Fig 2: Involvement of ferroptosis in the Mn-MOF-induced improved tumor immunity. (A-I) The numbers of CD4+ T cells (A), CD8+ T cells (B), CD8+CD69+ T cells (C), CD8+IFN-γ+ T cells (D), CD8+GzmB+ T cells (E), CD11c+CD80+ dendritic cells (F), CD11c+CD86+ dendritic cells (G), MDSCs (H) and Tregs (I) in tumor tissues of H22 tumor-bearing mice after intravenous injection of PBS, Mn-TCPP, MOF or Mn-MOF at Zr dosage of 5 mg/kg (the corresponding Mn dosage was 1 mg/kg) in the presence or absence of intraperitoneal injection of UAMC-3203 at 20 μg/kg dosage, followed by US irradiation (1.0 MHz, 1 W/cm2, 50% duty cycle) at tumors for 10 min. The data are presented as mean ± s.d. (n= 5). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: CD274-L2A-derived soluble PD-L1 acts as a receptor antagonist in the presence of transmembrane PD-L1.(A) Primary CD8+ T cells were labelled with Cell Trace Violet (CTV) and stimulated with CD3- and CD28-coated beads for 72 hr. T cells were co-cultured with parental HEK293T or HEK293T.CD274v1 transfected cells in the presence of conditioned media from HEK293T.CD274-L2A transduced cells or a PD-L1-blocking antibody. Mean (± SEM) proportion of CTVlo and GzmB+ cells of three healthy donors is shown. (B) Percentage of activated (CD25+CD69+) Jurkat cells in the presence of conditioned media from parental HBL-1 cells, IFN-γ-stimulated HBL-1 cells, and transduced B-3T3.CD274-L2A cells. Cells were stimulated with CD3- and CD28-coated beads for 24 hr, with 10 µg/mL of a PD-L1-blocking antibody added where indicated. Mean (± SEM) proportion from three independent experiments are shown. (C) Percentage of activated (CD25+CD69+) Jurkat.PDCD1 cells in the presence of conditioned media from parental HBL-1 cells, IFN-γ-stimulated HBL-1 cells, and transduced HEK293T.CD274-L2A cells. Cells were stimulated with CD3- and CD28-coated beads for 24 hr, with 10 µg/mL of a PD-L1-blocking antibody added where indicated. Mean (± SEM) proportion from three independent experiments are shown. (D) Percentage of activated (CD25+CD69+) Jurkat.PDCD1 cells following co-culture with parental HEK293T or HEK293T.CD274v1 transfected cells in the presence of conditioned media from HEK293T.CD274-L2A or a PD-L1-blocking antibody. Cells were stimulated with CD3- and CD28-coated beads for 24 hr. Mean (± SEM) proportion from three independent experiments are shown. (E) Percentage of activated (CD25+CD69+) Jurkat.PDCD1 cells following co-culture with HEK293T cells transfected with varying ratios of CD274v1 and CD274-L2A as shown. The concentration of the CD274v1 plasmid is kept constant across all conditions at 1000 ng. Cells were stimulated with CD3- and CD28-coated beads for 24 hr. Mean (± SEM) proportion from three independent experiments are shown.

Fig 4: ARIH1 promotes anti-tumor immunity via PD-L1 degradation.a Immunoblot of PD-L1 and ARIH1 (HA) in H1975 cells transfected with HA-ARIH1 (WT or Y392C); HA-tagged empty vector (HA-EV) was transfected as a negative control. b Co-IP analysis for the interaction of K48-ubiquitin, ARIH1 (HA), and PD-L1 in HEK293T cells transfected with Flag-PD-L1 and HA-ARIH1 (WT or Y392C) in the presence of 10 μM MG132 for 6 h. c 4T1 cells were infected with an empty vector (CTRL) or two different ARIH1-overexpressing lentiviral preparations (OE#1 and OE#2). ARIH1 and PD-L1 levels were determined by immunoblotting. d–f Tumor growth (d, e) of CTRL (n = 10) and ARIH1-OE cells (n = 11) in BALB/c mice and final tumor weights (f). Data represent means ± SEM, ***P < 0.001 (P = 0.0001), ****P < 0.0001. g, h Flow cytometry analysis for the tumor levels of CD8+ T cells (g) and CD8+GzmB+ T cells (h). Data represent means ± SEM, CTRL (n = 10) and ARIH1-OE (n = 5), ***P < 0.001 (P = 0.0006), **P < 0.01 (P = 0.0046). i–k 4T1 tumor xenograft growth in BALB/c mice (I, j) and final tumor weights (k) following treatment with ES-072 and/or anti-CTLA4 (n = 6-7). Vehicle = sodium carboxymethyl (CMC-Na). ES = ES-072. Data represent means ± SEM, *P < 0.05 (P = 0.01), **P < 0.01 (P = 0.0013), ***P < 0.001 (P = 0.0001), ****P < 0.0001. l, m Flow cytometry analysis for the tumor levels of CD8+ T cells (l) (n = 4–7) and CD8+GzmB+ T cells (m) (n = 4–7). Data represent means ± SEM. l **P < 0.01 (P = 0.0017), ***P < 0.001 (P = 0.0001), ****P < 0.0001. m *P < 0.05 (P = 0.0286), **P < 0.01 (P = 0.0012), ***P < 0.001 (P = 0.00099). n Immunobloting of the indicated tumor lysates. Source data are provided as a Source Data file.

Fig 5: SHP2 inhibition decreases tumor load in the 4T1 syngeneic mice model. (a) Tumor growth curve (expressed as tumor volume) of subcutaneous 4T1 tumors in 4T1 syngeneic mice with different treatments. ?T/?C of last time point was labeled. (b) Spider plot of tumor growth curve (expressed as tumor volume) of subcutaneous 4T1 tumors in 4T1 syngeneic mice with different treatments. (c) Flow cytometry analysis of the percentage of CD8+ T cells over CD45+ cells in tumor tissues with different treatments in 4T1 syngeneic model. (d) Flow cytometry analysis of the absolute number of CD8+ T cells per milligram tumor with different treatments in 4T1 syngeneic model. (e) Flow cytometry analysis of the percentage of Ki67+ CD8+ T cells over CD8+ cells in tumor tissues with different treatments in 4T1 syngeneic model. (f) Flow cytometry analysis of the percentage of GZMB+ CD8+ T cells over CD8+ cells in tumor tissues with different treatments in 4T1 syngeneic model. (g) IHC analysis of CD8 T cells in tumor tissues with different treatments from 4T1 syngeneic model. Y axis means the percentage of CD8+ cells over all the cells in the same slide. (h) MFI of the tumor surface MHC Class I with different treatments in 4T1 syngeneic model. (i) MFI of the tumor surface PD-L1 with different treatments in 4T1 syngeneic model. Flow cytometry data was analyzed and processed with FlowJo (Version 10.7.1, https://www.flowjo.com/solutions/flowjo/downloads/previous-versions).