Tim3 pe cy7

Anti (α)-mouse CTLA-4, α-mouse programmed death-ligand 1 (PD-L1), and mouse IgG2b isotype antibodies were purchased from BioXCell (West Lebanon, New Hampshire, USA). Mouse Ki67 Alexa Fluor 647, PD-1 PE, GR-1(Ly6G/Ly6C) Brilliant Violet 650, DX5 Dazzle 594, CD11c PE/Cy7, LAG3 Brilliant Violet 650, TIM3 PE/Cy7, CD3e PerCP/Cy5.5, CD11b FITC, CD45 APC/Fire 750, α-CD4 Brilliant Violet 421 and α-CD8a Brilliant Violet 605 were bought from BD Biosciences (San Jose, California, USA). Mouse F4/80 Superbright 702, FoxP3 PE and nestin monoclonal antibodies were purchased from Thermo Fisher. I-BET726 was purchased from Millipore Sigma (Burlington, Massachusetts, USA) and JQ1 was purchased from Tocris (Minneapolis, Minnesota, USA). Live/dead fixable aqua dead cell stain kit and Brilliant stain buffer were purchased from Thermo Fisher.

Tumor xenografts were cut into pieces and digested with collagenase IV (Sigma, USA) and DNAse I (Sigma, USA) for 30 min at 37°C. Then, cell suspensions were passed through a 70-μm strainer to remove undigested tissues. Erythrocytes were removed by Red Blood Cell Lysis Solution (Qiagen, USA). For cell surface staining, 1 × 10⁶ cells were incubated with anti-Fc receptor blocking antibody at 4°C for 30 min. For intracellular staining, 1 × 10⁶ cells were fixed and permeabilized using the Fixation/Permeabilization Solution Kit (BD Bioscience, USA) according to the manufacturer’s instructions. Then, cells were stained with the indicated antibodies for 30 min at 4°C. Flow cytometry was performed using a BD Influx cell sorter (BD Bioscience). Flow cytometry data were analyzed by FlowJo version 10 (FlowJo, USA). The flow cytometry antibodies used in our study were CD45 APC, CD4 PE, CD8a V450, CD25 APC-CY7, FoxP3 PE-CY7, IFN-γ PE-CY7, IL-4 APC-CY7, Gr-1 V450, Ly6C FITC, CD11 b PE, F4/80 APC-CY7, Tim-3 PE-CY7, PD-1 PerCP CY5.5, T-bet FITC, GATA3 APC, TNF-α APC-CY7, anti-Ki67 FITC, and anti-Granzyme B PE, all from BD Bioscience.