The HPV16-associated cervical cancer cell line, CaSki, and a HPV-negative epithelial cell line, PC3, were obtained from the China Center for Type Culture Collection (Wuhan, China). Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% newborn calf serum (NBCS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (North China Pharmaceutical Co., Ltd., Hebei, China) at 37°C in 5% CO₂. Purified plasmids MSCVPIG (Addgene, Cambridge, MA, USA) and MSCVPIG-sPD-1 (constructed in the lab) were stably transfected into PC3 cells (1×105/ml) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 2 weeks, positive clones were selected based on their resistance to G418 (800 mg/ml; North China Pharmaceutical Co., Ltd.), which were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and western blot analysis.

Stable transfection clones were seeded into 96-well plates (1×104 cells/well) and cultured in RPMI-1640 medium supplemented with 10% NBCS and 1% penicillin/streptomycin at 37°C and 5% CO₂. MTT reagent (5 mg/ml; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was added to the cells at different time-points (12, 24, 48, 72 and 96 h) and were incubated for 4 h. After discarding the supernatant, 100 µl dimethyl sulfoxide was added to each well and the plates were agitated at 37°C for 15 min. The absorbance of samples was recorded at 490 nm using a Multiskan Spectrum (Thermo Fisher Scientific, Inc.).

PC3 cells (0.5–1×106) were collected, washed with 1X phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde at 4°C for 30 min, and then incubated with 0.1% Triton X-100 (Sigma-Aldrich; Merck Millipore), containing 2% bovine serum albumin (BSA Sigma-Aldrich; Merck Millipore) at room temperature for 15 min. After blocking with 2% BSA at 37°C for 30 min, the cells were incubated with mouse anti-HPV16E7 (cat no. sc-6981) or rabbit anti-human PD-L1 (cat no. sc-50298) primary antibodies (dilution 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 30 min at 37°C, and then incubated with either fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse (cat. no. 715–005-151) or goat anti-rabbit (cat. no. 111-095-003) secondary antibodies (dilution 1:3,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 30 min at room temperature. Cells were resuspended in 500 µl 1X PBS and analyzed by fluorescence-activated cell sorting to determine the expression levels of E7 and PD-L1. The same protocol was used, with the exception of the use of an isotype control (cat. no. sc-2025 or sc-2027; Santa Cruz Biotechnology, Inc.) in place of the primary antibodies.

In order to perform cell cycle analysis, PC3 cells were collected, washed with 1X PBS and fixed with 70% ethanol for 1 h at 4°C. The cells were then treated with 0.1% Triton X-100 and 50 µg/ml RNase (Sigma-Aldrich; Merck Millipore), and labeled with 20 µg/ml propidium iodide (Sigma-Aldrich; Merck Millipore) for 30 min at room temperature. A total of 1×10⁴ cells were analyzed for each sample with flow cytometry using the Coulter EPICS flow cytometer (Beckman Coulter, Inc., Brea, CA, USA).

PC3 cells (1×106) were collected and the total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was synthesized using the SuperScript™ II reverse transcriptase (Invitrogen; Thermo Scientific, Inc.) at 65°C for 5 min, 42°C for 1 h and 70°C for 5 min, according to the manufacturer's instructions. PCR amplification was performed using specific oligonucleotide primers (Sangon Biotech Co., Ltd., Shanghai, China). β-actin was used as an endogenous control. HPV16E7 forward, 5′-GCTCAGAGGAGGAGGATGAAATAGA-3′ and reverse, 5′-CACAACCGAAGCGTAGAGTCAC-3′; PD-L1 forward, 5′-GCTATGGTGGTGCCGACTACA-3′ and reverse, 5′-CTTGATGGTCACTGCTTGTCCA-3′; β-actin forward, 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′ and reverse, 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. PCR reactions contained 25 ng cDNA template, 100 ng sense and antisense oligonucleotide primers, 2.5 µl Taq PCR buffer (Promega Corporation, Madison, WI, USA), 0.4 mM dNTP mixture, and 1U Taq polymerase (Promega Corporation, Madison, WI, USA) in a total reaction volume of 25 µl. Following an initial 3 min incubation step at 94°C, PCR was performed using 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and elongation at 72°C for 30 s. PCR products were separated by electrophoresis at 100 V for 40 min using a 2% agarose gel, and were detected by ethidium bromide staining.

