DC Subsets in BCG Infection Figure 2. Different levels of cytokine production by DC subsets. DC subsets were isolated from spleens of BCG infected (i.v.) mice at day 21 post-immunization as described in the Materials and Methods. A, purified DC subsets were analyzed by flow cytometry for purity. The 56105 sorted cells were co-stained with APC-conjugated anti-CD11c and PE-conjugated anti-CD8. The purities of the sorted iCD8+ DC (left) and iCD82DC (right) are indicated at the right upper corner and right lower corner respectively. B, freshly isolated iCD8+ DC and iCD82 DC subsets were cultured. The culture supernatants were harvested at 72 h and tested for IL-10 and IL-12p70 by ELISA. Data are shown as mean6SD of each group. One representative experiment of three independent experiments with similar results is shown.*p,0.05, **p,0.01. doi:10.1371/journal.pone.0009281.g002 mice at day 21 post immunization and were adoptively transferred i.v. to na¨ıve C57BL/6 mice. At 7 days after cell transfer, the recipient mice were challenged i.v. with BCG. Mice that received PBS (sham treatment) or DC subsets from naive mice with the same challenge infection were used as controls. Twenty-one days after challenge infection, mice were sacrificed and the bacterial loads in the lung and liver were measured. As shown in Fig. 4, a significant reduction in tissue bacterial loads were observed in the mouse group receiving iCD8+ DC compared with the mice without DC transfer (sham treated control mice). In contrast, although the recipients of iCD82 DC also appeared to have a trend of lower CFU in the lung and liver compared to PBS controls, the differences were not statistically significant. As controls, the recipients of either DC subsets from na¨ıve mice (nCD8+ DC or nCD82 DC) showed comparable levels of bacterial loads with the PBS treated control mice, therefore not generating protection. More remarkably, further histopathological analysis (Figure 5) showed that the recipient of iCD8+ DC only had mild pathological changes in the lung and liver. In contrast, the sham-treated mice and the recipients of iCD82 DC showed much more severe pathological inflammatory reactions. Interestingly, although both sham-treated mice and iCD82 DC recipients showed severe pathological reactions, their pattern of changes were different. Sham-treated mice showed multiple granulomas in both lung and liver tissues, characterized by dominant epithelioid cells (indicated by green arrows in Fig. 5) at centre with surrounding lymphocytes, neutrophil and monocytes in peribronchial and perivascular areas (lung) and peri-central vein areas of the hepatic lobules (liver). Figure 3. iCD8+ DC and iCD82 DC subsets generate different patterns of BCG-driven T cell cytokine production. BCG-specific CD4+ T cells (56105) cell from BCG-infected mice were co-cultured with iCD8+ DC or iCD82 DC (56104 cells/well) in the presence of HK-BCG. Culture supernatants were collected at 72 h. IFN-c and IL-4 were measured by ELISA. The experiment was repeated twice showing similar results. *p,0.05. doi:10.1371/journal.pone.0009281.g003 PLoS ONE | www.plosone.org 5 February 2010 | Volume 5 | Issue 2 | e9281