Cd8 pe cy5

For the in vitro tumor-cell challenge, 5–10×10⁵ tumor-infiltrating lymphocytes (TIL), which were isolated from tumor tissues or lymphoid organs, were cultured with OVA peptide (1 µl/ml) for 4–5 hours in the presence of 1 µl/ml of Brefadin A (Sigma), washed and incubated with rat anti-mouse CD16/CD32 mAb (2.4G2) to block nonspecific binding, and then stained with CD8-PE-Cy5 and IFNγ-FITC or control antibodies according to the manufacturer’s instructions (BD Pharmingen). Tumor antigen-specific CD8 T cells were identified by staining with OVA-tetramer (Beckman Coulter) and TRP-2 pentamer (ProImmune). Cells were analyzed using FACScan flow cytometer and FlowJo version X.10 (Tree Star, Ashland, OR) software.

PBMCs were plated at 100,000/well in a 96 well plate with 2ug/mL galectin-9, 10ug/mL anti-Tim3 neutralizing antibody (Clone 344801; R&D Systems, Minneapolis, MN) or both and incubated for 24 hours at 37°C and 5% CO₂. Blocking was determined by phospho-flow cytometry utilizing phospho-Y265 PE (Millipore, Billerica, MA) and Tim3 APC or secondary staining (R&D Systems, Minneapolis, MN). Cultures were assayed for apoptosis using the APO-DIRECT flow cytometry apoptosis method (Millipore, Billerica, MA) per manufacturers’ instructions. In brief, extracellular staining was carried out for 30 minutes with CD4-PE, CD8-PE-Cy5 and CD14-PE-Cy7 (BD Biosciences, San Jose, CA). Cells were fixed and incubated overnight at −20°C in 70% ethanol. Cells were washed and stained for DNA breaks by FITC tagged nucleotides, resuspended in FACS buffer and read on a Guava easyCyte flow cytometer (Millipore, Billerica, MA). 20,000 events were counted and apoptotic, dead, and live cells were compared between groups with and without galectin-9.

Cell surface and intracellular immunostaining analyses were performed using an Accuri C6 Flow Cytometer. NK cells and T cells were stained with the dye-conjugated mouse mAbs to human CD56-PE-Cy5 (Beckman Coulter), CD3-PE (eBioscience), CCR7-FITC (R&D Systems), granzyme B-PE (Invitrogen), and CD16-FITC, CD8-PE-Cy5, CD45RA-FITC, CD45RO-PE, and CD57-FITC (BD Biosciences). MDSCs were stained for CD11b-FITC, CD14-PE, CD33-APC, CD34-PE-Cy5, CD11c-PE, HLA-DR-PE, DC-SIGN-FITC, CD80-FITC, CD86-FITC, and CD83-PE (BD Biosciences and eBioscience), as well as IDO-A488 (R&D Systems), NOS2-PE (Santa Cruz Biotechnology), and COX1-FITC/COX2-PE (BD Biosciences). The corresponding mouse antibody isotype controls IgG1-FITC, IgG2b-FITC, IgG1-PE, IgG2a-PE, IgG1-PE-Cy5, IgG1-APC, and IgG1-A488 (BD Biosciences) were used, as appropriate. Before staining, the cells were treated for 20 min at 4°C in PBS buffer containing 2% human serum, 0.5% BSA, 0.1% NaN₃, and 1 μg/ml of mouse IgG (Sigma-Aldrich) to block non-specific binding. Cell permeabilization for intracellular staining was performed using the Foxp3 Fix/Perm Buffer Set (eBioscience), according to the manufacturer’s protocol. Cells were stained for 40 min at 4°C followed by washing with PBS buffer containing 0.5% BSA and 0.1% NaN₃, then fixed and stored in 4% paraformaldehyde until analysis.