![Figure 2. The delivery of IL-12 is polarized during the DC/NK cell cross-talk. (A-B) Preassembled stores of IL-12 in activated human and mouse DCs. Confocal microscopy study of human MD-DCs stimulated by LPS for 24 hours using anti-IL-12 mAb (A). In mDCs that do not form conjugates with NK cells or mDCs cultured alone staining with anti-IL-12 mAb highlighted a dotted cytosolic and membrane-associated localization of the cytokine. Cytofluorometric analyses of day 7 mouse BM-DCs stained with anti-mouse IL-12p70 mAb or isotype control antibody (blue line; B). (C) Close contacts between DCs and NK cells are required for IL-12 bioactivity on NK cells. Human mDCs and resting NK cells were cocultured together at a 1:10 DC/NK cell ratio in the upper chamber of a transwell containing resting NK cells alone in the lower chamber. Both compartments were harvested separately at 24 hours, incubated 45 minutes with an IFN-γ capture antibody (Miltenyi Biotech), and then stained with anti-CD3 (FITC), anti-CD56 Cychrome (CyC), and anti-IFN-γ (allophycocyanin [APC]) mAbs and examined by flow cytometry. The percentage of IFN-γ staining in the CD3-/CD56+ NK cell gate is depicted for NK cells cultured without mDCs, or together with mDCs separated or not by a transwell membrane, for a representative experiment (performed twice with similar results). (D-G) Selective IL-12 polarization in DCs at the DC/NK cell interface. mDCs were admixed with autologous resting NK cells or CD8+ CTL (LT11) clones. After fixation, cells were permeabilized and stained with anti-IL-12 (D-F) or anti-IL-15 (G) mAb. In panels D, F, and G Normaski (left) and fluorescent (middle) images are shown and in panel D, fluorescence intensity is depicted (right). In mDCs forming tight conjugates with NK cells, IL-12 selectively concentrated at the DCNK-IS interface (D). A deconvoluted 3-dimensional reconstruction of the mDC in close contact with an autologous NK confirmed the mobilization of IL-12 toward the DC/NK synapse (E). A total of 78% ± 4% of DCs interacting with NK cells relocalized their IL-12 to the cellular junction with NK cells However, such an IL-12 polarization toward the intercellular junction was not seen in mDC/LT11 conjugates (F). Lack of polarization of IL-15 at the DC/NK cell surface is shown in panel G.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/104/10/10.1182_blood-2004-01-0380/6/zh80220469130002.jpeg?Expires=1735523261&Signature=bY9ORH-sNCb9qZ~8B3O9KwmA~ljL6eIvWq2MSnTEe3XYBGGLSlnUunkh01SflOAyrqJpYa25CU31j4-ap0Fj5KTs--pajOH8ZxLeKdcS9H2hyEyF7gtmQWVSjk1Z-hLe1US9vbN-ugU9yGsFyq4Rh1n-MckV7WazlhelbYLoxGfD1RE6oc10wHmiRuduNCye9bVsFsNCk8c02QiIaF3ijkctTuaQ2cjKiguTU6m00LSMEjwWCEdKcUZAXoXudol38~N36ytI61ietIaPxzO6yk3MUj1bPxum-ufnxR62VM5m8fhnVpPkCUVkTA1koCv7eTjLkW~OxcCC2n-kfxiEDA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The delivery of IL-12 is polarized during the DC/NK cell cross-talk. (A-B) Preassembled stores of IL-12 in activated human and mouse DCs. Confocal microscopy study of human MD-DCs stimulated by LPS for 24 hours using anti-IL-12 mAb (A). In mDCs that do not form conjugates with NK cells or mDCs cultured alone staining with anti-IL-12 mAb highlighted a dotted cytosolic and membrane-associated localization of the cytokine. Cytofluorometric analyses of day 7 mouse BM-DCs stained with anti-mouse IL-12p70 mAb or isotype control antibody (blue line; B). (C) Close contacts between DCs and NK cells are required for IL-12 bioactivity on NK cells. Human mDCs and resting NK cells were cocultured together at a 1:10 DC/NK cell ratio in the upper chamber of a transwell containing resting NK cells alone in the lower chamber. Both compartments were harvested separately at 24 hours, incubated 45 minutes with an IFN-γ capture antibody (Miltenyi Biotech), and then stained with anti-CD3 (FITC), anti-CD56 Cychrome (CyC), and anti-IFN-γ (allophycocyanin [APC]) mAbs and examined by flow cytometry. The percentage of IFN-γ staining in the CD3-/CD56+ NK cell gate is depicted for NK cells cultured without mDCs, or together with mDCs separated or not by a transwell membrane, for a representative experiment (performed twice with similar results). (D-G) Selective IL-12 polarization in DCs at the DC/NK cell interface. mDCs were admixed with autologous resting NK cells or CD8+ CTL (LT11) clones. After fixation, cells were permeabilized and stained with anti-IL-12 (D-F) or anti-IL-15 (G) mAb. In panels D, F, and G Normaski (left) and fluorescent (middle) images are shown and in panel D, fluorescence intensity is depicted (right). In mDCs forming tight conjugates with NK cells, IL-12 selectively concentrated at the DCNK-IS interface (D). A deconvoluted 3-dimensional reconstruction of the mDC in close contact with an autologous NK confirmed the mobilization of IL-12 toward the DC/NK synapse (E). A total of 78% ± 4% of DCs interacting with NK cells relocalized their IL-12 to the cellular junction with NK cells However, such an IL-12 polarization toward the intercellular junction was not seen in mDC/LT11 conjugates (F). Lack of polarization of IL-15 at the DC/NK cell surface is shown in panel G.
