3D microscopy study of DR5 expression by CD4⁺ T cells.

<p><b>A.</b> CD4⁺ T cells were cultured overnight in presence or absence of PHA (2.5 µg/ml) and stained for DR5 (green), nuclear staining DAPI (blue) and CD4 (red) to study DR5 protein expression and localization. Unstimulated CD4⁺ T cells exhibited DR5 protein expression in the cytoplasm at close membrane proximity, defined by CD4 staining (upper panels). In contrast, PHA-activated CD4⁺ T cells expressed intracellular and membrane DR5. DR5/CD4 colocalization appears in yellow. To better visualize DR5 localization, a zoom of the dotted line defined zone from CD4/DR5/DAPI overlay picture was added (extreme right panels). <b>B.</b> DR5 analysis using 3D interactive surface plot. Previous 3D microscopy pictures were analyzed using 3D interactive surface plot. Representation allowed a clear visualization of intracellular DR5 (green) and membrane CD4 (red). Unstimulated CD4⁺ T cells harbored only intracellular DR5 inside the CD4 (membrane) delimitation. PHA stimulation of CD4⁺ T cells induced the export of DR5 protein at the surface delineated by CD4 staining (red) but some DR5 also remained intracellular. Yellow spots represent DR5/CD4 colocalization. <b>C.</b> FACS analysis of intracellular staining of DR5 and compilation of all plans from previous microscopic images were used to perform 3D reconstruction. This representation illustrates the difference of DR5 quantity between unstimulated and activated CD4⁺ T cells. PHA-stimulated cells expressed more DR5 (green) and show more colocalization (yellow) with CD4 than unstimulated cells. Each picture is representative of the vast majority of the observed cells on the slides.</p

3D microscopy quantification of DR5 expression by CD4⁺ T cells.

<p><b>A.</b> CD4⁺ T cells were cultured overnight in presence or absence of PHA (2.5 µg/ml) and stained for DR5 (green), nuclear staining DAPI (blue) and CD4 (red) to study DR5 protein expression and localization. 10 cells per experiment were quantified for their DR5 spot number in 3 independent experiments for a total of 30 cells per condition (untreated or PHA-stimulated). White arrows show some examples of the DR5 spots that were counted <b>B.</b> Total number of DR5 spots in 30 untreated and 30 PHA-stimulated cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032874#s2" target="_blank">Results</a> show the number of DR5 spots per cell <b>C.</b> Percentage of membrane DR5 expression in 30 untreated and 30 PHA-stimulated cells. P values (<i>p</i>) were determined using a two-tailed Student's <i>t</i> test (p<0.05 one star, p<0.01 two stars, p<0.001 three stars).</p

Apoptosis inhibition of CD4⁺ T cells by soluble DR5 and DR5 siRNA.

<p><b>A.</b> CD4⁺ T cells were cultured 24 h in presence of increasing concentration of PHA and with or without 5 µg/ml of DR5. Apoptosis was quantified using Annexin-V staining by flow cytometry. <b>B.</b> CD4⁺ T cells were cultured 24 h in presence of 5 µg/ml of PHA and with or without soluble DR5 (5 µg/ml). Apoptosis was quantified using Annexin-V staining by flow cytometry. <b>C.</b> Efficiency of siRNA transfection in CD4⁺ T cells. Cells were transfected with unlabelled or Alexa-488 scrambled siRNA overnight. Dot plots show transfection efficiency of CD4⁺ T cells by Scr-Alexa-488 siRNA (right panel). <b>D.</b> Transfection of CD4⁺ T cells with DR5 siRNA significantly inhibited DR5 expression by PHA-stimulated CD4⁺ T cells in contrast to scrambled (Scr) siRNA (left panel). Inhibition of DR5 expression at the surface of PHA-stimulated CD4⁺ T cells using DR5 siRNA also dramatically inhibited Annexin-V positive cells induced by PHA compared to Scr siRNA (right panel). <b>E.</b> Dot plot analysis of untransfected and transfected CD4⁺ T cells with scrambled siRNA (Scr siRNA), DR5 siRNA (DR5 siRNA). After overnight transfection, CD4⁺ T cells were cultured in absence (upper panels) or presence (lower panels) of PHA. Images show dot plots of DR5/Annexin-V staining. <b>F.</b> TRAIL, DR5 and CD69 FACS analysis by CD4⁺ T cells stimulated overnight in presence of anti-CD3 and anti CD28 antibodies. <b>G.</b> FACS dot plot analysis of CD69-Annexin-V and CD69-Topro-III staining of CD4⁺ T cells cultured in presence or absence of anti-CD3/CD28 activation overnight. Data of panels C, D, E, F and G are representative of 4 independent experiments. P values (<i>p</i>) were determined using a two-tailed Student's <i>t</i> test (p<0.05 one star, p<0.01 two stars, p<0.001 three stars).</p

PHA induced Apoptosis of CD4⁺ T cells.

