KIR2DL4 (CD158d), an NK Cell-Activating Receptor with Inhibitory Potential Mathias Faure and Eric O. Long This information is current as J Immunol 2002; 168:6208-6214; ; of September 25, 2021. doi: 10.4049/jimmunol.168.12.6208 http://www.jimmunol.org/content/168/12/6208 Downloaded from References This article cites 43 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/168/12/6208.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 25, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology KIR2DL4 (CD158d), an NK Cell-Activating Receptor with Inhibitory Potential Mathias Faure and Eric O. Long1 KIR2DL4 (CD158d) is an unusual member of the killer cell Ig-like receptor family expressed in all NK cells and some T cells. KIR2DL4 activates the cytotoxicity of NK cells, despite the presence of an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic tail. The role of this ITIM on the activating function of KIR2DL4, and whether it can provide inhibitory signals, is not known. Mutated forms of KIR2DL4 were engineered that lacked either the tyrosine in the ITIM or an arginine-tyrosine motif in the transmembrane region that is required for the activation signal. The activity of the mutated KIR2DL4 molecules was tested in a redirected lysis assay. The ITIM was not necessary for activation of lysis by KIR2DL4. The activation signal of KIR2DL4 was sensitive to inhibition by another ITIM-containing receptor. The activation-deficient mutant of KIR2DL4 inhibited the signal delivered by the activating receptor CD16. In pull-down experiments with GST fusion proteins, the tyrosine-phospho- Downloaded from rylated cytoplasmic tail of KIR2DL4 bound the Src homology 2-containing phosphatases 1 and 2, as did the tail of the inhibitory receptor KIR2DL1. Therefore, KIR2DL4 has inhibitory potential in addition to its activating function. The Journal of Immu- nology, 2002, 168: 6208–6214. atural killer cells are a fundamental component of the inhibition motif (ITIM)-containing receptors (5). In human NK immune system, providing a link between innate and cells, killer cell Ig-like receptors (KIR) and the lectin-like CD94/ http://www.jimmunol.org/ N adaptive immunity. NK cells respond quickly and effi- NKG2A receptor are mainly responsible for such inhibition. Some ciently to viral, bacterial, and parasite infections by producing cy- members of the KIR family carry two consecutive ITIM in their tokines and by engaging a variety of receptors that recognize li- cytoplasmic tail and bind to classical HLA class I molecules. The gands on infected cells (1, 2). In healthy organisms, the cytotoxic ITIM is defined by the consensus sequence I/V/LxYxxL/V (6). activity of NK cells is regulated by a balance of positive and neg- Following tyrosine phosphorylation, the tandem ITIM recruits and ative signals emanating from activating and inhibitory receptors, activates tyrosine phosphatases that dephosphorylate components respectively. Specific signaling pathways leading to the activation of the activation pathway. Src homology 2-containing phosphatase or the inhibition of NK cells remain to be fully depicted (3). (SHP)-1 is thought to be the major phosphatase recruited in vivo by guest on September 25, 2021 Several of the NK cell-activating receptors signal by association by KIR, but the related phosphatase SHP-2 also binds phosphor- 2 with immunoreceptor tyrosine-based activation motif (ITAM) - ylated ITIM in vitro and in vivo (7–10). Although the precise containing partner chains (4). For instance, CD16 associates with molecular target(s) of SHP-1 still remains to be determined, the ␨-chain, NKp46 associates with the FcR ␥-chain, while NKp44 ITAM-induced positive signals as well as signals from the acti- and KIR2DS pair with DAP12. The noncovalent association of the vating receptor 2B4 are sensitive to ITIM-mediated inhibition (3). ITAM-containing chains is controlled by a basic amino acid in the In contrast, the signaling pathway induced by the NKG2D/DAP10 transmembrane region of the receptor that is compensated by an receptor complex is not as sensitive to ITIM-mediated inhibition as acidic amino acid at a similar position in the adaptor protein. Thus, that induced by ITAM-containing receptors (11–14). the