NK-Activating Receptor KIR2DS4 MHC Class I-Independent

MHC Class I-Independent Recognition of NK-Activating Receptor KIR2DS4 Gil Katz, Roi Gazit, Tal I. Arnon, Tsufit Gonen-Gross, Gabi Tarcic, Gal Markel, Raizy Gruda, Hagit Achdout, Olga This information is current as Drize, Sharon Merims and Ofer Mandelboim of September 29, 2021. J Immunol 2004; 173:1819-1825; ; doi: 10.4049/jimmunol.173.3.1819 http://www.jimmunol.org/content/173/3/1819 Downloaded from References This article cites 31 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/173/3/1819.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on September 29, 2021 *average 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology MHC Class I-Independent Recognition of NK-Activating Receptor KIR2DS41 Gil Katz,* Roi Gazit,* Tal I. Arnon,* Tsufit Gonen-Gross,* Gabi Tarcic,* Gal Markel,* Raizy Gruda,* Hagit Achdout,* Olga Drize,† Sharon Merims,† and Ofer Mandelboim2* Natural killer cells are capable of killing tumor and virus-infected cells. This killing is mediated primarily via the natural cytotoxicity receptors, including NKp46, NKp44, NKp30, and by the NKG2D receptor. Killer cell Ig-like receptors (KIRs) are mainly involved in inhibiting NK killing (inhibitory KIRs) via interaction with MHC class I molecules. Some KIRs, however, have been found to enhance NK killing when interacting with MHC class I molecules (activating KIRs). We have previously demonstrated that KIR2DS4, an activating KIR, recognizes the HLA-Cw4 protein. The interaction observed was weak and highly restricted to HLA-Cw4 only. These findings prompted us to check whether KIR2DS4 might have additional ligand(s). In this study, we show that KIR2DS4 is able to also interact with a non-class I MHC protein expressed on melanoma cell lines and on a primary Downloaded from melanoma. This interaction is shown to be both specific and functional. Importantly, site-directed mutagenesis analysis reveals that the amino acid residues involved in the recognition of this novel ligand are different from those interacting with HLA-Cw4. These results may shed new light on the function of activating KIRs and their relevance in NK biology. The Journal of Immunology, 2004, 173: 1819–1825. atural killer cells are non-T, non-B lymphocytes that are In addition to the inhibitory KIR2Ds, activating KIR2Ds also http://www.jimmunol.org/ stained positively for CD56 and are negative for CD3. exist, which activate NK killing after interacting with HLA-C- N They comprise between 5 and 10% of PBL. NK cells expressing cells (15, 20). These activating KIR2Ds resemble the recognize and eliminate tumor and virus-infected cells (1). This inhibitory KIR2Ds in their extracellular domains, but contain a killing is mediated via the natural cytotoxicity receptors, including charged residue in their transmembrane domain (Lys) and also NKp46, NKp44, NKp30, and the NKG2D receptor (2, 3). In ad- lack any known motifs in their short cytoplasmic tail (21). The dition, NK cells also express inhibitory receptors that inhibit NK activating receptors are associated with the signal transduction activity (4–10). protein DAP12, which contains the ITAM motif in its cytoplasmic The inhibition of the killing mediated by NK cells is mostly domain (22). The ligands that are recognized by the activating achieved via the interactions of MHC class I proteins with inhib- KIR2Ds are only poorly defined. by guest on September 29, 2021 itory killer cell Ig-like receptors (KIRs).3 The inhibitory KIRs con- The KIR2DS4, an activating KIR2D, is a unique molecule. It sist of either two (2D) or three (3D) extracellular Ig-like domains contains a mixture of residues from both group 1 and 2 KIR2Ds. and interact with HLA-C or HLA-B molecules, respectively (4– For example, KFN, instead of MFN or KFK in positions 44–46, it 8). The inhibitory signal is transduced by ITIM motif, located in also expresses its own unique residues (23). We have recently the long cytoplasmic tail of the receptor (6, 8). Once phosphory- shown that KIR2DS4 interacts with HLA-Cw4, but not with HLA- lated, these ITIMs recruit Src homology 2-containing protein ty- Cw6 (24). The observed interaction was of low affinity, compared rosine phosphatase 1 and 2 (11, 12). By means of phosphorylation, with that of KIR2DL1, an inhibitory KIR2D (24). We suggested the recruited phosphatases become activated and interfere with NK that the presence of group 2 residues