![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=1724253825&Signature=oegCMLvtR5nrUgXr9cLF6rf6KHRHoEECUbFYG6AKeRr4zPqgK4Aqut0WB2d60Qr6m-uIs82J3g-bTT5NbkH9l06PjaTVGeh7z2ApGSYAa7~Cp-j-2~QC3mVFrTigDXCCpNpV~oXuZ2ynEMCzcoY3XVe3RR0-QiUXvY9uptT2IdiZAQU2YODDO8Phf6xyS1ChQVDfOAzeXocFc~BniUa-lpmTQmcvAe1FaL~Yykp99IVf8AfYoNkLVYTOscSk-0scNtcHPQVsHmhLt4jrPyP8~MhTK-thSqgSSG-55Vi3jiWN7FQdYOGVbGqe~hECx16pweWAd99SP3cNHuVzIHUKGg__&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.
