Surface and intracellular molecular staining

Transfected CT26 and CT26/MUC1 cells (2×10⁶ cells/well) were stimulated using IL-17A (10 ng/ml; R&D Systems China Co., Ltd.) and IFN-γ (10 ng/ml; R&D Systems China Co., Ltd.) at 37°C for 48 h, the cells were then harvested via centrifugation (250 × g; 4°C; 10 min) and counted. Subsequently, the cells were stained with PE anti-PDL1 at 4°C for 30 min (1:100; cat. no. 124308; clone 10F.9G2; BioLegend, Inc.). Fluorescence data were acquired using a CytoFLEX flow cytometer (Beckman Coulter, Inc.) and analyzed using Kaluza software v2.1 (Beckman Coulter, Inc.).

For the surface staining of tumor cells and immune cells, the tumor tissues from tumor-bearing mice or patients with colon cancer were weighed, homogenized and digested with hyaluronidase (2 mg/ml), collagenase type IV (2 mg/ml), and deoxyribonuclease (25 µg/ml; Sigma-Aldrich; Merck KGaA) for 50 min at 37°C. Immune cells were enriched with Ficoll reagent and stained with propidium iodide at 4°C for 5 min. After counting viable cells, the samples were incubated with an anti-CD16/CD32-blocking Ab (1:100; cat. no. Ab00123-23.0-BT; Absolute Antibody) at 4°C for 10 min and then stained at 4°C for 30 min with Brilliant Violet 510™ anti-mouse CD45 (1:100; cat. no. 103138; clone 30-F11; BioLegend, Inc.), Brilliant Violet 510™ anti-human CD45 (1:100; cat. no. 304036; clone HI30; BioLegend, Inc.), FITC anti-mouse CD4 (1:100; cat. no. 100509; clone RM4-5; BioLegend, Inc.), FITC anti-human CD4 (1:100; cat. no. 317408; clone OKT4; BioLegend, Inc.), FITC anti-mouse CD8a (1:100; cat. no. 100706; clone 53-6.7; BioLegend, Inc.), FITC anti-human CD8a (1:100; cat. no. 301050; clone RPA-T8; BioLegend, Inc.), APC anti-mouse CD279 (PD-1; 1:100; cat. no. 109112; clone RMP1-30; BioLegend, Inc.), APC anti-human CD279 (PD-1; 1:100; cat. no. 329908; clone EH12.2H7; BioLegend, Inc.), PE anti-mouse/human CD11b (1:100; cat. no. 101207; clone M1/70; BioLegend, Inc.), FITC anti-mouse Ly-6G/Ly-6C (Gr-1; 1:100; cat. no. 108406; clone RB6-8C5; BioLegend, Inc.), FITC anti-human CD66b (1:100; cat. no. 305104; clone G10F5; BioLegend, Inc.), PerCP/Cyanine5.5 anti-mouse F4/80 (1:100; cat. no. 123128; clone BM8; BioLegend, Inc.), PerCP/Cyanine5.5 anti-human CD68 (1:100; cat. no. 333813; clone Y1/82A; BioLegend, Inc.), APC anti-mouse CD274 (B7-H1, PD-L1; 1:100; cat. no. 124311; clone 10F.9G2; BioLegend, Inc.), APC anti-human CD274 (B7-H1, PD-L1; 1:100; cat. no. 329708; clone 29E.2A3; BioLegend, Inc.) or PE anti-human MUC1 (1:1,000, cat. no. ab213337; clone EP1024Y; Abcam). The live cells were gated. Fluorescence data were acquired using a CytoFLEX flow cytometer and analyzed using Kaluza software v2.1.

For the intracellular staining of immune cells, 1×10⁶ cells/ml were treated with Cell Activation Cocktail containing Brefeldin A (1:1,000; cat. no. 423304; BioLegend, Inc.). After 4 h, the cells were collected and stained with the aforementioned Brilliant Violet 510™- or FITC-conjugated mAbs against mouse or human CD45, CD4 or CD8 mAbs (BioLegend, Inc) at 4°C for 30 min. The cells were then fixed with Fixation Buffer (cat. no. 420801; BioLegend, Inc.) in the dark at room temperature for 20 min. Next, the cells were permeabilized with 1X intracellular Staining Permeabilization Wash Buffer (cat. no. 421002; BioLegend, Inc.) and incubated with PE-conjugated anti-IFN-γ anti-IL-17 or anti-granzyme B (BioLegend, Inc.) for 30 min at room temperature. For forkhead box P3 (Foxp3) staining, the fixed cells were treated using the True-Nuclear™ Transcription Factor Buffer Set (BioLegend, Inc.). The tumor-infiltrating lymphocytes (TILs) were gated. Fluorescence data were acquired on a Beckman CytoFLEX and analyzed using Kaluza software 2.1.