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. 2006 Mar;80(5):2194-205.
doi: 10.1128/JVI.80.5.2194-2205.2006.

Matrix protein mutant of vesicular stomatitis virus stimulates maturation of myeloid dendritic cells

Maryam Ahmed  1 , Kristina L Brzoza, Elizabeth M Hiltbold
Affiliations

Matrix protein mutant of vesicular stomatitis virus stimulates maturation of myeloid dendritic cells

Maryam Ahmed et al. J Virol. 2006 Mar.

Abstract

Matrix (M) protein mutants of vesicular stomatitis virus have recently been used as oncolytic viruses for tumor therapies and are being developed as vaccine vectors for heterologous antigens. Because dendritic cell (DC) maturation is an important correlate of tumor immunosurveillance and vaccine efficacy, we sought to determine the ability of a recombinant M protein mutant virus (rM51R-M virus) to mature DC in vitro. We have previously shown that rM51R-M virus is defective at inhibiting host gene expression in several cell lines compared to its recombinant wild-type counterpart, rwt virus. Therefore, rM51R-M virus allows the expression of genes involved in antiviral responses, such as the type I interferon (IFN) gene. Our results demonstrate that, in contrast to the rwt virus, rM51R-M virus induced the maturation of myeloid DC (mDC) populations, as indicated by an increase in the surface expression of CD40, CD80, and CD86 as well as the secretion of interleukin-12 (IL-12), IL-6, and type I IFN. In addition, mDC infected with rM51R-M virus effectively activated naïve T cells in vitro, whereas rwt virus-infected mDC were defective in antigen presentation. The inability of rwt virus to induce mDC maturation was correlated with the inhibition of host gene expression in rwt virus-infected cells. Our studies also indicated that the production of costimulatory molecules on mDC by rM51R-M virus was dependent on the type I IFN receptor, while maturation induced by this virus was largely independent of MyD88. These data indicate that rM51R-M virus effectively stimulates the maturation of mDC and has the potential to promote effective T-cell responses to vector-expressed antigens, activate DC at tumor sites during therapy, and aid in tumor immunosurveillance and destruction.

