A New TB Vaccine Strategy and B) or per lung post infection (C and D). Background staining from cells stimulated with medium alone has been subtracted. Data represent the mean+SEM of four mice per group with **, p,0.01 (2-way ANOVA with Bonferroni posttest). (E and F) The ratio between Ag85B (E) and TB10.4 (F) specific CD4 and CD8 T cells was calculated from A–D. doi:10.1371/journal.pone.0005139.g006 BrdU In experiments measuring proliferation, BrdU (0.8 mg/ml) was added to the drinking water to the mice for three days before sacrifice. Bacteria M.tb Erdman was grown at 37uC in suspension in Sauton medium (BD Pharmingen) enriched with 0.5% sodium pyruvate, 0.5% glucose, and 0.2% Tween 80. All bacteria were stored at Figure 7. The proliferative capacity and frequency of the recruited Ag85B and TB10.4 specific CD4 and CD8 T cells to the lungs. CB6F1 mice were vaccinated once with rH4 in cationic liposomes followed two weeks later by one Ad-H4 vaccination, and challenged by the aerosol route with virulent M.tb at week 10. The last three days before killing, BrdU (0.8 mg/ml) was added to the drinking water. Two weeks after challenge, mice were sacrificed and lung lymphocytes were stimulated with Ag85B or TB10.4 peptides prior to staining with anti-CD4, -CD8, -BrdU, and -IFNc (A–C). (A) Frequencies represent the percentage of BrdU+ CD4 or CD8 T cells out of total CD4 or CD8 T cells. Data represent the mean of +SEM of four mice per group with *, p,0.05 (Unpaired t-test, two-tailed). Dot plots, representative of four mice, are also shown depicting BrdU+ CD4 or CD8 T cells. (B and C) Frequencies represent the percentage of BrdU+ CD4 or CD8 T cells out of total IFN-c+ CD4 or CD8 T cells specific for either Ag85B (B) or TB10.4 (C). Data represent the mean+SEM of two mice per group with **, p,0.01 (Unpaired t-test, two-tailed). Background staining from cells stimulated with medium alone has been subtracted. (D and E) CB6F1 mice were vaccinated once with rH4 in cationic liposomes followed two weeks later by one Ad-H4 vaccination, and challenged by the aerosol route with virulent M.tb at week 10. The last three days before sacrificing the animals, BrdU (0.8 mg/ml) was added to the drinking water. Two weeks after challenge mice were sacrificed and lung lymphocytes were stimulated with TB10.4 peptides prior to staining with anti-CD4, -CD8, -BrdU, -IFN-c, TNF-a, and IL-2. The BrdU histograms represent a normalized overlay of all the cytokine producing CD4 (D) and CD8 (E) T cells, (IFN-c+IL-2+TNF-a+, IFN-c+TNF-a+, IFN-c+IL-2+, IFN-c+, IL-2+TNF-a+, IL-2+, and TNF-a+) (solid line), and all the non-cytokine producing CD4 and CD8 T cells (IFN-c2IL-22TNF-a2) (dashed line), and the percentages shown are the percentages of cytokine producing or cytokine non-producing T cells out of total T cells. Instead of displaying counts in the FACS histogram overlays, each histogram in the overlay has been normalized to its maximum counts (‘‘% of MAX’’ counts). Data represent the mean of +SEM of three mice per group. Background staining from cells stimulated with medium alone has been subtracted. doi:10.1371/journal.pone.0005139.g007 PLoS ONE | www.plosone.org 10 April 2009 | Volume 4 | Issue 4 | e5139