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

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

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

Macaque bone marrow pDC express lower CD123 and HLA-DR, display higher percentages of CD34+ and Ki67+ precursors than blood pDC, and are poor IFN-I producers.

<p>(<b>A</b>) CD34⁺ and Ki67⁺ pDC-precursor frequencies are higher in the bone marrow (BM) than in the blood (non-infected macaques, n = 6), and both CD123 and HLA-DR expressions are lower in BM pDC than blood pDC. From <b>left to right</b>: Percentage of CD34⁺ pDC precursors, percentage of Ki67⁺ pDC precursors, and CD123 and HLA-DR geometric MFI (gMFI) in BM and blood pDC. Dotplot showing Ki67 and CD34 expression by pDC in BM and blood, from one representative animal. (<b>B</b>) Bone marrow pDC produce less IFN-I in response to TLR-7/8 stimulation (R848) than blood pDC (non-infected macaques, n = 6) (<b>Left</b>). Most IFNα is produced by Ki67⁻ pDC and not by K67⁺ precursors (<b>Middle</b>). Representative dot plot showing that only Ki67⁻ pDC produce IFNα (<b>Right</b>). Wilcoxon's rank sum test was used for all comparisons of paired data.</p

Plasmacytoid DC produce IFNα in both lymphoid and mucosal compartments.

<p>Dotplot showing IFNα intra-cellular staining in gated pDC (CD45⁺HLA-DR⁺lin⁻CD123⁺) in different tissues. Cells were labeled <i>ex vivo</i> on fresh cells after 30 min incubation in 10 µg/mL brefeldin A in the absence of any stimulation. Data for two macaques sacrificed on day 10 p.i. and one uninfected control are shown. Mononuclear cells from BM, spleen, peripheral LN, mesenteric LN, ileum, and colon were extracted for FACS analysis. Frequencies of IFNα-pDC are indicated in bold and # indicates the number of pDC recorded for each file.</p

Plasmacytoid DCs are major contributors of IFNα production in peripheral lymph nodes during primary infection.

<p>(<b>A</b>) Plasmacytoid DC frequencies among CD45⁺ PLN leukocytes on days 9 and 35 and month 3 post-infection (n = 6) and in uninfected macaques (n = 7). (<b>B</b>) IFNα-producing pDC in peripheral lymph nodes of 9 macaques at various times after infection as assessed by IFNα intracellular staining. Freshly isolated cells were labeled at various times after infection without any additional <i>in vitro</i> stimulation, after 30 min incubation in the presence of 10 mg/mL Brefeldin A. (<b>C</b>) Dotplots for two representative infected macaques (#30717, #30978) with fluorescence minus one (FMO) shown as a negative control (left). Dotplot showing intracellular IFNα expression in the total live CD45⁺ leukocyte gate for one representative infected macaque at day 9 p.i., and one representative uninfected macaque (right) (<b>D</b>). The percentage of IFNα⁺ pDC correlates with relative SIVgag mRNA expression in peripheral lymph nodes (day 9 p.i., n = 9). Spearman correlation. (<b>E</b>) Log₁₀ (relative IFNα mRNA expression) plotted against the percentage of IFNα expressing pDC in PLN (day 9 p.i., n = 9). Spearman correlation. Values at different time points were compared with the Wilcoxon rank sum test. When baseline values were not available, data for infected macaques were compared with uninfected macaques using the Mann-Whitney rank test; p values are given if the differences are statistically significant.</p

Infection of six cynomolgus macaques with SIVmac251 and establishment of chronic infection.

<p>(<b>A</b>) vRNA load in plasma. (<b>B</b>) Evolution of total CD4 T cell numbers in the blood over time (percentage of baseline). (<b>C</b>) vRNA in RB. (<b>D</b>) vRNA in PLNs. ULD = under the limit of detection. P values are given for non-parametric Wilcoxon rank sum test and was considered significant if p < 0.05.</p