The murine HCC cell line hepa1-6 was obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM with 10% fetal bovine serum (Gibco, Gaithersburg, MD). The murine HCC cell line hepa1-6 has recently been authenticated by mouse cell line authentication service provided by Applied Biological Materials (Richmond, BC, Canada).
Mice and murine HCC models
C57BL/6 Itgax-IL-27p28f/f mice and C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I mice) were provided by Dr. Zhinan Yin (Jinan University, Guangzhou, China). All mice were housed in specific-pathogen-free facilities and in accordance with the National Animal Care and Use Committee. All animal experiments were approved by the Institutional Laboratory Animal Care and Use Committee of Soochow University (Suzhou, China).
Two murine HCC models were established in the study. To generate the orthotopic HCC model by hydrodynamic injection, 1 × 106 hepa1-6 cells in 2 ml PBS were injected via tail vein within 8 to 10 s. Mice were sacrificed 3 weeks later, and tumor nodules were counted. The DEN-induced spontaneous HCC model was generated by injecting 14-day-old C57BL/6 mice with 25 mg/kg DEN (Sigma-Aldrich, St Louis, MO). Eight months later, mice were sacrificed, and the livers were removed. Tumor size was measured, and the number of tumor nodules was counted.
For cell-surface staining, cells were blocked with CD16/32 FcR-block (BioLegend, San Diego, CA) for 15 min, and stained with fluorescent dye-conjugated mAb for 30 min at 4°C. For intracellular cytokine staining, cells were stimulated for 4–6 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of brefeldin A (10 µg/ml; BD Bio- sciences, San Jose, CA). Cells were stained for surface markers for 30 min, then fixed with 4% paraformaldehyde, permeabilized with 1% saponin (Sigma-Aldrich, St Louis, MO), and stained for cytokines for 30 min at 4°C. The antibodies used for FACS staining were FITC-anti-mouse-NK1.1 (PK136), PE/CF594-anti-mouse-CD3 (145-2C11), PE/CF594-anti-mouse-CD45 (30-F11), PerCP/Cy5.5-anti-mouse-CD4 (RM4–5), PerCP/Cy5.5-anti- mouse-NK1.1 (PK136), PE/CY7-anti-mouse-CD19 (1D3), APC/H7-anti-mouse-CD4 (GK1.5), and ACP/Cy7-anti-mouse-CD11b (M1/70) were purchased from BD Bioscience (San Jose, CA). The FITC-anti-mouse-CD8 (53–6.7), FITC-anti-mouse-I-A/I-E (M5/114.15.2), FITC- anti-mouse-CD4 (GK1.5), PE-anti-mouse-Ly6G (1A8), PE-anti- mouse-CD4 (RM4-5), PE-anti-mouse-NK1.1 (PK136), PE-anti-mouse-IFN-γ (XMG1.2), PE-Rat-IgG2b, kappa Isotype (RTK4530), PE/Dazzle594-anti-mouse-CD45 (30-F11), PerCP/Cy5.5-anti-mouse-TCRγδ (GL3), PerCP/Cy5.5- anti-mouse-IL-17A (TC11-18H10.1), PerCP/Cy5.5-anti-mouse-CD45.1 (A20), APC-anti-mouse-CD8 (53–6.7), APC-anti-mouse-CD4 (RM4-5), APC-anti-mouse-F4/80 (BM8), Alexa fluor 700-anti-mouse-CD45 (30-F11), APC-anti-mouse- IFN-γ (XMG1.2), PE/Cy7-anti-mouse-TNF-α (MP6-XT22), and PE/Cy7- anti-mouse-Ly6C(HK1.4), PE/Cy7-anti-mouse-CD44 (IM7), APC/Cy7-anti-mouse-CD11b (M1/70), APC/Cy7-anti-mouse-CD62L (MEL-14), Brilliant Violet421-anti-mouse CD8a (53 − 6.7), Brilliant Violet510-anti-mouse CD3ε (145-2C11), Brilliant Violet510-anti-mouse CD11c (N418) were purchased from BioLegend (San Diego, CA).
Neutralization of IL-27 in vivo.
To neutralize IL-27 in vivo, mice were injected i.p. with anti-IL-27p28 50ug (R&D Systems, Minneapolis, MN) per mouse immediately after the tumor cell inoculation on day 0 and one week later. Monoclonal goat IgG (Bio X Cell, Lebanon, NH) were used as isotype control.
Fecal pellets from WT and Itgax-IL-27p28f/f mice were collected, and each fecal pellet was resuspended in 1ml PBS. The suspension from each fecal pellet was used for oral gavage of one recipient mouse at 200ul per gavage. Mice were gavaged with WT or Itgax-IL-27p28f/f mice fecal suspension or PBS as control every other day for one week prior to tumor inoculation and once a week on day 7 and day 14 post tumor implantation.
