Figure 1.
C57BL/6 mice were infected with F-MLV. At the time indicated, the number of viral foci in the spleen was measured by focus-forming assay on Mus Dunni cells. Each bar represents the average of five mice. The data shown are representative of two independent experiments.
Figure 2.
F-MLV infection causes dendritic cell maturation in vivo.
C57BL/6 mice were infected with F-MLV. At the times indicated, dendritic cell populations in the spleen were analyzed for maturation by staining for CD80 and CD86. Each FACS plot is representative of five mice. Data from uninfected mice are shown in red, while infected mice are shown in blue. The data shown are representative of three independent experiments.
Figure 3.
Dendritic cells are required for immune control of F-MLV infection.
Lethally irradiated C57BL/6 mice that had been reconstituted with wild-type (WT) or CD11c-DTR bone marrow were infected intravenously with F-MLV. DT was injected intraperitoneally into both wild-type and CD11c-DTR mice every day from one day prior to infection until 14 days post-infection. At 14 days post-infection, spleens were harvested, the level of DC depletion analyzed (A), and the number of viral foci per spleen and spleen weight were measured (B). Each bar is the average of seven mice for each group. The data shown are representative of three independent experiments.
Figure 4.
Dendritic cells are required for the neutralizing antibody response to F-MLV.
(A) In dendritic cell-depleted F-MLV–infected mice, the CD8+ T cell response to F-MLV at 14 dpi was measured by staining splenocytes with the Db-GagL tetramer. Representative FACS plots of DbGagL staining gated on CD8+ cells are shown on the left. The proportion of CD8+ T cells that stained with GagL was calculated for each sample and is shown on the right. Each bar represents the average value of four mice. (B) In dendritic cell-depleted F-MLV–infected mice, the titer of F-MLV–neutralizing antibodies in the serum was measured. Numerical values represent the maximum dilution of serum that neutralized F-MLV infection of Mus Dunni cells. Each dot represents an individual mouse. The data shown are representative of three independent experiments.
Figure 5.
Myd88 is required for immune control of F-MLV.
Myd88 knockout mice and heterozygous littermates were infected with F-MLV. (A) At the times indicated, the viral titer in the spleen was measured. Each bar is the average of 4–8 mice. (B) At the times indicated, the weight of the spleen was measured. Each bar is the average of 4–8 mice. (C) The survival of Myd88 knockout mice and heterozygous littermates was measured over a 16-week period. The data shown are representative of two independent experiments.
Figure 6.
Dendritic cell maturation during F-MLV infection is partially dependent on Myd88.
Myd88 knockout mice and heterozygous littermates were infected with F-MLV. At 7 dpi, splenic DCs were stained for expression of CD80. The populations were gated as follows: CD11c+ B220− CD8α−, CD11c+ B220- CD8α+, and CD11cint, B220+ CD8α+. A representative histogram of CD80 staining from three independent experiments using 4–6 mice is shown for each population.
Figure 7.
T cell responses to F-MLV are partially dependent on Myd88.
Myd88 knockout mice and heterozygous littermates were infected with F-MLV for 14 days. (A) Splenocytes were stained with TCRβ, CD8α, and the DbGagL tetramer, and the proportion of TCRβ+ CD8+ cells that were GagL+ was measured. A representative FACS plot gated on TCRβ+ CD8+ cells is shown in the upper panel. For the bar graph in the lower panel, each bar is the average of four mice. (B) Splenocytes were stained for TCRβ, CD4, and intracellular interferon-γ. The proportion of TCRβ+ CD4+ T cells that expressed interferon-γ was measured. Each bar is the average of four mice. The data shown are representative of three independent experiments.
Figure 8.
Myd88 is required for a neutralizing antibody response against F-MLV.
Serum was isolated from the peripheral blood of F-MLV–infected Myd88−/− and Myd88+/− littermate controls at 2 wpi and 8 wpi. The titer of neutralizing antibodies in the serum was measured. Each dot represents an individual mouse. The data shown are from one of four independent experiments.
Figure 9.
F-MLV–specific IgG is absent from serum of infected mice lacking Myd88 or CD11c+ DCs.
Serum samples from naïve or infected mice were assayed for the presence of F-MLV–specific IgG, by incubation with a suspension of F-MLV–infected cells, followed by secondary staining for mouse IgG. The cells were then analyzed by flow cytometry. Representative stainings for Myd88 knockout mice (upper left) and CD11c+ DC-depleted mice (upper right) are shown. Mean fluorescence intensities (MFI) for the average of five mice were calculated for each condition and plotted (lower panel). The data shown are from one of two independent experiments.
Figure 10.
Transfer of serum from infected wild-type mice to Myd88 knockout mice rescues control of F-MLV.
Myd88 knockout mice or heterozygous littermates were infected with F-MLV. At 7 dpi, infected mice were injected intraperitoneally with 0.5 ml serum from uninfected C57BL/6 mice or from C57BL/6 mice that had been infected with F-MLV for 14 days. At 14 dpi, the levels of viral focus-forming units in the spleens of infected mice were analyzed. Each bar represents the average of 4–7 mice from one of two independent experiments.