αLy6G mAb treatments downregulate the antigenicity of tumor cells and activates CD8 ⁺ TILs in HCC-bearing C57BL/6J mice, while HCC-bearing Mrp8 ^Cre .TGFβ ^fl/fl mice respond to αPD-1 mAb treatment. (A) SiglecF⁻ and SiglecF^hi TANs were adoptively transferred alongside OVA-expressing RIL175 (left) or RIL175_shTgfbr1 cells (right). OVA/MHCI presentation by tumor cells. (B) Representative histograms and MHCI expression on NRAS/AKT tumor cells following αLy6G or isotype control mAbs treatments. (C) UMAP of tumor infiltrating CD8⁺T cells (CD8⁺TILs) in NRAS/AKT-injected C57BL/6J mice treated with αLy6G or Isotype mAbs. (D) Distribution of marker genes in different CD8⁺TIL subsets. (E) Expression of T cell receptor signaling related genes within the Teff_CD8 subset. (F–J) OVA-expressing NRAS/AKT HCC tumors were used to assess tumor antigen presentation and T cell recognition following αLy6G or isotype control mAbs treatments. Schematic outline of the experiment (F). Representative histograms and OVA/MHCI presentation by tumor cells (G). OVA/MHCI presentation by tumor-infiltrating CD11c⁺MHCII⁺ DCs (H). Proliferation of OT-1 cells 3 days after adoptive transfer into OVA-NRAS/AKT HCC mice (I). Tumor cells from OVA-NRAS/AKT HCC mice were co-cultured with OT-1 cells at the indicated ratios for 3 days. Representative histograms and proliferation index of OT-1 cells (J). (K–P) NRAS/AKT HCC was induced in Mrp8^Cre+ve.TGFβ^fl/fl mice (Mrp8^Cre.TGFβ^fl/fl) and Mrp8^Cre−ve.TGFβ^fl/fl littermates (W/T), followed by treatment with αPD-1 or isotype control mAbs. MHCI expression on tumor cells (K). Number of TCRβ⁺CD8⁺TILs per gram of HCC tissue (L). Proportion of PD-1⁺ cells among TCRβ⁺CD8⁺TILs (M). Frequency of proliferating TCRβ⁺CD8⁺PD-1⁺TILs determined by Ki-67 staining (N). Proportion of terminally exhausted cells (PD-1^hiCTLA4⁺Tox⁺Tcf1⁻) among TCRβ⁺CD8⁺TILs (O). Percentage of TCRβ⁺CD8⁺PD-1⁺TILs co-producing IFNγ and TNFα after in vitro anti-CD3/CD28 restimulation (P). Each symbol represents one mouse (A, B, G–I, and K–P), or the mean of tumor cells isolated from three mice (J). Data are mean ± SEM and represent two to three independent experiments (A, B, and G–P). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired Student’s t test (A, B, and G–I), two-way ANOVA (J), and one-way ANOVA (K–P).