Abortive versus productive viral infection of dendritic cells with a paramyxovirus results in differential upregulation of select costimulatory molecules

Abstract

Dendritic cells are the most potent antigen-presenting cell for priming naive T cells. Optimal activation of T cells requires that dendritic cells undergo a process of maturation resulting in the increased expression of costimulatory molecules, such as CD40, CD86, and CD80, and the production of cytokines. In this study we analyzed the effect of infection of dendritic cells obtained from two strains of mice, BALB/c and C57BL/6, with the paramyxovirus simian virus 5 (SV5). Our results show that C57BL/6 bone marrow-derived dendritic cells (BMDC) are much more permissive to infection with SV5 at a multiplicity of infection (MOI) of 10 PFU/cell compared to BALB/c BMDC, as determined by the production of viral proteins and progeny. However, infection of BALB/c BMDC with a higher MOI of 50 PFU/cell resulted in a productive infection with the production of significant amounts of viral proteins and progeny. Regardless of the permissivity to infection, both BALB/c and C57BL/6 BMDC efficiently upregulated CD40 and CD86. However, CD80 upregulation correlated with the level of expression of viral proteins and the production of viral progeny. While secreted alpha/beta interferon was required for increased expression of all three molecules, optimal CD80 expression was dependent on an additional signal provided by a productive viral infection. These findings provide evidence that the signals controlling the expression of costimulatory molecules following viral infection are distinct. Further, they suggest that the amount of virus encountered and/or the permissivity of a dendritic cell to infection can alter the resulting maturation phenotype and functional capacity of the infected dendritic cell.

FIG. 1.

SV5-matured C57BL/6 BMDC are much more efficient inducers of T-cell proliferation than rSV5-matured BALB/c BMDC. (A) C57BL/6 BMDC infected with rSV5 or treated with LPS for 24 h were pulsed with 10⁻¹⁰ M Ova peptide and then added to CFSE-labeled, Ova_257-264-specific TCR transgenic T cells for 3 days. (B) BALB/c BMDC infected with rSV5 or treated with LPS for 24 h were pulsed with 10⁻⁹ M listeria p60_217-225 peptide and then added to CFSE-labeled, listeria p60-specific TCR transgenic T cells for 3 days. (C) Table showing the analysis from the proliferation assay of T cells. The proliferation index is the average number of divisions in cells that divided and the division index is the average number of divisions the cell population has undergone. The proliferation profiles shown are representative of three independent experiments. Note: the peptide concentration used was the minimal concentration of peptide that gave detectable proliferation of antigen-specific T cells with SV5-matured DC.

FIG. 2.

rSV5-infected C57BL/6 and BALB/c BMDC produce a similar subset of proinflammatory cytokines. In vitro-cultured BMDC were mock-infected, LPS-treated, and infected with rSV5 at an MOI of 2, 10, or 50 or with UV-inactivated rSV5 (MOI = 10). Twenty-four hours after infection, supernatants from the cultures were harvested and cytokines were measured using ELISA (IL-12p40) or cytometric bead analysis (TNF-α and IL-6). IFN-α/β was measured using a biological assay based on vesicular stomatitis virus (VSV)-mediated cytolysis of NTCC L929 cells. Poly(I:C) was used as a positive control for IFN-α/β production by BMDC. (A) IL-12p40, (B) IL-6, (C) IFN-α/β, (D) TNF-α. The graphs show the average ± standard error of the mean of four independent experiments.

FIG. 3.

C57BL/6 and BALB/c BMDC infected with rSV5 efficiently upregulated the expression of CD40 and CD86, while CD80 is efficiently upregulated only on C57BL/6 BMDC. Day 7 in vitro-cultured C57BL/6 BMDC (A-C) or BALB/c (D-F) were infected for 24 h with rSV5 (MOI = 10), stained with antibodies to CD11c and CD40, CD86, or CD80, and analyzed by flow cytometry. LPS (200 ng/ml)-treated BMDC served as a positive control. Colors: isotype control (black histogram), mock-infected BMDC (green histogram), LPS-treated BMDC (red histogram), SV5-infected BMDC (blue histogram). The numbers shown in parentheses are the geometric mean fluorescence intensity (MFI). The histograms shown are representative of four independent experiments. Upregulation of costimulatory molecules following infection with rSV5 compared to mock-infected BMDC was statistically significant (average of four experiments). C57BL/6 BMDC: CD40 (P = 0.045), CD86 (P = 0.011), CD80 (P = 0.024); BALB/c BMDC: CD40 (P = 0.040), CD86 (P = 0.015).

FIG. 4.

CD80 expression continues to increase only on C57BL/6 BMDC between 24 and 48 h postinfection with rSV5. Day 7 BMDC from both strains of mice were mock infected, treated with LPS, or infected with rSV5 (MOI = 10) for 24 h or 48 h. Cells were then stained with antibodies to CD11c and CD80 and analyzed by flow cytometry. The graphs in the figure show the geometric mean fluorescent intensity of CD80 for CD11c⁺ cells for both C57BL/6 (A) and BALB/c (B) BMDC. The graphs show the average ± standard error of the mean of four independent experiments. P values were calculated using the paired t test. *, upregulation of CD80 on C57BL/6 BMDC following infection with rSV5 compared to mock-infected BMDC was statistically significant (24 h: P = 0.024; 48 h: P = 0.040).

FIG. 5.

