Effect of anti-CD25 mAb treatment on Treg compartment and on anti-mycobacterial Th1 responses.

<p>(A) Mice received, at days −2 and 11, i.p. injections of PBS or of 1 mg of the control rat IgG (CRL-1912) or of anti-CD25 mAb (PC61). At day 0, mice were immunized s.c. with 1×10⁶ CFU of BCG. (B) At day 21, percentages of FoxP3⁺ cells within CD4⁺ T cell splenocytes were determined. Results represent mean±SD obtained in individually studied mice (<i>n</i> = 3). * = statistically significant, as determined by Anova test, <i>p</i><0.001. (C) Frequency of IFN-γ producing T splenocytes in response to TB10.3 or TB10.4 in mice immunized by BCG and treated with anti-CD25 mAb. At day 21 post-immunization, serial dilutions of splenocytes from mice (<i>n</i> = 6), unvaccinated or immunized with BCG and injected with PBS, control IgG or PC61 mAb, as described in (A), were stimulated <i>in vitro</i> with 10 µg/ml of TB10.3:74–88 (left) or TB10.4:74–88 (right) to determine the frequency of specific CD4⁺ IFN-γ-producing T cells by ELISPOT. Horizontal bars and the boxes indicate the mean values and SD, respectively. Whiskers show the smallest and the largest values. * or ** = statistically significant, as determined by Student's <i>t</i> test, <i>p</i><0.02 or 0.01, respectively. <i>Ns</i> = no significant difference.</p

Effect of anti-CD25 mAb treatment on Th1 responses to immunodominant or subdominant regions of TB10.4 immunogen.

<p>Effect of anti-CD25 mAb treatment on Treg anti-mycobacterial Th1 responses to 15-mer overlapping peptides covering the entire sequence of TB10.4. Mice were treated with the control rat IgG (CRL-1912) (A) or with anti-CD25 mAb (PC61) (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002833#pone-0002833-g002" target="_blank">Figure 2A</a>. At day 21, splenocytes from individual mice were stimulated <i>in vitro</i> with 20 µg/ml of individual 15-mer overlapping peptides covering the entire sequence of TB10.4. IFN-γ secretion in the culture supernatant was assessed by ELISA at 72 h. Three mice were individually studied per experimental group and one representative individual is shown.</p

Effect of anti-CD25 mAb treatment on CD4⁺ T cells of BCG-immunized, <i>M. tuberculosis</i>-infected mice.

<p>(A) Protocol of i.p. anti-CD25 (PC61) mAb injections, s.c. BCG immunization, aerosol <i>M. tuberculosis</i> challenge and of study of T cell functions and mycobacterial load in the lung parenchyma of BALB/c mice. (B) Activated/effector CD4⁺ T cells in the lung parenchyma of these mice. At one month post-challenge, pooled lymphocytes from lung parenchyma of each experimental group were stained with a combination of allophycocyanine-anti-CD4, FITC-anti-CD45RB and PE-anti-CD27 mAbs and analyzed by cytofluorometry.</p

CD8⁺ T-cell responses in anti-CD25 mAb treated, BCG-immunized mice.

<p>(A) Representative CD8⁺ T-cell response to TB10.3 or TB10.4 in BALB/c mice immunized by BCG compared to control non-immunized mice. At day 21 post-immunization, splenocytes were stimulated <i>in vitro</i> with 10 µg/ml of TB10.3/4:20–28, containing a H-2K^d-restricted immunodominant epitope. Six days later, splenocytes were stained with a combination of FITC-anti-CD8, allophycocyanine-anti-CD44 and PE-conjugated H-2K^d pentamer complexed with TB10.3/4:20–28 peptide. Cells were then analyzed by cytofluorometry. (B) Comparison of CD8⁺ T-cell response to TB10.3 or TB10.4 in individual mice, injected with PBS, the control IgG or PC61 mAb and immunized with BCG, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002833#pone-0002833-g002" target="_blank">Figure 2</a>. Percentages of pentamer⁺ CD44⁺ cells within the CD8⁺ T-cell population are shown in individual mice. Results are pooled from two independent experiments. <i>Ns</i>, no significant difference as determined by Student's <i>t</i> test.</p

Effect of the attenuation of Treg on the protective capacity of BCG.

