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

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

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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:

4539Ð4547. 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 (14Ð18). 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, 15Ð20). 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 (6Ð9). 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 (26Ð31).

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 (34Ð36), 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). Cell culture and Abs Downloaded from The IL-2-dependent NK-like cell lines, NKL (a gift from Dr. M. Colonna, Redirected cytotoxicity assay Washington University, St. Louis, MO) and NK-92 (a gift from Dr. C. NK-92 cells were cultured with fresh IL-2-containing medium on the day Lutz, University of Iowa, Iowa City, IA), were cultured as previously de- before assay, and redirected cytolytic activity was tested against the scribed (18). Cells were passed with fresh IL-2 every 4 days. The murine ϩ Fc␥RII/III P815 murine mastocytoma cell line in a 4-hr 51Cr-release as- mastocytoma, P815, was cultured as previously described (18). Abs were say, as previously described (18). Cytotoxicity of NKL cells show low purified with protein G from the hybridomas for the anti-3DL1 mAb DX9 cytotoxicity against P815. Thus, the cytotoxicity of NKL was induced by and the anti-CD56 mAb B159.5.2, which were kindly provided by Dr. L. L. the anti-NKp46 (9E2) mAb at 5 ␮l of hybridoma supernatant per http://www.jimmunol.org/ Lanier (University of California, San Francisco, CA) and Dr. B. Perussia 200-␮l well. (Thomas Jefferson University, Philadelphia, PA), respectively. Hybridoma supernatant containing anti-NKp46 mAb (9E2) was kindly provided by Dr. M. Colonna. Target cell conjugate assay cDNA constructs Analysis of target cell conjugation was based upon the assay of Burshtyn et al. (12). P815 cells were cultured with fresh medium 1 day before assay. KIR cDNA constructs were ligated into the retroviral expression vector, NK-92 cells were restimulated with fresh IL-2-containing medium 1 day pBMN-NoGFP, derived from pBMN-IRES-EGFP (40), using BamHI and before assay and the cell numbers of all NK-92 cell cultures were equally NotI restriction sites as previously described (18). 3DL1 (NKAT3) cDNA adjusted at 8 ϫ 104 cells/ml in a 75-cm2 flask at that time, because cell (GenBank accession number, L41269) was obtained from Dr. M. Colonna. density and activation status influenced optimal conjugation. P815 and by guest on October 2, 2021 The deletion mutant of 3DL1 (272P⌬) lacking most of the cytoplasmic NK-92 cells were separately incubated for 30 min at 2 million/ml with domain was described previously (18). To mutate the membrane proximal prewarmed (37¡C) medium-containing either CellTracker Orange tyrosine residue (Y377F), the membrane distal tyrosine residue (Y407F), CMTMR (Molecular Probes, Eugene, OR) at 2 ␮M or CellTracker Green or both tyrosines (Y377F/Y407F) in the ITIM motifs, 3DL1 cDNA was CMFDA (Molecular Probes) at 0.2 ␮M, respectively. The stained cells amplified by PCR using the primers described in Table I. To make KIR/ were washed and incubated in prewarmed medium for 30 min at 2 million/ phosphatase chimeric receptors, PCR primers described in Table I were ml. The stained cells were then washed again. P815 cells were preincu- used to generate cDNAs encompassing the extracellular and transmem- bated at room temperature with anti-KIR3DL1 mAb (DX9; 2 ␮g/ml) for 15 brane domains of 3DL1 with 5Ј-BamHI and 3Ј-XbaI sites and subdomains min before effector cell addition. Conjugate formation was performed in a of human SHP-1 (from Dr. C. T. Walsh, Harvard Medical School, Boston, 96-well V-bottom plate by mixing 5 ϫ 104 effector and 5 ϫ 104 target cells MA) or human SHP-2 (a gift from Dr. B. G. Neel, Beth Israel Deaconess in a final volume of 100 ␮l per well. The plate was centrifuged at room Ј Ј ϫ Medical Center, Boston, MA) with 5 -XbaI and 3 -NotI sites. The BamHI- temperature for 1 min at 500 g and placed at 37¡Cin7%CO2 humidified XbaI fragment of 3DL1 and the XbaI-NotI fragment of each domain of atmosphere. Cells were recovered by gently pipetting, transferred into phosphatase were cloned into the pBMN-NoGFP vector. Human C459S round-bottom tubes (Falcon 2054; BD Biosciences), and immediately an- SHP-2 (mutated at the catalytic cysteine residue in the phosphatase domain alyzed on a FACScan (BD Biosciences) at each time point (10,000 events to serine; a gift from Dr. B. Neel) was transferred into the pT-Adv vector counted/assay point). Results are expressed as the percentage of NK cells

