Fig 2: Anticancer activity and improved tumor immune microenvironment of a combination of anti-PD-1 antibody and Met@Man-MPs in AOM/DSS-induced CAC mice.a Schematic schedule for anticancer experiment in AOM/DSS-induced CAC mice. b Numbers of tumors with size>3 mm and <3 mm in CAC mice after treatment with PBS, Man-MPs, Met@Man-MPs, anti-PD-1 antibody, a combination of Man-MPs and anti-PD-1 antibody, or combination of Met@Man-MPs and anti-PD-1 antibody at the anti-PD-1 antibody dosage of 100 µg per mouse and Met dosage of 10 mg kg-1 indicated in (a). Data are presented as mean ± s.d. (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). c–j Percentages of M1-like TAMs (c), M2-like TAMs (d), CD8+ T cells (e), CD8+CD69+ T cells (f), CD4+ T cells (g), CD4+CD69+ T cells (h), MDSCs (i) and Tregs (j) in colon tumors of CAC mice after treatment with PBS, Man-MPs, Met@Man-MPs, anti-PD-1 antibody, a combination of Man-MPs and anti-PD-1 antibody, or combination of Met@Man-MPs and anti-PD-1 antibody at the anti-PD-1 antibody dosage of 100 µg per mouse and Met dosage of 10 mg kg-1 indicated in (a). Data are presented as mean ± s.d. (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). Source data are provided as a Source Data file.

Fig 3: Activation of CD8+ T cell by peptides on SARS-CoV-2 Spike protein. (A, B) Mitomycin pretreated T2 cells were loaded with n-Sp1, 2, 6, 7, 11, 13, and 14 peptides and incubated with CD8+ T cell from healthy donors at a 1:1 ratio. Activation, cytotoxicity, and generation of epitope-specific CD8+ T cells were evaluated. Expression level of the T-cell activation marker CD69 was evaluated with flow cytometry after 16 h of stimulation. (B) Values indicate the percentage of CD69+CD8+ T cells, n = 3. (C, D) Expression level of the T-cell activation marker CD137 was evaluated with flow cytometry after 16 h stimulation. (D) Values indicate the percentage of CD137+CD8+ T cells. n = 3. (E, F) Epitope-specific CD8+ T-cell-mediated cytotoxicity was evaluated after 7 days of culture. The remaining CFSE-labeled T2 cells were calculated as survived target cells. (F) Values indicate the percentage of surviving T2 cells, n = 3. Apoptosis of T2 cells after 7 days culture. (G, H) Epitope-stimulated T-cell-mediated T2 apoptosis was calculated as the proportion of CFSE+AnnexinV+ cells. (H) Values indicate the percentage of CFSE+Annexin V+ T cells, n = 3. (I, J) The expression of IFN-? after epitope stimulation for 7 days. IFN-? was measured with intracellular staining flow cytometry. (J) Values indicate the percentage of IFN-?+CD8+ T cells, n = 6. (K, L) Epitope-specific CD8+ T-cell generation after 7 days of stimulation. Stimulated CD8+ T cells were stained with given epitope-based tetramer and measured with flow cytometry. (L) Values indicate the percentage of tetramer+ cells, n = 5. Day 0 ctrl: staining before stimulation; T2 ctrl: T2 without peptide loading; Pos ctrl: positive control, T2 loaded with influenza A M1 peptide M58-66 GILGFVFTL. Neg ctrl, negative control; T2 loaded with Zika virus P30-38 GLQRLGYVL peptides. (M, N) A representative dot plot showing the measurement of peptide specific CD8+ T cells in HLA-A2+ non-vaccinated and vaccinated donors by flow cytometry. (N) Values indicate the percentage of n-Sp1 tetramer+CD8+ T cells, n = 5 of non-vaccinated donors and n = 16 of vaccinated donors. The data are represented as the mean ± SD in three independence experiments, ***p = 0.001.

Fig 4: Antitumor activity by anti-B7H3×CD3 BiTE in combination with SOR in vitro. (A) Morphology of tumor cells after different treatments for 24 h. Scare bar, 100 µm. (B) Normal activation marker CD69 and CD25 expression on T cells. Human T cells were co-cultured with SKOV3 by adding 5 µM SOR alone or in combination with 5 µg/mL anti-B7H3×CD3 BiTE. T cells were collected 24 h and measured by 3-color flow cytometry. (C) Statistical analysis of the mean fluorescence intensity in figure B. *P < 0.05, **P < 0.01. (D) Infiltration and killing activity of T cells was detected using the 3D tumorsphere model after various treatments. SKOV3 tumoursphere were cocultured with T cells labelled CFSE. Scare bar, 50 µm. (E) Effect of different treatments on real-time cell cytotoxicity analyzer curves generated with SKOV3 cells.

Fig 5: The degradation of B7H3 mainly depends on the autophagy–lysosomal pathway.A, Western blot showing LC3-II, a marker of autophagy, and B7H3 expression in different breast cancer cell lines. B, immunofluorescence detection of LC3-II/LAMP1 and B7H3 in different breast cancer cell lines which showing B7H3 was enriched within autophagosomes and lysosomes. Scale bar, 25 µm. C, Western blot showing LC3-II and B7H3 expression in MCF-7 and BT-474 treated with or without 150 nM Baf A1 for 6 h, an inhibitor of lysosome. D, flow cytometry analysis showing B7H3 expression in MCF-7 and BT-474 treated with or without Baf A1. E and F, flow cytometry analysis showing the proportion of Granzyme B (GZB)+CD8+T cells (E) and CD69+CD8+T cells (F). The left showed the representative flow cytometry images, and right showed the statistical columns. All experiments were performed three times independently. For D–F, data were presented as means ± SD, *p < 0.05, **p < 0.01 by Student’s t test. B7H3, B7 homolog 3.