<p><b>A.</b>CD4⁺ T cells were purified from PBMC from healthy donors. <b>B.</b> Cells were cultured 24 h in presence of 2.5 µg/ml of PHA and observed by microscopy (20×). PHA-treated cells are aggregated in multicellular blast. <b>C.</b> After 24 h, cells were analyzed by FACS. CD4⁺ T cells were cultured in presence or absence of increasing concentration of PHA and apoptosis was quantified by cytometry using Annexin-V staining. <b>D.</b> Apoptosis of CD4⁺ T cells cultured in presence or absence of 2.5 µg/ml was quantified using late (7-AAD, Topro-III) apoptotic markers by cytometry. <b>E.</b> Dot plots analysis of unstimulated or PHA-activated CD4⁺ T cells showing Annexin-V/Topro-III or 7AAD/Topro-III double staining. <b>F.</b> Percentage of Annexin-V, 7-AAD, Topro-III expressed by CD4⁺ T cells cultured in presence or absence of PHA for 24 h analyzed by cytometry. <b>G.</b> CD69 activation marker and Topro-III double staining of untreated or PHA-activated CD4⁺ T cells. P values (<i>p</i>) were determined using a two-tailed Student's <i>t</i> test comparing unstimulated versus PHA stimulation (p<0.05 one star, p<0.01 two stars, p<0.001 three stars). Data of panels <b>C</b>, <b>D</b>, <b>E</b> and <b>G</b> are representative of 4 independent experiments.</p

Circulating levels of HMGB1-specific antibodies, sTRAIL, and IP-10 in HIV-1 infected patients.

<p>(A): Serum from healthy donors (n = 12) and HIV-1-infected patients (n = 67) were tested for their content in total (residual + HMGB1-bound) HMGB1-specific IgG antibodies with an in-house ELISA assay, as described in M&M. Median and IQR are shown on the left hand side diagram. Spearman correlation between residual HMGB1 and total anti-HMGB1 antibodies is shown on the right hand side. (B): Spearman correlation between sTRAIL and total anti-HMGB1 antibodies in viremic patients (n = 22) on the left hand side, and between sTRAIL and IP-10 (n = 67) on the right hand side. (C): Comparative levels of circulating sTRAIL in aviremic (n = 19) vs viremic (n = 22) HIV-infected patients on the left hand side; spearman correlation between sTRAIL and HIV proviral DNA in the whole group HIV-infected patients (n = 67).</p

Impact of aNK cells on the proinflammatory array of cytokines and chemokines released by pDCs in the context of HIV-1 infection.

<p>Cytokines (A, B) and chemokines (C, D) content were quantified by MAP technology in 24 hour cell-free culture supernatants of pDCs exposed to low (1 ng p24/ml) or high (20 ng p24/ml) concentration of HIV-1, and further cocultured or not with aNK cells. Heat-maps (A, C) from one representative experiment out of four conducted with different primary cell preparations are shown. Mean values ± SD for indicated cytokines and chemokines from six independent experiments are shown (B, D) (non-parametric Wilcoxon test).</p

HIV-1 increases the release of proinflammatory cytokines and chemokines by pDCs in a dose-dependent manner.

<p>Cytokines and chemokines content were quantified by MAP technology in 24 hour cell-free culture supernatants of pDCs incubated in medium, stimulated with CpG ((3μg/ml) or were exposed to increasing concentrations of HIV-1 (1, 10 and 20 ng/ml p24). Heat-maps (A-C) and Pie Charts (B-D) were used to visualize the broad array of cytokines (A-B) and chemokines (C-D). Analysis of the data was made using SPICE Software. Pictures from one representative experiment out of seven conducted with different primary cell preparations are shown. The mean values ± SD of seven independent experiments testing the impact of HIV-1 (20 ng/ml) stimulation on cytokine and chemokine release by pDCs are shown (E). A p-value ≤ 0.05 was considered significant; non-parametric Wilcoxon test was used.</p

HIV-1 triggers the translocation of TRAIL from the cytoplasm to the membrane of pDCs.

