Flow Cytometry Analysis of Tumor-Infiltrating Immune Cells

The details of staining and flow cytometry analysis protocols is according to our previously published methodology^{5 (link)}. Briefly, cells were incubated with Ghost Dye Violet 510 (Tonbo Biosciences, #13-0870, 1:1,000) to discriminate live/dead cells and washed with PBS containing 1% v/v FBS. After blocking Fc receptors with anti-mouse CD16/CD32 mAb (BD Biosciences, # 553142, 1:50) for 20 min, cells were incubated with target antibodies, including CD45-APC/Cy7 (BioLegend, #103116, 1:250), CD3-PerCP/eFluor 710 (eBioscience, #460032-80, 1:200), CD4-BV421 (BioLegend, #100438, 1:200), CD8-AlexaFlour700 (BioLegend, #100729, 1:500), and CD69-APC (BioLegend, #104513, 1:100). After staining, cells were washed twice in PBS containing 1% v/v FBS and fixed with 1% formalin in PBS. Data were collected using a BD LSR Fortessa flow cytometer and analyzed using FlowJo software (Version 10.5.3). The gating strategy for flow cytometric analysis of tumor samples was shown in Supplementary Fig. 7d.