Fig. 6: Immune responses induced by AMF-Bac.

A Illustration of potential immune responses induced by AMF-Bac. AMF induces the lysis of bacteria, and bacterial lysates may recruit and/or activate macrophages, neutrophils, NK cells, and/or DCs. The release of CD47nb blocks the “Don’t eat me” pathway and enhances the phagocytosis of tumor cells by macrophages. Besides innate immune responses, the adaptive immune responses may also be affected. The colons of BALB/c mice were inoculated with CT-26 cells (1 × 10⁵ cells/mouse) on day -7, and mice were treated with the indicated engineered bacteria (1 × 10⁸ CFU) by colon-specific administration on days 0 and 3, followed by AMF treatment (310 kHz and 23.8 kA/m) for 80 min at 24 h after colon administration. The Anti-CD47ab (20 mg/kg) and CD47nb (20 mg/kg) were i.p. injected on days 0 and 3 in G3 and G2/G6, respectively. Analysis of the immune responses was performed on day 9. B Flow cytometry analysis of the percentage of macrophages (F4/80⁺ cells) in tumors (n = 5 mice). C The ratio of M1 to M2 macrophages (F4/80⁺CD80⁺ cells vs. F4/80⁺CD206⁺ cells) in tumors (n = 5 mice). D Flow cytometry analysis of the expression of SIRPα in tumor-infiltrating macrophages (n = 5 mice, cf. Supplementary Figure 25B). E–I Flow cytometry analysis of tumor-infiltrating neutrophils (CD11b⁺Ly6G⁺ cells; E), NK cells (CD45⁺CD49b⁺ cells; F), CD8⁺ T cells (CD3⁺CD8⁺ cells; G), effector T cells (CD3⁺CD8⁺IFN-γ⁺ cells; H) and B cells (CD45⁺CD19⁺ cells; I) (n = 5 mice). J IFN-γ secretion by the splenocytes, as determined by ELISPOT assay after re-stimulation with a CT-26-specific antigen peptide (sequence: SPSYVYHQF; n = 4 mice). K, L Flow cytometry analysis of tumor-infiltrating, mature DCs (n = 5 mice). The percentages of CD11c⁺CD86⁺ (K) or CD11c⁺CD80⁺ (L) cells are shown. The data (B, C, E–I, K, L) are shown as the mean ± SD. Statistical analysis was performed by a two-tailed unpaired t test. ^*P < 0.05; ^**P < 0.01^{; ***}P < 0.001. Source data are provided as a Source Data file.