Canine Cell Line Culturing and Immune Modulation

The canine melanoma cell lines CMeC, LMeC, CMM-1, and CMM-2^{32 (link),33 (link)}, were cultured as described previously^{6 (link)}. The canine osteosarcoma cell lines POS^{34 (link)} and HMPOS^{35 (link)} were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 200 μg/mL streptomycin, and 200 U/mL penicillin (Thermo Fisher Scientific) at 37 °C under a 5% CO₂ atmosphere. Canine PBMCs were purified from heparinized blood obtained from healthy beagles by density gradient centrifugation on Percoll (GE Healthcare UK, Buckinghamshire, UK) and cultured as described previously^{6 (link)}. PBMCs were stimulated with 5 μg/mL Staphylococcal Enterotoxin B (SEB) (Sigma-Aldrich) and 1 μg/mL anti-canine CD28 antibody (eBioscience, San Diego, CA). In some experiments, cells were co-treated with 5 μM meloxicam (Sigma-Aldrich), 2.5 μM Prostaglandin E2 (Cayman Chemical), and/or 20 μg/mL canine chimeric anti-PD-L1 antibody c4G12^{11 (link)} as indicated. The same concentrations of DMSO (Nacalai Tesque, Kyoto, Japan) and dog IgG (Jackson ImmunoResearch, West Grove, PA) were used as negative controls.