Bifidobacterium species including B. bifidum, B. longum, B. lactis and B. breve were purchased from China General MicroBiological Culture Collection Center (BeiJing, China). Bacteria were cultured in an anaerobic chamber according to manufacturer's instructions. To confirm the compositions, bacteria were harvested and washed with PBS and centrifuged at 3000 rpm for 30 minutes. The pellet was sent for sequencing (GENEWIZ, Suzhou, China). For bacteria transplantation, a cocktail of Bifidobacterium species was resuspended in PBS at 5x109 CFU/ml. Each mouse was given 200µl of Bifidobacterium (1x109 CFU/mouse) by oral gavage with the same schedule as fecal transplant.
Fecal sample processing, sequencing, and analysis
Mice feces were collected into autoclaved EP tube and stored at − 80°C. Extracted DNA samples were amplified, DNA libraries were constructed, and the sequencing 16S rRNA was performed on Illumina MiSeq platform (GENEWIZ, Beijing, China). Sequences were grouped into operational taxonomic units (OTUs) using the clustering program VSEARCH (1.9.6) against the Silva 132 database pre-clustered at 97% sequence identity. The Ribosomal Database Program (RDP) classifier was used to assign taxonomic category to all OTUs at confidence threshold of 0.8. The RDP classifier uses the Silva 132 database which has taxonomic categories predicted to the species level. Base on the OUT analysis results obtained, Shannon index and Chao1 index, coordinates analysis (PCoA) were conducted by R package to display microbiome space between samples. A heatmap of the identified key variables was completed by the Heat map builder.
Bifidobacterium bacteria were harvested and centrifuged to collect the pellet. Bacterial 16S rRNA were amplified and sequenced (GENEWIZ, Beijing, China). Briefly, 16S rRNA was amplified from extracted DNA using broad-range bacterial primers 27F and 1492R. Amplified products from samples were verified by gel electrophoresis using the PCR reaction mixture in agarose gels. The 16Sr RNA was sequenced by Sanger Sequencing. The sequencing data was blasted in NCBI database for confirmation.
Activation of T cells with OVA peptide-pulsed TIP-DCs
TIP-DCs (CD45 + CD11b + Ly6C+) were sorted using a FACSAria III flow cytometer (BD Biosciences, San Jose, CA) and pulsed with 10 µg/ml OVA peptide for 2 hr at 37° in a total volume of 1 ml, then washed four times in RPMI-1640. In addition, CD8+ T cells from OT-I mice were separated by using mouse CD8 isolation cocktail kits, respectively (Stem cell, Vancouver, BC). CD8+ T cells in the suspension were then isolated and co-cultured with TIP-DC pulsed with or without OVA peptides (10 ug/ml, Sigma-Aldrich, St. Louis, MO) in a 96-well at a ratio of 3:1. After three days, production of IFN-γ and TNF-α by CD8 + T cells was analyzed by flow cytometry.
T cell proliferation assays
CD8+ T cells from OT-I mice were separated by using mouse CD8+ isolation cocktail kits, respectively (Stem cell, Vancouver, BC). Sorted CD8+ T cells were labeled with CFSE (ThermoFisher, Grand Island, NY) according to the manufacturer's instructions. Briefly, cells were resuspended in 50ml PBS and centrifuged for 5 minutes at 300g. Cells were then resuspended in 10 ml of 5 µM CellTrace CFSE staining solution and incubated in a 37°C water bath for 20 minutes. Then 40 ml serum-free RPMI-1640 medium was added, and cells were incubated for 5 minutes. After incubation, cells were centrifuged for 5 minutes at 300g and resuspended in serum-free RPMI-1640 medium. CFSE-labeled CD8+ T cells were seeded in anti-CD3 (5ug/ml, Biolegend, San Diego, CA) and anti-CD28 (2ug/ml, Biolegend, San Diego, CA) pre-coated 96-well plate and cultured in the presence of various concentrations of IL-27. After three day, CFSE expression on CD8+ T cells was analyzed by flow cytometry. In some experiments, CFSE-labeled CD8+ T cells were co-cultured with TIP-DCs pulsed with OVA peptides in 96-well plates at a ratio of 3:1. After three days, CFSE expression on CD8+ T cells was analyzed by flow cytometry.
Multiple comparisons were performed using one-way ANOVA with Tukey’s post-hoc test when comparing among all groups or Dunnett’s post-hoc test when comparing to control only. Comparisons between two groups were performed using Students’ unpaired t-test. All statistical analyses were performed with Graphpad Prism version 7 (GraphPad Software, San Diego, CA). Data were shown as mean ± SEM, and p < 0 .05 was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.