NK cell survival mediated through the regulatory synapse with human DCs requires IL-15Rα
Fabienne Brilot, Till Strowig, Susanne M. Roberts, Frida Arrey, Christian Münz
Fabienne Brilot, Till Strowig, Susanne M. Roberts, Frida Arrey, Christian Münz
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Research Article Immunology

NK cell survival mediated through the regulatory synapse with human DCs requires IL-15Rα

Abstract

DCs activate NK cells during innate immune responses to viral infections. However, the composition and kinetics of the immunological synapse mediating this interaction are largely unknown. Here, we report the rapid formation of an immunological synapse between human resting NK cells and mature DCs. Although inhibitory NK cell receptors were polarized to this synapse, where they are known to protect mature DCs from NK cell lysis, the NK cell also received activation signals that induced mobilization of intracellular calcium and CD69 upregulation. The high-affinity component of the receptor for IL-15, IL-15Rα, accumulated at the synapse center on NK cells, and blocking of IL-15Rα increased NK cell apoptosis and diminished NK cell survival during their interaction with DCs. Furthermore, IL-15Rα–deficient NK cells, obtained from donors with a history of infectious mononucleosis, showed diminished survival in culture with DCs. Synapse formation was required for IL-15Rα–mediated NK cell survival, because synapse disruption by adhesion molecule blocking decreased DC-induced NK cell survival. These results identify what we believe to be a novel regulatory NK cell synapse with hallmarks of spatially separated inhibitory and activating interactions at its center. We suggest that this synapse formation enables optimal NK cell activation by DCs during innate immune responses.

Authors

Fabienne Brilot, Till Strowig, Susanne M. Roberts, Frida Arrey, Christian Münz

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Figure 5

IL-15Rα was expressed by mDCs and NK cells, and IL-15Rα and IL-15Rβ accumulated, at the interface of NK cells with mDCs.

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IL-15Rα was expressed by mDCs and NK cells, and IL-15Rα and IL-15Rβ accu...
(A and B) imDCs and mDCs (A) and purified blood NK cells (B) were washed, stained with anti-IL-15Rα polyclonal Ab followed by Alexa Fluor 488– or Alexa Fluor 647–conjugated donkey anti-goat IgG, and analyzed by flow cytometry. DCs were gated on size, CD11c+, and CD83 (CD83+ for mDCs, CD83– for imDCs); NK cells were gated on size, CD3–, and CD56+. Representative data from 3 experiments are shown. (C) Cells were also lysed in buffer containing 1% NP-40, and proteins were separated by SDS-PAGE. IL-15Rα was then visualized by Western blotting with anti–IL-15Rα mAb followed by a goat anti-mouse-HRP secondary antiserum. Data are representative of 3 separate experiments. Immunofluorescence was performed with polyclonal anti–IL-15Rα followed by Rhodamine Red-X–conjugated donkey anti-goat IgG (red) or with IL-15Rβ mAb followed by Alexa Fluor 488–conjugated rabbit anti-mouse IgG (green). (D) IL-15Rα localization in unconjugated resting NK cells and DC/NK cell conjugates after 1 min of interactions. (E) Fluorescence intensities of IL-15Rα (red) and Cell Trace Far Red DDAO-SE–labeled NK cells (pseudocolored in blue) were plotted (bottom) along the trajectory illustrated above. The area of the synapse on the NK cell side is delimited by boxes. (F) Images of IL-15Rβ localization in unconjugated resting NK cells and DC/NK cell conjugates after 1 min of DC/NK cell coculture. White arrows denote DC/NK cell interface. Results are representative of 100–200 conjugates in at least 3 separate experiments. Scale bars: 5 μm.