed no significant differences (data provided by SABiosciences Corporation, Fredrick, MD).
Ice-cold acetone fixed 5 μm cryo-sections were blocked with 5% fetal bovine serum in PBS for 30 min at room temperature prior to incubation with biotinylated primary antibodies. The ABC Vectastain kit (Vector Laboraboratories, Burlingame, CA) was used for antibody detection, followed by Mayer's hematoxylin counterstaining and slide mounting in Permount (Fisher Scientific, Pittsburgh, PA). Images were obtained with an IX70 Olympus fluorescence microscope (Olympus, Corp. Tokyo, Japan), a DP70 Olympus camera, using its own software.
Acetone/methanol (2:1 v/v) fixed 5 μm cryo-sections were blocked with 5% fetal bovine serum in PBS for 30 min at room temperature prior to incubation with primary antibodies. Anti-CD8 antibody (clone 53-6.7, BD Biosciences, San Diego, CA), was incubated for 30 min after blocking. Tetramers (a kind donation from Dr. Luescher laboratory, Ludwig Institute for Cancer Research, Lausanne, Switzerland) were incubated for 2 hours at room temperature. Tissue was counterstained with 4'6-diamidino2-phenylindole (DAPI) (Sigma-Aldrich, Saint Louis, MO). For Granzyme B staining, sections were fixed in 4% formaldehyde after anti-CD8 staining and washing, washed, blocked and permeabilized with 5% FBS, 0.5% Triton X-100, 1 mg/ml rat IgG. Anti-Granzyme B (clone 16G6, eBiosciences, San Diego, CA) was incubated for 30 min at room temperature, followed by counterstaining with DAPI. Slides were mounted with Prolong (Invitrogen, Carlsbad, CA). Images were obtained with a BX61 Olympus fluorescence microscope (Olympus, Corp. Tokyo, Japan), a DP70 Olympus camera, using its own software.
Cell cycle analysis
TC-1 cells were seeded in a density of 5 × 104 cells/well in 24 well plates in 10% calf serum supplemented RPMI. Cells were treated with 0 to 300 ng/ml of IL-10 for 4 days, after which cells were harvested by trypsinization. After washing in PBS, cells were fixed with 3.7% buffered formaldehyde for 24 hours at 4°C. Cells were washed twice with PBS and incubated for 45 min at 37°C in a 0.1% (v/v) Triton X-100, 200 μg/ml DNase free RNase A (Sigma, St Louis, MO), 10 μg/ml propidium iodide (BD Biosciences, San Diego, CA). Cells were washed once before acquisition in a FACSCalibur (BD Biosciences, San Diego, CA).
Tumor growth kinetics experiments were tested by Mann-Whitney. Data from all other experiments were tested by t-test. In all cases, p < 0.05 was considered significant.