Table 1. Frequency of T cell subsets. T cells (%)*** IFN-c+ IL-2+ TNF-a+ IFN-c+ IL-2+ TNF-a2 IFN-c+ IL-22 TNF-a+ IFN-c+ IL-22 TNF-a2 IFN-c2 IL-2+ TNF-a+ IFN-c2 IL-2+ TNF-a2 IFN-c2 IL-22 TNF-a+ In total CD4 T cell (%)*** CD8 T cell (%) Ag85B CD4 0.44 0.00 0.34 0.03 0.37 0.00 0.51 1.69 CD8 0.06 0.01 1.13 0.81 0.03 0.00 0.66 2.69 15.1 18.0 Two weeks pre-infection* Two weeks post Infection** TB10.4 CD4 0.01 0.00 0.03 0.00 0.01 0.00 0.10 0.15 CD8 0.04 0.02 3.29 2.11 0.96 0.00 0.64 7.05 Ag85B CD4 0.40 0.01 1.99 0.11 0.30 0.00 2.03 4.84 CD8 0.04 0.00 1.68 0.27 0.00 0.11 0.25 2.35 25.1 19.4 A New TB Vaccine Strategy TB10.4 CD4 0.67 0.03 3.56 0.10 0.56 0.00 3.10 8.03 CD8 0.18 0.00 10.99 1.46 0.02 0.32 0.89 13.85 Two weeks pre-infection* Two weeks post Infection** Frequency of cytokine producing Ag85B and TB10.4 specific CD4 and CD8 T cells two weeks before and after M.tb challenge, as well as the frequencies of total CD4 and CD8 T cells, in rH4/Ad-H4 vaccinated mice. *The frequencies were obtained by FACS analysis on in vitro antigen stimulated splenocyte cultures from vaccinated mice (two weeks before challenge). **The frequencies were obtained by FACS analysis on in vitro antigen stimulated lung lymphocyte cultures from vaccinated and M.tb challenged mice two weeks after challenge. ***Background staining from cells stimulated with medium alone has been subtracted. doi:10.1371/journal.pone.0005139.t001 and 2–3% for Ag85B, indicating that the CD8 epitopes were indeed processed and presented in the lungs at the very onset of infection, and that CD8 T cells primed by Ad5 vector delivery, as well as the CD4 T cells, were recruited to the site of infection (pre- infection the numbers in the lung were less than 0.1%, data not shown) (Fig. 5). For both the CD4 and CD8 T cell subsets we observed more polyfunctional cells (IFN-c+TNF-a+ or IFN- c+TNF-a+IL-2+) in the rH4/Ad-H4 group following M.tb challenge compared non-vaccinated mice. This could indicate that this vaccination strategy resulted in a pool of polyfunctional T cells that was maintained after challenge, whereas the natural infection of unprimed mice tended to drive the activated T cells toward a state of terminal differentiation (IFN-c+) (Fig. 5) [25]. Interestingly, in the rH4/Ad-H4 vaccinated mice, we noted important differences between the CD4 and CD8 T cell recall responses. Thus, although we observed strong, local CD8 T cell memory, as measured by total CD8 T cell numbers, CD4 T cells preferentially accumulated (due to recruitment and/or expansion) at the site of infection (Fig. 6). In line with this, CD4 T cells with proliferative capacity showed higher accumulation at the site of infection than CD8 T cells two weeks post infection (although we can exclude that this bias in CD4 proliferation may in part reflect a bias inherent in the CD4 prime/boost mechanism). This was observed by analyzing the total pool of CD4 T cells, and also when studying TB10.4 and Ag85B antigen specific T cells (Fig. 7). Moreover, as for the phenotype of the proliferating cells, the majority of the cytokine (any combination of IFN-c, TNF-a or IL- 2) producing CD4 T cells belonged to the proliferating CD4 T cell population, in contrast to the cytokine producing CD8 T cells that where mainly non-proliferating (Fig. 7). Taken together, these data revealed that although both CD4 and CD8 T cells were primed through vaccination, there was an increased recruitment and proliferation of the CD4 T cells as compared to the CD8 T cells suggesting that this subset may be primarily involved in the recall response that results in early control of bacterial replication in vaccinated mice, and that increasing the CD4 T cell numbers in this stage of the infection is important. We are presently testing H4 in new adjuvants specifically selected for their ability to induce a strong CD4 T cell response. In conclusion, heterologous prime boost based on H4, produced an additive effect on the priming of CD4 and CD8 cells and in terms of the protective capacity of the vaccine, and therefore represent an interesting vaccine strategy against M.tb. However, compared to CD8 T cells, antigen specific CD4 cells were more polyfunctional, recruited at a higher rate, and proliferated more following infection with M.tb, indicating that although both CD4 and CD8 epitopes are clearly presented at the very onset of infection, CD4 and CD8 T cells respond very differently to live infection, in a manner which support the consensus that CD4 T cells play the major role during the early stages of an M.tb infection. Materials and Methods Ethics Statement The handling of mice were conducted in accordance with the regulations set forward by the Danish Ministry of Justice and animal protection committees by Danish Animal Experiments Inspectorate, and in compliance with European Community Directive 86/609 and the U.S. Association for Laboratory Animal Care recommendations for the care and use of laboratory animals. Animals Studies were performed with six-eight week-old female CB6F1 (C57BL/66BALB/c) mice from Harland Netherlands. Non-infected mice were housed in cages in appropriate animal facilities at Statens Serum Institut or at the Panum Institute, University of Copenhagen. Infected animals were housed in cages contained within laminar flow safety enclosures (Scantainer from Scanbur, Denmark) in a separate biosafety level 3 facility at Statens Serum Institut. All mice were fed radiation sterilized 2016 Global Rodent Maintenance diet (Harlan, Scandinavia) and water ad libitum. All animals were allowed a one- week rest before initiation of the experiments. PLoS ONE | www.plosone.org 8 April 2009 | Volume 4 | Issue 4 | e5139