CaSki cell (5×106) were collected and lyzed in 1X SDS sample buffer containing 5% β-mercaptoethanol (Sigma-Aldrich; Merck Millipore) at 4°C. The protein samples were denatured by heating at 95°C for 5 min, and 30 µg/well protein was separated in a 12% SDS-PAGE Bis-Tris gel (Invitrogen; Thermo Fisher Scientific, Inc.) in 1X MES SDS running buffer (Invitrogen; Thermo Fisher Scientific, Inc.), and then transferred to a polyvinylidene difluoride (PVDF) membrane (current, 250 mA; duration, 2 h). The PVDF membrane was blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBST; Sigma-Aldrich; Merck Millipore) at 4°C overnight. After rinsing with TBST, the membrane was incubated at room temperature for 2 h with a primary rabbit anti-human PD-1 antibody (cat no. sc-10295; Santa Cruz Biotechnology, Inc.) at 1:500 dilution in TBST and then washed three times with TBST. Proteins were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody at 1:3,000 dilution in TBST for 1 h at room temperature, before washing three times with TBST. The immunoreactive proteins were detected with a chemiluminescence substrate (Pierce; Thermo Fisher Scientific, Inc.), and the signals were captured on an X-ray film in a darkroom. The relative protein level in each sample presented in a bar graph was calculated based on the protein band density after normalizing to β-actin (cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) (1:1,000 dilution in TBST) for sample loading.

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of 20 healthy donors (Center Blood Bank of Yichang City, Yichang, China) by density gradient centrifugation (536 × g for 20 min) over Ficoll/Hypaque (Sigma-Aldrich; Merck Millipore), according to the manufacturer's instructions. PBMCs were resuspended to a final concentration of 1×106/ml in PBS with 0.1% BSA, before they were stained with 5 µmol/l 5-(−6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes; Thermo Fisher Scientific, Inc.) for 10 min at 37°C. The stain was quenched by incubating cells for 5 min in PBS containing 10% fetal bovine serum. Excess CFSE was removed by adding PBS and centrifuging (134 × g for 5 min). This wash-step was repeated twice. The CFSE-labeled PBMCs were co-cultured with stable transfection clone sPD1-PC3 cells at a ratio of 1:100. Following 2 days of culture, the PBMCs were harvested and washed three times, resuspended with 300 µl PBS, subjected to FACS analysis (Ex/Em=488/518 nm), and analyzed using CellQuest Pro software version 5.1 (BD Biosciences, San Diego, CA, USA) to determine the cell proliferation.

Using above PBMCs stimulated with tumor cells serving as effector cells, the target cells (1×104/well) were incubated at different effector-to-target ratios (1:1, 50:1 and 100:1). The following three control groups were established: PBMCs with 1% NP-40 (Sigma-Aldrich; Merck Millipore), denoted as the ‘maximum lytic group’; target cells, denoted as the ‘effector spontaneous release group’; and the remaining group containing phenolsulfonphthalein medium as the ‘blank control group’. All of the cells were incubated at 37°C for 6 h. Centrifuged supernatant (536 × g for 10 min; 50 µl) was collected to assess the quantity of lactate dehydrogenase by quantitative measurements using the Cytotox 96 Non-Radioactive Cytotoxicity assay kit (Promega Corporation) according to the manufacturer's instructions. The lytic activity of lymphocytes was assessed using the mean value from triplicate repeats according to the following formula: Percentage of specific lysis = (experimental release - spontaneous release)/(maximum release - spontaneous release ×100.

