Fig 2: FMD does not improve the efficacy of anti-PD-1/anti-CTLA4 combo therapy against B16 F10 melanoma tumor.A Schedule of tumor implantation and treatment for B16F10 syngeneic tumor models. B, C B16 Tumor growth in immunocompetent C57/BL6 syngeneic mice treated with isotype control and anti-PD-1/anti–CTLA4 and fed with standard diet or FMD (AL NT n = 12; FMD NT n = 12; AL PD1 CTLA4 n = 10; FMD PD1 CTLA4 n = 11). Analysis of tumor immune infiltrate by FACS: D CD8+ CD44+GzmB+ cytotoxic T cell (AL NT n = 4; FMD NT n = 3; AL PD1 CTLA4 n = 6; FMD PD1 CTLA4 n = 5); E CD8+ CD44+CD62L- effector memory T cell (AL NT n = 6; FMD NT n = 5; AL PD1 CTLA4 n = 6; FMD PD1 CTLA4 n = 5); F CD45+NKp46+GzmB+ NK cell (AL NT n = 6; FMD NT n = 5; AL PD1 CTLA4 n = 6; FMD PD1 CTLA4 n = 5). Statistical analysis was performed using one-way analysis of variance (ANOVA). P values were determined by one-way ANOVA with Tukey’s post analysis Differences were considered significant when P ≤ 0.05. All data are represented as mean ± SEM. Source data are provided as a Source Data file.

Fig 3: FMD does not improve immune-therapy efficacy against lung LLC1 cancer, but reduces immune-therapy related cardiac damage.A Schedule of tumor implantation and treatment for B16F10 syngeneic tumor models. B LLC1 Tumor growth in immunocompetent C57/BL6 syngeneic mice treated with isotype control, anti-OX40/anti-PD-L1 and anti-PD-1/anti–CTLA4 and fed with standard diet or FMD (AL IgG n = 13; FMD IgG n = 13; AL OX40PDL1 n = 12; FMD OX40PDL1 n = 14; AL PD1CTL4 n = 12; FMD PD1CTLA4 n = 13). Analysis of tumor immune infiltrate by FACS: C CD8+ on CD45+ T cells (n = 5); D GZMB+ on CD8+ T cells (n = 5); E CD4+ on CD45+ T cell (n = 5); F FOXP3+ on CD4+ T cells (n = 5); G H&E staining in the heart of C57/J mice bearing LLC1 lung tumor and treated with IgG or anti-OX40/anti-PD-L1 or anti-PD-1/anti-CTLA-4; H CD3 and CD8 staining in the heart of C57/J mice bearing LLC1 lung tumor and treated with IgG or anti-OX40/anti-PD-L1 or anti-PD-1/anti-CTLA-4 (3 tissue sections for each tumor; 5 tumors per each group); I Quantification of heart fibrosis and necrosis in the different experimental group; L Quantification of pro collagen 1α1 and MMP9 in the heart belonging to different experimental group (n = 5); M Quantification of CD3 and CD8 cell count in heart belonging to different experimental group (3 tissue sections for each tumor; 5 tumors per each group). Statistical analysis was performed using one-way analysis of variance (ANOVA) and Global Kruskal Wallis test. P values were determined by One-way ANOVA with Tukey’s post analysis. Differences were considered significant when P < 0.05. All data are represented as mean ± SEM. Source data are provided as a Source Data file.

Fig 4: Changes in PBMC profiles after immunization. Flow cytometric analysis of PBMC samples obtained from the different groups of mice, targeting CD68 (a monocyte/macrophage cellular marker), CD4 (a co-receptor for the T cell receptor and specific marker of T helper cells), CD8a (a co-receptor for the T cell receptor and specific marker of cytotoxic T cells), and CD335 (a cytotoxicity-activating receptor that mediates tumor cell lysis and distinguishes NK cells from other populations). The bar graphs show the percentage of CD68+ (A), CD4+ (B), CD8+ (C), double positive CD4+/CD8+ (D), and CD335+ (E) cells in PBMCs from the different groups of mice. Results are presented as the mean ± SEM (n = 4, ** p < 0.01, * p < 0.05, # p < 0.1).

Fig 5: FMD prevents heart inflammation associated with immune therapy administration by reducing cardiac immune cell infiltration.A CD3 and CD8 staining in the heart of C57/J mice bearing B16-F10 melanoma tumor and treated with IgG or anti-OX40/anti-PD-L1 or anti-PD-1/anti-CTLA-4. B Quantification of CD3 and CD8 cell count in heart belonging to different experimental group (3 tissue sections for each tumor; 5 tumors for each group). Global Kruskal Wallis test with post hoc Mann–Whitney U-test was used to analyse the overall difference between groups. Differences were considered significant when P-value < 0.05. All data are rapresented as mean ± SEM. Source data are provided as a Source Data file.