Tularemia Vaccination and BALT Figure 6. Germinal center B cells are exclusively found within BALT from LPS+rPorB vaccinated/LVS challenged mice. (A) Representative lung from na¨ıve negative control; (B) mediastinal lymph node from LPS+rPorB/challenged mouse as an internal positive control; (C) Lung from LPS vaccinated/challenged; (D, E) Lung from LPS+rPorB vaccinated/challenged mouse and (F) lung from LPS+PorB vaccinated/ unchallenged mouse. Cells markers are B220 for B cells (red- Alexa 549) and PNA to detect germinal center B cells (green- Alexa 488). Areas of color overlap indicate germinal centers (see arrows). Cell nuclei were stained with 49-6-Diamino-2-phenylindole (DAPI-blue). Representative pictures were taken with a 206 objective. doi:10.1371/journal.pone.0011156.g006 vaccinated intranasally with LPS+rPorB candidate presented accumulations of lymphoid cells around the bronchi and vessels of the lung parenchyma. For simplicity we will refer to these structures as BALT, although they are clearly distinct from the follicles constitutively present in the lungs of certain animal species. Immunofluorescence analysis of the follicles revealed highly organized structures in the lungs of the mice immunized with the LPS+rPorB mice and challenged with LVS. The presence of dense B cell areas containing proliferating B cells and germinal center B cells, surrounded by less dense T cell infiltrates, in vaccinated/challenged survivors, were indicative of BALT forma- tion. In contrast, scattered and disorganized B and T cell clusters were detected in lung tissue of control groups. Additional immunological factors induced by nasal vaccination and bacterial challenge were analyzed in BALT areas. Lung sections were stained for local antibody production and we found that IgG, IgA and IgM producing plasma cells were contained within BALT structures. IgG antibody producing plasma cells were more abundant than IgM+ and IgA+ cells. Given that some of them were positive for the cell proliferation marker PCNA, this may indicate that they are being directly stimulated by antigen. Considering that DCs were also present in BALT structures, it is also possible that these immune cells can induce factors like B cell- activating factor (BAFF), a proliferating inducing ligand (APRIL) and interleukin-6 (IL-6) that may promote expansion and survival of plasma cells [41,42]. According to the location of the PCNA clusters, it is likely that plasma cells are being generated in germinal centers (GCs) of BALT areas, and may play an important role in preventing dissemination of infection and promoting accelerated local bacterial clearance. The observation that GC-like structures were not present in draining lymph nodes (data not shown) may suggest that immunity is initiated and maintained more efficiently in the lung. We also observed the local production of homeostatic chemokines that play a pivotal role in the architectural maintenance of tertiary lymphoid structures (CXCL13 and CCL21) inside BALT structures. CXCL13 is thought to be mainly involved in the recruitment of CXCR+ B cells and critical for maintaining B cell follicle organization. In contrast, CCL21 attracts CCR7+ T cells and dendritic cells at the infection site. Attraction of na¨ıve CCR7+ cells, CCR7+ dendritic cells and central memory T cells could contribute to accelerated T cell priming and fast activation of memory T cells, enhancing the cellular immune response in the lung of vaccinated mice, and promoting faster bacterial clearance. CCL21 was also detected by ELISA in lung lysates and serum from mice euthanized at day 30 post-challenge. Increased chemokine levels were detected in the LPS+rPorB vaccinated/challenged group, similarly to previously reported studies on chronic tuberculosis infection and autoimmu- nity [30,43]. Overall our findings may suggest that local antibody production and cell mediated immunity, activated by vaccination with LPS+rPorB and further stimulated by LVS challenge, triggered the formation of BALT in an attempt to clear bacterial infection in the lung. As mentioned above, infection with LVS alone also induced accumulation of lymphocytes around bronchi [40], but after IF staining we observed a lower cellular organization of the areas (data not shown). This indicates that PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e11156