Table 1. Sequences of synthesized oligonucleotide primers to make constructs used in this study a

Primer Sequence

KIR3DL1 sense (BamHI) 5Ј-TCGACTGGATCCACCATGTCGCTCATGGTCGTCAGCATG-3Ј pBMN antisense 5Ј-GACCTTGCATTCCTTTGGCG-3Ј Y377F sense 5Ј-AGGAGGTGACATTCGCACAGTTGGATCA-3Ј Y377F antisense 5Ј-TGATCCAACTGTGCGAATGTCACCTCCT-3Ј Y407F sense 5Ј-ATACCATCTTGTTCACGGAACTTCCAAA-3Ј Y407F antisense 5Ј-TTTGGAAGTTCCGTGAACAAGATGGTAT-3Ј KIR3DL1 antisense (XbaI) 5Ј-TTGTTCTCTAGAGTCCTCGCTGTTGGC-3Ј SHP-1 CAT sense (XbaI) 5Ј-GTCTACTCTAGACAGCCGTACTATGCC-3Ј SHP-1 CAT antisense (NotI) 5Ј-ACGACAGCGGCCGCTAAAGATCTTCTTCAGAAATAAGTTTCTGTTCCTTCCTCTTGAGGGAACCCT-3Ј SHP-2 CAT sense (XbaI) 5Ј-CTACAATCTAGACAGCCCCTTAACACG-3Ј SHP-2 CAT antisense (NotI) 5Ј-ACGACAGCGGCCGCTAAAGATCTTCTTCAGAAATAAGTTTCTGTTCTCTGAAACTTTTCTGCTGTTGCATCAG-3Ј SHP-2 SH2 sense (XbaI) 5Ј-CTACAATCTAGAACATCGCGGAGATGGTTTC-3Ј SHP-2 SH2 antisense (NotI) 5Ј-CTGATGCGGCCGCTAACCCAATGTTTCCACCATAG-3Ј

