pDC depletion leads to increased bacterial loads and tissue inflammatory changes in the lungs.

<p>Mice were depleted of pDCs using anti-mPDCA Ab prior to and during the course of infection as described in the <i>Materials and Methods.</i> Control group mice received Rat IgG2b Ab. Following infection, the animals were monitored everyday for body weight changes (<b>A</b>). At day 9 p.i., the mice were sacrificed and the lungs were collected and quantitative assessment of bacterial loads was performed as described in <i>Materials and Methods</i>. <b><i>C,</i></b> Increased tissue pathological response in the lungs of pDC depleted mice compared to control mice as analysed by H&E staining. <b><i>D &E,</i></b> Shown are the graphs depicting differences in the percentage and absolute numbers of lung T cells (CD3+), Mφ, alveolar macrophages (F4/80+CD11c+ cells), cDCs, conventional DCs (F4/80−CD11c+ cells) and Gr, granulocytes (Gr-1+ F4/80−CD11c− cells). Results are shown as mean ± SD. Results of one representative (of three) experiment with four mice in each group are depicted. *, p<0.05, and ***, p<0.001.</p

<i>Cpn</i> infection activated pDCs induce Treg IL-10 production <i>in vitro</i>.

<p>pDC isolated from the lungs of <i>Cpn</i> infected (day 9 p.i.) and uninfected mice were co-cultured with CD4 T cells purified from <i>Cpn</i> immunized mice in the presence of SK-EB as described in the <i>Materials & Methods.</i> Following three days of co-culture, the cells were washed and analysed by flow cytometry. <b><i>A&B</i></b>, The cells were stained for intracellular Foxp3 expression. The analysis was performed on gated CD4+ cells. Representative histograms <i>(</i><b><i>A</i></b><i>)</i> are shown and the summary graph <i>(</i><b><i>B</i></b><i>)</i> provided. <b><i>C&D,</i></b> IL-10 production by Tregs; analysis was performed on CD4+Foxp3+ gated cells. Shown are representative dot plots <i>(</i><b><i>C</i></b><i>)</i> and a graphical summary <i>(</i><b><i>D</i></b><i>)</i> of the percentages of IL-10 producing Tregs. Data are shown as mean ± SD. Results of one representative (of three) experiment with five mice in each group are shown. *, p<0.05, **, p<0.01.</p

Reduced Treg numbers and lower IL-10 production by Tregs in pDC depleted mice.

<p>The frequencies of Tregs in the lungs and dLNs in pDC depleted and the sham-treated mice were analyzed following <i>Cpn</i> infection (day 9 p.i.). <b><i>A,</i></b> representative flowcytometry images show the percentages of CD4+Foxp3+ Tregs in the lungs and dLN<b>.</b> The absolute numbers of Tregs were depicted in the respective graphs. <b><i>B</i></b><i>,</i> Intracellular staining for IL-10; The dLN cells were cultured with SK-EB for 3 days and restimulated with PMA and ionomycin. The cells were stained first for surface markers, fixed, permeabilized and stained intracellularly for IL-10 as described in <i>Materials and Methods.</i> Shown are representative flowcytometry plots and the summary graphs for IL-10 production by Tregs (gated on CD4+Foxp3+CD8− cells). Data expressed as mean ± SD. Results are representative of three independent experiments with three mice in each group. **, p<0.01, ***, p<0.001.</p

Activation of pDCs following <i>Cpn</i> infection.

<p>C57BL/6 mice were intranasally infected with <i>Cpn</i> (3×10⁶ IFUs). Mice were killed at day 9 p.i., and lungs were aseptically collected. To analyse pDCs, lung cells were stained and analysed by flowcytometery as decribed in the <i>Materials and Methods</i>. pDCs were identified as mPDCA+ CD11c ^int/lo cells. <b><i>A,</i></b> pDC expansion after <i>Cpn</i> infection. Shown are representative dot plots with the percentages of pDCs in <i>Cpn</i> infected mice in comparison with uninfected mice (left) and the graphical summary for the absolute numbers of pDCs in the lungs. Total pDC number per mouse lung was calculated as % mPDCA+ CD11c ^int/lo cells x total number of cells per mouse lung/100. <b><i>B,</i></b> Expression of MHC II and costimulatory molecules CD40, CD80 and CD86 on gated pDCs (filled histograms) and isotype control (dotted line) are shown. The mean fluorescence intensity (<i>left</i>) and the percentages of positive cells (<i>right</i>) are indicated. <b><i>C,</i></b> pDCs purified (as described in <i>Materials and Methods</i>) from <i>Cpn</i> infected (PDC-Inf) and uninfected mice (PDC-N) were cultured in the presence or absence of <i>Cpn</i> (SK-EB). IL-12p40, IL-12p70 in the 72 hrs supernatants were measured by ELISA. IFNα production by pDCs was analysed by intracellular staining and the graph shows the percentages of IFNα producing cells. Results are shown as mean ± SD. At least three independent experiments with four mice in each group were performed and one representative experiment is shown. *, p<0.05, and ***, p<0.001, Student’s t test.</p

