Cell suspensions were washed and stained with Ghost Dye Violet 510 (Tonbo, 13-0870) in PBS according to the manufacturer’s protocol. After live/dead staining, cells were incubated with Fc block (purified anti-mouse CD16/32 antibody, clone 93, BioLegend) and stained with indicated surface markers at 4°C for 20 min. For cytokine staining, cells were restimulated with phorbol 12-myristate 13-acetate (PMA; 20 ng/ml; Sigma-Aldrich, P8139) and ionomycin (1 μg/ml; Sigma-Aldrich, I0634) for 4 hours in the presence of GolgiStop (BD Biosciences, 554724) and GolgiPlug (BD Biosciences, 555029) followed by surface marker staining. Cells were then fixed and permeabilized with either Cytofix/Cytoperm solution (BD Biosciences) for cytokine staining or the Foxp3/Transcription factor staining buffer set (eBioscience) for GzmB, Ki-67, CTLA-4, and transcription factors according to the manufacturers’ protocols. Cells were measured with BD LSRFortessa (BD Biosciences) or CytoFLEX LX (Beckman Coulter). Data were analyzed by FlowJo software (Treestar Inc.).

Antibodies (clone) used for the surface staining include CD4 (RM4-5), CD8α (53-6.7), CD11b (M1/70), CD11c (N418), CD19 (1D3), CD25 (PC61.5), CD44 (IM7), CD45 (30-F11), CD62L (MEL-14), CD137/4-1BB (17B5), CD223/LAG-3 (C9B7W), CD276/B7-H3 (EPNCIR122), CD279/PD-1 (RMP1-30), CD335/NKp46 (29A1.4), CD366/Tim-3 (RMT3-23), NK1.1 (PK136), Slamf6 (13G3), TCR-β (H57-597), rat anti-mouse IgG1 (A85-1), mouse anti-human IgG Fc (HP6017), goat anti-rabbit IgG H&L, and anti–hB7-H3 (185504). Antibodies (clone) used for the intracellular staining include CD152/CTLA-4 (UC10-4B9), Eomes (Dan11mag), Foxp3 (FJK-16 s), GzmB (GB11), IFN-γ (XMG1.2), Ki-67 (SolA15), T-bet (4B10), TCF1/TCF7 (C63D9), and TNF-α (MP6-XT212). All antibodies were purchased from BioLegend, eBioscience, BD Biosciences, R&D Systems, Abcam, or Cell Signaling Technology.

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