Fig 2: SVMAV promoted DC functions in vitro. (A) FITC fluorescence intensity of BMDCs with different treatments. (B) Frequency of CD11c+CD80+SIINFEKL+ BMDCs after different treatments. (C) Schematic illustration of the experimental design for cytolytic activity analysis of splenocytes primed by BMDCs. (D) The cytotoxicity of BMDCs treated with different formulations of vaccines was assessed by the LDH assays. (E) The viability of the remaining B16/F10-OVA cells was measured by the CCK8 assay. (F) Representative Photographs of the remaining B16/F10-OVA cells stained with crystal violet for different groups. Data are displayed as mean±SD. *P<0.05, **p<0.01, ****p<0.0001. BMDCs, bone marrow-derived dendritic cells; DC, dendritic cell; FITC, fluorescein isothiocyanate; LDH, lactate dehydrogenase; OVA, ovalbumin; SVMAV, self-assembling vehicle-free multicomponent antitumor nanovaccine.

Fig 3: Huaier enhances activation of DC cells. (A) The experimental design and cell culture process of bone marrow derived DC cells. (B) The populations of activated DCs (CD86high CD11c+) were presented via the FlowJo analyses. (C) Scheme of the vaccination assay in vivo. (D) 4T1 cells were pretreated with Huaier, or freeze-thawed, followed by inoculated s.c. into BALB/c mice as a vaccine (n = 6 for each group). A week later, mice were rechallenged with live 4T1 cells. (E) Individual tumor growth curves of tumor-bearing mice in three groups. s.c., subcutaneously; *p < 0.05; **p < 0.01.

Fig 4: DMF treatment differentially affects the infiltrated immune cells in ileum LP of EAE-affected mice. (A) DMF treatment does not significantly revert the increase in CD45+ cells in ileum LP induced by EAE. Confocal microscopy images of the ileum of naïve and EAE-affected mice treated or not with DMF (left panel) and quantification of the CD45+ cells (right panel). Slides were stained with anti-CD45 antibody (red) and with DAPI (blue). Scale bar: 50 µm. (B) DMF treatment of EAE-affected mice increases the percentage of CD11c+ cells, but has no effect on the increased CD4+ cell population in the ileum LP. FACS analysis of CD11c+ and CD4+ T cells in LP from naïve mice and at 4 days post onset from EAE-affected mice, treated or not with DMF. Average clinical scores of mice at time of the analysis: WT-EAE-affected mice: 3 ± 0.17; WT-EAE-affected mice treated with DMF: 2.75 ± 0.11. Results are shown as mean ± SEM of at least N = 3 independent experiments, with at least N = 3 mice per group per experiment. *P < 0.05.

Fig 5: SHV could suppress breast tumor in BLAB/c mice. (a) Analysis of apoptosis by flow cytometry in the injection site of mice after NIR irradiation (808 nm, 100 mW/cm2, 10 min). (b, c) Secretion levels of TNF-a (b) and IFN-? (c) in the blood and interstital fluid from immunized mice. (d) Activated DC ratio in inguinal lymph nodes for mice with different treatment (n = 3). APC anti-CD86 and PE anti-CD11c stained with CD86 and CD11c markers in DCs. (e) Cytotoxic T lymphocytes in interstitial fluid after treatment (n = 3). CD3 and CD8a as markers on CD8+ T cell surfaces stained with APC anti-CD8a and FITC anti-CD3. Experiments were repeated three times. (f) Activated B cell ratio in bone marrows for mice with different treatment (n = 3). PE anti-CD138 and FITC anti-CD45 stained with CD138 and CD45 markers in B cells. (g) Helper T cells in interstitial fluid after treatment (n = 3). CD3 and CD4 as markers on CD4+ T cell surfaces stained with Percp Cy5.5 anti-CD4 and FITC anti-CD3. Experiments were repeated three times. (h) Activated Central Memory T cell ratio in interstitial fluid for mice with different treatment (n = 3). PE anti-CD62L and APC anti-CD44 stained with CD62L and CD44 markers in Tcms. (i) Anti-4T1 IgG titer in spleen, blood, peritoneal fluid and interstitial fluid. (j) The growth curve of tumors in BLAB/c mice. (k) Representative photographs of tumors two weeks after injection of 4T1 cells in pre-immunized BLAB/c mice. (l) The body weight change curve of 4T1 tumor-bearing mice during the three weeks evaluation period. Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test (b, c, d, e, f, g, h, i and j), *P < 0.05; **P < 0.01; ***P < 0.001.