<p>Evaluation of the protective capacity of BCG in mice treated with anti-CD25 mAb, and challenged with <i>M. tuberculosis</i>, performed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002833#pone-0002833-g006" target="_blank">Figure 6A</a>. At one month post-challenge, mycobacterial load in the lungs of mice was determined by CFU counting. The number of CFU/lungs at day 1 post-challenge was between 100 and 200 in the experiment 1, and between 10 and 100 in the experiment 2. Horizontal bars and the vertical bars indicate the mean values and SD, respectively. Two independents experiments were performed and data from both of them were taken in account in statistical analyses performed by use of StatXact program and Permutation test. Ns = not significant, *, ** or *** = statistically significant with two-sided <i>p</i>-value <0.0420, 0.0046 or 0.0001, respectively.</p

Analysis of CD25⁺ FoxP3⁺ T cells in the lung parenchyma of <i>M. tuberculosis</i>-infected mice.

<p>(A) Percentages of CD25⁺ FoxP3⁺ cells within the CD4⁺ T-cell subset in lung parenchyma of untreated controls or of mice infected with ≈100 CFU of H37Rv by aerosol route and (B) number of CD4⁺ CD25⁺ FoxP3⁺ cells, in the lung parenchyma of control or <i>M. tuberculosis</i>-infected mice. Results are means±SD of three mice, studied individually. *, statistically significant (Student's <i>t</i> test, <i>p</i><0.02).</p

Innate responses of cord blood pDCs to viruses.

<p>Adult or neonatal whole blood (WB) (A and C), mononuclear cells (MNC) (A and C) or purified pDCs (A–C) were stimulated with CpGA (50 µg/ml for WB, 5 µg/ml for others in A), live IAV (1000 HAU/ml for WB, 10 HAU/ml for others), heat-inactivated IAV (HI-IAV in C) live HIV (10⁵ infected T cells in A) and live HSV-1 (10⁶ pfu/ml in A) for 24 hrs. Plasma or supernatants were collected and tested for IFN-α (A and C) or TNF-α, CCL3 and CCL4 (B). NA: non applicable. The number of donors is indicated for each group (n) as well as <i>P</i> values for adults and neonates comparison. In B, n = 8 for adult and n = 7 for neonates.</p

Survival fate of neonatal pDC following CpG activation.

<p>(A) IFN-α responses of neonatal pDCs to CpGA were plotted against the survival rate. Cell survival is shown by annexin V and propidium iodide staining (PI). (B) Neonatal pDCs were stimulated with CpGA or IAV in the absence or presence of IL-3 (10 ng/ml) or GMCSF (10 ng/ml) for 24 hrs. IFN-α was detected from supernatants.</p

IL-12p40 producing pDCs represent a small subset of CD2+pDC.

<p>(A) Neonatal and Adult MNCs were stimulated with IAV-HI CpGA then CD2⁺CD5⁺, CD2⁺CD5⁻ and CD2⁻CD5⁻ pDCs subsets were gated to analyze their capacity to produce IL-12 p40 detected by intracellular cytokine staining. CpGA stimulated mDC (BDCA1⁺CD20⁻CD14⁻), monocytes (CD14⁺) and B cells (CD20⁺) from adult PBMC were also stained for IL-12p40 intracellularly. Numbers indicate the percentage of cytokine-producing cells among totally gated cells. (B) Expression of co-stimulatory markers CD40, CD86 and HLADR on neonatal BDCA4⁺CD123⁺pDCs defined as CD2⁺CD5⁺, CD2⁺CD5⁻ and CD2⁻CD5⁻ subsets under steady state. Data are representative of 3 experiments.</p

Heterogeneity of neonatal pDCs.

<p>(A) The percentages CD123^hiBDCA2⁺ pDCs within CD45+ MNC were calculated from neonatal cord blood and adults. pDCs could be subdivided by CD2 and CD5, and the accumulated data for CD2⁺, CD2^- pDCs subsets and CD2⁺CD5⁺, CD2⁺CD5⁻ pDCs subsets are shown in (B). (C) The innate IFN-α production of sorted CD2⁺ and CD2⁻ pDCs subsets to CpGA, live IAV and HIV (the results are incated as ng/ml). (D) Neonatal and Adult MNCs were stimulated as indicated and CD2⁺CD5⁺, CD2⁺CD5⁻ and CD2⁻CD5⁻ pDCs subsets were gated to analyze their capacity to produce IFN-α detected by intracellular cytokine staining. Numbers indicate the percentage of cytokine-producing cells among gated cells. Data are representative of 5 experiments. The number of donors is indicated for each group (n) as well as <i>P</i> values for adults and neonates comparison.</p