Figure 1
Figure 1. The formation of the regulatory synapse between mature DCs and resting NK cells is a fast event and matures with DC-derived f-actin polymerization at the synapse. (A) CSFE-labeled mature DCs (green) were co-cultured with PKH26 labeled NK cells (red) in extracellular matrix. Conjugate formation was visualized by live cell imaging (see supplemental Video 1). Pictures were acquired every 5 minutes for at least 90 minutes. Arrows indicate the immunologic synapse. A representative conjugate is shown. The graph represents the duration of different synapses between DCs and NK cells. (B) Mature DC single cell cultures or mature DC/resting NK-cell cocultures were fixed after 1, 20 minutes or 120 minutes of interaction and f-actin was stained with bodipy conjugated phallacidin (green). DAPI was used to stain nuclear DNA (blue). Arrows indicate the synapse. The graph represents the quantification of f-actin staining intensity at the synapse compared with the staining at the opposite side of the same DC. Values were normalized to the values of molecule distribution in unconjugated cells, assigned as 1. (C) Mature DCs were allowed to conjugate with resting NK cells for 20 minutes. Cocultures were fixed and stained with bodipy conjugated phallacidin to stain f-actin (green) and anti-KIRs antibodies (red). The fluorescence intensities of KIRs and f-actin stainings were plotted along the indicated trajectory, from A to B. The synapse area in cellular conjugates is indicated by boxes. (D) Mature DCs were allowed to conjugate with resting NK cells for 20 minutes. Cocultures were fixed and treated for TEM analysis. Original magnifications are 4200× and 17 500×, respectively from left to right. The graph represents the size of several regions in the synaptic clefts of 5 DC/NK-cell conjugates. Images are representative of 1 experiment for TEM images and at least 3 independent experiments for the other techniques. Original magnifications are 100× for all the light microscopy images. Values on graph bars are medians and error bars represent interquartile ranges from the analysis of at least 100 conjugates from at least 3 independent experiments. ↑↑ indicates fold increase > 2. Scale bars are 10 μm unless specified otherwise.

The formation of the regulatory synapse between mature DCs and resting NK cells is a fast event and matures with DC-derived f-actin polymerization at the synapse. (A) CSFE-labeled mature DCs (green) were co-cultured with PKH26 labeled NK cells (red) in extracellular matrix. Conjugate formation was visualized by live cell imaging (see supplemental Video 1). Pictures were acquired every 5 minutes for at least 90 minutes. Arrows indicate the immunologic synapse. A representative conjugate is shown. The graph represents the duration of different synapses between DCs and NK cells. (B) Mature DC single cell cultures or mature DC/resting NK-cell cocultures were fixed after 1, 20 minutes or 120 minutes of interaction and f-actin was stained with bodipy conjugated phallacidin (green). DAPI was used to stain nuclear DNA (blue). Arrows indicate the synapse. The graph represents the quantification of f-actin staining intensity at the synapse compared with the staining at the opposite side of the same DC. Values were normalized to the values of molecule distribution in unconjugated cells, assigned as 1. (C) Mature DCs were allowed to conjugate with resting NK cells for 20 minutes. Cocultures were fixed and stained with bodipy conjugated phallacidin to stain f-actin (green) and anti-KIRs antibodies (red). The fluorescence intensities of KIRs and f-actin stainings were plotted along the indicated trajectory, from A to B. The synapse area in cellular conjugates is indicated by boxes. (D) Mature DCs were allowed to conjugate with resting NK cells for 20 minutes. Cocultures were fixed and treated for TEM analysis. Original magnifications are 4200× and 17 500×, respectively from left to right. The graph represents the size of several regions in the synaptic clefts of 5 DC/NK-cell conjugates. Images are representative of 1 experiment for TEM images and at least 3 independent experiments for the other techniques. Original magnifications are 100× for all the light microscopy images. Values on graph bars are medians and error bars represent interquartile ranges from the analysis of at least 100 conjugates from at least 3 independent experiments. ↑↑ indicates fold increase > 2. Scale bars are 10 μm unless specified otherwise.

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