Dendritic cell and natural killer cell cross-talk: a pivotal role of CX3CL1 in NK cytoskeleton organization and activation

Abstract

IntroductionNatural killer (NK) cell activation is initiated when NK cells form tight contacts with target cells. [1][2][3][4] However, this activation can be abrogated by interactions between major histocompatibility complex (MHC) class I molecules and inhibitory killer cell immunoglobulin-like receptors (KIRs). SHP-1 is then recruited to the KIR intracytoplasmic domain and a dominant inhibition of cytoskeletal and lipid raft polarization occurs, protecting MHC class I-positive targets from autologous NK cell-mediated cytotoxicity. [5][6][7][8][9][10][11] We have previously described another type of synapse (DCNK-IS) that regulates DC-mediated NK activation. 12 This dynamic network in which NK interacts with surrounding DC was also described in vivo during Leishmania major infection. 13 The DCNK-IS has been shown to enable optimal NK activation by IL-15 and to display a spatial organization distinct from the classic target/NK synapses. 12,14,15 However, the initial molecular events governing mature DC (mDC) and NK interactions remain to be identified. In particular, the mechanisms allowing autologous resting NK activation by mDCs in the presence of KIR on NK cells and self-MHC proteins on DCs, have not been studied. MethodsMice C57BL/6 mice were obtained from Janvier (Le Genest St Isle, France), and maintained in the IFR133 animal facility according to the guidelines of the Animal Ethics Committee. C57BL/6 CX3CR1-knockout mice 16 were provided by Bernhard Ryffel (GEM2358, CNRS, Orleans, France). Dendritic cell differentiationHuman DCs were generated from adherence-selected monocytes in AIMV complete medium containing 1000 IU/mL of both rhuGM-CSF and rhuIL-4 (Peprotech, Neuilly-sur-seine, Saint-Quentin, France). DCs were exposed to lipopolysaccharide (LPS; 1 g/mL, Sigma-Aldrich, France), characterized and used at day 6, as previously described. 12 Mouse bone marrowderived DCs (BMDCs) were differentiated as previously described. 12 Purification of NK cellsNK cells were negatively selected from Ficoll-separated peripheral blood mononuclear cells (PBMCs) using the Miltenyi NK-cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) Slide preparation and confocal microscopy mDCs (5 ϫ10 4 ) and NK cells (10 5 ) were mixed and spread onto poly-Llysine-coated slides (Sigma, France) for 25 minutes at 37°C. Cells were fixed and permeabilized with 0.2% sodium dodecyl sulfate. After 20 minutes of blocking in 20% fetal bovine serum and washing, cells were stained For personal use only. on May 12, 2018. by guest www.bloodjournal.org From with the appropriate mAbs. Stacks of confocal images were collected with an Olympus FV1000 laser scanning confocal microscope. Cell labeling and antibodiesPhalloidin (Molecular Probes, Eugene, OR) and cholera toxin ␤ subunit (CTX; Sigma-Aldrich, St Louis, MO) were used to detect polymerized F-actin and raft-associated GM1 gangliosides. NK cells were imaged using anti-KIR2DL1 (T-20; Santa Cruz Biotechnology, Santa Cruz, CA) and PT-100 (Cell Signaling Technology, Beverly, MA). Anti-...

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