Flow cytometry

Mouse tumors were excised and cut into 2–3 mm pieces to create single-cell suspensions. CT26.CL25 and B16-F10 tumors were dissociated with collagenase/hyaluronidase (STEMCELL Technologies, 07912) and murine tumor dissociation kit (Miltenyi, 130-096-730), respectively, following the manufacturers’ recommendation. Dissected LNs were mechanically disrupted to create single cells and washed once using flow running buffer (0.1% bovine serum albumin in PBS). All segregated cells were filtered using a 70 μm nylon cell strainer (Corning).

Resulted single cells were incubated on ice with anti-CD16/32 antibodies for 15 min. The cells were stained using Live/Dead Fixable Violet or Aqua dyes (Thermo Fisher) and surface-labeled with anti-CD8α BUV395 (clone 53-6.7; BD), anti-CD4 BV786 (clone GK1.5; BD), anti-CD3 BV786 or BV605 (clone 145-2C11; BD), anti-CD45 FITC or BV605 (clone 30-F11; BD), anti-PD-1 BV605 (clone J43; BD), anti-CTLA-4 PerCP/Cy5.5 (clone UC10-4B9; BioLegend), anti-Tim3 PE (clone B8.2C12; BioLegend), anti-LAG3 APC (clone C9B7W; BD), anti-CXCR3 BV650 (clone CXCR3-173; BioLegend), anti-CD11b BUV395 (clone M1/70, BD), anti-F4/80 Ax647 (clone T45-2342; BD), and anti-Ly6G BV711 (clone 1A8; BD) for 30 min at room temperature (see Supplementary Table ¹ and Supplementary Fig. ⁸).

For intracellular staining, cells were stimulated with cell activation cocktail containing phorbol myristate acetate (PMA)/ionomycin and Brefeldin A at 37 °C for 4 h prior to surface-staining as described above. The cells were fixed and permeabilized using the forkhead box P3 (FoxP3)/Transcription Factor Staining Buffer Set (eBiosciences) according to the manufacturer’s recommendation. Cells were stained with anti-FoxP3 PE (clone MF23; BD), anti-T-bet Ax647 (clone 4B10; BD), anti-IFN-γ APC (clone XMG1.2; eBioscience), anti-TNFα PerCP/Cy5.5 (clone MP6-XT22; BioLegend), or anti-granzyme B Ax647 (clone GB11; BioLegend). Detailed information pertaining to antibodies and panel used can be found in Supplementary Table ¹ and Supplementary Fig. ⁸.

All events were acquired immediately following sample processing using an LSR Fortessa flow cytometer (BD). All flow cytometric data analysis was performed using FCS Express 6 software package (De Novo Software).