CD2 accumulates more frequently at inhibitory synapses than at activating synapses.

<p>(A) Conjugates between IL-2 activated polyclonal human NK cells and 721.221-Cw15 cells or S2–LFA-3/Cw4 cells were formed and stained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003278#pone-0003278-g002" target="_blank">Figure 2</a>. Activating (Act.) and inhibitory (Inhib.) synapses were scored for clustering of CD2 at the zone of contact. The number of conjugates scored in each condition is indicated in parentheses. (B) Conjugates between activated NK cells and 721.221-Cw15 cells were allowed to form for 1 minute or 10 minutes as indicated, stained with the cyt42/43 antiserum and an anti-CD2 antibody as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003278#pone-0003278-g002" target="_blank">Figure 2</a>, and scored for CD2 clustering.</p

Surface level of CD11a is reduced at inhibitory synapses.

<p>IL-2 activated polyclonal human NK cells and S2 cells expressing ICAM-1 and peptide-loaded HLA-Cw4 were mixed at 37°C for 10 minutes, fixed, permeabilized, and stained with cyt42/43 and a mAb to CD11a followed by the appropriate secondary antibodies. (A) Confocal microscope <i>z</i>-series were obtained, and single sections are shown. The cell labeled #1, which shows KIR expression and clustering, represents an inhibitory synapse while cell #2, which lacks KIR2DL1 expression, displays an activating synapse. (B) The fluorescence intensity was scanned around the perimeter of conjugated NK cells. Profiles labeled 1 and 2 are from the corresponding cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003278#pone-0003278-g005" target="_blank">Figure 5A</a>. The third profile is another representative inhibitory synapse. The green and red lines represent the cyt42/43 and anti–CD11a fluorescence, respectively. Vertical red and blue lines mark the boundaries of cell contact as determined in DIC images. (C) Confocal <i>z</i>-stacks were used to create an <i>en face</i> view of the zone of cell contact in 2 inhibitory synapses. (D) The frequency of synapses displaying increased CD11a, reduced CD11a, or no change in CD11a intensity was determined for both activating and inhibitory synapses.</p

2B4 accumulates at both activating and inhibitory synapses.

<p>Activated NK cells were mixed with target cells at 37°C for 10 minutes, fixed, permeabilized, and stained with the cyt42/43 antiserum and a mAb to 2B4 followed by the appropriate secondary antibodies. (A) Mixed with .221-Cw15 target cells. Confocal microscope <i>z</i>-series were obtained, and single sections are shown. The cells labeled #1 and #3, which display KIR expression and clustering, represent inhibitory synapses while cell #2, which lacks KIR2DL1 expression, is an activating synapse. The NK cells without a number in the top image were not analyzed, because they did not appear to form tight conjugates with target cells. (B) The fluorescence intensity was scanned around the perimeter of conjugated NK cells. Profiles labeled 1, 2, and 3 are from the corresponding cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003278#pone-0003278-g004" target="_blank">Figure 4A</a>. The green and blue lines represent the cyt42/43 and anti–2B4 fluorescence, respectively. Vertical red and blue lines mark the boundaries of cell contact as determined in DIC images. (C) Confocal <i>z</i>-stacks were used to create an <i>en face</i> view of the zone of cell contact in 2 inhibitory synapses.</p

Detection of inhibitory synapses using the cyt42/43 antiserum.

<p>IL-2 activated polyclonal human NK cells were mixed with target cells for 10 minutes at 37°C, fixed, permeabilized and stained with the cyt42/43 rabbit polyclonal antiserum. NK cells and NK:target cell conjugates stained with the cyt42/43 antiserum were scored for KIR clustering. The number of cyt42/43-positive cell conjugates analyzed is given in parentheses over each bar. (A) Mixing with .221 and .221-Cw15 target cells, as indicated. (B) NK cells expressing KIR2DL2 and not KIR2DL1 mixed with .221-Cw3 and .221-Cw15 cells, as indicated. (C) Mixing with S2–Cw4 cells loaded with a peptide that is permissive for KIR2DL1 binding (peptide #1) or a peptide that is nonpermissive for KIR2DL1 binding (K8E), as indicated.</p

CD2 accumulates at both activating and inhibitory synapses.

<p>IL-2 activated polyclonal human NK cells were mixed with target cells at 37°C for 10 minutes, fixed, permeabilized, and stained with the cyt42/43 antiserum and a mAb to CD2 followed by the relevant secondary antibodies. Confocal microscope <i>z</i>-series were obtained. (A) Mixed with .221-Cw15 as indicated. A single confocal section is shown. The cell labeled #1, which displays KIR expression and clustering, represents an inhibitory synapse while cell #2, which lacks KIR2DL1 expression, displays an activating synapse. (B) Mixed with S2–LFA-3/Cw4 target cells, as indicated. A single confocal section is shown. (C) The fluorescence intensity was scanned around the perimeter of conjugated NK cells. Profiles labeled 1, 2, and 3 are from the corresponding cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003278#pone-0003278-g002" target="_blank">Figures 2A and 2B</a>. The green and red lines represent the cyt42/43 and anti–CD2 fluorescence, respectively. Vertical red and blue lines mark the boundaries of cell contact as determined in DIC images. (D) Confocal <i>z</i>-stacks were used to create an <i>en face</i> view of the zone of cell contact in 2 inhibitory synapses.</p

NK cell receptors percentage and MFI in SLE patients and controls.

<p>Cell surface expression of indicated molecules on NK cells within peripheral blood lymphocytes from controls (squares) and SLE patients (triangles) are given as percentage of total NK cells and MFI. Horizontal bars indicate median values. The <i>p</i> value is mentioned above a given NK cell subset when a statistically significant difference is observed between the two groups.</p

Percentage of leukocytes subsets and DAP12 expression in SLE and control patients.

<p>Both percentage of NK cells among total lymphocytes and percentage of the CD56^bright and CD56^dim subsets among NK cells are indicated. The percentage of dendritic cells, monocytes and neutrophils among the leukocytes are given. Statistically significant values are shown in bold.</p

F4 MAb recognizes DAP12 antigen in transfected RBL cells.

<p>Stable transfectants of the rat basophilic leukemia cell line (RBL) have been described earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006264#pone.0006264-Anfossi1" target="_blank">[31]</a>. In brief, RBL-CD158j and RBL-CD158j/DAP12 have been obtained by transfection with human cDNAs encoding CD158j alone or both CD158j and human DAP12, respectively. RBL cells were fixed by incubation with 2% paraformaldehyde at room temperature. Then the cells were permeabilized by Permwash treatment (Beckton Dickinson) before staining with 50 µg/ml irrelevant or F4 antibody on ice for 30 min. After three washes in cold PBS, the cells were incubated 1∶100 FITC-conjugated goat anti-rat immunoglobulins.</p

Number of NK cells in SLE and control patients.

<p>Absolute numbers and percentage of NK cells among total lymphocytes are indicated for the control group (n = 92) and the SLE group of 13 patients.</p

Patients characteristics.

<p>Quantification of total White blood cells (WBC), total lymphocytes (Ly) and NK cells are indicated in cells per µl. Treatment for each patient is shown: MMF for mycophenolate mofetil, H for hydroxychloroquine and P for prednisone (at time of analysis all patients received ≤10 mg/day of prednisone). Main biological characteristics of autoantibodies are shown: antinuclear antibodies (ANA) titer, type of ANA and titer of anti-dsDNA autoantibodies (N<7). The number of ACR criterias for each patient is indicated.</p