Cd80 l307.4

Fluorochrome-conjugated Abs against human B7-H1 (MIH1), PD-1 (EH12.2H7) and CD80 (L307.4) were purchased from BD Biosciences (Mountain View, CA), BioLegend (San Diego, CA), or eBioscience (San Diego, CA). To detect intracellular Survivin (clone 32.1, Novus), and Bcl-2 (Clone 50E3, Cell Signaling Inc.), tumor cells were incubated in Fixation Buffer (BioLegend) for 20 min at room temperature, followed by permeabilization using Permeabilization Wash Buffer (BioLegend). After staining with antibody, cells were washed three times with washing buffer before analysis. Apoptosis of tumor cells was analyzed by staining with Annexin V (BD Biosciences) and tetramethylrhodamine (TMRE; ethyl ester) (Invitrogen-Molecular Probes) for 10 min at room temperature. At least 100,000 viable cells were live gated on FACSCailbur (BD Biosciences) instrumentation. Flow cytometry analysis was performed using FlowJo software (Tree Star, Ashland, OR).

To measure DC activation markers, cells were fixated for 30 min with 4% paraformaldehyde (PFA, Electron Microscopy Sciences). For cell surface staining, cells were incubated in 0.5% PBS-BSA (Sigma-Aldrich) containing antibodies for 30 min at 4°C. To measure CTL responses, PBLs were harvested and stained for viability using Fixable Viability Dye eFluor 780 (eBioscience) for 5 min at 4°C and subsequently fixated for 10 min with 2% PFA. After fixation, cells were permeabilized using Perm/Wash solution (BD Biosciences) for 5 min 4°C and incubated with antibodies diluted in Perm/Wash solution for 10 min at 4°C. Single-cell measurements were performed with a FACS Canto flow cytometer (BD Biosciences). FlowJo V.10 software was used to analyze the data. Antibody clones used to analyze DC activation are CD86 (2331 (FUN-1)), CD80 (L307.4), and CD83 (HB15e) (all BD Pharmingen). For each experiment, live cells were gated on FSC (forward scatter) and SCC (side scatter) and analyzed further with the markers mentioned. Antibody clones used for cytotoxic T-cell responses are CD3 (UCHT1), CD8 (RPA-T8), IFN-γ (B27) (all BioLegend), perforin (dG9, eBioscience), and granzyme B (GB11, BD Pharmingen). For each experiment, live cells were selected and gated on CD3 and CD8 expressions. In this CD3⁺CD8⁺ population, cytokine expression was analyzed.