Permeabilisation solution

In brief, MDA MB 231 cells or Hs578T cells were cocultured with the activated CD8+T cells for 48 hours in the 6-well plates. The ratio of CD8+ T cells to MDA MB 231 cells or Hs578T cells was 5:1. Then, the percentages of CD8+ perforin+ T cells and CD8+ TNF-α+ T cells were analyzed by flow cytometry according to previous description.24 (link) To be specific, cells were collected and washed twice with PBS. Then, cells were incubated with the permeabilisation solution (BD Biosciences, San Jose, California, USA) containing PE-Cy7 anti-α-TNF-α (506323, Hengfei Biological Technology Co., Ltd., Shanghai, China) and PerCp-Cy5.5 anti-Perforin (303935, Yubo Biological Technology Co., Ltd., Shanghai, China) for 25 min. Later, CD8+ perforin+ T cells and CD8+ TNF-α+ T cells were detected with the flow cytometer (FACSCalibur, BD Biosciences, San Jose, California, USA). The percentages of CD8+ perforin+ T cells and CD8+ TNF-α+ T cells were analyzed by the FlowJo 9.2 software (Ashland, Oregon, USA).

Splenocytes were collected from mouse spleen and incubated at 37°C for 6 h in DMEM/F‐12 medium supplemented with 10% FBS. Golgi stop (1.5 μg/ml, BD Biosciences, USA) was added for the final 5 h of incubation to block cytokine secretion. Cells were harvested, washed and blocked with CD16/CD32‐blocking Ab (BD Bioscences, USA) for 5 min at room temperature, and stained with 0.4 μg/ml BV510‐labelled CD45, PerCP‐Cy5.5‐labelled CD3, FITC‐labelled CD8, PE‐Cy7‐labelled CD4, BV421‐labelled CD25 (BD Bioscences, USA) for 15 min at room temperature. Then cells were washed and incubated with permeabilisation solution (BD Bioscences, USA) for another 20 min at 4°C. Finally, cells were further labelled with 0.4 μg/ml Alexa 647‐labeleld IFN‐γ, PE‐labelled FOXP3 (BD Bioscences, USA) for 15 min at room temperature and analysed by flow cytometry. Tumour tissues were minced into small pieces and digested in collagenase type IV suspension (0.05 mg/ml, Worthington Biochem) for 40 min at 37°C. The resulting suspension was filtered through the 70 μm cell strainer. The extract was centrifuged at 500×g for 10 min after which the supernatant was removed. The mixture was re‐suspended in ACK lysis buffer to remove red blood cells and the rest cells were stained with different fluorescently‐labelled antibodies as described above.