a The restriction sites are highlighted in bold letters. Myc tag encoding sequences in SHP-1 CAT and SHP-2 CAT antisense oligos are underlined. The Journal of Immunology 4541 that formed conjugates with target cells as calculated by the ratio of two- sidual inhibition remains, even when both ITIM tyrosines are mu- color events to total effector cell events using FlowJo software (Tree Star, tated to phenylalanine. Woburn, MA). The initial stage of NK cell-mediated cytotoxicity is the Pervanadate treatment and cell lysis formation of stable conjugates between target and effector, and KIR engagement has been shown to disrupt conjugate formation NK cells were washed three times in HBSS (Life Technologies), stimulated ␮ (12). To determine whether the interaction of NK-92 with P815 with pervanadate (final concentration: 100 MNa3VO4 plus 0.015% H2O2), and lysed for 30 min on ice in 1 ml of 1% Triton X-100 lysis buffer cells was disrupted by engagement of the individual mutant per sample as previously described (18). Lysates were cleared by centrif- KIR, we quantitated conjugate formation using two-colored ϫ ugation at 20,800 g for 15 min at 4¡C. flow cytometry. We found that engagement of the mutant Immunoprecipitation and immunoblotting retaining only the N-ITIM of 3DL1 (YF) still significantly reduced conjugate formation (Fig. 1C), although, as in the Lysates were precleared twice for 30 min each at 4¡C with protein G- coupled agarose (Upstate Biotechnology, Lake Placid, NY). CD56 and cytotoxicity experiments (Fig. 1B and Table II), the inhibition KIR were sequentially immunoprecipitated for 90 min at 4¡C with was not as strong as that of the wild-type receptor. In contrast, B159.5.2 and DX9 mAbs (5 ␮g/each sample precoupled to 30 ␮l of protein the other mutants, FY or FF, were inefficient in disrupting G-agarose) as performed previously (18), separated on SDS-PAGE, trans- conjugate formation (Fig. 1C). These combined results demon- ferred to polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), and probed either directly with HRP-coupled 4G10 mAb (anti- strate similar patterns of inhibitory capacities for the mutants in phosphotyrosine, 1:10,000; Upstate Biotechnology) or initially with rabbit assays of both conjugate formation and redirected cytotoxicity, polyclonal anti-SHP-1 (1 ␮g/ml; Upstate Biotechnology) or anti-SHP-2 (1 with WT (YY) Ͼ YF ϾϾ FY Ͼ FF. ␮g/ml; Santa Cruz Biotechnology, Santa Cruz, CA) Abs and secondarily Biochemical analysis was next performed to assess PTP with HRP-coupled protein G (1:10,000; Calbiochem, La Jolla, CA). recruitment to phosphorylated 3DL1 ITIM mutants (Fig. 2). Downloaded from Vaccinia virus infections NK-92 cells expressing the wild-type and mutant forms of Purified recombinant vaccinia virus preparations generated with the plas- 3DL1 were treated with pervanadate to induce optimal tyrosine mid pSC65 containing cDNA of wild-type SHP-1 or DN-SHP-1 (C453S) phosphorylation before 3DL1 immunoprecipitation. In these (11) were kindly provided by Dr. E. O. Long (National Institutes of Health, studies, both SHP-1 and SHP-2 were recruited to the phosphor- Rockville, MD). Generation of the recombinant vaccinia virus encoding ylated wild-type form of the receptor, but SHP-1 was not C459S SHP-2 cDNA was performed as described (41). Aliquots of the recruited to any of the KIR with mutant ITIMs (Fig. 2), http://www.jimmunol.org/ recombinant virus preparations were dispersed by water bath sonication and stored at Ϫ70¡C before use. The titer of virus stocks was determined indicating that SHP-1 binding requires tyrosine phosphoryla- by plaque assay as described (41) (wild-type SHP-1, 1 ϫ 109 PFU/ml; tion of both ITIMs. In contrast, phosphorylation of only the C453S SHP-1, 0.25 ϫ 109 PFU/ml; C459S SHP-2, 1 ϫ 109 PFU/ml). N-ITIM (YF) of 3DL1 still recruited increased levels of SHP-2 NK-92 cells were infected with the recombinant vaccinia virus as described (Fig. 2A), which correlated with the stronger inhibitory prop- previously (18, 42). Vaccinia virus infections were monitored for SHP-1 or SHP-2 protein expression by immunoblot analysis of the infected cells. erties of that mutant KIR. In contrast, the receptor with muta- Viability of the infected cells was monitored by trypan blue staining before tion of the N-ITIM (FY) did not demonstrate increased SHP-2 mixing with target cells (Ͼ95% viable). binding in pervanadate-treated cells, despite strong tyrosine phosphorylation (Fig. 2A). Also, as we have previously ob- Phosphatase assay by guest on October 2, 2021 served with a phenylalanine mutant of the single N-ITIM in NK-92 cells stably transduced with KIR/SHP1 or KIR/SHP2 were lysed 2DL4 (18), the FY and FF mutants constitutively bound SHP-2, with Triton X-100 lysis buffer (lacking sodium orthovanadate, sodium flu- oride, and EDTA) and immunoprecipitated as described (18). Immunopre- even in unstimulated cells, which was most evident in immu- cipitates were washed three times with ice-cold 0.1% Triton X-100 buffer noprecipitates from greater numbers of cells (Fig. 2B). Inter- (same components as lysis buffer), washed again three times with 150 mM estingly, this constitutive SHP-2 association correlated with the NaCl/50 mM Tris-HCl (pH 7.5) and resuspended in 100 ␮l of reaction weak inhibitory properties of the FY and FF mutant receptors in buffer (12 mM p-nitrophenylphosphate (pNPP), 20 mM Tris-HCl (pH 7.5), the redirected cytotoxicity assays (Fig. 1B). These results indi- 100 mM NaCl, 1 mM DTT, 1 mM EDTA). After incubation at 37¡C for 1 h, the reaction was stopped by adding 17 ␮l of 2.5 N NaOH, and the cate that SHP-2 is constitutively associated with the unphos- amount of p-nitrophenyl released was determined from absorbance at phorylated N-ITIM of KIR and may contribute to early negative 410 nm. signaling and/or allow rapid high avidity recruitment upon ITIM phosphorylation. Importantly, the common theme among Results these mutant receptors is a direct correlation between SHP-2 The analysis of KIR with mutant ITIMs in human NK cells recruitment capacity and inhibitory functions (Fig. 1, B and C, To determine the enzyme recruitment properties of and functional and Table II). These results suggest that SHP-2 contributes roles for the individual ITIM tyrosines on classical inhibitory KIR substantially to the inhibitory properties of the mutant receptors in human NK cells, we have used retroviral transduction to express and is likely also contributing to the inhibitory function of wild-type and tyrosine to phenylalanine mutant forms of 3DL1 wild-type 3DL1. (WT) at the N-terminal (N)-ITIM (FY), C-terminal (C)-ITIM (YF), or both ITIMs (FF) (Fig. 1A, left panel). A truncated form of 3DL1, in which the cytoplasmic domain is deleted just before the DN-SHP-2 blocks inhibition of natural cytotoxicity by 3DL1 N-ITIM (272P⌬), was used as a negative control. These were ex- Because our experiments demonstrated efficient SHP-2 binding ca- pressed at comparable levels in the NK-like cell line, NK-92 (Fig. pacity by KIR and correlations between SHP-2 binding capacity 1A, right panel), and tested for capacities to inhibit cytotoxicity in and inhibitory function, we next tested whether expression of a a redirected assay against P815 target cells designed to specifically DN-SHP-2 protein (catalytically inactive) could abolish KIR in- engage only the 3DL1 inhibitory receptors. Retention of only the hibitory function. Vaccinia virus-mediated expression of DN- N-ITIM of 3DL1 was still strongly inhibitory (Fig. 1B, middle SHP-1 has been shown to block inhibition through KIR, which panel), in contrast to 3DL1/FF and 3DL1/FY, which demonstrated provides convincing evidence for SHP-1 involvement in this pro- weak but highly reproducible inhibition (Fig. 1B and Table II). cess (10, 11, 23). Therefore, we compared the impacts of recom- These results demonstrate that the N-ITIM is essential and suffi- binant vaccinia virus preparations expressing DN-SHP-1 (C453S), cient to contribute strong inhibitory function to 3DL1, while re- wild-type SHP-1 (WT-SHP-1) (11), and DN-SHP-2 (C459S; with 4542 SHP-2 MEDIATED INHIBITION BY KIR3DL1

