Pe conjugated anti mouse pd 1

T-cell suspensions were isolated from the spleen and lymph nodes, and were stained with antibodies against the following cell surface antigens: APC-conjugated anti-mouse CD3, FITC-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD8, PE-conjugated anti-mouse Gr-1, APC-conjugated anti-mouse CD11b, PE-conjugated anti-mouse TCR (eBioscience), PE-conjugated anti-mouse PD-1, and PE-conjugated anti-mouse CD152 (CTLA-4; BD Bioscience, San Jose, CA, USA). Tregs were detected from the spleen and lymph nodes using a Mouse Regulatory T-Cell Staining Kit (eBioscience) according to the manufacturer's instructions. DCs were isolated from the bone marrow and cells were incubated with FITC-conjugated anti-CD86, APC-conjugated anti-CD11C, PE-conjugated anti-MHC-II, FITC-dextran, PE-conjugated anti-CCR7, and anti-PD-L1 (eBioscience). The cells were analyzed by FACS, and the acquired data was performed with FlowJo Software, version 9.1 (Tree Star, San Carlos, CA, USA).

For flow cytometry, allophycocyanin (APC)-conjugated anti-mouse CD3 (BD Biosciences, Palo Alto, CA), phycoerythrin (PE)-conjugated anti-mouse PD-1, fluorescein isothiocyanate (FITC)-conjugated CD127, and PE-Cy5.5-conjugated anti-mouse CD4 or CD8 (BD Biosciences Pharmingen) monoclonal (m) Abs were used for flow cytometry. For the flow cytometric analysis, 10⁵ cells were labeled in a fluorescence-activated cell sorter (FACS) buffer (PBS/2% FCS/0.1% sodium azide), fixed in 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO), and analyzed on a FACSCalibur using CellQuest software (Becton Dickinson, Mountain View, CA).