Female BALB/c mice (6–8 weeks old) were purchased from the Center of Medical Experimental Animals of Xuzhou Medical College (Xuzhou, China). The animals were maintained under pathogen-free conditions. The mouse protocol was approved by the Animal Care and Use Committee of Xuzhou Medical College. The mouse H22 hepatocarcinoma and 293T cell lines were purchased from the Institute of Oncology (Beijing, China). The mouse H22 cell line was maintained by intraperitoneal (i.p.) passage in BALB/c mice.

The murine sPD-1 expression plasmid vector psPD-1 has been described previously (27). Briefly, the cDNA of the PD-1 extracellular domain was obtained from the whole cDNA of murine splenocytes by reverse transcription-PCR (RT-PCR). The sequences of the PCR primers specific to sPD-1 are as follows: sense 5′-GGTTCATAGAATTCCTGA AGGCGACACTGCC-3′, (underlined nucleotides indicate EcoR I site) containing a restriction site for endonuclease EcoRI, and antisense 5′-CCTGGTAAGCTTATTGAAA CCGGCCTTCTGG-3′ (underlined nucleotides indicate HindIII. site) containing a restriction site for endonuclease HindIII. The plasmid vector psPD-1 was constructed by insertion of the purified sPD-1 cDNA into plasmid pcDNA3.1 and then sequenced by Shenggong Biotechnology Co. (Shanghai, China). Plasmid pcDNA3.1 was a gift from Professor Chenzhi (Institute of Infectious Disease, Zhejiang University, China). The murine IL-21 expression plasmid vector pmIL-21 was provided by Professor Doujun (Department of Microbiology and Immunology, Southeast University, China).

In the in vitro culture, the transfection of psPD-1 or pmIL-21 into 293T cells was performed using Lipofectamine 2000 liposomes (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Stably transfected clones were selected using G418 (500 μg/ml).

In the in vivo animal studies, the transfection was performed by direct local injection. Briefly, BALB/c mice received an intramuscular (i.m.) injection of 100 μg naked plasmid in 100 μl saline at the inoculation site. The plasmids were injected every 3 days.

The BALB/c mice (8 per group) received a subcutaneous injection of 1×10⁵ H22 cells in the right hind limb to establish the hepatoma model. Day 1 post H22 cell inoculation, 100 μg plasmid DNA was injected directly into the muscle at the inoculation site in all treatment groups. In the combination treatment group, the mice received 50 μg pmIL-21 and 50 μg psPD-1 by i.m. injection. The plasmid or saline was injected every 3 days. The mice in the control groups received an equal amount of pcDNA3.1 or equal volume of saline. The mice were sacrificed on day 28 after injection of the H22 cells.

The sizes of the implanted tumors were measured every week with a ruler. The tumor volume was calculated on days 7, 14, 21 and 28 using the formula: V=1/2(a×b²) (V, tumor volume; a, length, b, width). TGI was calculated using the formula: (1−T/C) × 100% (T, tumor volume of the treated group; C, tumor volume of the control group).

Total RNA was obtained from the tumor marginal tissues of the tumor-bearing mice using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The relative quantities of the mRNAs of IL-2, IFN-γ and IL-10 were determined by RT-PCR using a Two Step RT-PCR kit (Tiangen Biotech Co., Ltd., Beijing, China); 30 PCR cycles were used for each sample and β-actin was used as the matched control. The primer sequences were as follows: IL-2 sense, 5′-ACCTTGCTAATCACTCC-3′, antisense, 5′-AAGTCCACC ACAGTTGCT-3′; IFN-γ sense, 5′-ATTGGCATAGATGTG GAA-3′, antisense, 5′-TCAAACTTGGCAATACTC-3′; IL-10 sense, 5′-ACCTGGTAGAAGTGATGC-3′, antisense, 5′-AAG GAGTTGTTTCCGTTA-3′; and β-actin sense, 5′-AGCGAG CATCCCCCAAAGTT-3′, antisense, 5′-GGGCACGAAGGC TCATCATT-3′.

The IL-21- and sPD-1-expressing 293T clones (2.0×10⁶/well) were cultured in 24-well plates for 24 h. The supernatants of the cultures were collected for IL-21 and sPD-1 detection. Murine IL-21 and sPD-1 ELISA kits (R&D Systems, Minneapolis, MN, USA) were used to identify the expression of IL-21 and sPD-1 proteins, respectively.

