A New TB Vaccine Strategy Figure 2. Cytokine frequencies and phenotypic profiles of specific CD8 T cells in Ad-H4, rH4, or rH4/Ad-H4 vaccinated mice one week after final vaccination. CB6F1 mice were vaccinated once with Ad-H4, twice with rH4 in cationic liposomes, or once with rH4 in cationic liposomes followed two weeks later by one Ad-H4 vaccination. Mice were sacrificed one week after the final vaccination and splenocytes were stimulated with Ag85B or TB10.4 peptides prior to staining with anti-CD8, -CD44, -IFN-c, -IL-2, and –TNF-a. (A and C) Frequencies represent IFN-c, IL- 2, or TNF-a producing CD44+CD8+ T cells out of total CD8 T cells specific for Ag85B (A) or TB10.4 (C). Background staining from cells stimulated with medium alone has been subtracted. Data represent the mean+SEM of a minimum of five mice per group with ***, p,0.001, **, p,0.01, and *, p,0.05 (2-way ANOVA with Bonferroni posttest). (B and D) IFN-c, IL-2, and TNF-a cytokine profiles of Ag85B (B) or TB10.4 (D) specific CD8 T cells shown as triple, double, or single positive CD8 T cells. The color code for the pies is the same as shown in Fig. 1E. Dot plots, representative of five mice, are also shown in (B) and (D), depicting IFN-c expressed by CD44+CD8+ cells after stimulation with Ag85B (B) or TB10.4 (D). (E) Cells were stimulated with Ag85B or TB10.4 in vitro for 72 hours and IFN-c levels in supernatants were assessed by ELISA. Data represent the mean+SEM of a minimum of five mice per group with ***, p,0.001, ** and p,0.01 compared with non-vaccinated group, unless otherwise indicated (2-way ANOVA with Bonferroni posttest). doi:10.1371/journal.pone.0005139.g002 were challenged with virulent M.tb six weeks after last vaccination, and two weeks after infection the mice were sacrificed. The two weeks post infection time point was chosen since the majority of cells in the lung at that time point are recalled T cells and not yet newly infection-primed T cells (Non vaccinated showed ,1% CD8 Ag85B or TB10.4 specific T cells and ,0.2% CD4 T cells) [23], which enabled us to measure recruitment of the pre-existing CD4 and CD8 T cells. Lung lymphocytes were stimulated in vitro with Ag85B and TB10.4, and expression of IFN-c, IL-2, and TNF-a in the CD4 and CD8 T cell subsets was analyzed by intracellular FACS. Two weeks post infection we observed a significant higher proportion of Ag85B and in particular of TB10.4 specific CD4 T cells in the lungs in the rH4/Ad-H4 group when compared to the non-vaccinated group (p,0.01) (Fig. 5A and C). There were also increased levels of CD8 T cells in the lungs of rH4/Ad-H4 vaccinated mice, in particular of the IFN-c+ TNF-a+ effector phenotype (Fig. 5B and D) [24,25]. Of all CD8 T cells 13.8% were specific for TB10.4 and 2.4% for Ag85B in the vaccinated group, and the numbers for CD4 T cells were 8.0% and 4.8%, respectively (Table 1). Thus, both CD4 and CD8 T cells were recruited to the lungs. Importantly, the CD8 T cells induced in the non-vaccinated mice showed a much higher frequency of single positive T cells, as compared to the vaccinated group, indicating the induction of terminally differentiated T cells PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e5139