PC3 cells (1×105/ml) were transiently transfected with the PD-L1-firefly luciferase reporter gene generated at the Institute of Molecular Biology of China Three Gorges University (Yichang, China). Following 48 h of transfection, luciferase activity was measured using the Dual-Luciferase^® Reporter assay system (E1910, Promega Corporation) according to the manufacturer's instructions. Luminescence was read from the 96-well plate using an Infinite™ 200 luminometer (Tecan, Männedorf, Switzerland).

Cervical carcinoma (40 patients) and normal cervical tissue (8 individuals) samples were obtained from the Department of Obstetrics and Gynecology, the Second Affiliated Hospital of China Three Gorges University (Yichang, China) between March 2010 and May 2011. All patients did not accept any pretreatment for six months prior to the study. The study met the criteria of the Ethics Committee of The Second Affiliated Hospital of China Three Gorges University (Yichang, China). All cervical tissue specimens were fixed with 10% neutral formalin, embedded with paraffin, and serially-sectioned at 5 µm. The sections were mounted onto the histostick-coated slides. A total of 4–5 adjacent sections were collected for histopathological analysis and immunohistochemical staining. The clinical histopathological diagnosis of all cases was performed according to the WHO (2003) standard (12).

The biotin-streptavidin complex kit (Boshide Biological engineering Ltd., Wuhan, China) was used for the immunostaining of PD-L1 and HPV16E7. All of the following agents were part of the kit, except the primary antibodies. According to the manufacturer's protocol the procedures were briefly as follows: Following de-waxing, inactivation of endogenous peroxidase activity and inhibition of cross-reactivity using normal non-immune goat serum, the sections were incubated at 4°C overnight with a diluted solution of the primary antibodies (as aforementioned). Localization of the primary antibodies was achieved by subsequent application of a biotin-conjugated IgG secondary antibody, and a streptavidin-conjugated peroxidase. Signals were visualized with diaminobenzidine, and cellular nuclei were counterstained with instant hematoxylin. Negative controls were established by replacing the primary antibody with normal isotype control (the kit supplied). The quantity of positive staining was analysed as follows: Opening the image analysis system, defining the negative and positive standard, followed by scanning the samples. The relative brightness values were obtained using the Image-Pro Plus 6.0 software (Media Cybernetics, Washington DC, USA).

Data were analyzed using SPSS software (version 13.0; SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. Differences between two groups were analyzed with Student's t-test or chi-square test. P<0.05 and P<0.01 were considered to indicate statistically significant differences.

In order to explore the correlation between HPV16E7 and PD-L1 expression, the present study examined their mRNA and protein expression levels in clinically diagnosed cervical cancer tissues. Immunohistochemical analysis demonstrated that the normal cervical epithelium rarely expressed PD-L1, while the majority of cervical cancer samples exhibited strong expression of PD-L1 (P=0.017; Table I). HPV16E7 expression was detected using immunohistochemistry and RT-PCR analyses. The results demonstrated that HPV16E7 was expressed at low levels in normal tissues and at high levels in cervical cancer tissues, with HPV16E7 protein primarily localized to the nucleus and cytoplasm of the cervical cancer cells (Fig. 1). Statistical analysis of these data indicated that HPV16E7 and PD-L1 protein expression was positively correlated (r=0.531) in cervical cancer tissues and reached statistical significance (P=0.043).

