Patients and tissue samples
One hundred thirteen peripheral blood samples were collected, including 42 from patients with cervical cancer, 35 from patients with high-grade squamous intraepithelial lesion (HSIL), and 36 from subjects with a normal cervix. In addition, we collected 11 pairs of cervical cancer and adjacent tissues. All patients with cervical cancer included in this study underwent primary surgery. All samples were collected from the Qilu Hospital of Shandong University. The study was approved by Qilu Hospital's ethics committee.
Cell isolation
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll (TBD Science, Tianjin, China) density gradient centrifugation. We obtained single cell suspensions from fresh tumour tissue. According to the manufacturer’s instructions, a gentle MACS C tube (Milteny Biotec, Bergisch Gladbach, Germany) was used for mechanical dissociation and a tumour dissociation kit (Milteny Biotec) was used for enzymatic hydrolysis. The digested cells were filtered through a 70 µm mesh, centrifuged with Ficoll (Solarbio, Beijing), and the monocytes were resuspended in RPMI 1640.
Cell culture
The mouse cervical cancer cell line U14 was obtained from the Academy of Medical Sciences (Beijing, China). U14 cells were cultured in DMEM nmented with 10% foetal bovine serum (all from Gibco, Grand Island, NY, USA), 50 U/mL penicillin and 50 mg/mL streptomycin (all from Solarbio Science & Technology, Beijing, China). CD8+ T cells were purified from PBMCs through positive selection using a kit (Milteny Biotec, Bergisch Gladbach, Germany). CD8+ T cells were stimulated with an anti-CD3/CD28 antibody (Stemcell, Canada) in T cell expansion medium (Stemcell, Canada). Activated CD8+ T cells were treated with 5 μg/mL CD155-Fc (R&D Systems), or 10 μg/mL CD155-Fc. Activated CD8+ T cells were cocultured with tumour cells at a 10:1 ratio. Next, 10 μg/ml anti-PD-1 mAb or 5 μg/ml anti-TIGIT mAb (R&D Systems) were added to the cells. We used α-human IgG1 (R&D Systems) as an isotype control. After 48 h, CD8+ T cells were collected to determine cytokine production using the T cell function assay.
Flow cytometry
PBMCs isolated from patients with cervical cancer or normal people were stained with fluorochrome-conjugated PE-conjugated-anti-CD8 (Elabscence, Wuhan, China) and PE-conjugated-anti-TIGIT-FITC (eBioscience) antibodies for 30 min at 4 °C. The samples were collected and flow cytometry was used for detection. For intracellular staining, cells stained with antibodies against cell surface markers were fixed and permeabilized with a fixation and permeabilization kit (BD Bioscience) for 20 min, and then treated with the fluorochrome-conjugated antibodies APC-conjugated-anti-TNF-α (eBioscience), and APC-conjugated-anti-IFN-γ (eBioscience), APC-conjugated-anti-GranzymeB (eBioscience) for intracellular staining at 4 °C for 30 min. Finally, the stained cells were analysed using a FACS Calibur flow cytometer (Becton Dickinson, USA), and the data were analysed using Flow Jo software.
Immunohistochemistry (IHC)
For immunohistochemical analysis, the sections were deparaffinized, and then citric acid buffer was used for heat-mediated antigen retrieval. For testing, we followed the manufacturer's instructions and use an immunohistochemistry detection kit (Zhongshan Jinqiao, Beijing, China). Sections were incubated at 4 °C overnight with primary antibodies in PBS (anti-human TIGIT, 1:100, Cell Signaling Technology, Danvers, MA; anti-human CD155, 1:100, Cell Signaling Technology Danvers, MA; anti-human CD8, Abcam, Cambridge, UK; anti-mouse CD8, 1:200, Cell Signaling Technology Danvers, MA). Sections were then incubated with a biotin-labelled goat anti-rabbit IgG secondary antibody for 10 min at 37 °C. Streptavidin-conjugated peroxidase was incubated with sections for 15 min at 37 °C before staining with DAB (Zhongshan Jinqiao, Beijing, China). Meyer's haematoxylin (Solarbio Science & Technology, Beijing, China) was used to stain sections for 5 min. The slices were sealed with neutral resin after they were dehydrated.
Multiplex immunohistochemistry (mIHC)
For the immunofluorescence analysis, we used multiple fluorescence immunohistochemical staining kits (Absin, Shanghai, China). Heat-mediated antigen retrieval and primary antibody incubation were performed using the same procedures as those described for immunohistochemistry. After an incubation with the secondary antibody for 10 min, the sections were incubated with the fluorescent staining amplification solution for 10 min at 37 °C. After three washes with TBST, sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Finally, an anti-fluorescence quenching agent was used to seal the slides.
