Dendritic cell-based vaccine prepared with recombinant Lactococcus lactis enhances antigen cross-presentation and antitumor efficacy through ROS production

Abstract

Introduction: Lactococcus lactis (L.L) is safe and can be used as vehicle. In this study, the immunoregulatory effect of L.L on dendritic cell (DC) activation and mechanism were investigated. The immune responses and antigen cross-presentation mechanism of DC-based vaccine prepared with OVA recombinant L.L were explored.

Methods: Confocal microscopy and flow cytometry were used to analyze the mechanism of L.L promoting DC maturation, phagosome membrane rupture and antigen presentation. The antitumor effect of DC vaccine prepared with L.L-OVA was assessed in the B16-OVA tumor mouse model.

Results: L.L significantly promoted DC maturation, which was partially dependent on TLR2 and downstream MAPK and NF-κB signaling pathways. L.L was internalized into DCs by endocytosis and did not co-localized with lysosome. OVA recombinant L.L enhanced antigen cross-presentation of DCs through the phagosome-to-cytosol pathway in a reactive oxygen species (ROS)- and proteasome-dependent manner. In mouse experiments, L.L increased the migration of DCs to draining lymph node and DC vaccine prepared with OVA recombinant L.L induced strong antigen-specific Th1 and cytotoxic T lymphocyte responses, which significantly inhibited B16-OVA tumor growth.

Conclusion: This study demonstrated that recombinant L.L as an antigen delivery system prepared DC vaccine can enhance the antigen cross-presentation and antitumor efficacy.

Keywords: antitumor efficacy; cross-presentation; dendritic cell-based vaccine; reactive oxygen species; recombinant Lactococcus lactis.

Figure 1

L.L enhanced GM-DC maturation by TLR2 signaling pathway. (A) GM-DCs were pretreated with mAb-TLR2 (blocking antibody) for 1 h, and then treated with LPS and L.L for 24 h. The expression of CD40 and CD86 were tested by flow cytometry. The IL-12p40 secretion was measured by ELISA. (B) GM-DCs were treated with L.L for 30 min and 120 min, then proteins of GM-DCs were isolated to detect their expression and phosphorylation by Western blot. (C) GM-DCs were pretreated with JNK inhibitor SP600125, ERK inhibitor U0126 and p38 MAPK inhibitor SB202190 for 45 min, 1h and 2 h, and then treated with LPS and L.L for 24 h. The expression of CD40, CD80 and IL-12p40 was measured. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to untreated group.

Figure 2

The expression of EGFP and its subcellular localization in GM-DCs. (A) Determination of growth curve of recombinant L.L. (B) Detection of EGFP expression in GM-DCs by flow cytometry and confocal laser scanning microscopy after co-cultured with recombinant L.L for 3 h, 6 h, 9 h, 12 h and 24 h. The time of the EGFP signal gating strategy is 6 h. (C) The co-localization of EGFP with EEA1 and LAMP1 was detected by confocal laser scanning microscopy. The higher magnification of areas in white boxes and quantification of colocalization were shown in right panel. * p < 0.05, ** p < 0.01; *** p < 0.001 compared to L.L group.

Figure 3

ROS production and amount of L.L-CFSE in GM-DCs. (A) The generation of ROS in GM-DCs was detected by flow cytometry after co-cultured with different concentrations of L.L for 6 h. (B) The fluorescence intensity of L.L-CFSE was detected by flow cytometry after the addition of DPI and NAC for 12 h. DPI inhibits ROS production in phagosomes. NAC inhibits all ROS production in cells. (C) CFSE subcellular localization and fluorescence intensity in GM-DCs were analyzed by confocal laser scanning microscopy after co-cultured with L.L-CFSE for 6 h in the presence or absence of DPI. The higher magnification of areas in white boxes and quantification of colocalization were shown in right panel. *** p < 0.001 compared to untreated group, or between indicated groups.

Figure 4

Subcellular localization of L.L-CFSE in GM-DCs. (A) Co-localization of L.L-CFSE and EEA1 after co-cultured with GM-DC for 2 h, 4 h and 6 h. (B) Co-localization of L.L-CFSE and LAMP1 after co-cultured with GM-DC for 2 h, 4 h and 6 h. The higher magnification of areas in white boxes and quantification of colocalization were shown in right panel.

Figure 5

Antigen processing of L.L-OVA by GM-DCs. (A) The expression of MHC I-OVApep on GM-DCs was detected by flow cytometry after co-cultured with different OVA recombinant L.L for 6 h, 12 h, 24 h, 36 h and 72 h. (B) The effects of DPI (ROS inhibitor) on the expression of MHC I-OVApep in GM-DCs after co-cultured with L.L-OVA were detected by flow cytometry. GM-DCs were pretreated with DPI for 12 h, then co-treated with L.L-OVA for 24 h. (C) The effects of MG132 (proteasome inhibitor) on the expression of MHC I-OVApep in GM-DCs after co-cultured with L.L-OVA were detected by flow cytometry. GM-DCs were pretreated with 10 μg/ml MG132 for 30 min and co-treated with L.L-OVA for 24 h, then another 10 μg/ml MG132 was added after 12 h. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to L.L group, or between indicated groups.

Figure 6

Antitumor effect in vivo. Mouse melanoma B16-OVA cells were subcutaneously injected into the right flank of C57BL/6 mice. (A) Mouse body weight, tumor volumes and tumor weight. (B) The frequencies of immune cells in spleens. CD3⁺CD19^- cells correspond to T cells; CD49b⁺ cells correspond to NK cells; CD4⁺CD25^-Foxp3⁺ cells correspond to iTreg; CD4⁺CD25⁺Foxp3⁺ cells correspond to nTreg. (C) OVA-specific cellular responses in inguinal LNs. (D) The correlation of CD4⁺IFN-γ⁺, CD8⁺IFN-γ⁺, CD8⁺Granzyme B⁺ and CD8⁺IFN-γ⁺Granzyme B⁺ T cells with tumor volumes. # p < 0.05; ## p < 0.01; ### p < 0.001 compared to L.L group or LPS+OVA group, * p < 0.05; ** p < 0.01; *** p < 0.001 between indicated groups.