Ly6c clone hk1.4

Aliquots of 1 × 10⁶ cells were stained with different monoclonal antibodies according to standard protocols. The cells were analyzed on a FACSVerse cytometer (BD Biosciences, San Diego, CA, USA). Fluorescent antibodies of CD45 (clone 30-F-11), CD3ε (clone 145-2C11), CD4 (clone GK1.5), CD83 (clone Michel-19), CD11b (clone M1/70), ly6c(clone HK1.4), F4/80 (clone BM8), B220 (clone RA3-6B2), NK1.1 (clone PK136), MHC-II (clone M5/114.15.2), CD11c (clone N418), CD69 (clone H1.2F3), and Ki67 (clone B56) conjugated with the corresponding fluorescent dyes (eBioscience, San Diego, CA, USA) were used in the experiments.

Fresh liver was perfused with 5 mL of saline through the portal vein at 100‐300 mg, then digested with Roswell Park Memorial Institute 1640 medium with deoxyribonuclease I and collagenase IV at 37^oC. After digestion, hepatocytes were pelleted and discarded by centrifugation at 50g for 5 minutes. Nonparenchymal cells were washed with Roswell Park Memorial Institute 1640 medium and pelleted by centrifugation at 350g for 15 minutes, then washed and blocked with 10% mouse serum for 30 minutes. Antibodies against cluster of differentiation 11b (CD11b; clone M1/70; Ebioscience, Hatfield, UK), Ly‐6C (clone HK1.4; Ebioscience), CD45.2 (clone 104; Ebioscience), F4/80 (dilution 1:50; clone BM8; Invitrogen), and Ly‐6G (clone 1A8; Biolegend, San Diego, CA) were added for 30 minutes in a dilution of 1:100 unless otherwise specified. Cell viability was assessed with Fixable Viability Dye eFluor780 (Ebioscience) according to the manufacturer's protocols. Cells were formalin‐fixed and analyzed by flow cytometry using a BD LSR Fortessa II (BD Bioscience, Oxford, UK).

Bone marrow cells were flushed from the femurs and tibias with PBS with a syringe. The spleen samples were processed through mechanical dissociation, and tumour tissues were processed into single-cell suspensions by dissociating the tissues enzymatically for 1 h with 1 mg/ml type I collagenase (Sigma-Aldrich) in the presence of 50 units/mL DNase (Sigma-Aldrich). The cells were lysed with red blood cell lysis buffer and filtered with a 100 μm membrane, further washed with 1% BSA in PBS and blocked by non-specific staining with Fc Block (anti-mouse CD16/32 mAb; BD Biosciences). The samples were then stained with fluorescence-conjugated antibodies against the surface markers CD45 (clone 30-F11, eBioscience), CD11b (clone M1/70, eBioscience), Ly6C (clone HK1.4, eBioscience), Ly6G (clone 1A8-Ly6g, eBioscience), CD3 (clone 145-2C11, eBioscience) and CD8 (clone 53–6.7, eBioscience) and detected using flow cytometry (LSR BD Fortessa).