Mouse blood serum was collected on day 28 after injection of the H22 cells. We used murine IL-21, sPD-1, IFN-γ, IL-2 and IL-10 ELISA kits (R&D Systems) to assess the levels of IL-21, sPD-1, IFN-γ, IL-2 and IL-10 in murine serum.

Muscle tissues, isolated 72 h after the i.m. injection of psPD-1 or pmIL-21, were incubated with lysis buffer and a protease inhibitor cocktail (EMD Biosciences, Inc., San Diego, CA, USA) at 4°C for 20 min. Western blot analyses were performed using standard techniques. Protein levels were quantitated using a Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Total protein (30 μg per lane) was run on 12% SDS-PAGE gel and transferred to a PVDF membrane. After blocking and washing, the membranes were incubated with antibodies against either IL-21 or sPD-1 in TBS-5% milk overnight at 4°C and then incubated with the appropriate secondary antibody. The proteins were detected using an enhanced chemiluminescence ECL kit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The expression of IL-21 or sPD-1 protein in the tumor tissues of the tumor-bearing mice treated with the sPD-1 and/or IL-21 gene were also detected as described above. Anti-mouse PD-1 polyclonal antibody was purchased from BioLend (USA) and anti-mouse IL-21 polyclonal antibody was purchased from ReliaTech GmbH (Wolfenbüttel, Germany).

A lactate dehydrogenase (LDH) release assay was performed to determine the cytotoxicity of the mouse splenocytes. The splenocytes were co-cultured with irradiated H22 cells for 7 days in the presence of 20 U/ml rIL-2 and then used as effector cells for the cytotoxicity assay. The H22 target cells were plated at 5×10³ cells/well in 96-well round-bottom plates and co-cultured with effector cells at various effector/target (E:T) ratios for 4 h at 37°C. After incubation, cytotoxic activity was detected by LDH release using the CytoTox 96 Nonradioactive Cytotoxicity assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The rate of specific target cell lysis was calculated using the following formula: [(Sample release -spontaneous release)/(Total release-spontaneous release)] × 100.

Flow cytometry was used to detect the numbers of CD8⁺ T cells and NK cells in the spleen. A single-cell suspension of splenocytes was prepared from the spleens of the tumor-bearing mice. The cells were then stained with fluorescein isothiocyanate (FITC)-labeled anti-CD8, phycoerythrin (PE)-Cy5 anti-CD3 and PE-anti-NK1.1. These fluorochromes were detected using a flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using CellQuest software. All antibodies used were purchased from BioLend (San Diego, CA, USA).

The mice were sacrificed 28 days after injection of the H22 cells. The tumors were surgically excised and fixed in 10% formalin. The formalin-fixed tissues were embedded in paraffin and then sectioned for H&E staining. The slides were viewed with a microscope at a magnification of ×400.

For descriptive statistics, values are expressed as the mean ± standard deviation. The statistical significance of differences between groups was assessed using the Student’s t-test. P<0.05 were considered to indicate a statistically significant difference. Statistical analysis was performed using the GraphPad Prism 4.0 statistical software package (GraphPad Software, Inc., La Jolla, CA, USA).

Following the transfection of the recombinant plasmids into 293T cells and the establishment of stably IL-21- and sPD-1-expressing 293T cells, an ELISA was performed to detect the secretion of IL-21 and sPD-1 proteins by the transfected cells. We successfully detected high expression levels of IL-21 (839.98±56.38 pg/ml) and sPD-1 (764.64±61.25 pg/ml) proteins in the culture supernatants. We also examined the expression of IL-21 and sPD-1 proteins in the peripheral blood of the tumor-bearing mice. The levels of IL-21 protein were higher in the mice treated with the IL-21 gene alone (357.54±60.36 pg/ml) or in combination with sPD-1 (238±53.44 pg/ml) than those in the mice treated with the sPD-1 gene (98.37±27.64 pg/ml) or plasmid pcDNA3.1 (82.15±19.87 pg/ml), and the levels of sPD-1 protein were significantly elevated in the mice treated with the sPD-1 gene alone (369.53±97.37 pg/ml) or in combination with the IL-21 gene (217.38±65.64 pg/ml) than those in the mice treated treated with the IL-21 gene (57.86±21.30 pg/ml) or plasmid pcDNA3.1 (49.31±17.25 pg/ml). Later, we examined the expression of IL-21 and sPD-1 in vivo following the injection of naked recombinant plasmid DNA. As shown in Fig. 1, the IL-21 and sPD-1 proteins were detected in the injected muscle tissues or tumor tissues by western blot analysis.

