Fig 2: ANGPTL2 attenuates IFN?-induced MHC-I expression via a5ß1 integrin. (A) Representative histograms showing cell surface expression of a5 integrin, ß1 integrin, a5ß1 integrin, and PIR-B in mouse tRCC cells. Shown is a representative of three independent experiments. (B) Representative histograms showing cell surface expression of a5 integrin in control KO (Control) and Itga5 KO (Itga5 KO1 and KO2) cells. Shown is a representative of three independent experiments. (C) Relative expression of transcripts encoding MHC-I (H2-K1 and H2-D1) and antigen presentation machinery-related molecules (B2m, Tap2, and Tapbp) in IFN?-treated or untreated control KO (Control) and Itga5 KO (Itga5 KO1 and KO2) cells (n = 4 per group). Levels in IFN?-untreated control cells were set to 1. Data are means ± SD. Statistical significance was determined by two-way ANOVA with Tukey's post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Representative histograms showing cell surface expression of H-2Kb and H-2Db in control KO (Control) and Itga5 KO (Itga5 KO1 and KO2) cells. Shown is a representative of three independent experiments.

Fig 3: Angptl2 deficiency accelerates T-cell-mediated tumor cell eradication by increasing IFN?-induced MHC-I expression in tumor cells. (A) Representative immunoblotting of ANGPTL2 in control KO (Control) and Angptl2 KO tRCC cell lines. Shown is a representative of two independent experiments. Hsc70 served as a loading control. (B) Relative expression of mRNAs encoding MHC-I (H2-K1 and H2-D1) and antigen presentation machinery-related molecules (B2m, Tap2, and Tapbp) in IFN?-treated or untreated control KO (Control) and Angptl2 KO (Angptl2 KO1 and KO2) cells (n = 4 per group). Levels in IFN?-untreated control cells were set to 1. Data are means ± SD. Statistical significance was determined by two-way ANOVA with Tukey's post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Representative histograms showing cell surface expression of H-2Kb and H-2Db in IFN?-treated or untreated control KO (Control) and Angptl2 KO (Angptl2 KO1 and KO2) cells. Shown is a representative of three independent experiments. (D) Representative histograms showing cell surface expression of SIINFEKL-bound H-2Kb in IFN?-treated or untreated Control-OVA, Angptl2 KO-OVA, and Angptl2 KO cells. Shown is a representative of three independent experiments. (E) Levels of indicated secreted T-cell cytokines (IFN? and TNFa) in culture supernatants of OT-I T cells, OT-I T cells co-cultured with Control-OVA, Angptl2 KO-OVA, or Angptl2 KO cells, and in corresponding tumor lines (n = 4 per group). Data are means ± SD. Statistical significance was determined by one-way ANOVA with Tukey's post hoc test. ***P < 0.001; n.s, not significant; n.d, not detected. (F) T-cell cytotoxicity assay with Control-OVA, Angptl2 KO-OVA, and Angptl2 KO cells (n = 6 per group). OT-I T cells were co-cultured 8 h with tumor cells at the indicated effector/target (E/T) ratios. Data are means ± SD. Statistical significance was determined by two-way ANOVA with Dunnett's post hoc test. **P < 0.01; ***P < 0.001.