<p>(A): pDCs were exposed to CpG (3 μg/ml) or increasing concentrations of HIV-1 (1 to 20 ng p24/ml) for 24 h. The expression of mTRAIL at the surface of pDCs was monitored by flow cytometry. The mean values ± SD of five independent experiments are shown (p-value ≤ 0.05 was considered significant; non-parametric Mann-Whitney test). (B): To characterize TRAIL and HMGB1 localization on pDCs exposed to HIV-1 (1ng/ml p24) or CpG (3 μg/ml), images were analysed using 3D interactive surface plot analysis. Deconvolution overlays of representative 2D red cross axis XY focal plan with XZ/YZ view of focal plane and projection overlay of cell stainings (analyzed by Metamorph software) are shown. (C): Flow cytometry analysis of mTRAIL expression and HMGB1 intracellular staining of pDCs cultured in medium, exposed to HIV-1 (1 ng p24/ml) or stimulated with CpG (3 μg/ml). Dot plots from one representative experiment out of three independent experiments are shown.</p

HIV-1 mediates phenotypic maturation of pDCs in a dose-dependent manner.

<p>(A): Phenotypic characterization of freshly sorted pDCs. The expression of HIV-1- receptor CD4, co-receptors CCR5 and CXCR4, maturation markers CD40, CD80, CD86, CD83, HLA-DR and chemokine-receptor CCR7 were analyzed by flow cytometry on gated CD123⁺CD303⁺ pDCs. (B): Sorted pDCs were either incubated with medium (NS) or stimulated with CpG (3μg/ml) for 24 hours and the expression of the indicated markers was analyzed. (C): pDCs were exposed to indicated concentrations of HIV-1 and the expression of the indicated markers was analyzed. The percentage of cells expressing the indicated marker and mean fluorescence intensity (in brackets) are indicated. (D): HIV-1-induced phenotypic maturation of pDCs was demonstrated following the identification of mature cells, i.e. CD123^brightHLA-DR^brightFSC^high cells. These results are representative of at least three different experiments conducted with primary cells from distinct donors.</p

HIV-1-induced phenotypic maturation of pDCs requires HMGB1 and IFN-α.

<p>(A): pDCs were exposed to CpG (3 μg/ml), increasing concentrations of HIV-1 (1 to 20 ng/ml), or incubated in medium (NS) during 24 hours. IFN-α concentration in culture supernatants was quantified with a specific ELISA. (B): The IFN-α inhibitor CB was added at 1 μM in cultures of pDCs stimulated with either control DNA (3 μg/ml), CpG (3 μg/ml) or HIV-1 (20 ng/ml p24), and IFN-α concentration in culture supernatants was quantified with a specific ELISA. (C): Inhibition by the IFN-α inhibitor CB (1 μM) of pDC maturation triggered by HIV-1 (1 or 20 ng/ml p24) or CpG (3 μg/ml). Mature pDCs were identified as CD123^brightHLA-DR^bright cells. Results from one representative experiment out of four conducted with primary cells from distinct donors are shown. (D): HMGB1 concentrations were measured in culture supernatants of pDCs exposed 24 hours to the indicated concentrations of HIV-1 with a specific ELISA. (E and F): Impact of neutralizing anti-HMGB1 antibodies (α-HMGB1 Abs) (10 μg/ml) or control IgG on pDC maturation following their culture in medium (NS) or their stimulation with CpG (3μg/ml) or HIV-1 (20 ng/ml) for 24 hours. Mature pDCs were identified as CD123^brightHLA-DR^bright or CD123⁺ CD83⁺ cells. Results from one representative experiment out of four conducted with primary cells from distinct donors are shown. Data shown in Figures A, B, D and F represent the mean values ± SD of at least 4 different experiments performed with distinct donors. Nonparametric Wilcoxon signed-rank test was used to compare the levels of IFN-α or HMGB1 released by pDCs with a p-value ≤ 0.05 being considered as significant.</p