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. 2014 Feb 21;4:22. doi: 10.3389/fcimb.2014.00022

Figure 3.

Figure 3

Comparison of different BMDM and DC vaccine vectors for protection against listeriosis. (A) C57BL/6 mice were vaccinated i.p. or not (NV) for 7 days with different BMDM or DC vaccine vectors (BMDM-LM-WT, BMDM-LLO, BMDC-LM-WT, BMDC-LLO, and BMDC-GAPDH) (1 × 106 cells) (n = 5 mice/vaccine vector) and challenged i.p. with 5 × 103 CFU LM-WT for 3 days. Results of spleens homogenates are mean ± SD of three different experiments (P < 0.01) (black bars in figure and left scale legend). Same vaccine vectors were incubated in vitro with SRBC (0.5% solution). Hemolytic units were defined as the dilution of the sample that caused 50% hemoglobin release from 200 ml of 0.5% SRBC. Controls were established for 0% hemolysis using empty DCs and for 100% hemolysis by incubating SRBC with distilled water. (B) C57BL/6 mice were vaccinated i.p. or not with empty DCs (DC-Ø) or with saline (NV) using the vaccine vectors: DC-LM lysate, DC-GAPDH1−22, DC-LLO189−201, DC-LLO91−99. We also vaccinated with the following control vectors: DC-LMWT, DC-LPS-LLO91−99, and DC-LPS-GAPDH1−22 protection results were as follows: 70, 92, and 98%. In several samples, positive selection of DC was performed with anti-mouse CD11c-coated magnetic beads and MACSTM separation columns, protection results of DC-LLO91−99 and DC-GAPDH1−22 after positive selection were as follows, 90 and 98%, respectively. Left plot corresponds to results of spleens homogenates that are the mean ± SD of three different experiments (P < 0.05). Surface expression of different markers was analyzed by FACS in the prepared DC vaccine vectors. Samples were acquired using FACSCanto flow cytometer and percentages of positive cells for each antibody are shown. Results are expressed as the mean ± SD of triplicate samples (P < 0.05). (C) Cytokines were measured in serum from vaccinated and NV mice (DC-Ø) from (B). We also included another DC vaccine vector (DC-LPS) as a positive control. Levels of proinflammatory cytokines (MCP-1, TNF-α, IFN-γ, IL-6, IL-12, or IL-10) were analyzed by using the CBA kit (Becton Dickinson) and flow cytometry. Results were expressed as cytokine concentration (pg/ml of mean ± SD, P < 0.05). IL-12 concentration for DC-Ø vaccination was 8 ± 0.03 pg/ml and for DC-LPS, 19 ± 0.2 pg/ml. IFN-γ concentrations of samples were as follows: DC-LLO91−99 (389 ± 12 pg/ml), DC-GAPDH1−22 (425 ± 11 pg/ml), DC-LMWT (178 ± 10 pg/ml), DC-LPS (200 ± 0.3 pg/ml). (D) Spleen cells obtained from homogenates after vaccination were stimulated for 5 h with GAPDH1−22 in the presence of brefeldin A for intracellular cytokine staining. GAPDH-peptide-stimulated spleen cell surface was stained for CD4 or CD8 and fixed and permeabilized using a cytofix/cytoperm kit. Stimulated cells were surface stained for CD4 or CD8 using anti-CD4+ FITC-labeled or anti-CD8+ APC-labeled and data gated to include exclusively CD4+ or CD8+ events, R2, and R3 gates, respectively. Flow histograms show the percentages of GAPDH1−22/CD4+ and IFN-γ producers (lower left) and GAPDH1−22/CD8+ and IFN-γ producers (lower right) (R2 and R3 gates). Experiments were performed in triplicate and results are expressed as the mean ± SD (P < 0.05).