phenotypic and functional alterations in DCs have been reported in individuals with mycobacterial infection[14]. In human, CD162mDC and CD16+mDC subsets have been found to be different in preferential activation of T memory cells, showing that CD16+mDC elicits stronger IFNc response than CD162mDC[15]. In mice, splenic CD8a+ and CD8a2 DC subsets have been extensively studied and some functional differences between the two subsets have been reported in several model systems including infections, although some studies showed overlapping functions of the two subsets [16–20]. In particular, we have reported that CD8a+ DC isolated from Chlamydia muridarum infected mice are more potent in inducing protection against challenge infection with the same Chlamydia species than CD82 DC[21]. Little is known about the functional involvement of DC subsets in BCG induced protective immunity against mycobacterial infection. In the present study, we examined the relative effectiveness of splenic CD8a+(CD8+) and CD8a2 (CD8)DC subsets in BCG- infected mice in generating type-1 T cell responses and protection against challenge infection with an intention to know more on the mechanism by which a host defenses against Mtb infection. The data showed that the distinct DC subsets primed by in vivo BCG immunization are significantly different in their potency to induce protective immunity against challenge mycobacterial infection. Specifically, we found that CD8a+ DC is much more potent than CD8a2 DC for inducing type-1 T cell responses in both in vitro and in vivo conditions. This difference is unlikely due to the potential difference of the subsets in uptaking mycobacterial organisms because quantitative RT-PCR test showed similar levels of bacterial messages in the isolated DC subsets. Rather, it is more likely due to the functional difference of the DC subsets in modulating T cell responses. Materials and Methods Mice and Culture Medium Female C57BL/6 mice were bred at the University of Manitoba breeding facility and hosted under specific-pathogen-free condi- tions. Animals were used in accordance with the guidelines issued by the Canadian Council on Animal Care. The animal experi- mental protocol was approved by the ethical committee of The University of Manitoba. Complete RPMI 1640 medium supplemented with 10% heat– inactivated fetal bovine serum, 1%L-glutamine, 25 mg/ml genta- mycin and 561025M 2-mercaptoethanol was used for cell culture. Organism The original BCG vaccine was produced by Aventid Pasteur Limited (Toronto, Ontario, Canada). For expansion of the vaccine, BCG was grown in the Middlebrook’s 7H9 broth (Difco Laboratories Inc., Detroit, MI) containing 0.2%(v/v) glycerol and 0.05%(v/ v)Tween-80 and supplied with 10%(v/v) Middlebrook ADC enrichment (Difco) for 21days. The number of BCG bacilli, counted as colony-forming units (CFU), was measured by plating diluted culture on plates of Middlebrook 7H11 agar (Difco) containing 0.5%(v/v)glycerol and supplied with Middlebrook OADC enrich- ment(Difco). The BCG stock was stored at 280uC until use. For inactivation of BCG, live BCG was put at 65uC for 1 hour, which led to complete killing of BCG confirmed by viability testing. Mice Immunization Mice were injected intravenously with 56105 CFU BCG in 200 mL sterile protein-free PBS and sacrificed at day 21 after infection. Spleens were aseptically isolated and treated as following: Briefly, the spleens were digested in 1.5 mg/ml collagenase D at DC Subsets in BCG Infection 37uC for 30 min and EDTA was applied at the last 5 min to disrupt DC-T complexes. The cell suspensions was then pipetted up and down several times and filtered. DC Subset Isolation For total DC (CD11c+) isolation, the splenocytes after RBC lyses with ACK lyses buffer(150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) were incubated with the MACS (Miltenyi Biotec, Auburn, CA) CD11c beads for 15 min at 4uC. The cells were washed and passed through the LS columns for selection of CD11c+ cells. For isolation of DC subset (CD11c+CD8+DC and D11c+CD82DC), MACS CD8+DC positive selection kit was used according to the manufacturer’s instructions. Briefly, spleen cells were incubated with cocktail of biotin-conjugated antibodies against antibodies (CD90, CD45R, CD49b), followed by incuba- tion with anti-biotin microbeads to deplete of T, B and NK cells. The CD8+DC subset was isolated from the T,B and NK cell depleted preparation by incubating it with CD8+ cell selecting beads for 30 min on ice and passing through LD columns. The CD82DC subset was further isolated from this preparation using CD11c column. The purity of the total DC and DC subset was .95% based on flow cytometry analyses. Flow Cytometric Analysis For analysing total DC, purified CD11c+ DCs (26106 the cells) were pre-incubated with anti-mouse CD16/32 mAb for 15 min to block FcR binding before staining with specific antibodies. Cells were then incubated with specific mAbs at 4uC for 30 min in dark. After washing with a staining buffer [Dulbecco’s PBS (Sigma- Aldrich) without Ca2+ and Mg2+ containing 2% heat-inactivated FCS and 0.05% NaN3], the cells were fixed and analysed using FACS Calibur flow cytometer (BD Biosciences) and the data were analyzed using Cell QuestTM software (Becton Dickinson). To analyzed surface marker expression on DC subsets, three color- staining was performed. CD11c-allophycocyanin (Hamster IgG) and anti CD8-PE-Cy7 (Rat IgG2a k) were used for gating DC subsets and FITC conjugated anti-CD80 (Hamster IgG), CD86 (Rat IgG2b k), CD40 (Rat IgG2a k) and MHC-II (Rat IgG2b) were used for the specific markers. The florescence-conjugated appropriate isotype controls antibodies were used as control. Intracellular cytokines were analyzed as we previously de- scribed[22]. Briefly, freshly isolated draining lymph node cells (26106 cells) were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma Chemical Co., USA) and 1 mg/ml ionomycin (Sigma) for 6 hours. 20 mg/ml brefeldin A (Sigma) was added in the last 3 hours in order to accumulate cytokines intracellularly. After washing with a staining buffer, cells were incubated with anti-mouse CD16/32 (Fc block, e-Bioscience) for 30 min on ice to block the FcR non-specific binding and subsequently stained for surface markers with PE-anti-CD4, FITC-anti-CD3e, PerCy-anti-CD8 or isotype controls for 30 min on ice. The cells were fixed and permeabilized with Cytofix/CytopermTM buffer (BD BioScience) for 20 minutes at 4uC and incubated with allophycocyanin anti-IFN-c or corre- sponding isotype controls (eBioscience) for 20 minutes on ice. The raw sample data were collected using a FACS Calibur flow cytometer (BD Biosciences) and the data were analyzed using FlowJo (BD Biosciences). Adoptive Transfer of DC Subsets and Challenge Infection Two models were used. For model 1, intravenous route was used. Purified CD8+ DCs and CD82 DCs from BCG infected mice were injected intravenously (i.v.) to syngeneic recipient mice at the amount of 56105 cells in 200 ml sterile protein-free PBS. PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9281