Fig 1.
IL-21 signaling is required for efficient establishment of MHV68 latency.
Mice were infected intranasally with 1,000 pfu of MHV68-H2bYFP and splenocytes were harvested at the indicated time points. (A) Representative flow plots showing identification of YFP+ B cells at d16 post-infection. Gates were drawn based on the distribution of splenocytes from wt mice infected with wt MHV68 to distinguish YFP+ cells from background fluorescence. Flow plots were gated on CD3-, CD4-, CD8-, B220+ cells. (B) Frequency of YFP+ B cells. Results are compiled from 3 independent experiments at each time point with 3–6 mice per group. Each symbol represents an individual mouse, and the horizontal lines represent the mean frequency of infected B cells. ns = not significant; ****, p<0.0001.
Table 1.
Average number of YFP+ cells per spleen.
Fig 2.
IL-21 is required for generation of infected PCs at late time points during the onset of latency.
Mice were infected intranasally with 1,000 pfu of MHV68-H2bYFP and splenocytes were harvested at the indicated time points. (A) Representative flow plots showing the gating strategy to identify plasma cells. Plots were gated on CD3-, CD4-, CD8- splenocytes. Plasma cells were defined as B220neg-lo, CD138hi (red gated area). (B) Quantitation of the percentage of plasma cells present at the indicated time points. ns = not significant, **, p = 0.001. (C) Representative flow plots showing the percentage of infected cells that had a plasma cell phenotype (red gated area). Flow plots were gated on CD3-, CD4-, CD8-, YFP+ splenocytes and plasma cells were defined as in (A). (D) Quantitation of the percentage of YFP+ cells that had a plasma cell phenotype. ns = not significant; **, p = 0.0003; ***, p = 0.0025. Results in (B and D) were compiled from 3 independent experiments at each time point with 3–6 mice per group. Each symbol represents a single mouse and the horizontal lines represent the mean frequency.
Fig 3.
IL-21 signaling is required for efficient viral reactivation from splenocytes.
To determine the frequency of splenocytes capable of reactivating virus, limiting dilution analysis was performed on splenocytes from mice infected intranasally with 1,000 pfu of MHV68-H2bYFP and harvested at days (A) 14, (B) 16, (C) 18 and (D) 20 post-infection. Serial dilutions of splenocytes were plated on MEFs and the percentage of wells positive for cytopathic effect (CPE) was determined 14 days post-plating. Mechanically disrupted splenocytes were plated in parallel to detect the presence of preformed infectious virus. Results are from 3 independent experiments at each time point with 3–6 mice per group.
Table 2.
Correlation between infected plasma cells and reactivating cells.
Fig 4.
Reduced germinal center response in IL-21R-/- mice.
Mice were infected intranasally with 1,000 pfu of MHV68-H2bYFP and splenocytes were harvested at the indicated time points. (A) Representative flow plots showing reduced germinal center B cells in IL-21R-/- mice. Flow plots were gated on CD3-, CD4-, CD8-, B220+, and germinal center B cells were defined as CD95hi GL7hi. (B) Quantitation of the percentage of B cells that have a germinal center phenotype at the indicated time points. d14, ns = not significant; d16, p = 0.0052; d18, p = 0.0462; d20, p = 0.0178. (C) Gating strategy to determine the percentage of germinal center B cells that are infected based on YFP expression. Germinal center B cells are defined as in (A). (D) Quantitation of the percentage of germinal center B cells that are infected at the indicated time points. d14, ns = not significant; d16, p = 0.0001, d18 and d20, p< 0.0001. (E) Comparison of the increase in total germinal center B cells (left graph) versus the increase in the percentage of infected germinal center B cells (right graph). Results in B, D and E are from 3 independent experiments at each time point with 3–6 mice per group. Error bars represent SEM.
Fig 5.
Reduced frequency of infected B cells with a germinal center phenotype in IL21-R-/- mice.
Mice were intranasally infected with 1,000 pfu of MHV68-H2bYFP and splenocytes were harvested at the indicated time points. (A) Representative flow plots of YFP+ germinal center B cells (black dots) overlaid onto the total B cell population (gray), showing that a reduced percentage of YFP+ B cells have a germinal center phenotype in IL-21R-/- mice. Flow plots were gated on CD3-, CD4-, CD8-, B220+, YFP+, and germinal center B cells were defined as CD95hi GL7hi. (B) Quantitation of the percentage of YFP+ B cells that have a germinal center phenotype. Each symbol represents a single mouse. Results are from 3 independent experiments at each time point with 3–6 mice per group. d14, p = 0.0252; d16, p = 0.0001; d18 and d20, p<0.0001.
Fig 6.
Reduced infection in germinal centers in IL-21R-/- of mice.
Representative spleen sections from mice infected intranasally with 1,000 pfu of MHV68-H2bYFP and harvested at day 16 post-infection. Mice were injected intraperitoneally with Edu 5 hours before spleens were harvested to detect proliferating centroblasts in the dark zone of germinal centers. Sections were co-stained with anti-IgD to delineate the mantle zone surrounding germinal centers and anti-FDC-M1 to detect the follicular dendritic cell network in light zone in sections from (A) wt mice and (B) IL-21R-/- mice. Arrows indicate Edu+ YFP+ cells in IL-21R-/- mice.
Fig 7.
Altered GC B cell populations in IL-21R-/- mice.
Mice were infected intranasally with 1,000 pfu of MHV68-H2bYFP and splenocytes were harvested at the indicated time points. (A) Representative flow plots showing germinal center B cells populations. Plots are gated on YFP- or YFP+ germinal center B cells (CD3-, CD4-, CD8-, B220+, CD95hi, GL7hi). Centroblasts are defined as CXCR4hi, CD86lo-neg and centrocytes are defined as CXCR4lo-neg, CD86hi. Quantitation of (B) uninfected and (C) infected centroblast and centrocyte populations. *, p<0.05; **, p<0.01; ***, p<0.001; ****,p<0.0001.
Fig 8.
Requirement for IL-21 signaling in establishment of MHV68 infection is intrinsic to B cells.
IL21R+/+/IL21R-/- mixed bone marrow chimeric mice were infected intranasally with 1,000 pfu of MHV68-H2bYFP and spleens were harvested at the indicated days. (A) Total number of Ly5.1+ IL21R+/+ and Ly5.2+ Il21R-/- splenocytes harvested. (B) Percentage of B cells with a germinal center phenotype. Cells were gated on Ly5.1+ or Ly5.2+, B220+ CD3-, CD4-, CD8- and germinal center B cells were defined as CD95hi GL7hi. (C) Percentage of splenocytes with a plasma cell phenotype. Cells were gated on CD3-, CD4-, CD8- and plasma cells were defined as B220neg-lo CD138hi. (D) Percentage of splenocytes that were MHV68 infected (YFP+). Cells were gated on CD3-, CD4-, CD8- splenocytes. **, p = 0.0029; ****,p<0.0001.