Flow cytometry analysis of specific CD8 T cells and tumor cells

Heparinized blood was lysed with RBC Lysis Buffer (BioLegend). Subcutaneous MC38 tumors were excised, cut into small pieces and digested in RPMI containing 1 mg/ml Dispase (StemCell Technologies), 0.8 mg/ml Collagenase D (Roche), and 0.01 mg/ml DNase (StemCell Technologies) for 20 minutes at 37°C. The digest was filtered with a 70 µm cell strainer. Fcγ receptors IIa and III on white blood cells or tumor cells were blocked with 1.0 µg of TruStain fcX antibody (BioLegend). Antigen-specific CD8 T cells were labeled with M38-Kb MHC Dextramers (Immudex) and afterward labeled with fluorescent CD8a (53–6.7, BioLegend), CD4 (GK1.5, BioLegend), CD3ε (145–2 C11, BioLegend) and CD45 (30-F11, BioLegend). Dead cells were excluded with DAPI. CD8 T cells and tumor cells (CD45-cells) were phenotyped for PD-1 (29 F.1A12, BioLegend), CD44 (IM7, BioLegend), CD62L (MEL-14, BioLegend), CD127 (A7R34, BioLegend), CD25 (PC61, BioLegend) and PD-L1 (10 F.9G2, BioLegend). For intracellular staining of FoxP3, Fixation Buffer (BioLegend), Intracellular Staining Perm Wash Buffer (BioLegend), and anti-FoxP3 (MF-14, BioLegend) were used. SIINFEKL peptide-MHC class I complexes were detected with the monoclonal anti-Ova257-264 – mouse H-2Kb antibody (25-D1.16, BioLegend).