DC Subsets in BCG Infection Figure 5. Significantly milder pathological changes in recipients of iCD8 +DC following intravenous challenge infection. Mice were treated/challenged as described in the legend to Figure 4 and analyzed for histopathological changes in lung and liver at day 21 post challenge infection by H&E staining. Low magnification(x20) and high magnification (x100) were shown respectively. Green arrows indicate typical epithelioid cells. doi:10.1371/journal.pone.0009281.g005 tissues also showed higher IL-12 and IFN-c levels in the lung (Figure 7B) and liver (Figure 7C) of the iCD8+ recipient mice compared to sham control groups and iCD82 DC recipient mice. Serum antibody analysis showed low titers of BCG specific IgG2a and IgG1 antibodies in all the groups and no significant difference was observed among the groups (data not shown). To further examine the local T cell responses, we performed intracellular cytokine staining of T cells from the draining lymph nodes following intranasal challenge infection. As shown in Figure 8, the adoptive transfer of iCD8+ DC induced more IFN- c producing T cells than iCD82 DC. In particular, recipients of iCD8+ DC mounted more than two fold higher IFN-c producing CD8 T cells than the sham-treated mice. Similarly, more IFNc– producing CD4 T cells were also found in the recipients of iCD8+ DC. In contrast, adoptive transfer of iCD82 DC failed to enhance IFNc response by either CD4 or CD8 T cell. Taken together, the results suggest that iCD8+ DC has strong ability to promote type-1 T cell responses to mycobacterial challenge infection, which may be the basis for its strong capacity to generate protection against both systemic and local challenge infections. DC Subsets Isolated from BCG Infected Mice Show Similar Levels of BCG Messages A question which needed to be addressed was if the differences observed above for the DC subsets isolated from infected mice in generating protective immunity was due to the potential difference of the subsets in BCG loads, thus the amount of antigens carried. To answer this question, we further analyzed the bacterial loads of the CD8+DC and CD82DC subsets from BCG infected mice using quantitative RT-PCR. As shown in Figure 9, the BCG mRNA levels in the two DC subsets demonstrated in real-time PCR analysis were comparable. The in vitro culture of either DC subsets showed negative results for viable BCG. The data suggest that the DC subsets are not significantly different in carrying BCG and possibly its antigens. Discussion This report assessed the capacity of DC subsets in BCG infected mice in generating protective immunity against mycobacterial challenge infection. The data revealed that CD8+ DCs, but not CD82DCs, from the infected mice are able to induce protection and reduce pathological reactions in the local tissues of the recipient mice. This conclusion is supported by the facts that the recipients of iCD8+ DCs exhibited significantly less bacterial growth, much milder histopathological changes and significantly stronger type-1 immune responses compared to the mice without cell transfer or received iCD82DCs. To our knowledge, this is the first report showing the different capacity of DC subsets primed by in vivo immunization by BCG in reducing bacterial loads and pathological reactions following systemic and local mycobacterial challenge infection. Several published studies have used DCs which are infected by BCG in vitro or are loaded with mycobacterial antigens to investigate the role of DC in inducing T cell immune responses and protection using cell adoptive transfer approaches [24,25]. Demangel et al found that bone marrow-derived DCs that were infected with BCG showed increased expression/production of MHC class II antigens, co-stimulatory molecules and immune regulatory cytokines. Intratracheally adoptive transfer of these PLoS ONE | www.plosone.org 7 February 2010 | Volume 5 | Issue 2 | e9281