The Journal of Immunology TNFR-Associated Factor 6 and TGF-b–Activated Kinase 1 Control Signals for a Senescence Response by an Endosomal NK Cell Receptor Sumati Rajagopalan, Elizabeth C. Lee, Matthew L. DuPrie, and Eric O. Long The endosomal innate receptor CD158d (killer cell Ig-like receptor 2DL4) induces cellular senescence in human NK cells in response to soluble ligand (HLA-G or agonist Ab). These senescent NK cells display a senescence-associated secretory phenotype, and their secretome promotes vascular remodeling and angiogenesis. To understand how CD158d initiates signaling for a senescence response, we mapped the region in its cytoplasmic tail that controls signaling. We identified a conserved TNFR-associated factor 6 (TRAF6) binding motif, which was required for CD158d-induced NF-kB activation and IL-8 secretion, TRAF6 association with CD158d, and TRAF6 recruitment to CD158d+ endosomes in transfected cells. The adaptor TRAF6 is known to couple proximal signals from receptors such as endosomal TLRs and CD40 through the kinase TGF-b–activated kinase 1 (TAK1) for NF-kB–dependent proinflammatory responses. Small interfering RNA–mediated silencing of TRAF6 and TAK1, and inhibition of TAK1 blocked CD158d-dependent IL-8 secretion. Stimulation of primary, resting NK cells with soluble Ab to CD158d induced TRAF6 associ- ation with CD158d, induced TAK1 phosphorylation, and inhibition of TAK1 blocked the CD158d-dependent reprogramming of NK cells that produces the senescence-associated secretory phenotype signature. Our results reveal that a prototypic TLR and TNFR signaling pathway is used by a killer cell Ig-like receptor that promotes secretion of proinflammatory and proangiogenic mediators as part of a unique senescence phenotype in NK cells. The Journal of Immunology, 2014, 192: 714–721. atural killer cells carry out their effector functions through of fetal trophoblast cells by responding to HLA-G, a nonclassical their cytolytic activity and cytokine secretion and have MHC molecule produced by these fetal cells in the early weeks of N important roles in immune defense and in reproduction pregnancy (8). Endosomal signaling for a senescence response and (1, 2). CD158d (killer cell Ig-like receptor [KIR]2DL4) is a unique sustained SASP may promote the vascular remodeling required for member of the KIR family and expressed by all NK cells and successful placentation. In support of this, a senescence signature was a subset of T cells (3). Unlike most other KIR family members that seen in a retrospective analysis of microarrays of decidual NK cells are expressed at the cell surface, CD158d resides primarily in endo- isolated from first-trimester abortions that were compared with pe- by guest on October 1, 2021. Copyright 2013 Pageant Media Ltd. somes, from where it generates proinflammatory/proangiogenic ripheral blood NK cells (6, 9). signals in response to its ligand, soluble HLA-G (4). A signaling How CD158d initiates NF-kB signaling for a senescence re- cascade involving the DNA damage kinase DNA-dependent protein sponse is not known. In this study, we show that a short stretch of kinase, catalytic subunit, Akt, and NF-kB is initiated upon CD158d amino acids in the cytoplasmic tail of CD158d recruits the adaptor engagement (5). This endosomal signaling results in the induction TNFR-associated factor 6 (TRAF6), which is an essential node in of cellular senescence in primary, resting NK cells and the produc- TLR signaling. We demonstrate that this recruitment of TRAF6 tion of a characteristic senescence-associated secretory phenotype regulates NF-kB signaling in response to KIR2DL4 and that its (SASP) (6). The secretome of these metabolically active senescent interacting partner kinase TAK1 (TGF-b–activated kinase 1) is re- NK cells contain factors, including cytokines and chemokines, that quired for the senescence response induced by KIR2DL4. Thus, we http://classic.jimmunol.org promote vascular remodeling and angiogenesis. NK cell repro- show the unexpected usage of signaling effectors of the TLR family gramming toward an SASP has implications at sites of HLA-G ex- by an endosomal innate immune receptor on NK cells. pression, such as in early pregnancy and certain tumors (7). NK cells, which are abundant at the implantation site, may sense the invasion Materials and Methods Cell culture HEK293T cells were obtained from American Type Culture Collection and Downloaded from Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, cultured in DMEM containing 10% FBS. Cells were transiently transfected National Institutes of Health, Rockville, MD 20852 using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s Received for publication September 6, 2013. Accepted for publication November 10, instructions. 