Expression of ligands for NK cell receptors on HHV8-infected cells.

<p>(A) Expression of the indicated ligand was measured by flow cytometry on uninfected (open histograms) and HHV8-infected (gray histograms) HMEC cells (left panels) and KS-derived cells (right panels). Dotted lines represent staining with control isotypes. Histograms are representative of 3–6 independent experiments performed in each cell line. B) K3 and K5 mRNA levels in uninfected or HHV8-infected HMEC and KS-derived cells. Results show the expression level of the K3 and K5 transcripts relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA used as endogenous control, and represent the mean of 2 independent experiments. C) Consecutive sections of paraffin-embedded KS biopsies were stained with antibodies specific for HLA-1 (W6/32), MICA/B (SR99), or isotype control. Representative staining in 2 patients with classical KS out of 5 studied is shown. Staining with hematoxylin-eosin (HE) shows characteristic fascicles of spindle-shaped tumor cells forming the walls of slit-like vascular spaces, associated with mononuclear cell infiltrates. Original magnification x10 (Pt1), x40 (Pt2).</p

NK cell subset distribution in HHV8-infected subjects and controls.

<p>Summary graphs of statistical dot plots with medians (horizontal black bars) in the different study groups showing: (A) the percentage of CD3−CD56+ NK cells among PBMCs and the proportion of CD56bright and CD56 dim among NK cells; (B) the percentage of CD3−CD56+ NK cells expressing HLA class I-specific NK cell receptors. P values were not significant (n.s) after adjustment for multiple comparisons (Kruskall-Wallis test with Dunn's post test).</p

Decreased NK cell lytic capacity in patients with active KS.

<p>(A) Comparative analysis of CD107a expression on NK cells from healthy controls (HC, n = 10) and classical KS patients (n = 13, including 3 active and 10 resolved KS). Box and whisker plots show the median and 25–75^th percentiles of CD107a expression, in the absence of stimulation (unst) and after stimulation with SV2G or HHV8-SV2G target cells. Horizontal bars indicate minimum and maximum values. (B) The capacity to degranulate in the presence of K562 target cells is significantly decreased in patients with active KS (n = 6) compared to healthy controls (HC, n = 18) and patients with resolved KS (n = 12). (C) K562-induced NK cell degranulation is inhibited in the presence of anti-NKG2D blocking antibody. Data are mean ± SEM of results in 3 healthy controls, 3 patients with resolved classical KS and 2 patients with active classical KS. (D) Correlation between the expression level of NKG2D and the respective K562-induced degranulation of NK cells in 15 KS patients (active/resolved). Cells were analyzed on a LSRFortessa flow cytometer. Correlation coefficient (r) and P values are indicated. (E) Comparative analysis of K562-induced CD107a degranulation and levels of NKG2D, NKp30 and NKp46 in NK cells from 3 patients with active classical KS analyzed before and one year after successful local treatment.</p

Alterations of NK cell receptor expression in HHV8-infected individuals.

<p>(A) Summary graphs of statistical dot plots with medians (horizontal black bars) showing expression (mean fluorescence intensity, MFI) of NKp30, NKp46, CD161, DNAM-1 and NKG2D receptors on gated CD3−CD56+ NK cells in the different study groups. Cells were analyzed on a FACSCalibur flow cytometer. P values<0.05 after adjustment for multiple comparisons (Kruskall-Wallis test with Dunn's post test) are indicated. * P<0.01, ** P<0.005, *** P<0.0001. (B) Box and whisker plots showing the median and 25–75^th percentiles of NKG2D expression in healthy controls (n = 35), patients with active KS (n = 10, all classical HIV- KS) and patients with resolved KS (n = 35, including 21 classical HIV- KS and 14 HIV+ KS). Horizontal bars indicate the minimal and maximal values. ** P<0.01, *** P<0.001. (C) Representative flow cytometry analysis in healthy controls (upper panel), asymptomatic HHV8 carriers (middle panel) and patients with active classical KS (lower panel). Control isotypes are shown as dotted lines.</p

Effect of PGE2 on NK cell phenotype.

<p>(A) Levels of VEGF, TGFβ, IL-8 and PGE2 in sera from healthy controls, patients with resolved KS and patients with active KS. P values<0.05 after adjustment for multiple comparisons (Kruskall-Wallis test with Dunn's post test) are indicated. (B) Control PBMCs cells were exposed to IL-8 (100 ng/ml), VEGF (35 ng/ml), TGFβ (10 ng/ml) or PGE2 (100 ng/ml) for 48 h, after which expression of the indicated NK cell receptors was evaluated. Untreated cells are shown as open histograms, and treated cells as gray histograms. Control isotype is shown as thin lines. (C) Dose-dependent PGE2-mediated down-modulation of NKG2D levels. Data are presented as percent of inhibition of NKG2D MFI, and indicate the value means from 3 independent experiments.</p

Coordinate decrease of NKp30, NKp46 and CD161 expression in HHV8-infected individuals.

<p>Positive correlations between the proportions of NK cells expressing the indicated receptors in HHV8-infected individuals. Spearman rank correlation (r) and P values are indicated.</p

Predicted trajectories of mean √CD4 cell counts since HIV diagnosis according to blip status during the period of HIV control (81 HIV controllers, 1668 CD4 measurements).

<p>Predicted trajectories of mean √CD4 cell counts since HIV diagnosis according to blip status during the period of HIV control (81 HIV controllers, 1668 CD4 measurements).</p

Evolutionary relationships between the HIV-1 <i>env</i> genes in the eight donor/recipient pairs.

<p>The evolutionary history was inferred using the Neighbor-Joining method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069144#pone.0069144-Boeras1" target="_blank">[38]</a>. The optimal tree with the sum of branch length = 2.01912678 is shown. The tree is drawn to scale, with branch length in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069144#pone.0069144-Whitney1" target="_blank">[39]</a> and the unit is the number of base substitutions per site. Codon positions included were 1^st+2^nd+3^rd+noncoding. All positions containing gaps and missing data were eliminated from the dataset. There were a total of 230 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069144#pone.0069144-Redd1" target="_blank">[40]</a>. For each recipient, viruses isolated from PBMC-derived DNA (•) and plasma RNA (○) are represented, with a different color for each donor/recipient pair. Asterisks indicate branches with bootstrap values greater than 98%.</p

Comparison of baseline characteristics between HIV controllers and patients enrolled in the ANRS SEROCO Cohort and still alive in 2007.

<p>Data are median [IQR] or n (%);</p><p>*SSA/C  =  SubSaharan Africans or Caribbeans;</p><p>**Following HIV diagnosis.</p

Comparative <i>Highlighter</i> analyses of <i>env</i> diversity in a donor-recipient HIV-1 transmission pair.

<p>(A) Recipient#1 shows evidence of infection with a single virus. (B) Donor#1 was the chronically infected partner of recipient#1. The same reference amplicon, a V3 RNA sequence from recipient plasma, was used to depict the viral diversity in both individuals.</p