Tularemia Vaccination and BALT Figure 8. Antibody producing cells and homeostatic chemokines in BALT areas at 30 days post-challenge. (A) Representative lungs from mouse vaccinated with LPS+rPorB and challenged with LVS. The PCNA-CD3 combined stain was conducted to differentiate between nuclear (PCNA+) and surface (CD3+) staining. The upper left panel shows GC (cluster is outlined by the white dashed line) shown by the presence of cells with large PCNA+ nuclei (red nucleus staining). IgG producing proliferating plasma cells (indicated by the yellow arrows-main panel and insert) are detected with an anti-mouse IgG antibody (green cytoplasm) and PCNA (red nuclei), and few CD3+ cells (red surface) were identified in the same location. The upper middle panel shows IgM producing proliferating plasma cells (indicated by the yellow arrows-main panel and insert), identified by PCNA+ stain (red nuclei) and a dense IgM+ antibody signal (green cytoplasm). IgM+ B cells had a distinctive signal localized exclusively on the surface (green surface), and some CD3+ cells (red surface) were also detected. In the upper right panel, few IgA producing plasma cells (green cytoplasm) are present, but no proliferation is evident. Several CD3+ cells (red surface) were identified in the same area. In the lower left panel, some CXCL13 signal (red surface) co-localizes with the FDC network (combination of CD21, CD35 and FDCM1 markers-green) in the BALT area. The lower middle panel shows B cell clusters (green surface) and a CCL21+ high endothelial venule like structure (HEV) (red surface) as indicated by the yellow arrowhead. In the lower right panel, CCL21+ cells (red surface), resembling lymphatics or endothelial cells are shown by the white arrows. All sections were viewed with a 206 objective, and a 406 objective for the inserts in the first two upper panels. (B) Levels of CCL21 in lung lysate and serum are expressed as picograms (pg) per milliliter (ml) of lung lysate or serum. * (P,0.05). doi:10.1371/journal.pone.0011156.g008 respiratory tularemia. Overall, it is important to keep in mind the diversity of lung lymphoid structures (classic BALT versus iBALT) in different physiological and pathological settings, including autoimmunity, long lasting antigenic stimulation and chronic bacterial disease [46], as well as the different animal species they are detected in, for a better understanding of their role in the induction of local protective immunity. One additional interesting finding was the high stability of BALT that was detected at late time points after intranasal challenge in survivor mice, contrasting with the absence of lymphoid aggregates in lungs from moribund mice (data not shown). Although we showed pictures of BALT taken at 30 days post-infection, these organized structures were still found at 120 days after infection. Bacterial cultures of lung lysates were negative at these late time points, suggesting that factors other than chronic infection must play a role in BALT persistence. Similarly, another study reported that iBALT persisted in murine lungs even after the Influenza virus had apparently been cleared [47]. One explanation for this phenomenon is that protein and peptide antigens could remain in these areas for extended periods, continuously PLoS ONE | www.plosone.org 10 June 2010 | Volume 5 | Issue 6 | e11156