presence of a charged residue in the transmembrane region of an immunoreceptor indicates a potential for an activating function. Among KIR family members, KIR2DL4, which was assigned Other NK cell-activating receptors, such as 2B4 (CD244) and the new CD designation CD158d (15), displays unusual charac- CD2, carry signaling motifs in their own cytoplasmic tail (4). teristics. First, KIR2DL4 carries a single ITIM (the tyrosine of the To override potentially harmful activation signals delivered by second ITIM is substituted by a cysteine residue). Second, in ad- ITAM-containing molecules, NK cell activation is suppressed by dition to the presence of an inhibitory motif, KIR2DL4 contains a inhibitory signals transduced by immunoreceptor tyrosine-based charged residue in its transmembrane region, suggesting the pos- sibility of an activating function for this receptor (16). These two properties may explain earlier observations on the function of Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, KIR2DL4. Indeed, both inhibitory (17) and activating (18) func- National Institutes of Health, Rockville, MD 20852 tions have been described for KIR2DL4. Third, the mRNA of Received for publication February 1, 2002. Accepted for publication April 17, 2002. KIR2DL4 is present in every NK cell, in contrast to all other KIR The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance that are expressed in overlapping subsets of NK cells (19, 20). with 18 U.S.C. Section 1734 solely to indicate this fact. Fourth, the two Ig-like extracellular domains of KIR2DL4 and of 1 Address correspondence and reprint requests to Dr. Eric O. Long, Laboratory of KIR2DL5 have the unusual D0-D2 configuration, in contrast to the Immunogenetics, National Institute of Allergy and Infectious Diseases, National In- usual D1-D2 and D0-D1-D2 organization of Ig domains in other stitutes of Health, Twinbrook II, 12441 Parklawn Drive, Rockville, MD 20852-1727. E-mail address: [email protected] KIR2D and KIR3D receptors, respectively. Finally, KIR2DL4 2 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation binds to cells expressing HLA-G (20, 21), a nonclassical HLA motif; HA, hemagglutinin; IRS-1, insulin receptor substrate-1; ITIM, immunoreceptor class I molecule expressed by trophoblast cells that invade the tyrosine-based inhibition motif; KIR, killer cell Ig-like receptor; rADCC, redirected Ab-dependent cellular cytotoxicity; SHIP, Src homology 2-containing inositol 5Ј- maternal decidua (22), by activated monocytes (23, 24), by thymic phosphatase; SHP, Src homology 2-containing phosphatase; Vac, vaccinia virus. epithelial cells (25), and by certain tumor cells (26). Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 6209 In the present study, mutated KIR2DL4 receptors lacking either proteins expressed in the same cell. Phosphorylation of the GST fusion the charged residue in the transmembrane region or lacking the proteins was confirmed by Western blot with 4G10 mAb (Upstate Bio- cytoplasmic ITIM were tested for their ability to activate or to technology). No tyrosine phosphorylation of GST fusion proteins isolated from BL21(DE3) bacteria was detected. inhibit NK cells. We report that KIR2DL4 lacking the ITIM is still activating NK cell cytotoxicity and that KIR2DL4 lacking the Pull-down experiments, depletion, and immunoprecipitation charged residue in the transmembrane region can deliver inhibitory A total of 90 ϫ 106 IL-2-activated human NK cells was radiolabeled with signals. [35S]methionine (0.1 mCi/5 ϫ 106 cells/1 ml; Amersham, Arlington Heights, IL) for 7 h. The cells were then lysed in lysis buffer (150 mM Materials and Methods NaCl, 50 mM Tris, pH 7.4, 2 mM EGTA, 1% Triton X-100, 1 mM sodium Cells and Abs orthovanadate), and, following preclearing with purified GST, the total cellular protein lysate was aliquoted and incubated with each described 6 Human NK populations were purified as described (27). Briefly, NK cells GST fusion protein (5 nmol/10 ϫ 10 cells) in a final volume of 1 ml. After were isolated from human PBL using the magnetic activated cell sorter NK a 2-h incubation at 4°C, 50 ␮l glutathione-Sepharose (Pharmacia Biotech, cell isolation kit (Miltenyi Biotec, Auburn, CA). Human NK cells were