Mice
6- to 8-week old female C57BL/6 and BALB/c mice were purchased from the National Cancer Institute (Frederick, MD). TLR9−/− mice [25] were purchased from the Mutant Mouse Regional Resource Center (Bar Harbor, ME). All animal procedures were performed in accordance with protocols approved by the Johns Hopkins Institutional Animal Care and Use Committee and in accordance with recommendations for the proper use and care of laboratory mice.
Cells
Generation of TC-1 tumor cell line [26] and HPV16-E7-specific CTLs (recognizing epitope aa49–57 of E7) [27] has been described previously. EG7 cells (a derivative of EL4 lymphoma cells transduced with Ova) and CT26 (mouse colon carcinoma line) were obtained from ATCC (Manassas, VA). Cells were authenticated by short tandem repeat DNA fingerprinting. Cells were maintained at 37 °C under 5% CO2 atmosphere in RPMI-1640 medium supplemented with 10% fetal bovine serum, 50 U/ml penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 2 mM non-essential amino acids.
Quantification of DNA concentration
For in vivo DNA concentration measurement in the TC-1 model, 105 TC-1 cells were inoculated subcutaneously into C57BL/6 mice (5 per group). At days 5 and 8 after tumor challenge, naïve or TC-1 tumor-bearing mice were treated intraperitoneally with cisplatin (5 mg/kg) or PBS control. At days 5, 7, and 9 after tumor challenge, serum was collected from mice, and DNA concentration was determined with the Quant-iT PicoGreen dsDNA kit (Invitrogen, Carlsbad, CA).
For in vivo DNA concentration measurement in the CT26 or EG7 model, 106 CT26 or EG7 cells were inoculated subcutaneously into BALB/c or C57BL/6 mice (5 per group), respectively. After 11 or 14 days, naïve or tumor-bearing mice were treated intraperitoneally with cisplatin (5 mg/kg) or PBS control. At days 11, 13, and 15 after tumor challenge, serum was collected from mice, and DNA concentration was determined with the Quant-iT PicoGreen dsDNA kit.
Tumor treatment experiments
For experiments in the TC-1 model, TC-1 cells (1 × 105 per animal) were inoculated subcutaneously into C57BL/6 or TLR9−/− mice (10 per group). On days 5, 8, and 11 after tumor challenge, mice were administered with 5 mg/kg of cisplatin or doxorubicin intraperitoneally, with or without concurrent intratumoral injection of 20 μg of E7 peptide (aa43–62). PBS administrations were used as controls. Tumor growth was monitored by palpation and visual inspection twice per week. For experiments involving the use of DNase I, 2000 U of DNase I (Invitrogen, Carlsbad, CA) or PBS control were injected intravenously in concurrent with cisplatin and E7 peptide administration on days 5, 8 and 11 after tumor challenge.
For experiments in the CT26 model, CT26 tumor cells (2 × 105 per animal) were inoculated subcutaneously into BALB/c mice (10 per group). On days 5, 8, and 11 after tumor challenge, mice were treated intratumorally with 20 μg of AH1-A5 peptide (SPSYAYHQF), intraperitoneally with cisplatin (5 mg/kg body weight), and/or 2000 U of DNase I intravenously. PBS injections were used as controls. Tumor growth was monitored by palpation and visual inspection twice per week.
For experiments in the EG7 model, EG7 tumor cells (2 × 106 per animal) were inoculated subcutaneously into C57BL/6 or TLR9−/− mice (10 per group). At 10, 13, and 16 days post tumor challenge, mice were administered with cisplatin (5 mg/kg) or PBS intraperitoneally, together with direct Ova peptide (20 μg) (aa241–270, SMLVLLPDEVSGLEQLESIINFEKLTEWTS) injection into the tumor. Tumor growth was monitored by palpation and visual inspection twice per week.
Quantification of antigen-specific T cells
PBMCs were collected 1 week after the last drug/peptide injection. Erythrocytes were lysed in ammonium chloride-potassium bicarbonate buffer, and leukocytes were pulsed ex vivo with relevant peptide (1 μg/ml) (e.g., E7 aa49–57, Ova aa258–265, or AH1 aa6–14) overnight in the presence of Brefeldin A (BD Biosciences). Cells were stained with PE-labeled α-CD8 mAb (BD Biosciences), fixed and permeabilized with Cytofix/Cytoperm reagent (BD Biosciences), and then stained with FITC-labeled anti-IFN-γ mAb (BD Biosciences). The frequency of IFN-γ+ CLTs was examined by flow cytometry via FACSCalibur device (BD Biosciences), as previously described [28]. For tetramer binding analysis, PBMCs were co-stained with FITC-labeled anti-CD8 mAb (BD Biosciences) and PE-labeled H-2Db tetramer loaded with HPV-16 E7 epitope (aa49–57; RAHYNIVTF) (Beckman Coulter, Hialeah, FL), and then examined by flow cytometry. For analysis of tumor-infiltrating E7-specific CTLs, tumor tissue was excised from tumor-bearing mice, minced, and passed through a 100 μm strainer. Single cells were co-stained with FITC-labeled α-CD8 mAb and PE-labeled E7-Db tetramer and examined by flow cytometry. All data analysis was performed on gated lymphocyte populations (as defined by FSC/SSC features) using FlowJo software (Tree Star, Ashland, OR).
Analysis of APCs
To monitor the effects of cisplatin on infiltration of APCs into the tumor, 105 TC-1 cells were inoculated subcutaneously into wildtype or TLR9−/− C57BL/6 mice (5 per group). On days 5 and 8 following tumor challenge, mice were administered intraperitoneally with cisplatin (5 mg/kg) or PBS control. 24 h after the final drug injection, tumor tissue was excised. To process excised tumor tissue into single cells, excised tumor tissues were minced and washed 2 times with PBS and then digested with dispase (500 U/ml) (Godo Shusei, Tokyo, Japan) at 37 °C for 20 min. Fragments were centrifuged at 150×g for 5 min; the supernatant was then discarded, and the pellet was resuspended in 5 ml of PBS and homogenized to single cells. The cells were then passed through a 100 μM mesh stainless wire sieve and washed 2 times with 20 ml of PBS. Cells were then resuspended in PBS and stained with APC-labeled anti-CD11c mAb (BD Pharmingen, San Diego, CA). To detect maturation of APCs, cells were co-stained with FITC-labeled anti-CD40, CD80, or CD86 mAb (BD Pharmingen) and then examined by flow cytometry.
To detect migration of antigen-loaded APCs into lymph nodes, TC-1-bearing wildtype or TLR9−/− mice were treated with cisplatin intraperitoneally, FITC-labeled E7 antigen intratumorally, and/or DNase I intravenously as described in the tumor treatment experiment section. 2 days after the last treatment administration, draining lymph nodes were harvested and homogenized in RPMI-1640 medium in nylon mesh bags. Erythrocytes were lysed with ammonium chloride and washed twice with RPMI-1640 medium. Cells were stained with APC-labeled anti-CD11c mAb, and the frequency of FITC+ CD11c+ cells was examined by flow cytometry.
Statistical analysis
All data presented in this study are expressed as mean ± SD and are representative of 3 independent experiments performed. At least 3 samples per group were included in each of these experiments. Flow cytometry data and results of tumor treatment experiments were evaluated by analysis of variance (ANOVA) and the Tukey-Kramer test. Individual data points were compared by Student’s t-test. Event-time distributions for mice were compared by the Kaplan-Meier method and the log-rank test. P values < 0.05 were considered significant.