SiglecF ^hi TANs downregulate the antigenicity of tumor cells via TGFβ, and their removal sensitizes HCC mice to anti-PD-1 mAb treatment. (A) Distributions of Siglecf-Hi–like TAN signature score in HCC patients from the IMbrave150 and GO30140 clinical trials. (B) MHCI expression by IFNγ-treated RIL175 cells co-cultured with SiglecF⁻ or SiglecF^hi TANs, with or without Vactosertib (Vac.). (C and D) Antigen processing and presentation signature score was calculated from an imputed tumor-specific gene expression profile (tumor antigen presentation signature) in patients treated with Atezolizumab and Bevacizumab from the IMbrave150 and GO30140 clinical trials. Correlation between tumor antigen presentation signature and Siglecf-Hi–like TAN signature scores (C). Distributions of tumor antigen presentation signature score in responder (R) and non-responders (NR) (D). (E and F) OVA-expressing 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). Representative histograms and OVA/MHCI presentation by tumor cells (E). Tumor cells were co-cultured with OT-1 cells. Representative histograms and proliferation index of OT-1 cells (F). (G and H) NRAS/AKT HCC Mrp8^Cre+ve.TGFβ^fl/fl mice (Mrp8^Cre.TGFβ^fl/fl) and Mrp8^Cre−ve.TGFβ^fl/fl littermates (W/T) were treated with αPD-1 or isotype control mAbs for 2 wk. Representative images of HCC livers and liver-to-body weight ratio. The scale bar represents 1 cm (G). Representative images of AFP⁺ tumors (brown) and tumor nodule counts. Scale bar represents 100 μm (H). (I) NRAS/AKT HCC mice were treated with isotype control mAbs or αLy6G mAbs or αPD-1 mAbs or both at the indicated time. Survival curve (n = 10/group). (J–Q) Mice were sacrificed 2 wk following treatment. Representative images of HCC livers and liver-to-body weight ratio. Scale bar represents 1 cm (J). Representative images of AFP⁺ tumors (brown) and tumor nodule counts. Scale bar represents 100 μm (K). MHCI expression on tumor cells (L). Number of CD8⁺TILs (M). Proportion of PD-1⁺cells among CD8⁺TILs (N). Proportion of terminally exhausted cells (PD-1^hiCTLA4⁺Tox⁺Tcf1⁻) in CD8⁺TILs (O). Frequency of Ki-67⁺ CD8⁺PD-1⁺TILs (P). Percentage of CD8⁺PD-1⁺TILs co-producing IFNγ and TNFα after in vitro anti-CD3/CD28 restimulation (Q). (R–T) CD8⁺T cell depletion abrogated the beneficial effects of αLy6G mAb treatment in anti-PD-1 mAb administrated mice. Survival curve (n = 7–10/group) (R). Mice were sacrificed 2 wk following treatments. Liver-to-body weight ratio (S). Tumor nodule counts (T). Each symbol represents TANs isolated from two to three mice (B), one mouse (E, G, H, J–Q, S, and T), or the mean of tumor cells isolated from three mice (F). Data are mean ± SEM and are pooled from three independent experiments (B), or represent two to three independent experiments (E–T). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Wilcoxon rank-sum test (A and D), one-way ANOVA (B, G, H, J–Q, S, and T), unpaired Student’s t test (E), two-way ANOVA (F), and Kaplan–Meier analysis (I and R).