In our experiment, Ag85A (5 μg/ml) and ConA (10 μg/ml) were used as a specific stimulator MK-1775 in vitro and a polyclonal stimulator of T cells, respectively. As shown in Fig. 3, a low background level of T cell proliferation was observed in vector control group and pcDNA3-ub group. A significant increase in T cells proliferation (P < 0.01) was observed in pcDNA3-Ag85A group compared with vector group or pcDNA3-ub group. The ubiquitinated Ag85A DNA vaccine significantly enhanced Th cell proliferation responses compared with non-ubiquitinated Ag85A DNA vaccine (P < 0.05). As a specific indicator of CD4+ T cell activation, the cytokines were also detected. Th1 cytokines (IL-2,

IFN-γ) and Th2 cytokines (IL-4, IL-5 and IL-10) are major parameters in our understanding of the polarization of immune responses. Th1 immune responses this website are thought to drive induction of cellular immunity, whereas Th2 immune responses preferentially drive humoral immunity. In this study, the level of IFN-γ and IL-4 was examined. As demonstrated in Fig. 4, the level of IFN-γ was significantly higher in Ag85A DNA vaccine group than that in pcDNA3 group or in pcDNA3-ub group. The secretion of IFN-γ significantly increased in UbGR-Ag85A fusion DNA vaccine group (P < 0.01) compared with Ag85A DNA vaccine group. However, the level of IL-4 was lower in fusion DNA vaccine group than that in non-fusion

vaccine group (P < 0.01). In Ag85A DNA vaccine group, the level of IFN-γ was higher than that of IL-4, which indicated the Ag85A DNA vaccine elicited a Th1-profile immune response. The ub fusion DNA vaccine increased the secretion of IFN-γ and decreased the level of IL-4, which demonstrated that the ub fusion enhanced the Th1-type immune response. As IFN-γ is clearly a key molecule in the anti-tuberculosis protective response, the role of CD4+ and CD8+ T cell for secreting IFN-γ was investigated by intracellular staining. As shown in Fig. 5, the frequency of IFN-γ+ CD4 T cells and IFN-γ+ CD8 T cells was higher in Ag85A DNA vaccine group than those in pcDNA3 vector group or in pcDNA3-ub group. The frequency of IFN-γ+ CD8

T cells was much higher in the spleen of the UbGR-Ag85A fusion DNA vaccine group than that in Ag85A DOK2 DNA vaccine group (P < 0.01). Although to a lesser extent, the frequency of IFN-γ+ CD4 T cells was also higher in the UbGR-Ag85A fusion DNA vaccine group, compared with the Ag85A DNA vaccine group (P < 0.05). Overall, UbGR-Ag85A fusion DNA vaccine induced more antigen-specific CD8+ T cells than CD4+ T cells. These results indicated that UbGR-Ag85A fusion DNA vaccine activated CD4+ and CD8+ T cells, particularly CD8+ T cells. Cytotoxic T cell responses were determined with a LDH release assay, after in vitro restimulation, against the target cell line P815-Ag85A, which stably expressed the Ag85A protein. P815 cell was used as a negative control. As shown in Fig.