BCG immunization induced the expansion of CD8⁺ DC which expressed higher levels of co-stimulatory molecules compared with CD8^- DC.

<p>Mice (C57BL/6, n = 4/group) were infected i.v. with 5×10⁵ CFU of BCG and sacrificed at 21 days after immunization. Total DCs from infected and naïve 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 naïve (nDC) mice. B, purified DC were gated on either CD11c⁺ CD8⁺ DC (CD8⁺DC) or CD11c⁺CD8⁻ DC (CD8⁻ 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.</p

Effects of different DC subsets adoptive transfer on Th1-related cytokine production by recipient mice following challenge infection.

<p><b>A:</b> The recipient mice (C57BL/6, n = 4/group) of i.v. adoptive transferred iCD8⁺ DC or iCD8^- DC subsets and PBS treated control mice were challenged i.v. with BCG and sacrificed at day 21 post challenge as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009281#s2" target="_blank">Materials and Methods</a>. A, splenocytes were cultured at the concentration of 7.5×10⁶ cells/well using HK-BCG as stimulator. Culture supernatants were harvested at 72 h and the cytokines were measured by ELISA. B&C, lungs and livers were homogenized in 10 ml cold protein-free D-PBS and centrifuged. The cytokines levels in the lung (B) and liver (C) were measured by ELISA. Data are presented as mean±SD of each group. One representative experiment of three independent experiments is shown. *p<0.05; **, p<0.01.</p

Different levels of cytokine production by DC subsets.

<p>DC subsets were isolated from spleens of BCG infected (i.v.) mice at day 21 post-immunization as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009281#s2" target="_blank">Materials and Methods</a>. A, purified DC subsets were analyzed by flow cytometry for purity. The 5×10⁵ sorted cells were co-stained with APC-conjugated anti-CD11c and PE-conjugated anti-CD8. The purities of the sorted iCD8⁺ DC (left) and iCD8⁻DC (right) are indicated at the right upper corner and right lower corner respectively. B, freshly isolated iCD8⁺ DC and iCD8⁻ 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 mean±SD of each group. One representative experiment of three independent experiments with similar results is shown.*p<0.05, **p<0.01.</p

Adoptive transfer of iCD8⁺DC, but not iCD8⁻ DC, reduced bacterial growths in recipients following either i.v. or i.n. challenge infection.

<p>DC subsets were sorted from BCG-infected (i.v.) mice or naïve mice and adoptively transferred to syngeneic recipients (C57BL/6, n = 4/group) by i.v. (A&B, 5×10⁵ DC/mouse) or i.n. routes (C, 2×10⁵ DC/mouse). Mice were challenged with BCG through i.v. (A&B) or i.n. (C) routes, respectively, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009281#s2" target="_blank">Materials and Methods</a>. 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 mean±SD of each group. One representative experiment of three independent experiments is shown. * p<0.05;**, p<0.01.</p

Intranasal adoptive transfer of iCD8⁺DC, but not iCD8⁻DC, enhanced IFN- γ production by T cell.

<p>Mice were adoptively transferred i.n. with iCD8⁺DC or iCD8⁻DC subsets followed by the challenge with BCG through i.n route as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009281#s2" target="_blank">Materials and Methods</a>. Draining lymph nodes was isolated aseptically and single cell suspension was prepared in cold staining buffer. Cells were co-stained with FITC-anti CD3ε, PE-anti CD4, PerCP-anti CD8 Abs and stained for intracellular IFN-γ using allophycocyanin -conjugated anti-IFNγ Ab as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009281#s2" target="_blank">Materials and Methods</a>. Cells were gated on CD3ε⁺T cells, CD3ε⁺CD4⁺T cell and CD3ε⁺CD8⁺T cell respectively and the percentage of positive cells for IFN-γ is indicated in upper right corner (A). Pooled data in each group are shown as mean±SD (B). One representative experiment of two independent experiments is shown. *p<0.05; **, p<0.01.</p

iCD8⁺ DC and iCD8⁻ DC subsets generate different patterns of BCG-driven T cell cytokine production.

<p>BCG-specific CD4⁺ T cells (5×10⁵) cell from BCG-infected mice were co-cultured with iCD8⁺ DC or iCD8⁻ DC (5×10⁴ cells/well) in the presence of HK-BCG. Culture supernatants were collected at 72 h. IFN-γ and IL-4 were measured by ELISA. The experiment was repeated twice showing similar results. *p<0.05.</p

DC subsets from BCG-infected mice show similar BCG load.

<p>A real-time RT-PCR using the green fluorescent dye SYBR Green I was applied to determine the relative concentration of BCG mRNA in iCD8⁺DC and iCD8⁻DC. The BCG mRNA in different DC subsets was amplified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009281#s2" target="_blank">Materials and Methods</a>. The BCG mRNA level was normalized to GAPDH mRNA in different DC subsets demonstrated as <i>ΔCt</i>. <i>ΔCt</i> = <i>Ct</i> (BCG)–<i>Ct</i> (GAPDH), the threshold cycle of a BCG and the threshold cycle of the corresponding GAPDH in the same sample.</p

Pro-inflammatory cytokine/chemokine levels in local tissues in mice treated with different DC subsets following challenge infection.

<p>The mice were treated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009281#pone-0009281-g004" target="_blank">Fig. 4</a> and the levels of IL-6, MIP-1α and TNF-α proteins in the homogenates of lung and liver tissues were measure by ELISA.</p

Chlamydial Lung Infection Induces Transient IL-9 Production Which Is Redundant for Host Defense against Primary Infection

<div><p>IL-9/Th9 responses are recently found to be important for innate and adaptive immunity particularly in parasitic infections. To date, the study on the role of IL-9 in bacterial infections is limited and the reported data are contradictory. One reported function of IL-9/Th9 is to modulate Th1/Th17 responses. Since our and others’ previous work has shown a critical role of Th1 and Th17 cells in host defense against chlamydial lung infection, we here examined the role of IL-9 responses in <i>Chlamydia muridarum</i> (Cm) lung infection, particularly its effect on Th1 and Th17 responses and outcome infection. Our data showed quick but transient IL-9 production in the lung following infection, peaking at day 3 and back to baseline around day 7. CD4+ T cell was the major source of IL-9 production in the lung infection. Blockade of endogenous IL-9 using neutralizing antibody failed to change Interferon-γ (IFN-γ) and IL-17 production by cultured spleen mononuclear cells isolated from Cm infected mice. Similarly, in vivo neutralization of IL-9 failed to show significant effect on T cell (Th1 and Th17) and antibody responses (IgA, IgG1 and IgG2a). Consistently, the neutralization of IL-9 had no significant effect on disease process, including body weight change, bacterial burden and histopathological score. The data suggest that IL-9 production following chlamydial lung infection is redundant for host defense against the intracellular bacteria.</p></div

Impaired antibody responses in ICOS-KO and ICOS-YF mice.

<p>Lung homogenates were collected at day 14 post-infection and the <i>Chlamydia</i>-specific IgG2a, IgG1 and IgA titers were measured by ELISA as described in <i>Materials and Methods</i>. Data are presented as mean ± SD (7 ICOS-WT, 4 ICOS-KO, and 7 ICOS-YF mice) from one representative experiment out of three independent experiments with consistent results.* <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p