We measured the length and width of the implanted tumors and calculated the tumor volume on days 7, 14, 21 and 28 after the injection of the H22 hepatoma cells into the BALB/c mice. As shown in Fig. 2, treatment with the IL-21 gene alone significantly inhibited tumor growth at 14, 21 and 28 days after H22 cell injection, compared with tumor growth in mice receiving sPD-1 gene therapy or naked plasmid pcDNA3.1 (P<0.05). However, the combination of IL-21 and sPD-1 treatment showed the most potent suppression of tumor growth with a TGI of ≥70%. The sPD-1 treatment also resulted in a slight inhibition of tumor growth with a TGI of ≤20%. Therefore, the suppression of tumor growth by combined IL-21 and sPD-1 treatment was much stronger than that by IL-21 treatment alone (P<0.05). These data suggest that sPD-1 enhanced the IL-21-mediated antitumor responses.

We next examined the CTL cytotoxic activities of splenocytes from the tumor-bearing mice injected with pmIL-21 alone or with a combination of pmIL-21 and psPD-1 on day 28 after tumor inoculation. Fig. 3 shows that the cytotoxic activities of the splenocytes to H22 tumor cells were significantly enhanced in the pmIL-21 group and in the psPD-1 group compared with the control vector pcDNA3 treatment group (P<0.01). However, the cytotoxicity of the splenocytes was further enhanced in the IL-21/sPD-1 combination group, compared with the IL-21 and sPD-1 gene single treatment groups (P<0.01). Thus, IL-21 and sPD-1 each mediate the cytotoxic function of splenocytes, and a combination of IL-21 and sPD-1 showed synergistic antitumor CTL cytotoxicity.

To further study the antitumor immunity trigged by pmIL-21 gene therapy, we counted the numbers of CD8⁺ T cells and NK cells in the splenocytes of the tumor-bearing mice. Fig. 4 demonstrates that the percentages of CD8⁺ T cells and NK cells were significantly increased in the pmIL-21/ psPD-1 combination group compared with the single-gene (IL-21 or sPD-1) treated groups (P<0.05). Treatment with IL-21 gene alone also resulted in significantly increased quantities of CD8⁺ T cells and NK cells compared with the control group (P<0.05). Moreover, the percentage of CD8⁺ T cells in the combined treatment group was higher than that in the IL-21 treatment group (P<0.05), which supports the synergistic efficacy of the sPD-1 and IL-21 treatments.

Cytokines are important in immune responses. To assess the expression of cytokines associated with the antitumor immunity induced by IL-21 alone or in combination with sPD-1 treatment in tumor-bearing mice, we examined not only the serum levels of IL-2, IFN-γ and IL-10 by ELISA but also the transcription activities of IL-2, IFN-γ and IL-10 genes in tumor marginal tissues by RT-PCR. We found that in the combination treatment group the expression level of IFN-γ mRNA was markedly upregulated, but the expression level of IL-10 mRNA was significantly decreased compared with those in the single-gene treatment and control groups (P<0.05; Fig. 5). The changes of IL-2 and IFN-γ expression in the IL-21 treatment group showed similar trends to those in the control group. This was confirmed by ELISA detection of the cytokine levels in serum (Fig. 5). The sPD-1 blockade of PD-1 resulted in slight increases of IL-2 or IFN-γ expression levels but a significant decrease in IL-10 expression level relative to those in the control group (P<0.05).

To further investigate the antitumor effects of IL-21 and sPD1 treatment, tumor tissues were fixed and stained. Fig. 6A and B reveals that, consistent with the increased number of tumor-infiltrating immunocytes, increased numbers of necrotic or apoptotic tumor cells were found in the tumor sites derived from the tumor-bearing mice challenged with pmIL-21 alone or in combination with psPD-1. By contrast, tumor cells showed active growth and evident nucleic division in tumor sites derived from the tumor-bearing mice challenged with psPD-1 or pcDNA3.1 (Fig. 6C and D). These findings suggest that IL-21 alone or combination with sPD-1 is able to trigger the cellular immune response to the H22 hepatocellular carcinoma.