A New TB Vaccine Strategy Figure 3. Functional characterization of the vaccine induced T cells. The specific lysis of TB10.43-11, TB10.420-28, and TB10.470-88 loaded cells were determined in an in vivo cytotoxicity assay. Unloaded (CFSElow) and TB10.43-11, TB10.420-28, or TB10.470-88 (CFSEhigh) loaded splenocytes from na¨ıve mice were transferred into Ad-H4 (A), rH4 (B), or rH4/Ad-H4 (C) vaccinated mice. (A–C) The amount of splenocytes killed in vivo by cytotoxic T cells specific for either of the TB10.4 target peptides was observed as a reduction in the CFSEhigh population Percent specific lysis was calculated and is shown in the far right graphs. Non-specific cytotoxicity from non-vaccinated mice has been subtracted (Non-specific killing in non-vaccinated mice of either CFSEhigh or CFSElow cells were from 5–15%). doi:10.1371/journal.pone.0005139.g003 by the infection alone (Fig. 5B and D). Interestingly, in the rH4/ Ad-H4 group more than 50% of all the cytokine producing CD4 T cells had a more memory-like phenotype (IFN-c+IL-2+TNF-a+, IL-2+TNF-a+ or IL-2+) [24,25], in comparison with only 10–20% of all the CD8 T cells (Table 1). To analyze whether there was a more extensive recruitment, or expansion, of CD4 versus CD8 T cells, we next quantified the cell proportions of Ag85B and TB10.4 specific CD4 or CD8 T cells before and after infection. To determine the numbers of the antigen specific cells, all the subpopulations were added to give the total number of T cells expressing any combination of IFN-c, TNF-a, and IL-2 (Table 1). Pre-infection (two weeks pre-infection) we observed equal amounts of Ag85B specific CD4 and CD8 T cells in the vaccinated group in the spleen (Fig. 6A). However, 2 weeks post infection there were 2K times more Ag85B CD4 T cells than CD8 T cells in the lungs (Fig. 6C and E). As for TB10.4 specific T cells, around 50 times more TB10.4 specific CD8 T cells were observed pre-infection in the spleen than CD4 T cells (TB10.4 CD4/CD8 ratio of 0.025) (Fig. 6B). However, after infection we found almost the same numbers of TB10.4 CD4 and CD8 T cells in the lungs (CD4/CD8 ratio of 0.80) (Fig. 6D and F). This demonstrated that the CD4 T cells in both the Ag85B and TB10.4 pool of T cells were initially recruited/expanded more than the CD8 T cells, even though both cell types were clearly recruited to the lung at the onset of the infection. The proliferative capacity of the recruited T cells at the site of infection Since CD4 T cells were preferentially recruited to the infection site, we next evaluated if these infiltrating CD4 T cells were the result of an increased turn-over rate of CD4 versus CD8 T cells. The vaccinated and challenged mice were given BrdU in the drinking water three days before sacrifice to measure the degree of proliferation of CD4 and CD8 T cells at two weeks post infection. Of all CD4 T cells 31.5%67.3 had incorporated BrdU, whereas this number was 11.3%61.3 (p,0.05) for CD8 T cells (Fig. 7A). Stimulation with TB10.4 or Ag85B followed by gating on the IFN- c positive antigen specific T cells showed that also for antigen specific T cells the CD4 T cell population showed the highest proliferation (Fig. 7B and C). Gating on all combinations of T cell subtypes expressing one or more of the cytokines IFN-c/IL-2/ TNF-a clearly demonstrated that the majority of proliferating CD4 cells were cytokine expressing, in contrast to the non- cytokine producing CD4 T cell population, which did not proliferate (IFN-c2IL-22TNF-a2, Fig. 7D). In contrast, for the PLoS ONE | www.plosone.org 5 April 2009 | Volume 4 | Issue 4 | e5139