in positions 67–70 (GPMM activity. instead of SRMT) weakens the interaction between KIR2DS4 and The inhibitory KIR2Ds can be divided into two subgroups: HLA-Cw4. We further demonstrated that the W14 residue of groups 1 and 2, interacting either with Lys80 or Asn80 of the HLA-Cw4 is critical for the recognition by KIR2DS4 (24). HLA-C molecules, respectively (13, 14). Group 1 is characterized These results were puzzling, first because KIR2DS4 could only by the presence of MFN and SRMT residues in positions 44–46 interact in a very restricted specificity, and second because the and 67–70, respectively. In group 2, the KFK and GPMM residues interaction was of low affinity. We, therefore, investigated whether are located in these positions (15–19). the KIR2DS4 might have an additional ligand(s). In this study, we demonstrate binding of KIR2DS4 to a non-class I MHC protein, *Lautenberg Center for General and Tumor Immunology, Hadassah Medical School, expressed on melanoma cell lines and on a primary melanoma. The Hebrew University, Jerusalem, Israel; and †Sharett Institute of Oncology, Hadassah interaction observed was also functional, resulting in NK-mediated Medical Center, Jerusalem, Israel killing of the target cells expressing this, yet unidentified ligand. Received for publication February 17, 2004. Accepted for publication May 26, 2004. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 O.M. is supported by research grants from the Israel Cancer Research Fund, the Cells and mAb Israel Science Foundation, and the Israel Cancer Association (20042031-B). The cell lines used in this work are the human B lymphoblastoid cell line 2 Address correspondence and reprint requests to Dr. Ofer Mandelboim, Lautenberg 721.221 and other human melanoma lines, all of which are MHC class I Center for General and Tumor Immunology, Hadassah Medical School, Jerusalem negative. Transfection of 721.221 cells with the cDNA of various class I 91120, Israel. E-mail address: [email protected] MHC molecules was performed by electroporation, as previously described 3 Abbreviation used in this paper: KIR, killer cell Ig-like receptor. (13). Primary NK cells (NK clones) were isolated from PBL using the Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 1820 NOVEL LIGAND FOR KIR2DS4 human NK cell isolation kit and the autoMACS instrument (Miltenyi Bio- tec, Auburn, CA). mAb used in this work are mAb W6/32, directed against MHC class I molecules; mAb HP3E4 (a kind gift from M. Lopez-Botet, DCEXS Uni- versitat Pompeu Fabra, Barcelona, Spain), directed against KIR2DL1, KIR2DS1, and KIR2DS4 (24–26); and mAb EB6 (Immunotech, West- brook, ME), directed against KIR2DL1 and KIR2DS1 (24, 25). The anti- CD99 mAb 12E7 (a kind gift from A. Bernard, Hoˆpital de L’Archet, Nice, France) was used as a control. Cytotoxic assays The cytotoxic activity of primary NK cells against the various targets was assessed in 5-h 35S release assays, as previously described (13). In exper- iments in which mAb were included, the final mAb concentration was 5 ␮g/ml or 1/2 dilutions in cases in which the mAb were used as tissue culture supernatants. Ig fusion proteins The Ig fusion proteins used in this work are CD99-Ig, KIR2DL1-Ig, and KIR2DS4-Ig (wild type and mutants) (24). Briefly, the sequence encoding the extracellular portion of the receptor was amplified by PCR from cDNA isolated from human NK clones. These PCR-generated fragments were Downloaded from cloned into a mammalian expression vector, containing the Fc portion of human IgG1. The construct was transfected into COS-7 cells, and the protein produced was purified using protein G column. Point mutations in the cDNA coding for the KIR2DS4 were performed by PCR. Flow cytometry Cells were stained either with mAb or Ig fusion proteins. Second reagents Ј http://www.jimmunol.org/ were FITC-conjugated F(ab )2 goat anti-mouse IgG (ICN Biomedicals, Ј Aurora, OH) and PE-conjugated F(ab )2 goat anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), directed against mAb and Ig fusion proteins, respectively. mAbs were used at a final concentration of 3 ␮g/ml, and Ig fusion proteins at 100 ␮g/ml. The staining proce- dure was as follows: 30,000 cells were washed once in FACS medium (1ϫ ␮ PBS, 0.5% BSA, 0.05% NaN3) and then incubated in 100 l of FACS medium containing either mAb or Ig fusion proteins for 1 or2honice (4°C), respectively.

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