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Figures

FIG. 1.
FIG. 1.
rM51R-M virus is defective at inhibiting host gene expression in mDC. mDC were infected with rwt or rM51R-M virus at a multiplicity of infection of 10 or 1 PFU/cell or were mock infected as a control. At 8 and 24 h postinfection, cells were labeled with a 15-min pulse of [35S]methionine (100 μCi/ml) and harvested. Lysates were subjected to SDS-PAGE, and labeled proteins were quantitated by phosphorimaging. (A) Representative image from analysis of rwt and rM51R-M virus-infected mDC. (B) Host protein synthesis was determined from images similar to that shown in panel A for regions of the gel devoid of viral proteins between the L and G proteins. The results are shown as percentages of the mock-infected control value and are the means ± standard errors of three independent experiments. (C) Cells were infected with the rwt and rM51R-M viruses at multiplicities of 0.01, 0.1, 1, and 10 PFU/cell or were treated with LPS, and live cells were measured by an MTT assay at 24 h postinfection. Data are expressed as percentages of the cell viability of untreated cells and are the means ± standard errors of four experiments. (D) Viability of mDC infected with rwt or rM51R-M virus at a multiplicity of 1 or 10 PFU/cell for 8 or 24 h. Data are means ± standard errors of three experiments.
FIG. 2.
FIG. 2.
mDC are semipermissive for rwt and rM51R-M virus infections. The efficiency of G protein surface expression on mDC during VSV infection was determined by flow cytometry analysis of mDC infected with the rwt and rM51R-M viruses at a multiplicity of 1 PFU/cell for 24 h. (A) Representative histograms depicting increases in G protein expression in rwt and rM51R-M virus-infected cells. (B) The geometric mean fluorescence intensity of each sample was determined. Data are expressed in arbitrary values and are the means ± standard errors of four or five experiments. (C) Viral growth analysis in mDC. Cells were infected with rwt or rM51R-M virus at multiplicities of 0.01, 0.1, and 1 PFU/cell. At 24 h postinfection, supernatants were collected to determine the amounts of progeny virus by a plaque assay. Data are the means ± standard errors of three experiments.
FIG. 3.
FIG. 3.
Quantitation of cell death and CD11c expression following infection with rwt and rM51R-M viruses. mDC were infected with rwt and rM51R-M viruses at multiplicities of 0.1 PFU/cell (rwt virus) and 1 PFU/cell (rwt and rM51R-M viruses). At 24 h postinfection, cells were labeled with antibodies to CD11c and incubated with 7AAD. The images shown are representative dot plots depicting the forward and side scatter of each population and CD11c versus 7AAD staining within the low-side-scatter gate. The percentage of 7AAD-positive cells in the gated CD11c+ population was determined. Data represent the means ± standard deviations of three experiments.
FIG. 4.
FIG. 4.
rM51R-M virus induces expression of costimulatory molecules on mDC. mDC were infected with rwt or rM51R-M virus at multiplicities of 0.01, 0.1, and 1 PFU/cell or were treated with LPS or loxoribine. At 24 h postinfection or posttreatment, the cell surface expression of CD40, CD80, and CD86 was measured by flow cytometry. (A) Representative histograms depicting expression of costimulatory molecules by rwt and rM51R-M viruses (1 PFU/cell). The geometric mean fluorescence of each sample was determined and used to quantitate the increase in CD40 (B), CD80 (C), and CD86 (D) expression over that in untreated cells (NT). Data are the means ± standard errors of three or four independent experiments.
FIG. 5.
FIG. 5.
Cytokine expression induced by rM51R-M virus. mDC were infected with rwt and rM51R-M viruses at different multiplicities or were treated with the indicated TLR agonist [LPS or poly(I-C)] for a total of 24 h. Culture supernatants were removed, and the presence of IL-12 p40 (A), IL-6 (B), and TNF-α (C) was measured by ELISA. Data are expressed in pg/ml of cytokine secreted into the supernatant. Type I IFN in the supernatant was measured by a bioassay based on the reduction of VSV cytopathic effects (D). The IFN concentration (in IU/ml) was quantitated by comparing the results to those in cells incubated with serial dilutions of an IFN standard. Data were normalized to the percentage of viable cells and are the means ± standard errors for three or four experiments.
FIG. 6.
FIG. 6.
T-cell-activating capacity of rM51R-M virus-infected mDC. mDC were either infected with rM51R-M virus (1 PFU/cell), treated with LPS, or left untreated (NT) and then were pulsed with the indicated doses of OVA peptide antigen for 24 h. CFSE-labeled OT-1 cells (naïve, OVA-specific transgenic T cells) were then cultured with the DC for an additional 72 h. T cells were harvested and stained intracellularly for IFN-γ and TNF-α and analyzed by flow cytometry. (A) Histograms of CFSE fluorescence for the indicated DC treatments at the intermediate antigen dose, 0.1 ng/ml OVA peptide. (B) Division indices for each treatment at three antigen doses. The data shown are means and standard deviations for triplicate cultures. (C) Percentage of live, CD8+ T cells that produce both IFN-γ and TNF-α. Data are means and standard deviations for triplicate cultures and are representative of three independent experiments.
FIG. 7.
FIG. 7.
Maturation of mDC by rM51R-M virus occurs independently of MyD88. mDC derived from wt or MyD88−/− mice were infected with rM51R-M virus and treated with LPS or poly(I-C) for 24 h. The cell surface expression of CD40 (A), CD80 (B), and CD86 (C) was determined by flow cytometry. Data represent the means ± standard errors of three to five experiments. The production of IL-12 p40 (D), IL-6 (E), and TNF-α (F) in the supernatants was determined by ELISA. The data shown are representative of three experiments.
FIG. 8.
FIG. 8.
Maturation of IFNR−/− mDC by rM51R-M virus. mDC derived from wt or IFNR−/− mice were infected with rM51R-M virus and treated with LPS or poly(I-C) for 24 h. The cell surface expression of CD40 (A), CD80 (B), and CD86 (C) was determined by flow cytometry. Data represent the means ± standard errors of three to five experiments. The production of IL-12 p40 (D), IL-6 (E), and TNF-α (F) in the supernatants was determined by ELISA. The data shown are representative of three experiments.
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