Cd155 fc

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.

Anti-CD3 antibody (145-2X11, R&D systems, Minneapolis, MN, USA) together with recombinant mouse CD155.Fc or the control.Fc (Sino Biological, Beijing, China) or PD-L1. Fc or the control.Fc (R&D systems) or HVEM.Fc (R&D systems) were covalently attached to Dynabeads M450 Tosylactivated (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Anti-CD3 antibody together with control.Fc was used for the control. For each 10⁷ beads, 1 μg of anti-CD3 antibody (20% of total protein) and 4 μg of CD155.Fc, PD-L1.Fc, HVEM.Fc or control.Fc (80%) were used.