Tularemia Vaccination and BALT Figure 3. Lymphoid aggregates in lungs of vaccinated mice were detected at 30 days post-challenge (arrows). (A) Representative lung from na¨ıve mouse was used as a negative control; (B) Lung from LPS vaccinated/challenged; (C and D) Lung from LPS+rPorB vaccinated/challenged mouse and (E) Lung from LPS+rPorB vaccinated/unchallenged mouse. Formalin fixed, paraffin embedded sections were stained with H&E and representative pictures were taken with a 106 objective. doi:10.1371/journal.pone.0011156.g003 unchallenged mice 30 days after infection, morphometric analysis of lymphoid structures was conducted by calculating the average size of individual follicles, or the entire area occupied by multiple follicles. The average size of whole lymphoid follicles was significantly larger in lungs of mice vaccinated with LPS+rPorB 2 and subsequently challenged (,50,000 mm ), than in mice that 2 were only vaccinated (,13,000 mm ); differences were significant with a P value,0.01 (Figure 7A). B cell follicles were also slightly bigger in the vaccinated/challenged group (,13,800 mm2) com- 2 pared with mice that only received the vaccine (,10,800 mm ), though this difference was not significant (Figure 7A). The total area occupied by lymphoid follicles was larger in the LPS+rPorB/ challenged group (,2,000,000 mm2) as compared to total area in 2 LPS+rPorB/unchallenged group (,207,000 mm ), although these differences were not statistically significant (Figure 7B). A similar pattern was observed for the average area occupied by B cell lymphoid aggregates that was increased in vaccinated/challenged mice (,354,000 mm2) than in mice that were vaccinated but not challenged (,33,800 mm2) (Figure 7B). Morphometric analysis for PBS- and rPorB-vaccinated groups was not conducted because all challenged mice had died by day 30, and no lymphoid aggregates were found in their lungs at the moribund stage. Results for the LPS-vaccinated group are also not shown because only one mouse per group survived bacterial challenge, and thus could not be included in the statistical analysis of the morphometric study. At day 30, dendritic cells (DCs) were found within the BALT follicles from mice vaccinated with LPS+PorB and challenged, as shown in figure 7C. Cells stained with either CD11b, CD11c, or a combination of both cell markers, were found and confirmed to be DCs by their typical appearance characterized by cytoplasmic prolongations (Figure 7C, yellow arrows). We were also able to identify CD11c+ high macrophages, featuring a rounder and more defined shape (Figure 7C, white arrow). We analyzed antibody generating plasma cells in lungs of challenged mice that had previously been vaccinated and euthanized at day 30. IgG+, IgA+ and IgM+ cells were detected in areas of BALT, and outside the lymphoid structures (data not shown). IgG+ plasma cells were more abundant than IgM+ and IgA+ plasma cells and some were proliferating (red nucleus stained by PCNA and green cytoplasm stained for IgG, indicated by the arrows in the upper left panel of figure 8A). IgM+B cells were also present, as shown by the signal localized exclusively on the surface (upper middle panel, figure 8A). No proliferating T cells were detected, but clusters of PCNA+ cells (red nuclei) indicating germinal centers (GC) and individual T cells (red surface) were found in the tissue (upper panels, figure 8A). The production of homeostatic chemokines, concomitant to the development of BALT in lungs of vaccinated mice, was analyzed locally by immunofluorescence and measured in serum and supernatants from lung homogenates, 30 days post-challenge. We were able to detect a CXCL13 signal that co-localized with follicular dendritic cells (detected by CD21, CD35 and FDCM-1 markers- lower left panel, figure 8A), and CCL21 signal in a structure presenting the characteristic morphology of a high endothelial venule, located in the middle of the B cell follicle (lower middle panel, figure 8A). CCL21 was also detected in cells with an oval morphology compatible with endothelial cells or lymphatic precursors (lower right panel, figure 8A). By ELISA assay, the levels of CCL21 in lung lysates and serum were elevated above baseline in the group vaccinated with LPS+rPorB and then challenged with LVS, as compared with unvaccinated unchal- PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11156