DC Subsets in BCG Infection Figure 1. BCG immunization induced the expansion of CD8+ DC which expressed higher levels of co-stimulatory molecules compared with CD8- DC. Mice (C57BL/6, n = 4/group) were infected i.v. with 56105 CFU of BCG and sacrificed at 21 days after immunization. Total DCs from infected and na¨ıve mice were purified using the MACS CD11c+ isolation column. Purified DCs were co-stained with APC-conjugated anti-CD11c, PE-Cy7-conjugated anti- CD8 and FITC-conjugated Ab specific for one of the surface markers (CD80, CD86, CD40 or MHCII). The surface marker expression (solid lines) or matched Ab isotype control (shaded histogram) are shown respectively. All histogram were based on 10,000 cells satisfying a gate set of forward vs side scatter light histogram. A, purified DCs were gated on CD11c positive cells showing CD8+ DC population in infected (iDC) and na¨ıve (nDC) mice. B, purified DC were gated on either CD11c+ CD8+ DC (CD8+DC) or CD11c+CD82 DC (CD82 DC) and the surface molecules on the DC subsets were shown. The percentages of positive cells and mean fluorescence intensity (MFI) for the molecules were shown at the top and bottom lines respectively at the right upper corner of each histogram. doi:10.1371/journal.pone.0009281.g001 To further analyse the cytokine production by the DC subsets, we purified iCD8+ DC and iCD82 DC using MACS columns. As shown in Figure 2A, the sorted DC subsets were in high purity. To assay the cytokines production pattern of the DC subsets, the purified iCD8+ DC and iCD82 DC were cultured and the cytokine levels in the 72 h culture supernatants were measured by ELISA. As shown in Figure 2B, iCD8+ DC produced significantly higher level of IL-12p70 than iCD82 DC, which produced more IL-10. Taken together, the results indicate that BCG immunization leads to a preferential expansion of CD8+ DC which shows a different profile of co-stimulatory surface molecules and cytokines from CD8- DC. iCD8+DC Are More Efficient in Promoting Th1 (IFNc) Cytokine Production While iCD82 DC Are More Efficient in Promoting Th2 Cytokine (IL-4) Production by T Cells Primed with BCG In Vivo Since the analyses shown above on co-stimulatory molecule expression and cytokines production suggest a functional differ- ence of the DC subsets isolated from BCG immunized mice, We performed experiments to address this by co-culturing iCD8+ DC and iCD82 DC subsets separately with CD4 T cells isolated from BCG primed mice. As shown in Fig. 3, iCD8+ DC promoted dominant Th1-cytokine (IFNc) production, while the iCD82DC induced a dominant Th2 (IL-4) cytokine response by antigen- specific CD4 T cells. The experiments using an in vitro approach demonstrated that the DC subsets were functionally different in directing BCG antigen-specific T cells. Adoptive Transfer of CD8+ DC, but Not CD82 DC, Enhanced Bacterial Clearance and Reduced Pathological Reactions in the Infected Tissues Following Challenge Infection To further examine the functional difference of the DC subsets, we adoptively transferred the iCD8+ DC and iCD82 DC subsets and tested protection in the recipients of the different DC subsets to challenge infection. DC subsets were purified from BCG-infected PLoS ONE | www.plosone.org 4 February 2010 | Volume 5 | Issue 2 | e9281