dNK cells are cytotoxic against HCMV-infected autologous fibroblasts.

<p>Decidual fibroblasts were kept uninfected or infected for 48 h with HCMV. dNK cell cytotoxicity was determined by ⁵¹Cr-release assay after 18 hours of contact at different E/T ratios. (A) Fibroblasts were infected with VHLE clinical isolate (n = 3), (B) cells infected with AD169 laboratory strain of HCMV (n = 5). (C) Analysis of dNK cell cytotoxicity from a cohort of 10 <i>decidua</i> samples at the 50 to 1 ratio. (D) dNK cells were pre-incubated with anti-FasL or (E) anti-TRAIL blocking mAbs at the final concentration of 10 µg/ml for 20 min and cytotoxicity was monitored after 18 h. Control (CTRL), lysis performed in the presence of IgG control. Each data point is calculated as the mean lysis ± S.D. from at least five independent experiments done in replicate tissue culture wells. Statistical comparisons of mean lysis of uninfected versus HCMV-infected were performed using two-way ANOVA test. ***, <i>p</i><0.001; **, <i>p</i><0.01; ns, not significant, <i>p</i>>0.05.</p

Polarization of the MTOC and lytic granules to the immune synapse formed with HCMV-infected fibroblasts.

<p>Uninfected (AD169⁻) or HCMV-infected (AD169⁺) decidual fibroblasts (F) plated on glass coverslips were incubated with autologous dNK cells (dNK) for 20 min at 37°C. (A) Representative images of maximum intensity projection. Microtubules (α-tubulin in green), lytic granules containing Perforin (red), HCMV-IE antigen (blue). Arrowhead points to the MTOC polarization (aster). Bar represent 20 µm. (B) Left cartoon shows schematic representation of the immunological synapse (IS), D (distance in µm). The center of IS was defined as the center of the contact zone between dNK and target cell (see cartoon, red line). Zero on the Y axis (µm) represents synaptic area; blue dot represents the microtubule organizing center (MTOC) and microtubules are in green. The MTOC polarization (Right graph) defined by the distance between the MTOC and the center of IS formed with uninfected (AD169⁻) and HCMV-infected (AD169⁺) fibroblasts. Distances were calculated for 50 conjugates from five independent experiments. Statistical analysis was performed using unpaired Student's <i>t</i>-test. ***, <i>p</i><0.001. (C) Percentage of conjugates showing polarized perforin containing granules to the NKIS. Results from 5 independent conjugations were averaged, values represent means and S.D.s. At least 300 conjugates were analyzed. Statistical analysis was performed using unpaired Student's <i>t</i>-test. *** <i>p</i> = 0.0002. (D) Kinetic of CD107a cell surface expression was analyzed by flow cytometry on dNK cells that were in contact with uninfected or AD169-infected autologous fibroblasts. Values presented in the bar graphs are mean values calculated from three independent experiments done in triplicates at the ratio 1 to 1. Error bars are SEM. Statistical comparisons were performed using unpaired Student's <i>t</i>-test, ** <i>p</i><0.01.</p

dNK cells infiltrate infected placental tissues.

<p>(A) Two-color 3D-images of chorionic <i>villi</i> explants organ cultures established from first trimester trophoblast either uninfected (AD169⁻) or HCMV-infected (AD169⁺). Nuclei were stained with dapi (cyan). Infiltrating dNK cells (red). Lower and side panels show orthogonal XZ and YZ slices, respectively. Images are from two-photon Z-stack (total of 200 µm). Scale bars = 100 µm. (B) Two-color IHC of 6-µm-thick sections from paraffin-embedded whole placental biopsies of HCMV⁺ pregnancy termination. HCMV⁺ and HCMV⁻ tissue sections are presented. Representative immunostaining of HCMV-IE (blue, alkaline phosphatase staining) and CD56⁺ NK cells (brown, peroxidase staining) (n = 2).</p

HCMV infection modulates dNK cells cytokine/chemokine production.

<p>dNK cells were stimulated with uninfected (AD169⁻, gray) or AD169-infected (AD169⁺, black) autologous decidual fibroblasts for 24 h. Cytokines were quantified in the supernatants using a 42-multi-plexed cytokine assay. Representative histograms from selected cytokines-chemokines that showed significant differences are presented. (A & B) Soluble factors that are produced at high levels by dNK cells. (C) Soluble factors that are produced at low levels by dNK cells. Concentrations are given as differences between secretions of dNK cell in presence of uninfected or infected fibroblasts and the corresponding uninfected or infected fibroblasts. Normalized data points are given as mean ± S.D. calculated as from four independent experiments. Statistical comparisons were performed using Mann & Whitney test. **, <i>p</i><0.01; *, <i>p</i><0.05.</p

Exposure to infected cells modulates dNK cell receptor repertoire expression.

<p>dNK cells were co-cultured with autologous fibroblasts that were either uninfected or infected with HCMV-AD169 for 48 h. dNK cells were stained for surface expression of the indicated receptor using fluorochrome-conjugated antibodies and analyzed by flow cytometry. Representative FACS histograms gated on CD56^pos CD3^neg dNK cells are shown (n = 5). Specific receptors are indicated by the arrow. dNK cells in contact with uninfected fibroblasts are represented by black line, dNK cells in contact with HCMV-infected fibroblasts are represented by shaded gray. Dotted gray line represents isotype-matched control Ig. One representative histogram out of five independent experiments is shown.</p