FIGURE 1. The N-terminal tyrosine (Y377) of 3DL1 is essential for strong inhibition of cytotoxicity in human NK cells. A, Schematic representations of various 3DL1 constructs with tyrosine (Y) to phenylalanine (F; asterisks) mutations at the cytoplasmic N- and C-ITIM sequences, YAQL and YTEL, respectively. The cytoplasmic domain of 3DL1 was deleted just before the N-terminal tyrosine (272P⌬). D0, D1, and D2 are highly conserved Ig- like domains in the KIR family. TM and CY represent transmembrane and cytoplasmic domains, respectively. Stable transduced NK cell lines were established and surface expression of 3DL1 was compared Downloaded from using PE-labeled DX9 mAb (thick lines) vs staining of the 3DL1-negative parent cell line (dotted lines). B, Redirected cytotoxicity assay using NK-92 cells transduced with the receptors described in A. The stable transduced NK-92 cells were

tested for their abilities to lyse P815 in the http://www.jimmunol.org/ absence of mAb (E) or in the presence of 1 ␮g/ml anti-3DL1 mAb (DX9; F). Results represent one of six independent experiments, showing identical patterns of inhibition. C, Conjugate formation using NK-92 cells described in A against P815 cells in the absence of mAb (E) or in the presence of anti-3DL1 mAb at 1 ␮g/ml (DX9; F). Re- sults represent one of seven independent experiments. by guest on October 2, 2021

