The mice only received the PBS (sham-treatment) or DC subsets from naive mice were used as control groups. At day 7 after adoptive transfer, the mice were challenged with 56105 CFU BCG i.v. and were sacrificed at day 21 after challenge. The lungs and livers were homogenized in 10 ml PBS and plated in serial dilutions onto the Middlebrook 7H11 agar with Middlebrook ADC enrichment. The culture was allowed to proceed for 21 days at 37uC in an atmosphere of 9% CO2 and the number of CFUs was counted. For model 2, the DC subsets were adoptively transferred to recipient mice by intranasal (i.n.) route at the amount of 26105 cells in 40 ml sterile protein-free PBS. Two hours later, the recipient mice were challenged with 26105 CFU BCG i.n. and killed at day 21 after challenge infection. The BCG CFU in the lung homogenates were measured as above. Histopathological Analysis Lung and liver tissues of the mice with challenge infection were routinely fixed in 10% buffered formalin, embedded in paraffin, sectioned by a microtome, stained by hematoxylin and eosin (H&E) and examined under a light microscope. Infiltrating inflammatory cells were identified based on cellular morphology and characteristics. Cytokine Response Analysis For analysing cytokine production by cultured spleen cells, single spleen cells (7.56106 cells) were cultured in 1 ml complete medium for 72 h in the presence of heat-killed (HK)-BCG (7.56105 CFU). Cytokines in the supernatants were measured using ELISA. For testing cytokine levels in local tissues, the lung and liver tissues were homogenized in the 10 ml cold PBS. The supernatant were collected after centrifugation and the cytokine were measured by the ELISA. To test the spontaneous cytokines production by the freshly isolated DC subsets, the DC subsets cells were cultured with complete medium using 96-well culture plates at 56105 cells/well for 72 hours. The level of IL-12p70 and IL-10 in the culture supernatants were measured by ELISA. DC Subsets–T Cell Co-Culture To directly assess the T cell modulating abilities of DC subsets, purified DC subsets were cultured with CD4 T cells isolated from BCG-infected mice as we previously described[22]. CD4 T cells were isolated from the spleen using the CD4 T cell isolation column from MACS. The purity of CD4 T cells were .95%. DC subsets (16105 cells/well) and CD4 T cells (16106 cells/well) were co-cultured in 96-well plates in the presence of HK-BCG (56104 CFU/well) in 200 mL complete RPMI medium for 72 hours and the concentration of IFN-c and IL-4 in the supernatants were measured by ELISA. Quantitation of BCG Gene Expression in DCs by Real-Time RT-PCR To analyze the BCG gene expression in the isolated DC subsets from BCG infected mice, real-time RT-PCR were performed. cDNA synthesis is performed using total mRNA primed with random primer. The thermal profile for RT was incubated at 25uC for 5 min, 50uC for 1 hour. Inactivation of the reaction was done by heating at 70uC for 15 min. The real-time RT-PCR was carried out on the MiniOpticonTM System using an iGTM SYBR Green Supermix (BioRad Laboratories, Hercules, California, USA).The PCR was performed in a 48-well plate in a reaction volume of 25 ml containing 12.5 ml 2x iGTM SYBR Green Supermix Mix, 200 nM each of forward and reverse primer, DC Subsets in BCG Infection 0.5 ml template cDNA, added nuclease-free water to a final volume of 25 ml. The thermal profile for PCR was 95uC for 10 min, followed by 40 cycles of 95uC for 30 s and 58uC for 30 s, and 72uC for 60 s. Fluorescent signals were read and the data were collected at each annealing temperature. For each sample, the amplification plot and the corresponding dissociation curve were examined. The melting curve analysis, determining the specificity of the reaction, was carried out immediately after the final PCR cycle by measuring the changes in fluorescence during slowly heating the amplicon/probe heteroduplex. The threshold cycle (Ct) used in the real-time PCR quantification was defined as the PCR cycle number that a noticeable increase in reporter fluorescence above a baseline signal. The efficiency of target gene and GAPDH is similar and a comparative Ct method was used for calculations[23]. GAPDH was used as an endogenous control gene for normalization of the amount of RNA added to the reactions. DCt = Ct(BCG) - Ct(GAPDH), which means the difference between the threshold cycle of BCG and the threshold cycle of the corresponding GAPDH in the same sample. For each experiment, a negative control of nuclease-free water and a positive control (with known Ct value) were run in triplicate. The specific primers used in quantitative real-time PCR were: BCG IS6110 (123 bp): 59CCTGCGAGCGTAGGCGTCGG; 39CTC- GTCCAGCGCCGCTTCGG. GAPDH (191 bp): 59AACGAC- CCCTTCATTGAC, 39CACGACTCATACAGCACCT. Statistical Analysis One-way ANOVA (One-way analysis of variance) and further Newman-Keulse test were used to determined statistic significance among groups. CFU of BCG was converted to logarithmic values and analyzed using the ANOVA test. The value of P,0.05 was considered significant. Results BCG Immunization Induces Preferential Expansion of CD8+ DC Subset Which Exhibits Differential Expression of Surface Markers and the Production of Cytokines Compared to CD82 DC Subset To analyze the effect of BCG immunization on DC subset, we measured CD8 molecule expression on the total DC (CD11c+ cells) isolated from the spleens of BCG infected and naive C57BL/ 6 mice. As shown in Figure 1A, DCs isolated from BCG infected mice showed a higher percentage of CD8+ subpopulation than the DCs from na¨ıve mice (37% vs 21%), suggesting a preferential expansion of CD8+ DC following BCG immunization. Since the function of DC in modulating immune responses is largely dependent on their expression of co-stimulatory molecules and the production of cytokines, we further analyzed the surface markers on the DC subsets by three-color staining (CD11c, CD8 and a particular surface marker). As shown in Figure 1B, in comparison with the CD82 DC isolated from BCG infected mice iCD82 DC, the CD8+ DC isolated from the mice with the same infection iCD8+ DC expressed higher CD80 (65.66% vs 17.43%), CD86 (57.43% vs 30%) and CD40 (44% vs 35%) molecules. Similar differences were observed in comparison of the density (mean fluorescence intensity, MFI) of these molecules expressed on the surface of these cells. Although the iCD8+ DC and iCD82 DC subsets showed similar MHC-II in percentage (97% vs 95%), the MFI of MHC-II was much higher in iCD8+ DC. Similar pattern of differences in expression levels of surface markers were found in na¨ıve DC subsets, but the absolute levels in the na¨ıve mice were lower than those of infected mice, suggesting a significant impact of immunization on both DC subsets. PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9281