The expression of HPV16E7 and PD-L1 demonstrated a positive correlation in cervical carcinoma tissues. In order to further explore this correlation, a PC3 cell line stably expressing HPV16E7 (PC3-E7) was established by transfecting the pcDNA3.1 (−)-E7 plasmid or an empty vector control (PC3-3.1), and the expression levels of PD-L1 were measured. PC3-E7 clones demonstrated the oncogenic characteristics of enhanced viability, a significant reduction in the number of cells at the G0/G1 phase, an accumulation of cells at S phase, and no obvious difference at G2/M phase when compared with the empty vector controls (Fig. 2A-C). Flow cytometry analysis and a luciferase reporter system were used to detect PD-L1 expression in order to investigate whether PD-L1 expression may be regulated by HPV16E7. The results demonstrated that PD-L1 was overexpressed in the PC3-E7 clones when compared with the empty vector control cells (Fig. 2D). The luciferase assay results demonstrated that fluorescence values in the PC3-E7 cells were significantly higher than the control cells (P=0.022; Fig. 2E). These results suggest that HPV16E7 promoted PD-L1 expression in PC3-E7 cells. PD-L1 expressed on the surface of tumor cells inhibits lymphocyte activity through binding to PD-1 on lymphocytes (13). Consistent with these observations, the proliferation rate of lymphocytes was significantly reduced following co-culture with PC3-E7 cells compared with PC3-3.1 cells (Fig. 2F and G).

In order to further investigate the correlation between HPV16E7 and PD-L1, HPV16E7 was knocked down using RNA interference in the CaSki cell line, which had undergone integration of HPV16 genes, and PD-L1 expression levels were detected. A pSilencer™ 2.1-U6-E7siRNA plasmid was constructed and stably transfected into CaSki cells. Positive clones were identified by flow cytometry and denoted as CaSki-E7si cells. Empty vector control-transfected cells were denoted as CaSki-si-ctl. Suppression of HPV16E7 expression in CaSki-E7si cells was confirmed by flow cytometry analysis (Fig. 3A). The viability of CaSki-E7si cells was significantly suppressed compared with the controls [P=0.041 (48 h); P=0.028 (72 h); P=0.008 (96 h); Fig. 3B]. Flow cytometry analysis of cell cycle was conducted subsequent to E7 siRNA treatment. It was demonstrated that the cell cycle distribution was altered in the CaSki-E7si cells. The percentage of cells in the sub-G1 phase increased from 2.1% in the untreated control to 33.6% following E7 siRNA expression (P=0.018; Fig. 3C). Both flow cytometry and luciferase reporter system analysis demonstrated that PD-L1 expression was significantly reduced in CaSki-E7si cells (P=0.041; Fig. 3D; P=0.027; Fig. 3E). Following co-culture with lymphocytes, CaSki-E7si cells significantly stimulated lymphocyte proliferation when compared with CaSki-si-ctl cells (P=0.039 Fig. 3F and P=0.035; Fig. 3G). According to the results presented in the current study, HPV16E7 overexpression in cervical cancer may lead to PD-L1 upregulation and mediate the escape of tumors from the immune system.

Due to the observation that HPV16E7 suppressed lymphocyte proliferation and cytotoxic activity potentially via upregulating PD-L1 expression in cervical cancer, the authors hypothesized that inhibition of the PD-L1/PD-1 signaling pathway may improve immunotherapy in cervical cancer. PD-1 expressed on lymphocytes binds to PD-L1 on tumor cells and mediates negative regulation of the immune response (13). Therefore, the recombinant plasmid sPD-1 was constructed and stably transfected into CaSki cells. Empty vector controls were denoted as CaSki-PIG. CaSki-sPD-1 cells were determined to express sPD-1 by RT-PCR and western blotting analyses (Fig. 4A). Following co-culture of lymphocytes with CaSki-sPD-1 cells, lymphocyte proliferation was significantly elevated when compared with CaSki-PIG cells (P=0.038; Fig. 4B). The activated lymphocytes exhibited significantly enhanced cytotoxic activity when they were co-cultured with CaSki-sPD-1 cells compared with CaSki-PIG cells (P=0.042; Fig. 4C). These results demonstrated that inhibition of the PD-L1/PD-1 signaling pathway may partially restore the T cell response and enhance antitumor immunity.