a myc epitope tag) on 3DL1 inhibitory function. NK-92 cells ex- PTP (10- to 50-fold above endogenous; Fig. 3A and Ref. 18). The pressing wild-type 3DL1 were infected with purified vaccinia binding of the exogenous DN-SHP-2 protein to 3DL1 was dem- preparations at PFU titers that produced robust levels of exogenous onstrated by immunoprecipitation and immunoblotting analysis, confirming that DN-SHP-2 is recruited to phosphorylated 3DL1 in vivo (Fig. 3B). In a redirected cytotoxicity assay against P815 Table II. Statistical analysis of the capacities of mutant KIR3DL1 target cells (Fig. 3C), KIR inhibition was unchanged by expression receptors to inhibit cytotoxicity from multiple experimentsa of WT-SHP-1 (Fig. 3C, upper panels). However, DN-SHP-1 completely abrogated wild-type KIR-mediated inhibition (Fig. 3C, % of Control Cytotoxicity Mean-Anti- middle panels). As predicted from our biochemical results, expres- Receptors No. of experiments Mean-control (ϮSD) KIR3DL1 (ϮSD) sion of DN-SHP-2 also completely blocked KIR-mediated inhibi- KIR3DL1 6 100 (3.2) 32.8 (21.4)* tion (Fig. 3C, lower panels), providing direct evidence that SHP-2 FY 6 100 (8.5) 67.5 (10.3)* is recruited to KIR during inhibitory signaling in vivo. YF 6 100 (3.8) 41.6 (13.4)* We next tested the impacts of the DN-PTP expression on inhi- FF 6 100 (3.7) 78.5 (9.3)* bition through the mutant KIR (WT, YF, FY, and FF). We ex- 272P⌬ 4 100 (3.8) 95.2 (10.3) pected that the inhibitory function of these SHP-2-binding KIR a Statistical analysis was performed on cytotoxicity data from multiple experi- would be blocked by DN-SHP-2, but not DN-SHP-1. Indeed, the ments by converting the data at a 10:1 E:T ratio within each experiment to percentage of control cytotoxicity (without Ab). Within each experiment the duplicate or tripli- DN-SHP-1 was not effective in blocking any of these modified cate data points in the absence of Ab of each cell line were averaged, and the average inhibitory receptors (Fig. 4A and FF, data not shown). In agree- was designated 100% of control cytotoxicity. Every data point for each transduced ment with our biochemical data, we therefore conclude that these cell line within each experiment was converted to this percent of control cytotoxicity, and a Student’s t test was performed to compare the aggregate data points from Ab mutant KIR lacking specific ITIMs inhibit NK cell cytotoxicity in untreated and anti-KIR3DL 1-treated conjugation conditions for each transduced cell a SHP-1-independent manner. Unexpectedly, we were unable to line. Statistical analysis was performed on the Excel X program for Macintosh (Mi- crosoft, Redmond, WA). demonstrate blockade of inhibition through any of these mutant p Ͻ 0.05 compared to nontreated controls. KIR by DN-SHP-2 (Fig. 4B); rather expression of the DN-SHP-2 ,ء The Journal of Immunology 4543

FIGURE 2. Biochemical analysis of SHP-1 and SHP-2 recruitment to differentially phosphorylated KIR. A, SHP-2 recruitment can be seen in the mutant KIR with an intact tyrosine-phosphorylated N-terminal ITIM (Y407F) as well as with wild-type 3DL1. Receptor-transduced NK-92 cells (40 million cells/sample) were treated with pervanadate for 10 min, lysed, and sequentially immunoprecipitated with anti-CD56 as a control (data not shown) and then anti-3DL1 mAbs. Samples were analyzed on 10% SDS-PAGE gels under reducing conditions. Sequential immunoblot analysis was performed with anti-phosphotyrosine and anti-SHP-2. B, SHP-2 constitutively binds to the mutant ITIMs. Several forms of KIR were immunoprecipitated and processed (80 million/sample) as in A. Samples were sequentially immunoblotted with anti-SHP-1 and anti-SHP-2.

dramatically reduced the basal cytolytic capacity of NK-92 cells prompted us to try an alternative approach to answer whether Downloaded from expressing 3DL1/FY, 3DL1/YF, and 3DL1/FF (Fig. 3C and data SHP-2 indeed contributes directly to mutant KIR inhibitory not shown), but not those expressing wild-type 3DL1 (as in Fig. function. 3C). This is an intriguing observation, which may reﬂect the pos- sibility that DN-SHP-2 interferes with a broad spectrum of func- The SHP-2 catalytic domain inhibits natural cytotoxicity tions in human NK cells, including positive roles of SHP-2, such We demonstrated that KIR phosphorylated only at the N-ITIM as IL-2 receptor signaling (43Ð45). We cannot yet explain why strongly inhibit natural cytotoxicity and recruit only SHP-2 (Fig. http://www.jimmunol.org/ cytolytic capacity was selectively disrupted by DN-SHP-2 expres- 1B and 2). In addition, vaccinia virus-expressing DN-SHP-2 sion only in cells expressing these mutant KIR, but the result blocks normal 3DL1 inhibition of natural cytotoxicity (Fig. 3C). by guest on October 2, 2021

