Src Homology Region 2-Containing Protein Tyrosine Phosphatase-2 (SHP-2) Can Play a Direct Role in the Inhibitory Function of Killer Cell Ig-Like Receptors in Human NK This information is current as Cells of October 2, 2021. Sei-ichi Yusa and Kerry S. Campbell J Immunol 2003; 170:4539-4547; ; doi: 10.4049/jimmunol.170.9.4539 http://www.jimmunol.org/content/170/9/4539 Downloaded from References This article cites 66 articles, 41 of which you can access for free at: http://www.jimmunol.org/content/170/9/4539.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 October 2, 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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Src Homology Region 2-Containing Protein Tyrosine Phosphatase-2 (SHP-2) Can Play a Direct Role in the Inhibitory Function of Killer Cell Ig-Like Receptors in Human NK Cells1 Sei-ichi Yusa and Kerry S. Campbell2 The inhibitory forms of killer cell Ig-like receptors (KIR) are MHC class I-binding receptors that are expressed by human NK cells and prevent their attack of normal cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatase, Src homology region 2-containing protein tyrosine phosphatase (SHP)-1, to phosphorylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs). However, the functional significance of parallel re- cruitment of a SHP-1-related phosphatase, SHP-2, to KIR ITIMs has not been addressed. In the present study, our results with Downloaded from mutant forms of a classical KIR, KIR3DL1, show a direct correlation between SHP-2 recruitment and functional inhibition of target cell conjugation and cytotoxicity. In addition, KIR3DL1 inhibition of target cell cytotoxicity is blocked by overexpression of a dominant-negative form of SHP-2. Finally, KIR3DL1 fused directly with the catalytic domain of SHP-2 inhibits both target cell conjugation and cytotoxicity responses. These results strongly indicate that SHP-2 catalytic activity plays a direct role in inhibitory KIR functions, and SHP-2 inhibits NK cell activation in concert with SHP-1. The Journal of Immunology, 2003, 170: 4539–4547. http://www.jimmunol.org/ atural killer cells play an important role in immune sur- ment of these inhibitory receptors has been shown to block natural veillance by detecting and spontaneously lysing MHC cytotoxicity, Ab-dependent cellular cytotoxicity (10, 11), and ad- N class I (MHC-I)3-deficient tumor cells and virus-infected hesion toward target cells (12). cells that have escaped detection by CTL. NK cell activation is KIR possess two immunoreceptor tyrosine-based inhibitory mo- controlled by a dynamic balance of opposing signals from activat- tifs (ITIMs; (I/V)xYxx (L/V)) (13, 14), which contain tyrosine ing and inhibitory receptors (1, 2). Improved understanding of the residues that are critical elements for mediating inhibitory function receptors and intracellular pathways that control NK cell actions is (14–18). Phosphorylated tyrosines in the ITIMs of inhibitory re- important for developing methods to manipulate NK cells and en- ceptors serve as docking sites to recruit Src homology region 2 by guest on October 2, 2021 hance their therapeutic use in treating cancer, virus infection, and (SH2) domain-containing phosphatases, such as the protein ty- graft-vs-host disease (3, 4). NK cell activation is triggered by any rosine phosphatases (PTP), SH2-containing PTP (SHP)-1 and one of several receptor-coupled pathways, including NKp46, SHP-2, and the SH2-containing inositol 5Ј-phosphatase (SHIP). NKp44, NKp30, NKG2D, 2B4, LFA-1, CD2, and CD69 (5). Thus, Several groups have reported that tyrosine-phosphorylated KIR NK cell activation is not controlled by a single clonotypic Ag ITIMs recruit the PTP, SHP-1 (11, 15–20). SHP-1 is a cytosolic receptor, such as Ag receptors on T or B cells. PTP that contains two SH2 domains and a catalytic domain. KIR- Conversely, NK cell activation is abrogated by MHC-I-binding mediated reversal of activation-induced signaling events, such as inhibitory receptors. Inhibitory killer cell Ig-like receptors (KIR) phosphorylation of TCR ␨-chain, ␨-chain-associated protein-70, are the major classical MHC-I-binding inhibitory receptors ex- phospholipase C-␥, linker for activation of T cells, and SH2 do- pressed on human NK cells and subsets of T cells (6–9). Engage- main-containing leukocyte protein of 76 kDa (10, 21, 22), suggests that KIR use PTP activity for their negative signaling function. The Division of Basic Science, Fox Chase Cancer Center, Institute for Cancer