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

Human Dex harbour functional IL-15Rα and synergize with IL-15 for NK cell proliferation <i>in vitro</i> and IFNγ production <i>in vitro.</i>

<p><i>A.</i><i> Immunoblotting of IL-15Rα from Dex and DC lysates</i>. Western Blot analysis on 10–40 µg of protein lysates obtained from immature DC (iDC), Dex or Tex (exosomes from Mel888 melanoma cell line) using anti-IL-15Rα mAb. Positive controls included anti-HLA-DRα, -TSG 101 and -HSC 70 Abs. Representative immunoblots of two normal volunteers are depicted (NV1 and NV2). Molecular weights are indicated on the left lane. B. <i>Proliferative effects of recombinant IL-15 and Dex on NK cells</i>. CFSE-labeled NK cells were cultured with or without 10 µg autologous Dex or allogenic Tex in complete medium containing 0.5 ng/ml of human recombinant IL-15. At day 6 of culture, NK cell proliferation was determined by flow cytometry and the number of divisions were counted and depicted. A representative experiment out of two is shown. C–D. <i>Synergistic effects between Dex and recombinant IL-15 for NK cell triggering</i>. NV's PBL were cultured without (white histograms) or with (black histograms) 10 µg autologous Dex and increasing concentrations of human recombinant IL-15. NK (CD56⁺ CD3⁻) cells were then analysed for CD69 expression by flow cytometry (C) or supernatants were harvested to measure IFNγ levels in EIA (D). The graphs depict means±SEM of % of CD69 expressing NK cells in 3 experiments (C) or IFNγ concentrations in 4 experiments (D). * p<0.05, ** p<0.01 and ns: non significant.</p

Human Dex harbour functional NKG2D ligands.

<p>A–B. <i>Immunoblot detection of NKG2D ligands on Dex</i>. (A) 10–40 µg of protein lysates from immature DC (iDC), Dex or tumor cells (K562, GIST) were assayed in western blot analyses using anti-ULBP-1 and MICB Abs on whole protein lysates (A) (this result was similar in 6 Dex preparations from 6 different healthy donors) or on each fraction of distinct density (B) following ultracentrifugation of 400 µg of Dex proteins on a continuous density gradient. Controls included anti-HLA-DRα, TSG 101, hsc 70 Abs and anti-γ tubulin Abs are also depicted. Molecular weights are indicated on the left lane. Note that the density of the ULBP-1 positive Dex fractions is approximately 1.16 to 1.19 g/ml i.e. the assumed density flotation of Dex. C. <i>Dex express cell surface NKG2D ligands</i>. Flow cytometry analyses of the surface expression of NKG2D ligands (empty histograms) on beads coated-Dex using rhNKG2D-Fc chimera or a mix of anti-human MICA/B, ULBP-1, ULBP-2 and ULBP-3 mAbs and appropriate secondary Abs. Similar stainings of iDC and K562 cells (a negative and positive control respectively). Filled histograms represent stainings with isotype matched mAbs. D. <i>Engagement of NKG2D receptors on NK cells triggered by Dex</i>. NK cells were incubated 40 hrs with medium or 10 µg autologous Dex or irrelevant pelleted proteins (cell debris) coated onto MaxiSorp™ wells, and then stained with anti-CD3 APC, anti-CD56 CyC, anti-NKG2D PE mAb. Flow cytometry analyses revealed the mean % (±SEM) of NKG2D expressing NK cells in three independent experiments performed in triplicate wells. E. <i>NKG2D-dependent NK cell activation by Dex in vitro</i>. Identical setting as in D. but using rhNKG2D-Fc fusion proteins or Ig-Fc controls to determine CD69 (left panel) and NKG2D (right panel) expression on NK cells in flow cytometry analyses. * p<0.05.</p

Mouse Dex promoted NK cell proliferation: a role for IL-15Rα.

<p>A. <i>Dex induce NK cell influx in draining lymph nodes</i>. Enumeration of CD3⁻ NK1.1⁺ cells (NK cells) in the draining lymph node following intradermal inoculation of 10 µg of mouse Dex, or 3×10⁵ immature DC (iDC), or 10 µg of irrelevant pelleted proteins (cell debris) or PBS. B. <i>NK cells enter cell cycle following Dex inoculation</i>. Proportion of BrdU⁺ CD3⁻ NK1.1⁺ cells (NK cells) (left panel) or BrdU⁺ CD3⁺ NK1.1⁻ cells (T cells) (right panel) in the draining lymph node following intradermal inoculation of 10 µg of PBS or mouse Dex in the presence of anti-IL-15Rα blocking mAb (αIL-15Rα) or isotype control mAb (Isotype). The graphs depict the means of absolute numbers or percentages±SEM of the data from 4 pooled experiments.* p<0.05, ** p<0.01 and ns: “non significant”.</p

Mouse Dex promoted NK cell activation: a role for NKG2D.

<p>A. <i>Dex induced NK cell activation in the draining lymph node</i>. Absolute numbers of CD3⁻ NK1.1⁺ CD69⁺ cells in the draining lymph node following inoculation of 10 µg of mouse Dex, or 3×10⁵ iDC or 10 µg of irrelevant pelleted proteins (cell debris) or PBS. Each dot represents the result in one mouse. B. <i>Dex stimulated splenic NK cytotoxicity</i>. Killing assays on splenocytes against YAC-1 targets at 200∶1 and 50∶1 (not shown) after intradermal inoculations of 10 µg of Dex every other week for 8 weeks and sacrifice 48 hrs after the last immunization, or 24 hrs after a single subcutaneous injection of 10 µg of CpG ODN. C. <i>Therapy with unpulsed Dex reduced number of metastases</i>. Intradermal inoculation of 20 µg of exosomal proteins or PBS 5 days after intravenous injection of 3.10⁵ B16F10 tumor cells. Depletion of NK cells was achieved using 3 administrations of anti-NK1.1 mAbs or isotype control mAbs. Mice were sacrificed on day 10 for enumeration of lung metastases. D. <i>A role for NKG2D in Dex-mediated NK cell triggering</i>. Enumeration of CD3⁻NK1.1⁺ cells (left panel) and CD3⁻ NK1.1⁺CD69⁺ cells (right panel) in the draining lymph node following intradermal inoculation of PBS or 10 µg of mouse Dex in the presence of anti-NKG2D blocking mAb (αNKG2D) or control isotype mAb. The graphs depict the data of 4 pooled experiments (2 for B.). Means and SEM are shown. * p<0.05, ** p<0.01, *** p<0.001 and ns: “non significant”.</p