FIGURE 3. Transient expression of the DN forms of SHP-1 and SHP-2 blocks 3DL1 inhibition. A, Expression of the exogenous C459S SHP-2 using recombinant vaccinia virus. NK-92 cells were infected with the virus preparation at different PFU concentrations. The whole cell lysates (0.5 million cells/lane) were separated by 10% SDS-PAGE gels under reducing conditions and sequentially probed with anti-Myc followed by anti-SHP-2. B, The DN-SHP-2, C459S SHP-2, binding to 3DL1 in vivo. The 3DL1-transduced NK-92 (60 million) cells were infected with vaccinia virus possessing C459S SHP-2 cDNA at 10 PFU/cell, and stimulated without or with pervanadate for 10 min. KIR were immunoprecipitated with anti-KIR mAb (DX9), separated by 10% SDS-PAGE, and blotted sequentially with anti-phosphotyrosine (4G10), anti-Myc, and anti-SHP-2 Abs. C, Redirected cytotoxicity assay using P815 as a target. Recombinant vaccinia virus preparations encoding either wild-type SHP-1 (upper panels), C453S SHP-1 (middle panels), or C459S SHP-2 (lower panels) were used to infect 3DL1-expressing NK-92 cells at 10 PFU/cell (right panels). Aliquots of infected cells were used in a redirected cytotoxicity assay in the presence of either anti-CD56 (B159.5.2; E) as a control or anti-3DL1(DX9; F) mAbs (1 ␮g/ml). 4544 SHP-2 MEDIATED INHIBITION BY KIR3DL1

tive effector enzyme to mediate inhibition. Therefore, we also generated 3DL1 chimeras in which the cytoplasmic domain consists of the two SH2 domains of SHP-2 (Fig. 5A) and expressed them in NK-92 cells as before (Fig. 5B). However, the chimera with SHP-2 SH2 domains did not exhibit any inhibitory (or activating) capacity in the redirected cytotoxicity assay, thereby indicating that a free SHP-2 SH2 domain does not provide adaptor function to recruit another negative effector enzyme (Fig. 5A, right panel).

