Fig 2: V9302 enhanced CD8+T-cells infiltration and activation by promoting autophagy to reduce B7H3 expression in EO771 mouse model.A, autophagy marked by LC3-II immunostaining in EO771 tumors treated as indicated. Scale bars, 50 µm. B and C, B7H3 expression in EO771 tumors was detected by immunostaining (B) and flow cytometry (C). Scale bars, 50 µm . D, flow cytometry analysis of CD8+ T cells percentage in EO771 tumors treated as indicated. E, representative images of CD8 immunostaining in EO771 tumors treated as indicated. Scale bars, 50 µm. F, flow cytometry analysis of GzB+ CD8+ T cells percentages in EO771 tumors. IgG, rat IgG2a; P, anti-PD-1 antibody; V, V9302; P + V, V9302 plus anti-PD-1. For A, B, and E n = 3 per group, for C, n = 5 per group, for the rest, n = 4 per group. Data were presented as means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t test.

Fig 3: Autophagy, B7H3 expression, and CD8+T cells infiltration profile in breast cancer. Representative images of CD8+ T cell infiltrated areas (A), B7H3 expression (B) and LC3-II dots (C) by IHC in breast cancer tissue. Scale bar, 50 µm . D, the relationship between B7H3 expression and CD8+T cell infiltration percentage. E, the relationship between autophagy and B7H3 expression. Tumor samples were obtained from 65 patients with invasive breast carcinoma at the Sixth Affiliated Hospital, and data were presented as means ± SD; *p < 0.05 by Mann–Whitney U test. B7H3, B7 homolog 3; IHC, immunohistochemistry.

Fig 4: NAC blocks V9302 modulated ROS- autophagy-B7H3 axis in MCF-7.A, cell viability assay showing 5 mM NAC reversed the inhibitory effects of different concentration of V9302 (V). B, Ki67 staining showing 5 mM NAC restores cell proliferation inhibited by different concentration of V9302 (V). C, apoptosis assay by annexin V-FITC/PI staining showing 5 mM NAC decreased the percentages of apoptotic cells when treated with different concentration of V9302 (V). D, the apoptosis of MCF-7 cells was determined by Western blotting for cleaved and total PARP with different dose of V9302 (V) with or without 5 mM NAC treatment. E, flow cytometry assay probed by DCFDA showing 5 mM NAC stopped ROS generation in MCF-7 exposing to different dose of V9302 (V). F, flow cytometry analysis showing 5 mM NAC led to the upregulation of B7H3 expression in MCF-7 when treated with different concentration of V9302 (V). G, Western blot showing NAC reduced LC3-II expression and led to B7H3 accumulation in MCF-7 when treated with different concentration of V9302 (V). H and I, cytometry analysis showing 5 mM NAC suppressed V9302 (V) induced CD8+ CTLs activation indicated by the proportion of Granzyme B (GZB)+CD8+T cells (H) and CD69+CD8+T cells (I). For A, n = 5 independent experiments, for C and H, n = 4 independent experiments, for the rest, n = 3 independent experiments. Data were presented as means ± SD. **p < 0.01; ***p < 0.001 by Student’s t test. NAC, N-acetylcysteine.

Fig 5: An overview schematic demonstrating that V9302 enhanced CD8+T cell activation by promoting B7H3 degradation via autophagy–lysosome pathway and potentiated the antitumor effect of anti-PD-1 immune therapy.