The B16 melanoma cell line was maintained in our laboratory at the Chinese PLA General Hospital (Beijing, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Thermo Trace Ltd, Melbourne, Australia) at 37°C in an atmosphere of 95% air and 5% CO₂.

Male 6–8-week-old C57BL/6 mice were purchased from Vital River Biotechnology Co., Ltd. (Beijing, China). Animals were maintained in micro-isolator cages in specific pathogen-free conditions. They were handled under aseptic conditions following a protocol approved by the Institutional Animal Care and Use Committee of the Chinese PLA General Hospital (Beijing, China). All studies were approved by the Animal Study Committee of the Chinese PLA General Hospital.

The mice were randomly divided into three groups. Each group contained more than six mice. Animals were injected intraperitoneally twice on days 1 and 15, with 50 μg B16 cell lysate antigen or 50 μg B16 cell lysate plus 50 μg poly I:C, 1.5% Al(OH)₃ or with PBS. After the final immunization, the C57BL/6 mice were inoculated intraperitoneally with 1×10⁵ melanoma cells suspended in 100 μl PBS.

B16 cells were collected and washed three times with phosphate-buffered saline (PBS) buffer. Eight snap freeze-thaw cycles between liquid N₂ and 37°C were conducted. The cells were centrifuged at 500 × g to obtain the lysate, which was then filtered with a 70-mm Falcon filter (BD Biosciences, Erembodegen, Belgium). Coomassie blue staining method was used to measure the amount of protein and was performed according to the manufacturer’s instructions (Benda Biotechnology, Co., Shanghai, China). The lysate was separated and kept frozen in liquid N₂ until required.

Cell proliferation levels were determined using the MTT assay (21). One week after the final immunization, single-cell suspensions from the mice in each group were prepared under sterile conditions. Red blood cells (RBCs) were lysed using lysis buffer containing 0.75% NH₄Cl in Tris-buffer. Cell concentrations were adjusted to 3×10⁶ cells/ml in DMEM supplemented with 10% FBS. Samples (100 μl) of the suspensions were dispensed into 96-well round-bottom culture plates (Costar, Tewksbury, MA, USA) and incubated with 10 μg/ml B16 melanoma cell lysate for 48 h at 37°C in a 5% CO₂ humid incubator.

Splenocytes from the immunized mice were cultured in six-well plates for 24 h at 37°C in the presence of 5% CO₂, with or without 10 μg/ml B16 cell lysate. Total RNA was extracted with an RNApure kit (Bioteke, Beijing, China) and retrotranscribed with murine leukemia virus (MLV) reverse transcriptase (RT) (Invitrogen Life Technologies). PCR amplifications were performed using a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) and each sample was tested in triplicate. Thermal cycling conditions were 40 cycles of 12 sec at 95°C and 1 min at 60°C using SYBR-Green (Invitrogen Life Technologies). β-actin was used as the internal reference gene. The primers used were as follows: Interferon-γ (IFN-γ), 5′-CAGCAACAGCAAGGCGAAA-3′ and 5′-CTGGACCTGTGGGTTGTTGAC-3′; β-actin, 5′-AGAGGGAAATCGTGCGTGAC-3′ and 5′-CAATAGTGATGACCTGGCCGT-3′.

Seven days after the final immunization, single cell suspensions were performed for spleen T-cell subtype analysis. RBCs from 50 μl heparin-treated orbital blood were lysed with RBC lysis buffer (eBioscience, San Diego, CA, USA). Lymphocytes were stained with 100 μl PBS plus 1% bovine serum albumin and 0.1% NaN₃ together with 5 μl fluorescein isothiocyanate-conjugated anti-CD3 monoclonal antibody (mAb) followed by simultaneous staining with 5 μl phycoerythrin anti-CD4 or anti-CD8 mAb, and then incubated for 20 min at 4°C. Flow cytometry was performed using CellQuest software and a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA), with FlowJo software (Tree Star Inc., Ashland, OR, USA) used for data analysis.

