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

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

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

Cord blood pDCs are the main contributor for IFN-α response.

<p>(A) Neonatal cord blood was left untreated or exposed to IAV, for 6 hrs and stained intracellularly for IFN-α. pDCs are identified by CD123 and BDCA4 and monocytes by CD14 expression. (B) Total CBMC (1), or CBMC depleted of pDCs (2) or of monocytes (3) were stimulated with CpGA, live IAV and HI-IAV as in Fig. Plasma or supernatants were collected and tested for IFN-α (A). Depletion efficiency is shown for pDCs identified by CD123 and CD45RA and monocytes by CD14 and CD11b expression. One experiment representative of three is shown.</p

Comparative distribution of patient’s demographic, clinical and immunological characteristics in the derivation and validation sets.

<p>The main characteristics of the patients are listed here for the time point of enrollment during PHI (M0). For each parameter (except gender), the median values and the range are indicated. Plasma IP-10 concentrations were measured by ELISA at M0. For the derivation set, data are shown for those 45 patients out of 46, whose IP-10 was quantified by ELISA. The validation set comprised 88 patients. There was no significant difference between the two sets for any parameter (p>0.11). W: women, M: men, N: number of patients in each set.</p

Plasma protein levels at M0 according to disease progression profiles.

<p>The cytokine profiles at M0 are shown for each group of patients: 16 rapid progressors (<b>A</b>), 19 progressors (<b>B</b>), and 11 slow progressors (<b>C</b>). Color code and statistical analyses are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046143#pone-0046143-g002" target="_blank">Figure 2</a> (corrected threshold, p<0.005). The dotted horizontal line corresponds to the value in healthy donors. (<b>D</b>) Comparison of protein concentrations between the 3 groups of patients (SP, P and RP). Four representative cytokines are shown. The cytokines increasing significantly over groups were IP-10 and IL-10 (Cuzick’s test, p<0.007). When comparing the groups two by two, out of 28 proteins tested, the levels were different only for IP-10 (M&W test,***: p<0.005).</p

Immunological, virological and clinical characteristics of rapid progressors versus progressors and slow progressors assembled within patients of the derivation and validation sets.

<p>The derivation set comprised 46 patients (except for IP-10, where values based on the ELISA were available for only 45 patients). The validation set corresponded to 88 patients for all markers. Differences between rapid progressors and the other patients are indicated with the presence of a p value (M&W test). P values below 0.05 were considered to be significant. There was no significant difference regarding the time of enrollment at M0 (estimated days post-infection). (A) CD4⁺ T cells at M0 and M12; (B) Viremia at M0 and M12; (C) number of estimated days post-infection at M0; (D) Plasma IP-10 concentration at M0. M = month.</p

Plasma protein levels in HIV-1 infected patients.

<p>(<b>A</b>) The levels of 28 proteins in the plasma of 46 acutely infected patients (M0) are expressed as fold change compared to the levels in healthy donors (N = 17). The order of the proteins is presented according to their function (pro-inflammatory, adaptive, IFN-inducible, chemoattractants, hematopoietic and anti-inflammatory). The anti-inflammatory cytokines are presented on the right side of the figure. The dotted horizontal line at Y = 1 corresponds to the value in healthy donors. The boxes represent the median and the 25^th and 75^th percentile, with the line in the middle of the boxes corresponding to the median value. Colored boxes stand for the cytokines, whose levels were significantly different from healthy donors: blue boxes when p<0.05 (as it was the case for IL-1β) and red boxes when p<0.008 (M&W test). (<b>B</b>) Protein concentrations at M0, M1 and M6 of the soluble factors that were elevated at M0. The data are expressed in pg/ml, HD: healthy donors, M: months. Cytokine concentrations below the limit of detection were arbitrarily set at the level of the limit of detection. Dot-plots marked with one asterisk (*) (p<0.05) or two asteriks (**) (p<0.008) represent the cytokines, whose levels were significantly different from healthy donors (M&W test).</p

Cytokines predictive of immunological set-point levels. (A–E)

<p>Cytokine concentrations in plasma at M0 have been plotted against CD4⁺ T cell counts and T cell activation (CD3⁺CD8⁺CD38⁺HLA-DR⁺) at M6. Six patients, including 4 who were treated at M6, were excluded from the analysis at M6. T cell activation levels were available for 19 patients at M6 (4 SP, 7 SP, 8 RP). The correlations were thus analyzed in 40 patients regarding CD4⁺ T cell counts and viral load and for 19 patients regarding T cell activation. (<b>A</b>) IP-10 levels at M0 plotted against T CD4⁺ counts at M6. (<b>B</b>) IP-10 levels at M0 plotted against T cell activation at M6. (<b>C</b>) IL-18 levels at M0 plotted against T cell activation at M6. (<b>D)</b> TGF-β1 concentrations at M0 plotted against T cell activation at M6. The red line indicates that both the Spearman correlation and the linear regression analysis were significant. <b>(E)</b> Regression analysis for evaluation of the capacity of CD4⁺ T cell counts, VL and cytokines at M0 to predict rapid disease progression. Values obtained for both the derivation set (Luminex and ELISA) and the validation set are shown. Median values of CD4⁺ T cell counts, VL and cytokine levels were used: VL > = 5 log; CD4<570 cells (derivation set); CD4<546 cells (validation set); IP-10> = 869 pg/ml (derivation set, Luminex); IP-10> = 247 pg/ml (derivation set, ELISA); IP-10> = 232 pg/ml (validation set, ELISA).</p