Discussion The majority of previously published reports have focused on the role of SHP-1 in KIR function, and that literature confirms that phosphorylation of both ITIMs is required for SHP-1 recruitment (10, 11, 14Ð20). Although two published reports have previously described SHP-2 binding to phosphorylated KIR ITIM peptides (19, 25), only the work of Bruhns et al. (15) has previously ana- FIGURE 4. Transient expression of the DN form of SHP-1 does not lyzed SHP-2 recruitment to KIR when expressed in rat mast cells block inhibition through mutant KIR. Recombinant vaccinia virus prepa- or mouse B cells. In the current studies and our recent report (18), rations encoding C453S SHP-1 (A) or C459S SHP-2 (B) were used to we have used retroviral transduction to express mutant and wild- infect NK-92 cells that stably express the wild-type (WT; upper panels), Downloaded from Y377F (FY; middle panels), or Y407F (YF; lower panels) forms of 3DL1 type forms of 3DL1 and 2DL4 in NK cell lines to directly study by retroviral transduction. The KIR-expressing NK-92 cells were infected their inhibitory functions on NK cell cytotoxicity and their SHP- with recombinant vaccinia virus preparations at 10 PFU/cell. Aliquots of 1/SHP-2 recruitment capacities. Our results confirm and extend the infected cells were used in a redirected cytotoxicity assay in the presence work of Bruhns et al. (15) and our previous report (18) to show that of anti-CD56 (B159.5.2; E) as a control or anti-3DL1 (DX9; F) mAbs at phosphorylation of the N-ITIM of KIR is essential and sufficient 1 ␮g/ml. for strong inhibitory function (Fig. 1) and SHP-2 recruitment (Fig. 2), but phosphorylation of both ITIMs is required to recruit SHP-1 http://www.jimmunol.org/ (Fig. 2). Importantly, we have also shown that the capacities of Although these results strongly implicate SHP-2 in KIR inhibition, mutant forms of 3DL1 to recruit SHP-2 correlate with their inhib- SHP-2 more commonly contributes to activation signals than in- itory functions. Interestingly, we have shown that SHP-2 associ- hibitory signals in other receptor systems (46Ð52). Therefore, to ates weakly with mutant KIR in a phosphotyrosine-independent directly test whether the catalytic domain of SHP-2 can inhibit NK manner (Fig. 2). This supports our previous work (18), suggesting cell activation, we generated receptors in which we replaced the that SHP-2 is constitutively associated with the unphosphorylated cytoplasmic domain of 3DL1 with the catalytic domains of SHP-1 N-ITIM and may be poised at the receptor for rapid high avidity or SHP-2. This is a strategy similar to that used to study the binding upon ITIM phosphorylation. Previous work has ruled out by guest on October 2, 2021 detrimental effect of SHP-1 on TCR signaling (53Ð55). Our chi- a role for SHIP in KIR function (14, 23), and we have not detected meric receptors mimic conditions under which only a single ef- recruitment of SHIP to KIR in our studies (data not shown). There- fector enzyme is recruited, thereby excluding functional influences fore, the phosphorylation status of KIR ITIMs determines SHP-1/ from the other effector enzyme (Fig. 5A). NK-92 cells were trans- SHP-2 recruitment capacities, which ultimately correlate with induced with these chimeric receptors and cells expressing similar hibitory function. levels of receptor were sorted (Fig. 5B). We confirmed that im- The most convincing evidence for SHP-1 involvement in KIR munoprecipitates of each chimera exhibit PTP catalytic activity, function is derived from experiments demonstrating that expres- because they hydrolyzed pNPP to similar degrees (Fig. 5C). When sion of DN-SHP-1 in NK cells blocks KIR-mediated inhibition of tested for inhibitory capacity in redirected cytotoxicity experi- cytotoxicity responses (10, 11, 23). However, we have long con- ments against P815 target cells, the chimeras containing the cata- sidered that high avidity bivalent binding of DN-SHP-1 to the lytic domains of both SHP-1 and SHP-2 demonstrated substantial phosphorylated KIR ITIMs would likely outcompete not only the inhibition (Fig. 5D). Engagement of the chimeras containing the functional endogenous SHP-1, but also any other negative effector catalytic domains of both SHP-1 and SHP-2 also significantly in- protein that might be physiologically recruited to mediate inhibi- hibited the conjugate formation between NK-92 and P815 cells tion. Because our biochemical evidence showed SHP-2 recruit- (Fig. 5E), indicating that either PTP has the ability to regulate cell ment to KIR, we expressed DN-SHP-2 in NK cells and showed adhesion, when recruited to KIR ITIMs. These important results that it also effectively blocks the inhibitory function of wild-type provide direct evidence that the PTP catalytic activities of either KIR (Figs. 3 and 4). To our knowledge, this is the first report of the SHP-1 or SHP-2, when recruited to the cytoplasmic domain of abrogation of function through any inhibitory receptor by expres- KIR, can mediate negative signaling toward NK cell cytotoxicity sion of DN-SHP-2. Using DN-SHP-1, we also showed that inhi- responses. In addition, the chimeric receptor containing the phos- bition by the mutant forms of KIR is SHP-1-independent (Fig. 4). phatase domain of SHP-2 also inhibited the cytotoxicity induced Unfortunately, due to technical complications, we were unable to by NKp46 engagement on the surface of another NK-like cell line, use DN-SHP-2 to block the inhibitory signal from mutant KIR NKL, in a redirected cytotoxicity assay (Fig. 5F), showing that the (Fig. 4). Importantly, however, our results demonstrating the ca- inhibitory role of SHP-2 is functional in the contexts of two dif- pacities of DN-SHP-1 and DN-SHP-2 to block wild-type 3DL1 ferent NK-like cell lines. function, strongly indicate that both SHP-1 and SHP-2 play roles One readily apparent physical difference between SHP-2 recruit- in normal inhibitory KIR function. ment to wild-type 3DL1 (with two ITIMs) and SHP-2 recruitment Inhibition of cytotoxicity responses by the functionally related to 2DL4 or 3DL1/YF (with only the N-ITIM) is the likely exis- Ly-49A receptor is greatly impaired, but not completely abrogated, tence of a free SH2 domain in the latter situations. We hypothe- in SHP-1-deficient (motheaten) murine NK cells (56). It is possible sized that this free SH2 domain might recruit an additional nega- that SHP-2 is mediating the residual inhibition through Ly-49 in The Journal of Immunology 4545 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. A chimeric form of 3DL1 with the cytoplasmic domain replaced by the phosphatase domain of SHP-2 can inhibit natural cytotoxicity against