Survival curves of the animals treated with different protocols were plotted according to the Kaplan-Meier method. Statistical significance in different treatment groups was compared using the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

In order to detect whether the splenocytes of the mice immunized with B16 cell lysates plus poly I:C had an elevated antigen-specific proliferation rate, the cell numbers were measured by MTT assay. Ten days after the final immunization, spleens from mice in each group were removed and the rate of splenocyte proliferation was measured. After in vitro stimulation with B16 lysates for 24 h, the splenocytes from the mice immunized with B16 lysates in combination with poly I:C were significantly more numerous than those in either control group (P<0.01; Fig. 1). The cells treated with Concanavalin A (ConA) were used as positive controls and untreated cells were used as negative controls.

T-cell subsets were also analyzed. The percentages of T helper cells (CD4⁺CD3⁺ T lymphocytes) and cytotoxic T cells (CD8⁺CD3⁺ T lymphocytes) were determined by flow cytometry. As shown in Fig. 2, the mice immunized with B16 cell lysate plus poly I:C contained a higher percentage of CD3⁺CD4⁺ T lymphocytes in their peripheral blood than that of the mice injected with PBS. In addition, the frequency of CD8⁺CD3⁺ T lymphocytes in the peripheral blood appeared to increase in the mice immunized by B16 cell lysates plus poly I:C compared with that of the mice injected with PBS (Fig. 3).

C57BL/6 mice were immunized twice with B16 cell lysates in combination with poly I:C, the antigen alone, or PBS. Ten days after the final immunization, spleens were removed and splenocytes of single cell suspension were prepared. In order to compare the cell-mediated immune responses among the three groups, splenocytes from the immunized mice were in vitro-stimulated with B16 antigen for 24 h and IFN-γ mRNA levels were analyzed using qRT-PCR (amplification curve shown in Fig. 4A). The mean relative IFN-γ mRNA expression in the mice immunized with B16 cell lysate plus poly I:C was significantly higher than that in the mice immunized with the B16 cell lysate only (P<0.01; Fig. 4B). IFN-γ protein levels were also examined by ELISA. As expected, the mean IFN-γ production in the mice immunized with B16 cell lysate was higher than that in the antigen-immunized group following in vitro stimulation with B16 cell lysates (Fig. 4C).

To demonstrate the cytotoxic activity of splenocytes from the immunized mice for B16 cells, the release of cytosolic lactate dehydrogenase (LDH) into the culture medium by a damaged B16 melanoma cell line was tested. Ten days after the final immunization, damage to the membranes of B16 melanoma cells was evaluated in a 24 h cytotoxicity assay by measuring LDH release. LDH release assays were performed with splenocytes as effector cells and B16 melanoma cells as target cells. The effector-to-target cell ratios were 5:1, 10:1 and 20:1. As shown in Fig. 5, the CTL response was significantly higher in the mice immunized with B16 lysate plus poly I:C than that in those immunized with B16 lysate alone (P<0.01) or PBS (P<0.01). B16 melanoma cells and splenocytes from the immunized mice did not release any LDH when measured at 24 h. These were cultured alone in DMEM medium and were used as negative controls.

To assess in vivo antitumor responses in the immunized mice, the survival rates were evaluated in the immunized groups. Following the final immunization, all the mice received an intraperitoneal challenge of 1×10⁵ B16 melanoma cells. The results revealed that subcutaneous immunization of C57BL/6 mice with B16 cell lysate plus poly I:C conferred improved protection against B16 melanoma cells than did immunization with B16 cell lysate or PBS alone. The survival rate of the B16 plus poly I:C group was significantly higher than that of the B16 lysate group and PBS group (P=0.029 vs. B16 lysate group, P=0.003 vs. PBS group), as shown in Fig. 6. Also the antitumor effects in the mice immunized by B16 antigen plus poly I:C were significantly greater than those in mice immunized by B16 antigen plus Al₂(OH)₃, which is used as the positive adjuvant in market.