Immune adjuvant effect of a Toxoplasma gondii profilin-like protein in autologous whole-tumor-cell vaccination in mice

Abstract

Profilin-like protein in Toxoplasma gondii (TgPLP) is a Toll-like receptor (TLR) agonist. In this study, we investigated whether TgPLP has an adjuvant effect on immune function in autologous whole-tumor-cell vaccine (AWV) treatment. Mice vaccinated with AWV together with recombinant TgPLP protein had smaller CT26 tumors and increased survival. TgPLP treatment strongly increased the production of IL-12 through MyD88 signaling and several chemokines, including CCL5, CCL12, and XCL1, in bone marrow-derived macrophages (BMMs). In addition, TgPLP increased the phagocytosis of tumor cells by BMMs and promoted immune cell mobility on a tumor-matrigel scaffold. TgPLP triggered immune responses as demonstrated by increased expression of antigen presenting cell markers (MHC class I and II, B7.1, and B7.2) in BMMs and increased IL-12 and IFN-γ expression in mice. Mice vaccinated with AWV and TgPLP had more immune cells (CD4+ and CD8+ T cells, natural killer cells, and macrophages) in the spleen and higher total IgG and IgG2a concentrations in the blood than mice vaccinated with AWV alone. These findings suggest that TgPLP is a TLR-based vaccine adjuvant that enhances antitumor immune responses during vaccination with AWV.

Keywords: Toll-like receptor; Toxoplasma gondii; antitumor; profilin; vaccine.

Figure 1. Tumor reduction in CT26-tumor-bearing BALB/c mice vaccinated with AWV and/or TgPLP

Survival rate (%) and changes in tumor size (mm³) in mice vaccinated with AWV and/or TgPLP were investigated and compared with tumor-bearing mice without vaccination with AWV and/or TgPLP. A. Survival rates (%) in each experimental group were designated by the ratio compared with normal mice (all normal mice survived during the experimental period). B. Tumor size (mm³) in each experimental group between day 18 and 32 after tumor induction. Tumor sizes in mice vaccinated with TgPLP+AWV were significantly decreased compared with tumor-bearing mice without vaccination from day 18 when tumor size was monitored. * indicates statistical significance (p < 0.05).

Figure 2. IL-12 production and mRNA expression of the IL-12 subunits (p35 and p40) in BMMs after TgPLP treatment

A. IL-12 production was significantly increased by the addition of TgPLP (1 μg/ml) (p < 0.05). B. The induction of IL-12-p35 and -p40 expression reached peak levels 12 hours after TgPLP treatment as shown by real-time PCR analysis. C. IL-12 production induced by TgPLP treatment was dependent upon MyD88 signaling. MyD88 siRNA in BMMs decreased the production of IL-12. * indicates statistical significance (p < 0.05).

Figure 3. Cytokine and chemokine array of BMMs treated with TgPLP

BMMs were incubated with TgPLP (1 μg/ml) for 24 hours. The concentration of cytokines in the culture supernatant was analyzed using a cytokine array kit. Data represent a relative expression rate (%) compared with values in untreated BMMs.

Figure 4. Increased phagocytosis in BMMs after TgPLP treatment

A. EGFP⁺-CT26-cells were exposed by UV for 10 min, and then used as target cells. The increase in phagocytic ability of BMMs after TgPLP treatment was observed by fluorescence microscopy. Green colored spots represent EGFP⁺-CT26-cell-engulfed BMMs. The green colored spots increased in LPS- and TgPLP-treated BMMs. B. Wild-type CT26 cells were exposed by UV for 10 min, and then used as target cells. The increase in phagocytic ability of BMMs in the presence of LPS or TgPLP was observed by FACS analysis. Phycoerythrin (PE)-conjugated antibody against F4/80 and FITC-conjugated Annexin V were used for the observation of double positive signals. The data was represented by median fluorescence index (MFI), and showed the increase in BMMs that CT26 cells had been phagocytosed.

Figure 5. Increased BMM mobility (in vitro) and cell infiltration into a matrigel TM scaffold (in vivo) after TgPLP treatment

A. The mobility assay was performed to investigate the change of chemotactic BMM movement after LPS or TgPLP treatment. B. The degree of cell infiltration in mice was investigated using matrigel mixed with AWV and/or TgPLP and visualized by H&E staining and DAPI-fluorescence. C. The degree of cell infiltration was measured by the infiltrated cell thickness, which was visualized by H&E staining and measured using ImageJ. * indicates statistical significance (p < 0.05).

Figure 6. Increase in antigen presenting cell surface markers after TgPLP treatment

BMMs were cultured with LPS or TgPLP and the level of cell surface markers, MHC1, MHC2, CD80 and CD86, were measured by FACS analysis. Data are represented as a median fluorescence index (MFI). TgPLP is more effective than LPS at inducing antigen-presentation marker expression.

Figure 7. Immune characteristics of BALB/c mice vaccinated with AWV and/or TgPLP

After vaccination with AWV and/or TgPLP, mouse sera were collected and cytokines (IL-12 and IFN-γ) and immunoglobulins (total IgG and IgG2a) were measured using ELISA kit. Data represent mean ± SD (n = 5). * indicates statistical significance (p < 0.05).

Figure 8. Spleen cell phenotypes of BALB/c mice vaccinated with AWV and/or TgPLP

After vaccination, spleens were harvested and cell populations were examined by FACS analysis. Cell numbers were calculated as the ratio of each cell population (CD19⁺, DX5α⁺, F4/80⁺, CD4⁺ or CD8⁺) to total splenocyte number. Data present mean ± SD (n = 5). * indicates statistical significance (p < 0.05).