Research, most convincing evidence for SHP-1 involvement is the blockade Philadelphia, PA 19111 of inhibition through KIR by expression of dominant-negative Received for publication November 20, 2002. Accepted for publication February (DN) SHP-1 in human NK cells (10, 11, 23). Nevertheless, it is 28, 2003. possible that overexpression of DN-SHP-1 also blocks recruitment The costs of publication of this article were defrayed in part by the payment of page of alternative SH2-containing negative effector proteins to phos- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. phorylated KIR ITIMs. SHP-2 is a widely expressed cytoplasmic PTP, which shares 1 This work was supported by National Institutes of Health Grant CA83859 (to K.S.C.). The research was also supported in part by National Institutes of Health high structural and primary sequence homology with SHP-1 (24). Centers of Research Excellence Grant CA06927 and an appropriation from the Com- Interestingly, three previous reports have described recruitment of monwealth of Pennsylvania. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute. SHP-2 to phosphopeptides corresponding to the two ITIMs of KIR 2 Address correspondence and reprint requests to Dr. Kerry S. Campbell, Division of (19, 25) or to chimeric forms of KIR expressed in B cells and mast Basic Science, Fox Chase Cancer Center, Institute for Cancer Research, 7701 cells (15), while our group has recently demonstrated that SHP-2 Burholme Avenue, Philadelphia, PA 19111. E-mail address: [email protected] binding to the single ITIM of the KIR2DL4 (2DL4) cytoplasmic 3 Abbreviations used in this paper: MHC-I, MHC class I; KIR, killer cell Ig-like receptor; domain correlates with strong inhibitory function (18). Paradoxi- ITIM, immunoreceptor tyrosine-based inhibitory motif; SH2, Src homology region 2; PTP, protein tyrosine phosphatase; SHP, SH2-containing PTP; SHIP, SH2-containing cally, SHP-2 has been thought to be primarily a positive regulator inositol 5Ј-phosphatase; DN, dominant-negative; pNPP, p-nitrophenylphosphate. of cell proliferation when recruited to numerous receptors (26–31). Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 4540 SHP-2 MEDIATED INHIBITION BY KIR3DL1 In contrast, some evidence supports an inhibitory role for SHP-2 (Clontech Laboratories, Palo Alto, CA) using XhoI and NotI sites to gain upon binding certain ITIM-containing inhibitory receptors, such as a BamHI site and then ligated into the SalI and BglII restriction sites of CTLA4 (32, 33), platelet endothelial cell adhesion molecule/CD31 pSC65. All PCR were performed using Platinum Pfx DNA polymerase ␣ (Life Technologies, Grand Island, NY), and the integrity of all constructs (34–36), SHP substrate-1/signal regulatory protein- 1 (37), was confirmed by sequencing. FDF03 (38), and PZR (39). Although previous biochemical evidence demonstrated that Retroviral transduction into NK-92 cell line SHP-2 can be recruited to KIR, the functional significance of this recruitment has not been previously addressed. The current work The retrovirus-mediated transduction was performed with modifications from a previous description (18). Supernatants containing virus were cocul- was undertaken to evaluate whether SHP-2 plays a direct func- tured with the NK-92 cell line for8hinthepresence of Lipofectamine Plus tional role in KIR-mediated inhibition. In addition to showing a reagent in a six-well plate. The plate containing virus and NK-92 was correlation between inhibition and SHP-2 binding to mutant forms centrifuged at 1000 ϫ g for 45 min twice during the 8-h incubation and of KIR3DL1 (3DL1), we have observed that overexpression of a then complete ␣-MEM medium containing human IL-2 (18) was added for 3 days before sorting. The transduced NK cells were sorted for expression catalytically inactive SHP-2 mutant reverses KIR inhibition of NK of 3DL1 using the 3DL1-specific PE-conjugated DX9 mAb (BD Phar- cell cytotoxicity. Finally, direct fusion of the phosphatase domain Mingen, San Diego, CA) in the Fox Chase Cancer Center Cell Sorting of SHP-2 to KIR results in a receptor that inhibits natural cyto- Facility (Philadelphia, PA). These sorted cells stably expressed the recep- toxicity and conjugate formation. tors for at least 1 mo. Uniform exogenous 3DL1 expression in Ͼ98% of the transduced cell population was confirmed at the time of every experiment using PE-conjugated DX9 mAb by flow cytometric analysis on a FACScan Materials and Methods analyzer (BD Biosciences, Mountain View, CA).