Investigating Antitumor Immunity in Mice

Six-week-old female C57BL/6N mice were purchased from Japan SLC (Shizuoka, Japan) and kept in a specific pathogen-free environment. The animal use proposal and experimental protocols were reviewed and approved by The University of Tokyo Animal Care and Use Committee (ID:P15-125) and all animal procedures were conducted in accordance with institutional guidelines. The YTN2 and YTN16 cell lines [6 (link)] were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque, Kyoto, Japan) with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 100 μg/mL streptomycin, 100 U/mL penicillin (Nacalai Tesque), and MITO+ serum extender (Corning, Corning, NY, USA). Anti-PD-1 (RMP1-14), PD-L1 (10F9G2), CTLA-4 (9H10), and CD8α (53–6.7) mAbs were from Bio X Cell (Lebanon, NH, USA). DimerXI:Recombinant Soluble Dimeric Mouse H-2D^b:Ig (H-2D^b dimer), DimerXI:Recombinant Soluble Dimeric Mouse H-2K^b:Ig (H-2K^b dimer) were from BD Biosciences (Franklin Lakes, NJ, USA). FITC-conjugated anti-CD3ε, PerCP/Cyanine5.5-conjugated anti-CD4, APC/Cyanine7-conjugated anti-CD8, APC-conjugated anti-IFN-γ and Pacific Blue-conjugated anti-CD45 mAbs were from BioLegend (San Diego, CA, USA).