DC Subsets in BCG Infection Figure 6. Pro-inflammatory cytokine/chemokine levels in local tissues in mice treated with different DC subsets following challenge infection. The mice were treated as described in the legend to Fig. 4 and the levels of IL-6, MIP-1a and TNF-a proteins in the homogenates of lung and liver tissues were measure by ELISA. doi:10.1371/journal.pone.0009281.g006 infected DCs induced a potent protection against aerosol Mtb challenge, similar to those induced by in vivo BCG immunization [24]. Gonzalez-Juarrero et al reported that lung DCs pulsed with Ag85 (LDC-Ag85) in vitro were able to prime naive CD4+ T cells in vivo after adoptively transfer [25]. However, the LDC-Ag85 recipients were not more resistant to Mtb challenge than those receiving DCs pulsed with an irrelevant protein, instead, showing more serious consolidation in the lung. Comparing to these reported studies, our study using DCs primed/infected with BCG in vivo addressed the function of DCs in a more physiological and relevant way. Most importantly, our data not only showed the critical role of DCs in BCG-induced protection against mycobac- terial challenge infection, but the different capacity of DC subsets in this matter. In theory, a potential possibility for the difference of the DC subsets in generating protective immunity is because the DC subsets are different in uptaking and/or hosting BCG thus leading to differential immune responses. This concern is reasonable because some early studies have shown a poor phagocytic capacity of CD8+DC in certain infections [26,27]. However, our analysis on BCG gene expression in the two DC subsets showed similar levels of BCG mRNA in the isolated DC subsets (Figure 9). This is consistent with a reported study showing the similar efficiency of CD8+ DC and CD8- DC in acquiring mycobacteria-derived antigens [28]. This is also consistent with other reports which demonstrate a similar efficacy of the CD8+ DC and CD82 DC subsets in phagocytosis [29,30]. Therefore, the difference in immune responses generated by the adoptive transfer of the DC subsets observed in our study is more likely reflecting a functional difference of the subsets in activating/modulating T cell responses rather than their difference in handling the microbes/antigens, especially considering the dramatic difference of the DC subsets in the expression of co-stimulatory molecules and the production of immunoregulatory cytokines. The expression of co-stimulatory cell surface molecules and the production of cytokines have been shown to be the most important bases by which DCs modulate the function/polarization of T cell responses. Our data showing the significant differences of the DC subsets in the expression and production of these molecules and cytokines fit well with the functional difference of the DC subsets in inducing the different types of immune response and protection. In our study, a higher proportion of iCD8+ DC expressed costimulatory surface markers CD80 (65.66% vs. 17.43%), CD86 (57.43% vs. 30%) and CD40 (44% vs. 35%) compared with PLoS ONE | www.plosone.org 8 February 2010 | Volume 5 | Issue 2 | e9281