Upregulation of costimulatory molecules on C57BL/6 and BALB/c BMDC is dependent on live virus. Day 7 C57BL/6 (A) and BALB/c (B) BMDC were infected with live or UV-inactivated rSV5 (UV-rSV5, MOI = 10). LPS-treated and mock-infected BMDC were used as positive controls. Twenty-four hours after infection, cells were stained with antibodies to CD11c and CD40, CD86 or CD80 and analyzed using flow cytometry. The level of expression of these molecules on CD11c⁺ cells is shown. The graphs show the average ± standard error of the mean of four independent experiments. P values were calculated using the paired t test. *, C57BL/6 BMDC: CD40 (P = 0.030), CD86 (P = 0.012), CD80 (P = 0.025); BALB/c BMDC: CD40 (P = 0.045), CD86 (P = 0.040).

FIG. 6.

Binding of secreted IFN-α/β to its receptor is required for the upregulation of CD40, CD80, and CD86. BMDC from IFN-α/βR^−/− and control mice (129S1/SvImJ) were mock infected, treated with LPS, or infected with rSV5. Twenty-four hours later, cells were stained for CD11c and CD40, CD80, or CD86 and analyzed using flow cytometry. The graphs in the figure show the geometric mean fluorescence intensity of CD40, CD86, and CD80 for CD11c⁺ cells. The graphs show the average ± standard error of the mean of three independent experiments.

FIG. 7.

rSV5 infection of BALB/c BMDC does not prevent LPS-induced maturation of BMDC. BALB/c BMDC were LPS treated, mock infected, rSV5 infected, or treated with combinations of LPS (200 ng/ml) and rSV5 (MOI = 10). LPS and rSV5 were added concurrently to BMDC for 24 h, or LPS was added 24 h following infection with rSV5. The graph shows the geometric mean fluorescence intensity of CD80 for CD11c⁺ cells for BALB/c BMDC. The graphs show the average ± standard error of the mean of three independent experiments. P values were calculated using the paired t test. *, upregulation of CD80 on BALB/c BMDC treated with LPS and rSV5 (P = 0.009) and rSV5 for 24 h followed by LPS (P = 0.003) was statistically significant relative to that of rSV5-infected BMDC.

FIG. 8.

Newly synthesized SV5 proteins are readily detected in significant quantities in C57BL/6 BMDC but not in BALB/c BMDC following infection with rSV5. BMDC from C57BL/6 (A) or BALB/c (D) mice were cultured in vitro for 7 days as described in Materials and Methods. On day 7, cells were mock infected (M) or infected with rSV5-wt or rSV5-GFP (MOI = 10). At 24 h postinfection, cells were stained with a primary hamster anti-mouse antibody to CD11c, followed by the secondary antibody (goat anti-hamster Alexa-568) and immunofluorescence was analyzed. The images shown are representative of three independent experiments. P815 cells, C57BL/6 BMDC (B), or BALB/c BMDC (E) were either mock infected (M) or infected with live or UV-inactivated rSV5 (MOI = 10). Two and 24 hours after infection, cells were lysed and analyzed by Western blotting for SV5 P protein and cellular actin. The blot shown is representative of three independent experiments. Note: The amount of SV5-infected P815 lysate used was 15 μg, whereas 30 μg of the other samples were loaded. (C) C57BL/6 BMDC were infected on day 7 with live or UV-inactivated rSV5-GFP-Ova (MOI = 10). Twenty-four hours after infection, C57BL/6 BMDC were cocultured with naive OT-1 TCR transgenic CD8⁺ T cells (specific for Ova_257-264 peptide) at a ratio of DC:T cells of 1:10. Twenty-four hours following coculture, IFN-γ present in the supernatant was measured by ELISA. (F) BALB/c BMDC were infected on day 7 with live or UV-inactivated rSV5-I10 (MOI = 10). At 24 h postinfection, BMDC were cocultured with an I10-specific high avidity CTL line at a ratio of DC:T cells of 1:10. Twenty-four hours following coculture, IFN-γ present in the supernatant was measured by ELISA. The graphs show the average ± standard error of the mean of three independent experiments. P values were calculated using the paired t test. *, a significant decrease (P = 0.029) in the amount of IFN-γ produced by CD8⁺ cocultured with C57BL/6 BMDC infected with UV-inactivated rSV5-GFPOva relative to IFN-γ produced by CD8⁺ T cells cultured with BMDC infected with live rSV5-GFPOva.

FIG. 9.

Upregulated CD80 expression on BALB/c BMDC following infection with a high MOI of 50 PFU/cell correlates with significant expression of viral proteins. (A) BMDC from BALB/c mice were mock infected (M) or infected with live or UV-inactivated rSV5 (MOI = 50). Twenty-four hours after infection, cells were lysed and analyzed by Western blotting for SV5 P protein and cellular actin. The blot shown is representative of three independent experiments. (B) Day 7 in vitro-cultured BALB/c BMDC were infected for 24 h with rSV5 (MOI 10 and 50 PFU/cell), stained with antibodies to CD11c and CD80 and analyzed by flow cytometry. Mock- and LPS (200 ng/ml)-treated BMDC served as negative and positive controls, respectively. The graphs show a representative of three independent experiments. P values were determined using the paired t test. *, upregulation of CD80 on BALB/c BMDC infected with rSV5 at an MOI of 50 PFU/cell (P = 0.001) relative to mock-infected cells.