Fixation and permeabilization buffer kit

Tumors were sufficiently chopped using scalpels and then digested with 0.2 mg/mL collagenase intravenous and 0.1 mg/mL DNase I at 37°C for 1 hour, and then passed through a 70 µm strainer to determine the presence of the infiltrating T cell population. Mouse-specific antibodies including anti-CD3, anti-CD4, anti-CD8, anti-CD11b, and anti-Ly6G antibodies were purchased from BD Pharmingen. Anti-IFN-γ and PD-1 antibodies were purchased from eBioscience. The human-specific antibody against PD-L1 was purchased from BD Pharmingen. Major histocompatibility complex (MHC) Class I (H-2Kb) antibody, MHC Class II (I-A/I-E) antibody, human MHC Class I/ HLA-ABC antibody (W6/32) and human MHC Class II/ HLA-DR antibody (LN3) were purchased from eBioscience. Intracellular staining for IFN-γ was performed after stimulation with PMA at 37℃ for 4–6 hours as described in the protocol of the Fixation and Permeabilization Buffer Kit (BD Bioscience). For detection of intracellular IFN-γ, brefeldin A was used to block secretion of cytokines during the last few hours of the stimulation. Cells were then subjected to flow cytometry.

We obtained PBMCs from patients with high or low levels of TSLP by density centrifugation using Ficoll‐Paque and cultured these cells in RPMI 1640 supplemented with 10% FBS. Cells were resuspended at a density of 0.5 × 10⁵ cells·mL⁻¹, and 100–200 μL of cell suspension was stimulated with cell stimulation cocktail (plus protein transport inhibitors; 1.5 μL·mL⁻¹ of cell suspension; BD Biosciences). After stimulation in vitro, these cells were permeabilized with fixation and permeabilization buffer kit (BD Biosciences) and stained for 30 min at 4 °C in flow tubes with directly conjugated antibodies. The cells were fixed with 4% paraformaldehyde. The antibodies used for the flow cytometry analysis included anti‐human CD4‐allophycocyanin (560158; BD Biosciences), anti‐human IFN‐γ‐PeCy7 (557844; BD Biosciences), anti‐human IL‐4 (554516; BD Biosciences), anti‐human forkhead box P3 (Foxp3)‐Alexa Fluor 488 (560459; BD Biosciences) and anti‐human IL‐10‐phycoerythrin (554498; BD Biosciences). The cells were separated with a multicolor flow cytometer (BD Company, Franklin lakes, NJ, USA), and the fluorescence‐activated cell signaling data were analyzed by flowjo 10.0 software (BD Bioscience, San Jose, CA, USA).