DC Subsets in BCG Infection Figure 4. Adoptive transfer of iCD8+DC, but not iCD82 DC, reduced bacterial growths in recipients following either i.v. or i.n. challenge infection. DC subsets were sorted from BCG-infected (i.v.) mice or na¨ıve mice and adoptively transferred to syngeneic recipients (C57BL/6, n = 4/group) by i.v. (A&B, 56105 DC/mouse) or i.n. routes (C, 26105 DC/mouse). Mice were challenged with BCG through i.v. (A&B) or i.n. (C) routes, respectively, as described in Materials and Methods. All mice were sacrificed at day 21 post challenge infection. Homogenized lung or liver tissues were measured for BCG CFU. The CFUs of BCG were converted to logarithmic values and presented as mean6SD of each group. One representative experiment of three independent experiments is shown. * p,0.05;**, p,0.01. doi:10.1371/journal.pone.0009281.g004 Multinucleate giant cells were also observed in the sham-treated mice. In contrast, the inflammation in iCD82 DC recipients was diffused without notable granulomas and lack of typical epitheloid cells. The infiltrating cells in these mice were mainly neutrophils and mononuclear cell. Since tuberculosis is mainly a pulmonary disease, we further analysed the protective function of the DC subsets using a model of intranasal challenge infection. DC subsets were adoptive transferred to recipient mice followed by i.n. challenge infection with live BCG. Similar to what observed in the i.v. infection model, the recipients of iCD8+ DC showed lest organism growth (Figure 4B) and mildest pathological changes (not shown) in the lung among the groups, exhibiting very few inflammatory cells following intranasal challenge. In contrast, evident pathological changes were found in the sham treated control group (multiple granuloma and heavy inflammation) and iCD82 DC recipients (disseminated heavy inflammation). Taken together, the results indicate that only the CD8+ DC subset from infected mice can generate significant protective immunity in vivo. Moreover, since granuloma formation is a way for host to control infection (although not very efficient), the failure of iCD82 DC recipients to have granuloma formation instead showing heavily diffused inflammation suggest it may promote an inflammatory reaction which is not protective. Since proinflammatory cytokine and chemokine responses contribute to the inflammation of local tissues and correlate with the degree of pathological reactions in some circumstances, we further measured the MIP-1a (CCL3), IL-6 and TNF-a concentration in the lungs and liver. In consistence with the lowest degree of inflammation in the iCD8+DC recipients (Figure 5), this group of mice showed the lowest levels of IL-6, MIP-1a and TNF-a response in the local tissues (Figure 6). In contrast, the recipient of iCD82DC showed highest levels of these cytokines, even higher than the sham-treated, infected mice. Potentially, in the infected tissues many different types of inflammatory cells can produce these cytokines/chemokine including, but not limited to macrophages, DCs, and lymphocytes. The higher levels of these molecules in the lung of the iCD82DC recipients suggest a non-protective or less protective severe inflammation in the lung, which is not efficient for clearing the infection but contributes to pathological changes. This data are consistent with the severe and diffused inflammation in the iCD82DC recipients (Figure 5). The data suggest that the transfer of iCD8+DC, but not iCD82DC, can reduce the pathological inflammatory responses in the local tissues. The Adoptive Transfer of iCD8+ DC, but Not iCD82 DC, Enhances Type 1 Immune Responses Following Challenge Infection Since the type of immune responses plays a critical role in protection against mycobacterial infection, we further examined the T cell cytokine patterns and antibody responses in the recipients of different DC subsets in order to elucidate the mechanism underlying the difference in protection. As shown in Figure 7A, significant difference in the production of BCG driven type-1 related cytokines (IFN-c and IL-12) by bulk cultured spleen cells was observed between the different groups following challenge infection. Specifically, the recipient of iCD8+ DC showed significantly higher IFN-c production than the control mice without cell transfer. Analysis of cytokine patterns in the local PLoS ONE | www.plosone.org 6 February 2010 | Volume 5 | Issue 2 | e9281