P815 cells. A, Amino acid sequences of the junctions and C termini of the KIR/PTP chimera constructs. The cytoplasmic domain of 3DL1 (after S367) was replaced by catalytic (CAT) or SH2 domains of SHP-1 or SHP-2 using XbaI restriction sites (resulting amino acids underlined). Dots represent sequence identity with 3DL1. Only the initial N-terminal amino acids (starting with R212 of SHP-1 CAT, Q214 of SHP-2 CAT, and T2 of SHP-2 SH2) and C-terminal amino acids (ending with K597 of SHP-1 CAT, R593 of SHP-2 CAT, and G207 of SHP-2 SH2) of the PTP domains are shown here (intervening sequences marked by arrows). Myc epitope-tags are indicated in bold. B, Flow cytometric analysis of transduced NK-92 cells stained with anti-3DL1 (DX9) mAb (thick line). C, Dephosphorylation of pNPP by anti-3DL1 immunoprecipitates from transduced NK-92 populations. Dephosphorylation was determined from the absorbance measurement of p-nitrophenyl generation at 410 nm. Recombinant SHP-1 (4 ␮l; Upstate Biotechnology) was used as a positive control. D, NK-92-derived transfectants stably expressing KIR/phosphatase chimeric receptors were tested for their cytotoxicity against P815 targets in a redirected cytotoxicity assay. Transduced NK-92 cells were incubated with P815 in the absence of mAb (E), or in the presence of anti-CD56 (B159.5.2; ¹) as a control or anti-3DL1 (DX9; F) mAbs at 1 ␮g/ml. E, Conjugate formation using NK-92 cells transduced with KIR/phosphatase chimeric receptors against P815 cells in the absence of mAb (E) or in the presence of anti-3DL1 mAb at 1 ␮g/ml (DX9; F). Results represent one of two independent experiments. F, The KIR/phosphatase chimeric receptors efﬁciently inhibited the activation by NKp46 in another NK cell line, NKL. The NKL cell line was transduced with the KIR/SHP-2 CAT (left panel) or KIR/SH2 (right panel) constructs. NKL cytotoxicity against P815 was induced in the presence of anti-NKp46 mAb (E) and inhibited when combined with anti-KIR3DL1 mAb (F) as compared with the low cytotoxicity in the absence of Abs (¹). motheaten NK cells. In contrast, it must be considered that the domain, thereby mimicking only SHP-2 recruitment to KIR. Engage- Ly-49 receptor is structurally very different from KIR, and there- ment of this KIR/SHP-2 chimeric receptor inhibited both target cell fore, a role for SHP-2 in its function awaits formal proof. Unfor- conjugation and cytotoxicity in a redirected assay in two NK-like cell tunately, SHP-2-deﬁcient mice are embryonic lethal, and embry- lines (Fig. 5). A chimera with the catalytic domain of SHP-1 could onic stem cells from these mice are completely incapable of also inhibit NK cell activation, while a chimera with the SH2 domains supporting lymphocyte development (57). Nonetheless, the moth- of SHP-2 exhibited no inhibitory capacity. These results demonstrate eaten results demonstrate that another effector enzyme is also me- direct inhibitory capacity by both SHP-1 and SHP-2 phosphatases diating negative signaling through Ly-49 as well. when recruited to the target cell interface in NK cells. To directly address the capacity of SHP-2 catalytic activity to SHP-1 and SHP-2 share 60% sequence identity (amino acids) mediate KIR inhibition, we generated a chimeric KIR in which the and very high homology in secondary and tertiary structure (24, cytoplasmic domain was substituted with the SHP-2 phosphatase 58). However, differential charge distribution in the catalytic clefts 4546 SHP-2 MEDIATED INHIBITION BY KIR3DL1 and numerous functional studies indicate that their substrates seem 16. Burshtyn, D. N., A. S. Lam, M. Weston, N. Gupta, P. A. M. Warmerdam, and to be distinct (59Ð64). Upon KIR engagement, recruitment of E. O. Long. 1999. 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