pDC deficiency leads to inflammatory type effector CD8 T and CD4 cell responses after <i>Cpn</i> infection.

<p>Lung T cells as described in the legends to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083463#pone-0083463-g004" target="_blank">Figure 4</a> were analyzed by multi-color intracellular cytokine staining. Shown are representative flowcytometry dot plot images and summary graphs depicting the nature of cytokine responses by T cells. Analysis was performed on gated CD3+ CD8+ cells and CD3+CD4+ cells. Note increased inflammatory type-1 (IFNγ+TNFα+) CD8 <i>(</i><b><i>A</i></b><i>)</i> and CD4 T cells <i>(</i><b><i>B</i></b><i>)</i> and inflammatory Th2 type (IL-4+ TNFα+) CD4 T cells <i>(</i><b><i>C</i></b><i>)</i> in the lungs of pDC depleted mice compared to the control group mice. At least three independent experiments were performed and the data from one representative experiment is depicted. Data expressed as mean ± SD. *, p<0.05, and ***, p<0.001.</p

pDC deficiency <i>in vivo</i> leads to altered cytokine responses.

<p><b><i>A</i></b>, pDC depleted and the sham-treated mice (four mice/group), after <i>Cpn</i> infection were sacrificed 9 days postinfection and the lungs and draining (mediastinum) lymph nodes were collected. The draining lymph node (dLN) cells were cultured in the presence of SK-EB as described in <i>Materials and Methods</i>. Cytokine levels (IFN-γ, TNFα, IL-4 and IL-10) in 72-h dLN cell culture supernatants <i>(</i><b><i>A</i></b><i>)</i> and lung homogenates <i>(</i><b><i>B</i></b><i>)</i> were measured by ELISA. Data are presented as the mean ± SD of each group. Results of one of the three experiments with similar results are shown. *, <i>p</i><0.05, **, <i>p</i><0.01, and ***, <i>p</i><0.001; Student’s t test.</p

<i>In vitro</i> neutralization of toxins from <i>C</i>. <i>difficile</i> clinical isolates by mAbs using xCELLigence^® test.

<p><i>In vitro</i> neutralization of toxins from <i>C</i>. <i>difficile</i> clinical isolates by mAbs using xCELLigence^® test.</p

Survival after lethal rTcdA challenge in mice.

<p>Kaplan-Meier plot of survival following lethal challenge with rTcdA alone, or treatment with CANmAbA4 at either 250 μg or 50 μg dose in comparison to the CDA1 anti-TcdA and polyclonal (pAb anti-TcdA) control. Following lethal challenge, mice (n = 10) were monitored and sacrificed according to approved protocols. Statistical analysis (Log-rank Test) using GraphPad prism 5 indicated that all antibody treated groups had statistically significant higher survival rate compared to control group (rTcdA alone) (P<0.001). There is no significant difference in survival rate among antibody treated groups.</p

Summary of humanized (IgG1/κ) anti-toxin mAbs and murine progenitor from which they were derived.

<p>Summary of humanized (IgG1/κ) anti-toxin mAbs and murine progenitor from which they were derived.</p

Survival after lethal rTcdB challenge in mice.

<p>Kaplan-Meier plot of survival following lethal challenge with rTcdB alone, or treatment with CANmAbB4 or CANmAbB1 at either 250 or 75 μg doses in comparison to the anti-TcdB rabbit polyclonal (pAb anti-TcdB) control. Following lethal challenge mice (n = 10) were monitored and sacrificed according to approved protocols. Statistical analysis (Log-rank Test) using GraphPad prism 5 indicated that all antibody treated groups had statistically significant higher survival rate compared to control group (rTcdA alone) (P<0.001). While CANmAbB4 treated animals (both 250 μg and 75 μg) showed statistically significant higher survival rate in comparison with CANmAbB1 (250 μg) (P<0.01).</p