Real-time quantitative RT-PCR (qRT-PCR)
TRIzol reagent (Invitrogen) was used to extract total RNA from cells. After total RNA was quantified using spectrophotometry, reverse transcription was performed using the PrimeScript RT kit (Accurate Biology, Hunan, China). Real-time PCR was performed using SYBR Premix Ex Taq (Accurate Biology, Hunan, China) and a 7900HT fast real-time PCR system (Applied Biosystems, Waltham, MA, USA). The primer sequences for the TIGIT, PD-1, LAG3, Tim3 and β-actin genes are shown in the Additional file 1: Table S1. The mRNA level of a specific gene was normalized to β-actin.
Western blot
After washing the cells three times with PBS, they were lysed on ice in radioimmunoprecipitation analysis buffer (RIPA; Beyotime, China Institute of Biotechnology, 1% phenylmethylsulfonyl fluoride (PMSF), and 1% NaF for 30 min. The samples were centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected. Next, the proteins were separated on SDS–PAGE gels and transferred to a PVDF membrane (Merck Millipore, Burlington, Massachusetts, USA). The membrane was incubated with primary antibodies against β-actin (1:1000, Cell Signaling Technology), TIGIT (1:1000, Cell Signaling Technology), SHIP-1 (1:1000, Cell Signaling Technology), ERK (1:1000, Cell Signaling Technology), p-ERK (1:1000, Cell Signaling Technology), p-IκBα (1:1000, Cell Signaling Technology), p-NF-κBP65 (1:1000, Cell Signaling Technology) overnight at 4 °C, and then incubated with the appropriate secondary antibody. Image J software (National Institutes of Health) was used to analyse relative protein levels, and β-actin was used as an endogenous control.
Immunoprecipitation
The cells were placed in lysis buffer (Beyotime Biotechnology, China), lysed on ice for 30 min, and centrifuged at 15,000 rpm for 15 min at 4 °C. Ten milligrams of antibody were incubated with 1000 mg of protein supernatant at 4 °C overnight. The supernatant was collected and incubated with Protein A/G Sepharose beads (Santa Cruz, USA) for 6 h. The beads were washed three times and boiled before the immunoprecipitated protein was detected using western blotting.
Cas9-sgRNA knockout
Cas9 and single-guide RNA (sgRNA) lentiviruses were designed and constructed by OBiO Technology Company (Shanghai, China). A lentivirus containing Cas9 and sgRNA (sg-scramble, sg-CD155) was introduced into U14 cells. The sgRNA sequence used was ATTCGACAGGCGTCTTGGGAGGG. After 48 h, the transfected cells were selected in a medium containing puromycin. Compared with the control group, the silencing efficiency in U14 cells was approximately 99%.
In vivo treatments
Female C57BL6 mice (18–22 g, 4–6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. for this study. U14 cells were trypsinized, resuspended in PBS, and 200 ml (1 × 107 cells) of the cell suspension were subcutaneously injected into the right armpit of each mouse. For in vivo blockade, 3 days after the injection of the cell suspension, mice were randomly allocated to the anti-PD-1 mAb (100 μg, clone BE0188, BioXcell, West Lebanon, USA), anti-TIGIT mAb (100 μg, clone 1G9, BioXcell, West Lebanon, USA), anti-TIGIT mAb + anti-PD-1 mAb and IgG (mouse IgG1, clone MOPC-21, BioXcell) groups. Intraperitoneal injections of the blocking antibody and isotype control were administered three times a week. Mice were subcutaneously inoculated with 1 × 106 U14-NC-CD155 or U14-KO-CD155 cells to investigate the antitumor effects of the target CD8+ T cells.
Statistical analysis and bioinformatics analysis
GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA) was used to analyse statistical significance. The results are presented as the mean ± standard deviation. The two-tailed Student’s t test was used for statistical comparisons between two independent groups; a nonparametric test was used for data that did not conform to a normal distribution. Gene expression and clinical annotation data were downloaded from GEO (Gene Expression Omnibus) and TCGA (The Cancer Genome Atlas). The “limma” package was used to analyse differentially expressed genes between cervical cancer and normal tissues. For all experiments, a p value less than 0.05 indicated a significant difference (P value are listed as follows: *P < 0.05, **P value < 0.01, ***P < 0.001).