Fig 2: (a) Schematic depiction of the experimental approach for the abscopal effect of the PDM MNs. (b) Photos of primary tumors on day 12. (c) Tumor-growth curves of primary tumors, (d) the relative tumor weight, and (e) the relative fold change of tumor volume. (f) Photos of distal tumors on day 12. (g) Tumor-growth curves of distal tumors, (h) the relative tumor weight, and (i) the relative fold change of tumor volume. (j) The percentage of the matured DCs (CD40+CD11c+MHC II+) in lymph nodes. (k) The percentage of the CD8+ T cells (CD3+ CD8+) in primary tumor tissue. (l) The percentage of the GZMB+ CTLs (GZMB+ CD8+) in primary tumor tissue. (m) The percentage of the CD8+ T cells (CD3+ CD8+) in distal tumor tissue. (n) The percentage of the GZMB+ CTLs (GZMB+ CD8+) in distal tumor tissue. The significance was defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: (a) Immunofluorescence staining of CRT (green) in tumor tissue and (b) the relative quantified results. Scale bar =100 µm. (c) Immunofluorescence staining of ß-catenin (green) in tumor tissue and (d) the relative quantified results. Scale bar =100 µm. (e) The CCL4 level in tumor tissue. (f) Immunofluorescence staining of DCs (CD40+CD11c+) in tumor tissue and (g) the relative quantified results. Scale bar: 100 µm. (h) The percentage of the matured DCs (CD40+CD11c+MHC II+) in lymph nodes. (i) The percentage of the CD8+ T cells (CD3+ CD8+) in tumor tissue. (j) The percentage of the GZMB+ CTLs (GZMB+ CD8+) in tumor tissue. (k) Immunofluorescence staining of GZMB+ CTLs (GZMB+ CD8+) in tumor tissue and (l) the relative quantified results. Scale bar: 100 µm. (m) IFN-?, (n) IL-6, and (o) IL-12 levels in sera and tumor tissue of mice. The significance was defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 4: Inhibition of SIRT2 activity enhances macrophage stimulation in the lungs and spleen of infected animals.(A) After overnight stimulation with CSA, the cells isolated from the lungs of infected C57BL/6 animals were surface stained with CD11b (APC/Cy7), CD11c (APC), CD80 (FITC) and MHC-II (PE) followed by flow cytometry analysis. Gating strategy employed and flow cytometry dot plots of CD11b+CD80+, CD11b+MHCII+, CD11c+CD80+, CD11c+MHCII+ cells in the lungs of infected mice (B–E) Percentage of (B) CD11b+CD80+, (C) CD11b+MHCII+, (D) CD11c+CD80+ and (E) CD11c+MHCII+ cells in the lungs. (F–I) Percentage of CD11b+CD80+, CD11b+MHCII+, CD11c+CD80+, CD11c+MHCII+ cells in the spleen of infected mice. Data is representative of two independent experiments with five mice per group. Each graph represents mean ± SD (n = 5). ***p<0.0005.Figure 7—source data 1.AGK2 treatment enhances macrophage stimulation in the lungs and spleen of infected animals.Source data for Figure 7B–I.

Fig 5: Trained Immunity Shapes the Transcriptional Profile of TANs and Neutrophils from ß-Glucan-Trained Mice Suppress Tumor Growth via ROS Production(A–E) WT mice were treated with ß-glucan or PBS and after 7 days were inoculated with B16-F10 melanoma cells. TANs (CD45+CD11c-CD11b+Ly6c-Ly6g+) were sorted and RNA sequencing analysis was performed (n = 5 mice in the PBS group; n = 4 mice in the ß-glucan group) 14 days after the tumor injection. In (A), (C) and (E), false discovery rate (FDR) = 0.05.(A) Differential gene expression in TANs from mice pre-treated with ß-glucan compared with TANs from PBS-treated mice. Volcano plot showing the distribution of the adjusted p values (-log10(padj)) and fold changes (log2 fold change).(B) GSEA for genes related to a gene set previously implicated in the TAN1 phenotype (Shaul et al., 2016) was used to analyze the transcriptomic effect induced in TANs by ß-glucan administration in mice. Abbreviation is as follows: NES, normalized enrichment score.(C) Top 10 enriched canonical pathways identified by IPA in TANs from ß-glucan-treated mice compared to PBS-treated mice.(D and E ) Shown in (D) is a GSEA for genes related to ROS pathway, and in (E) is a heatmap of genes involved in ROS metabolic process in TANs from ß-glucan-treated mice as compared with TANs from PBS-treated mice.(F) WT mice were treated with ß-glucan or PBS and after 7 days were inoculated with B16-F10 melanoma cells. Staining for ROS in TANs (CD45+CD11b+Ly6g+) was performed by flow cytometry 14 days after the tumor cell injection. Relative mean fluorescence intensity (MFI) is shown. MFI of ROS was measured and expressed in relation to the PBS group, set as 1. Data are presented as mean ± SEM (n = 15 per group).(G) Splenic neutrophils were isolated from mice 7 days after injection with ß-glucan or PBS. Neutrophils were co-cultured with luciferase expressing B16-F10 cells for 24 h. Tumor cell survival was assessed by measuring luminescence (n = 5 per group).(H) Experimental scheme.(I) As indicated in (H), WT or NCF1-deficient mice were injected with ß-glucan or PBS, and after 7 days splenic neutrophils were isolated and were adoptively transferred together with B16-F10 cells into WT recipients. Tumor volume is shown. Data are presented as mean ± SEM (n = 7 mice in the PBS WT group; n = 12 mice in the ß-glucan WT group; n = 11 mice in the PBS NCF1-deficient group; n = 14 mice in the ß-glucan NCF1-deficient group).*p < 0.05, ****p < 0.0001.See also Figure S2.