Interleukin 2 (il 2)

After centrifuging the cell suspension for 10 minutes at 300 xg (RT), each cell pellet was resuspended in T-cell medium (2 x 10⁶ cells/mL) and the cultures rested in a 5% CO₂ and 37°C incubator for 90 minutes. Four culture conditions were created per subject: in the presence of either GSK2794776A or GSK2794778A [17 (link)] and in Th17 driving medium [T-cell medium containing 10 ng/mL IL-1β (Cell Guidance Systems #GFH167AF), 20 ng/mL IL-6 (BioVision #6464–10), 10 ng/mL IL-23 (BioVision #6470–10), 2 ng/mL TGFβ (BioVision #6479–10), 1 ng/mL IL-2 (BioVision #6461–10), 2 μg/mL anti-IL-4 (BioLegend #500815), 2 μg/mL anti-IFNγ (BioLegend #506513)] or in T-cell maintenance medium. Isolated naïve CD4⁺ T cells were then distributed into a 6-well plate containing each of the four conditions stated above in a cell density of 1 x 10⁶ cells/mL. Simultaneously, Dynabeads Human T-Activator CD3/CD28 (Life Technologies #11131D) were prepared according to the manufacturer instructions and resuspended in T cell medium. Bead suspension was then added to each well to give a final ratio of 1 bead: 50 cells, as described by Purvis et al (2010) [18 (link)]. Cells were incubated for 6 days in a 5% CO₂ and 37°C.
Significant differences between groups were determined using unpaired t-tests. Analyses were performed using Microsoft Excel and a p-value < 0.05 was considered statistically significant.

Blood samples for biochemical measurements were collected from each animal prior to and at the end of the experiment. Samples were obtained from the tail vein, collected into tubes containing ethylenediaminetetraacetic acid or heparin, and plasma was separated by centrifugation at 1000× g for10 min at 4 °C. Plasma samples were stored at −80 °C until further use. Plasma total blood urea nitrogen (BUN), creatinine, alanine transaminase (ALT), aspartate aminotransferase (AST), blood sugar, and lactic dehydrogenase (LDH) were measured using a SPOTCHEMTM automatic dry chemistry system (SP-4410; Arkray, Shanghai, China). Enzyme-linked immunosorbent assays (ELISAs) were performed to determine the plasma levels of C-reactive protein (CRP; Abcam Inc., Cambridge, MA, USA), high mobility group box 1 (HMGB1; LifeSpan Biosciences, Inc., Seattle, WA, USA), transforming growth factor β1 (TGF-β1; Abcam Inc., Cambridge, MA, USA), SDF-1α (R&D Systems Inc., Minneapolis, MN, USA), interferon-γ (INF-γ; Abcam Inc., Cambridge, MA, USA), and interleukin-2 (IL-2; Abcam Inc., Cambridge, MA, USA).

OT-I TCR Transgenic mice were purchased from the Walter and Eliza Hall Institute (Melbourne, Australia). Splenocytes were harvested from the spleen of OT-I mice and stimulated by incubation with 300 ng/mL of SIINFEKL (OVA peptide) (Sigma-Aldrich) for 24 hr to expand CD8⁺ OT-I T cells. After washing to remove the peptide, cells were cultured in media supplemented with IL-2 at 100 U/mL (Abcam) for an additional 2 days. T cells were subsequently isolated from the mononuclear layer using Ficoll separation and cultured with IL-2 for an additional 3 days before use in co-culture assays. Successful enrichment of a CD8⁺ T cell population was confirmed by flow cytometry staining for CD45, CD3 and CD8.

YTN5 and YTN16 cells were transfected with FOXM1 siRNA for 24 h and then exposed to 20 ng/mL IFNγ (Abcam, #9922) for 24 h. YTN5 and YTN16 cells were then trypsinized (Gibco, #15400-054) and seeded at 1 × 10⁵ in a 24-well plate and then pulsed with 1 ng/mL OVA peptide (Sigma, #S7951-01mg) for 3 h. Simultaneously, spleens were removed from C57BL/6-Tg (TcraTcrb) mice and splenocytes were treated with 1X Red Blood Cell lysis buffer (eBioscience, #00-4333-57) for 5 min. CD8⁺ T Cells were isolated using the MojoSort™ Mouse CD8⁺ T Cell Isolation Kit (Biolegend, #480035) and resuspended in RPMI-1640 medium (Corning, #45000-396) containing 20% FBS (Omega Scientific, #FB-02). CD8⁺ T cells were incubated with 100 U/mL IL2 (Abcam, #ab9856) for 24 h, followed by 300 ng/mL OVA peptide for 24 h. CD8⁺ T cells were spun down at 1500 × g for 5 min and resuspended at 4 × 10⁵ cells/100 μL of RPMI-1640 medium (Corning, #45000-396) containing 20% FBS (Omega Scientific, #FB-02). Media from YTN5 and YTN16 cells was removed and replaced with 400 μL of fresh culture media, followed by 100 μL of CD8⁺ T cell containing media. The co-cultured cells were incubated for 24 h. The media and suspended CD8⁺ T cells were removed, and alive cancer cells were stained with 1% crystal violet as described above.

VIP primer was synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). EcoR I and Hind III were obtained from MBIFermentas (Vilnius, Lithuania). Cell culture medium and supplements were purchased from Gibco (Mountain View, CA, USA). pcDNA3.1, Lipofectamine 2000 and TRizol were obtained from Invitrogen Corp. (Carlsbad, California, USA). The VIP ELISA assay kit was purchased from Solarbio (Shanghai, China). TNF-αELISA assay kit, IL-2 ELISA assay kit, IL-4 ELISA assay kit and the 5-bromo-2-deoxyuridine (BrdU) cell proliferation assay kits were purchased from Merck MilliporeCorp (Darmstadt, Hesse, Germany). Antibodies of VIP, TNF-α, IL-2, CollagenII, OPG, MMP-13 and ADAMTS-5 were purchased from Abcam (Cambridge, MA, USA). Antibodies of Vimentin, p65, p-p65, IκBα, p-IκBα, β-actin and IgG were purchased from Cell Signaling Technology. Inc. (Danvers, MA, USA).