The attenuated hepatocellular carcinoma-specific Listeria vaccine Lmdd-MPFG prevents tumor occurrence through immune regulation of dendritic cells

Abstract

Immunotherapy is a promising treatment for liver cancer. Here, we tested the ability of the attenuated hepatocellular carcinoma-specific Listeria vaccine (Lmdd-MPFG) to treat hepatocellular carcinoma (HCC) in a mouse model. Immunization with the vaccine caused a strong anti-tumor response, especially in mice reinfused with dendritic cells (DCs). In mice that were also administered DCs, tumor suppression was accompanied by the strongest cytotoxic T lymphocyte response of all treatment groups and by induced differentiation of CD4+ T cells, especially Th17 cells. Additionally, the Lmdd-MPFG vaccine caused maturation of DCs in vitro. We demonstrated the synergistic effect of TLR4 and NLRP3 or NOD1 signaling pathways in LM-induced DC activation. These results suggest that the Lmdd-MPFG vaccine is a feasible strategy for preventing HCC.

Keywords: Lmdd-MPFG; PRRs; dendritic cells; hepatocellular carcinoma.

Fig. 1. LM promotes maturation of BMDCs and induces MPFG-specific T cells

Lmdd-MPFG was compared with WT L. monocytogenes, LPS, and a control (no stimulation), for its potency to promote maturation of BMDCs. DCs were harvested 24 hours after infection with LM (at a MOI of 20), and the expressions of CD80 and CD86 were assessed by flow cytometry, and the peak with gray filled represents the control (A and B). At the same time, supernatants from control or L. monocytogenes–infected DC cultures were collected for measurements of IL-12p70 and TNF-α by ELISA (C). DCs from HLA A2.1+ or HLA A2.1– donors were generated and infected as above, followed by co-culture with allogeneic SPCs for 4 days. Proliferation (D) was measured by ³H thymidine incorporation, and stimulation indices (SI) were calculated as described. CD8⁺ TIL function was analyzed by an IFN-γ ELISPOT assay (E). Standard assays were then developed according to the kit manual, and the numbers of spot-forming colonies were calculated. Results were collected from at least three independent experiments. Individual data and mean values are shown. Statistically significant differences are indicated as determined by Student's t-test (*p<0.05, **p<0.01, ***p< 0.001).

Fig. 2. TLRs and NLRs in LM-promoted dendritic maturation

BMDCs were collected for 24 h treatment (control, LPS, LM, and LM-HK). Messenger RNA levels of NLRs (A) and TLRs (B) were detected in each group by quantitative real-time PCR. Protein extracts were prepared and relative NLR and TLR protein levels were detected by western blot assays (C). Densitometry values relative to internal controls are displayed in the histograms (D). Activation of several signaling pathways was also analyzed by western blot (E). Summary statistics are depicted in the histogram (F). All data are presented as the mean ± SEM (*p<0.05, **p<0.01, ***p< 0.001).

Fig. 3. Cross-presentation of NLRs and TLRs in LM-treated DCs

DCs were harvested after 24 hours of LM infection (at a MOI of 10) (or of incubation without LM) and then treated to silence or stimulate NLRP3 expression with corresponding receptor agonists (LPS, TLR4 agonist; MDP, NOD1/2 agonist; MSU, NLRP3 agonist) for 6-8 h as described in the Materials and Methods section. Expression of the functional DC molecules CD80 and CD86 was assessed by flow cytometry, and the peak with gray filled represents the control (A and B). Supernatants were collected for quantification of IL-12p70, IL-β, TNF-αand IFN-γ by ELISA (C). Protein levels of NOD1, NLRP3 and TLR4 (D) and expression of NF-kB and caspase-1 (E) were detected by western blot. GAPDH was used as the internal control, and summary statistics are depicted in the histograms. Each data point represents the mean ± SEM from three independent experiments (*p<0.05, **p<0.01, ***p< 0.001).

Fig. 4. Combined treatment with DCs and LM immunization plays a role in tumor suppression in vivo

Subcutaneous tumors were generated by injecting mice with Hepa1-6-MPFG tumor cells; corresponding control cells were injected into mice to generate controls. Mice were then immunized with PBS, Lmdd-MPFG, DC+LM or SiNLRP3DC+LM (n=5/group). Tumor sizes were measured every 3 days, and tumors were isolated on day 45. The tumor sizes and weights were measured and compared between groups (A). The tumor growth rates (B) and long-term tumor-free survival times (C) of mice in the vaccination group are shown. Error bars show SEM. Long-term tumor-free survival at day 45. Each data point represents the mean ± SEM from four single tumors. Mouse spleens were isolated, and SPCs were purified and cultured. Quantitative real-time PCR was used to detect expression of NOD1, NLRP3, TLR4 and of the downstream molecules caspase-1, IL-1β and IL-18 in SPCs (D). (E) SPCs were cultured overnight, and then secretion levels of IL-12p70, IL-1β, TNF-α and IFN-γ in the culture supernatant were measured by ELISA. A western blot assay was used to detect the activation of NF-kB and caspase-1 in SPCs (F). GAPDH was used as a loading control. Each data point represents the mean ± SEM from three independent experiments (*p<0.05, **p<0.01, ***p< 0.001).

Fig. 5. Combined DC and LM treatment increases immune system effects in spleen

SPCs were extracted from tumor-bearing mice of each vaccination groups (n=5), pooled and stained for DC or T cell markers. CD80 and CD86 expression and frequency of mature DCs in the SPCs were analyzed by flow cytometric analysis of cells from the vaccinated or control group, and the peak with gray filled represents the PBS group as control (A and B). CD4+ and CD8+ T cells in SPCs were detected by flow cytometric analysis, and the frequencies of each were analyzed in histograms (C and D). Each data point represents the mean ± SEM from at least three independent experiments (*p<0.05, **p<0.01, ***p< 0.001).

Fig. 6. Combination therapy increases the number of Th17 cells within the tumor

TILs were separated from the treated mice in each group. CD4+ and CD8+ expression in TILs were evaluated by flow cytometry (A and B). CD4+ T cell differentiation was observed, TILs were stained with antibodies to detect IL-1- and IL-17A-producing cells and Treg cells by flow cytometry (C and D). Foxp3 was detected using intracellular antibody staining. Representative flow cytometry dot plots from each group show Foxp3 expression. Cumulative data for the percentage of positive cells in the gated CD4+ T cell subsets of TILs are illustrated in the dot plots. Data were analyzed in histograms of at least three independent tests and then analyzed by Student's t-test (*p<0.05, **p<0.01, ***p< 0.001).

Fig 7. DCs with immature phenotypes and functional defects in HCC patients

(A) TILs were isolated from 10 HCC tissues with paired adjacent tissues, and expression of CD80 and CD86 was assessed by flow cytometry. (B) Peripheral blood DCs were obtained from 10 HCC patients and 3 normal people. Proportions of functional DCs were detected by flow cytometry. (C) IHC staining of CD11c in human HCC and adjacent tissues. Statistical analysis of positive cells per field of each index of the slides stained with CD11c is shown in the histogram. Original magnification, 100×. Data presented are from at least five independent slides. (D) Peripheral blood DCs were infected by LM at a MOI of 20; real-time PCR was used to determine the expression of NOD1, NLRP 3 and TLR4 in HCC or normal peripheral blood DCs. All data represent the mean ± SEM from at least three independent experiments (*p<0.05, **p<0.01, ***p< 0.001).