Characterization of the immunogenic region of huCCR6.

<p>The reactivity of the anti-huCCR6 mAbs <b>(A)</b> 2E5, 11E10 and 11A9 or <b>(B)</b> 53103, MM0066, G034E3 and 11A9 was compared to those of a control mAb (Ctr-mAb) by ELISA using plate-adsorbed N1-18, N19-47 and c-PA peptides. One representative analysis of three independent experiments carried out in duplicate.</p

Competition by mAbs for binding to huCCR6.

<p>Binding of anti-huCCR6 mAbs (1μg/mL) to huCCR6-CHO cells, in the absence (light grey histograms) or the presence of N1-18 peptide, at the concentration of 0.1 μg/mL (dark grey histograms) or 1 μg/mL (black histogram) was determined by flow cytometry. The <i>x-axis</i> represents fluorescence (four-decade log scale), and the <i>y-axis</i> represents the relative cell number. Histograms of cells stained with a control mAb (white histograms, nearest the <i>y-axis</i>) are superimposed over histograms of cells stained with the indicated anti-huCCR6 mAbs. One representative analysis from three independent experiments is shown. The histogram overlay were performed using Flowing Software vs 2.5.</p

Immunization strategies to generate mouse anti-huCCR6 mAb.

<p><b>(A)</b> Mice (n = 4) were injected three times i.p. at the indicated days with N19-47 or cPA peptides in the presence of Complete (CFA) or Incomplete (IFA) Freund’s Adjuvant. Serum was collected and the Ab specificity was determined at day 37 after the first immunization. <b>(B)</b> huCCR6-NIH-3T3 cells were used for two i.p. injections in mice (n = 4) at a 14 day interval and for a final i.v. boost injection at day 45, 4 days before generation of hybridoma cells.</p

Functional characterization of anti-huCCR6 mAbs.

<p><b>(A)</b> Binding titration of the 2E5 and 11E10 mAbs on huCCR6-CHO and huCCR2-CHO cells, analyzed by flow cytometry assay. After plotting the data (log(agonist) vs. normalized response, GraphPad Prism software), the EC50 was 1.911 μM for 2E5 and 0.6708 μM for 11E10. Mean values ± SEM of duplicate samples are shown in the graph. One representative analysis in duplicate from two independent experiments is shown. <b>(B)</b> Competitive binding of AF647-CCL20 (80 ng/mL) on huCCR6-CHO cells in presence of anti-CCR6 mAbs or a control mAb (Ctr-mAb) (300 μg/mL) or huCCL20 (8 μg/mL)). Maximum binding (without competitor) was used as 100% for normalization with GraphPad Prism software. Mean values ± SEM of pooled data from two independent experiments are shown in the graph. <b>(C,D)</b> Kinetics of calcium mobilization from internal stores in huCCR6-CHO cells following stimulation with <b>(C)</b> mAbs (100 μg/mL) or huCCL20 (80 ng/mL) and <b>(D)</b> inhibition of huCCL20-mediated Ca2+ flux (160 ng/mL) after an incubation for 15 min of the cells with mAbs (300 μg/mL) or huCCL20 (80 ng/mL). Maximum peak signal (without competitor) was used as 100% for normalization with GraphPad Prism software. One representative analysis of three independent experiments. Curves represent the mean values of triplicate measurements.</p

Specificity of mAbs generated by immunization with huCCR6-expressing cells.

<p>The specificity of the 2E5, 5G4 and 11E10 mAbs in the corresponding hybridoma supernatants was determined by flow cytometry, using <b>(A)</b> CHO-K1 cells, transfected with huCCR6 or huCCR2, respectively and <b>(B)</b> human CCR6⁺ and CCR6^- CD4⁺ T cells. The <i>x-axis</i> represents fluorescence (four-decade log scale), and the <i>y-axis</i> represents the relative cell number. Dark grey histograms from control cells are superimposed over light grey histograms of huCCR6⁺ cells. One representative analysis from four independent experiments is shown. The histogram overlay were performed using Flowing Software vs 2.5.</p

Characteristics of healthy donors and patients.

<p>Characteristics of healthy donors and patients.</p

Association of KIR2DL1 expression and DENV-2 infection.

<p>Results are grouped according to type of infection: DENV-2, CHIKV or co-infected by CHIKV and DENV-2 (CHIKV/DENV-2), and compared to non-infected Gabonese healthy controls (NI). Samples from infected patients were collected in early acute (EA; day (D)0-D3), late acute (LA; D12-D15) and convalescent (C; D>30) stages post-onset of symptoms. Frequency of KIR2DL1 (A) and KIR2DL2/2DL3 (B) gated on CD3^-CD56⁺ NK cells. Dot-plots show only cells from EA patients. * p<0.05, ** p<0.001.</p

Cytolytic markers and functional activity of NK cells in patients infected with DENV-2, CHIKV, co-infected with CHIKV and DENV-2 (CHIKV/DENV-2), and non-infected and healthy Gabonese controls (NI).

<p>Patients were tested at early acute (EA; day (D)0-D3) stage post-onset of symptoms. (A) Intracellular staining of perforin and granzyme-B cytolytic markers on non-activated CD3^-CD56⁺ NK cells. (B) CD107a expression on CD3^-CD56⁺ NK cells stimulated by K562 target cells. Results are shown for an effector/target (E/T) cell ratio of 1/1. (C) Intracellular staining of IFN-γ⁺ in CD3^-CD56⁺ NK cells after IL-12 plus IL-18 overnight stimulation. *: p<0.05; ***: p<0.0001.</p

Frequencies and characteristics of NK cells in patients infected with DENV-2, CHIKV, co-infected with CHIKV and DENV-2 (CHIKV/DENV-2), and non-infected and healthy Gabonese controls (NI).

<p>Patients were sampled in early acute (EA; day (D)0-D3), late acute (LA; D12-D15) and convalescent (C; D>30) stages post-onset of symptoms. Frequencies of CD3^-CD56⁺ NK cells within the CD45⁺ lymphocyte gate (A), frequencies of early activation marker CD69 (B) and NKp30, NKG2A, CD161, CD57 cell-surface receptors (C) on CD3^-CD56⁺ NK cells are represented. * p<0.05, ** p<0.001.</p

Hierarchical clustering analysis of 9 NK-cell markers from patients infected with DENV-2 (DENV; Green; n = 15), CHIKV (CHIK; Blue; n = 19), co-infected with CHIKV and DENV-2 (COIFN; Orange; n = 9), and non-infected healthy Gabonese controls (NI; Purple; N = 15).

<p>Patients were sampled in early acute (EA; day (D)0-D3), late acute (LA; D12-D15) and convalescent (C; D>30) stages post-onset of symptoms. Hierarchical clustering analysis was performed with the Genesis program (software available at <a href="http://www.genome.tugraz.at/" target="_blank">www.genome.tugraz.at</a>), as previously described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004499#pntd.0004499.ref014" target="_blank">14</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004499#pntd.0004499.ref025" target="_blank">25</a>]. Each column is dedicated to a definite NK marker. The color of each square reflects the percentage of expression of the corresponding marker in each individual. The values measured for samples were color displayed and rank ordered considering the healthy donors’ median as a reference: green color indicates inferior to median, and red color indicates superior to median, with values ranging from -3 to +3.</p