293T–2DL4-GFP cells are HEK293T cells that were stably 2013. transfected with a plasmid encoding a fusion protein of 2DL4 and GFP (4). This work was supported by the Intramural Research Program of the National Insti- Human polyclonal NK cells were isolated from PBLs from anonymous tute of Allergy and Infectious Diseases, National Institutes of Health. donors at the National Institutes of Health (NIH) Department of Trans- fusion Medicine, under an NIH Institutional Review Board–approved pro- Address correspondence and reprint requests to Dr. Eric O. Long, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National tocol, with informed consent. NK cells were isolated using the Negative Institutes of Health, Twinbrook II/201E, 12441 Parklawn Drive, Rockville, MD Selection Human NK Cell Enrichment Kit (Stem Cell Technologies). NK 2 + 20852. E-mail address: [email protected] cells were .97% CD3 and CD56 . Freshly isolated NK cells (resting NK) were cultured in Iscove’s modified DMEM containing 10% human Abbreviations used in this article: Ac, acidic; Ar, aromatic; cIgG, control IgG; cIgM, control IgM; KIR, killer cell Ig-like receptor; NIH, National Institutes of Health; serum without added IL-2 or feeders. Resting NK cells were incubated PBST, PBS plus 0.1% Tween-20; PCC, Pearson correlation coefficient; SASP, with control IgG1 (MOPC21) or agonist anti-KIR2DL4 (mAb #33) at senescence-associated secretory phenotype; siRNA, small interfering RNA; TAK1, 10 mg/ml for 16 h. NKL cells (a gift from M. Robertson, Indiana School TGF-b–activated kinase 1; TRAF6, TNFR-associated factor 6; wt, wild-type. of Medicine, Indianapolis, IN) were cultured in RPMI 1640 medium www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302384 The Journal of Immunology 715 containing 10% FCS, 1% glutamine, 1% sodium pyruvate, and 200 U/ml Pearson value of zero indicates no colocalization, whereas a value of one rIL-2. NKL cells were rested for 48 h in the absence of IL-2 and in reduced indicates perfect colocalization of all pixels. FCS (2%) prior to stimulation with anti-2DL4 Abs. RNA interference Abs and reagents HEK293T cells were transfected with small interfering RNAs (siRNAs; The following Abs were used: anti-TRAF6: H-274 (used for immuno- Dharmacon) at a final concentration of 100 nM using Lipofectamine 2000 blotting and immunocytochemistry) and D-10 (used for immunoprecipi- (Invitrogen). The siRNAs used were: ON-TARGETplus SMARTpool siRNA tation); HRP-conjugated anti-rabbit and anti-mouse Abs from Santa Cruz (consists of four individual siRNA oligonucleotides) for TRAF6 and TAK1, Biotechnology; anti-actin Ab from BD Biosciences; Alexa Fluor 488– ON-TARGETplus individual siRNA duplexes for TRAF6 (J-004712-09-0005) conjugated anti-HA (6E2), anti-TAK1, anti–phospho-TAK1 (Thr187), and TAK1 (J-003790-19-0005), and two custom synthesized individual siR- anti–phospho-p38 MAPK (Thr180/Tyr182), anti–phospho-IkBa (Ser32/36), NAs for Triad3A (Triad3A siRNA #1: sense, 59-AAGUGCUCAGUAGU- and anti-MOPC21 Abs from Cell Signaling Technology; anti-KIR2DL4 CAGGACAUU-39 and antisense, 59-UGUCCUGACUACUGAGCACUUUU- mAb (MAB2238) from R&D Systems; anti-KIR2DL4 mAbs #33 (IgG1), 39; Triad3A siRNA #2: sense, 59-UCUGGACCGAUCCCACUGAAUU-39 and #36 (IgM), and #64 (IgM) were produced in our laboratory (10); anti-HA antisense, 59-UUCAGUGGGAUCGGUCCAGAUU-39). ON-TARGETplus and Alexa Fluor 594–conjugated anti-HA (16B12) Abs from Covance; nontargeting siRNA pool (consists of four individual nontargeting siRNAs) anti-GFP and anti-rabbit Alexa Fluor 564 Abs from Molecular Probes; was used as a negative control in all siRNA experiments. Forty-eight hours and anti-Triad3A Ab from Abcam. HRP-conjugated Protein-A was from BD after transfection with siRNA, HEK293T cells were transfected with 0.5 mg Biosciences and was used for Western blot detection of immunoprecipitated 2DL4 plasmid DNA. At 24 h after 2DL4 transfection and 72 h from the start cell extracts. For immunocytochemistry experiments, ProLong Anti-fade kit of siRNA transfection, the culture medium was tested for the production of and Image-iT FX Signal Enhancer (used to improve signal-to-noise ratio of IL-8 by ELISA. Cell lysates from the siRNA-transfected cells were analyzed immunolabeled cells) were purchased from Molecular Probes. TAK1 in- by immunoblotting to assess knockdown efficiency of target proteins. hibitor (5Z)-7-Oxozeaenol was obtained from Tocris Bioscience. Immunoprecipitation and immunoblotting Molecular constructs Cells were washed with ice-cold PBS and lysed in 1% Nonidet P-40 lysis The HA-tagged wild-type (wt) 2DL4 in the pDisplay vector (Stratagene) buffer containing 50 mM Tris, 140 mM NaCl, and protease-inhibitor was previously described (4). Mutations in the cytoplasmic tail of 2DL4 mixture (Roche). Cell lysates were precleared with control IgG (cIgG) were generated from the pDisplay-2DL4 expression vector using a Stra- and protein G-agarose beads for 1 h, followed by incubation with various tagene Quick Change site-directed mutagenesis kit according to the Abs at 4˚C. Protein G-agarose beads were then added and allowed to bind manufacturer’s instructions. The following primers were used: forward, for 1 h at 4˚C. Immunoprecipitates were collected by brief centrifuga- 59-GAGGACTCTGATGAAGCAGACCCTCAGGAGGTG-39 and reverse, tion, washed six times, and eluted by boiling in 23 LDS sample buffer 59-CACCTCCTGAGGGTCTGCTTCATCAGAGTCCTC-39 to generate a (Invitrogen).