Fig 2: IR-780 induces immunogenic cell death (ICD) in vivo. (A) Immunofluorescence detection of CRT, CD3, and CD8 expression in tumor sections; scale bar = 20 μm. The percentages of tumor-infiltrating (B) CD3+, (C) CD4+, and (D) CD8+ T cells were analyzed by flow cytometry. And the activation of T cell populations was measured by detecting the percentage of (E) CD4+CD69+ and (F) CD8+ CD69+ T cells populations. (G) The activation of CD80+CD86+ DCs was analyzed by flow cytometry. (H) Schedule representation of different treatment-mediated antitumor vaccination effects in CT26 tumor models. (I) After subcutaneous injection of different treated CT26 cells (10 μM IR-780, 1 μM MTX, and 20 μM CIS, respectively) in the left flank of mice, the tumor volumes in the right flank of each group mice were detected and compared (tumor volume = length × width 2 × 0.5). (J) The frequency of tumor-free mice in each group after vaccinated with different pretreated CT26 cells (n = 10). All the data are presented as mean ± SD. *p < 0.05; **p < 0.01 as comparing with control group.

Fig 3: In vivo immunotherapy of BMS. (a) Quantified signal intensity after different formulation treatment at 16 d. (b) The body weight of GBM bearing mice in the progress of treatment. (c) Representative propotion of CD80 and CD86 cells expression after different formulation (PBS, SAL, BM, BMS) treatment. (d) The quantitative analysis of CD80, CD86 cells in GBM tissues after treatment according to Fig. 6c. (e) Representative propotion of CD163 and CD206 cells expression after different formulation (PBS, SAL, BM, BMS) treatment by flow cytometry. (f) The quantitative analysis of CD163, CD206 cells in GBM tissues after treatment according to Fig. 6e. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: G2A-activation does not alter proinflammatory markers in bone marrow-derived macrophages. (A) G2A-mediated macrophage migration. Data are shown as mean ± S.E.M. (n = 3–4). Migration of BMDMs from wild type and G2A−/− mice was not induced (control) or induced by serum and 9-HODE. Unpaired two-tailed Student‘s t-test. **p < 0.01 as compared to control. (B) RT-PCR analysis for G2A expression in BMDM, differentiated for 7 days with 20 ng/ml M-CSF and stimulated with 100 ng/ml LPS or 1 μM 9-HODE for 24 h. Data are shown as 2(−ΔΔCT), mean ± S.E.M. (n = 4). (C–H) Marker expression by bone marrow-derived macrophages from wild type mice after stimulation with 100 ng/ml LPS or 1 μM 9-HODE for 24 h. CD80 and CD86 expression were analyzed by FACS, nitrite using the Griess test and IL1β, IL6, and TNFα by ELISA. Data are shown as mean ± S.E.M. (n = 4–5). One-Way ANOVA with Bonferroni post-hoc Test *p < 0.05, **p < 0.001, ***p < 0.0001, ****p < 0.00001.

Fig 5: DsCystatin inhibits LPS or Borrelia Burgdorferi-stimulated activation of mouse bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs). (A,B) DsCystatin inhibits LPS-induced cytokine production: (A) cytokine mRNA level as determined by quantitative real-time PCR and normalized with mouse β-actin gene. (B) Cytokine protein level as determined by enzyme-linked immunosorbent assay. (C,D) DsCystatin inhibits B. burgdorferi-induced cytokine production at mRNA (C) and protein (D) levels. Cells that were not stimulated with LPS or B. burgdorferi were served as negative controls (NC). Results are expressed as mean + SEM of three triplicate samples. *p < 0.05 and **p < 0.01 (t-test). The representative results from at least three independent experiments are shown. (E,F) DsCystatin downregulates the surface expression of CD80 and CD86 on LPS stimulated BMDMs (E) and BMDCs (F). (G) DsCystatin inhibits OVA cleavage in BMDCs. Cells were pre-incubated for 3 h at 37°C in the presence or absence of 4 µM DsCystatin and further incubated with DQ-OVA (1 µg/ml) for 2 h at 37°C. OVA fragmentation was analyzed by flow cytometry. The representative results from three independent experiments are shown.