For cell cycle, the harvested cells were incubated with DNA staining and permeabilization solution (away from light, RT, 30 min). The samples were tested on CytoFLEX system and analyzed by Modifit software.

For apoptosis analysis, the harvested cells were resuspended gently with binding buffer and Annexin V-FITC staining solution (away from light, 4 °C, 15 min). After incubating with propidium iodide (PI) solution for another 5 min, the cells were analyzed on CytoFLEX system.

For the investigation of cytotoxic T cell infiltration, single-cell suspensions were prepared and the cells were stained with fluorescence-labeled antibodies against CD3, CD45, CD4 and CD8 (BD Pharmingen). The data were acquired on FACS Aria TM III Cell Sorter and analyzed with FlowJo (version 10.7.1). The antibodies were presented in Additional file 1: Table S2.

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