2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN- Γ Release in Human NK Cells Involves Distinct Pathways

2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN- γ Release in Human NK Cells Involves Distinct Pathways

This information is current as Samuel S. Chuang, Pappanaicken R. Kumaresan and of October 2, 2021. Porunelloor A. Mathew J Immunol 2001; 167:6210-6216; ; doi: 10.4049/jimmunol.167.11.6210 http://www.jimmunol.org/content/167/11/6210 Downloaded from

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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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN-␥ Release in Human NK Cells Involves Distinct Pathways1

Samuel S. Chuang, Pappanaicken R. Kumaresan, and Porunelloor A. Mathew2

2B4 (CD244), a member of the CD2 subset of the Ig superfamily receptors, is expressed on all human NK cells, a subpopulation of T cells, basophils and monocytes. 2B4 activates NK cell mediated cytotoxicity, induces secretion of IFN-␥ and matrix metalloproteinases, and NK cell invasiveness. Although there has been several molecules shown to interact with 2B4, the signaling mechanism of 2B4-mediated activation of NK cells is still unknown. In this study, we found cross-linking of 2B4 on YT cells, a human NK cell line, results in the increased DNA binding activity of activator protein-1 (AP-1), an important regulator of nuclear gene expression in leukocytes. We investigated the possible role of various signaling molecules that may be involved in the activation of lytic function of YT cells via 2B4. Treatment of YT cells with various speciﬁc inhibitors indicate that 2B4-stimulation of YT cells in spontaneous and Ab-dependent cytotoxicity is Ras/Raf dependent and involves multiple MAPK signaling pathways (ERK1/2 and p38). However, only inhibitors of transcription and p38 inhibited 2B4-mediated IFN-␥ release indicating distinct Downloaded from pathways are involved in cytotoxicity and cytokine release. In this study we also show that 2B4 constitutively associates with the linker for activation of T cells (LAT) and that 2B4 may mediate NK cell activation via a LAT-dependent signaling pathway. These results indicate that 2B4-mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways. The Journal of Immunology, 2001, 167: 6210Ð6216. http://www.jimmunol.org/

atural killer cells recognize and kill virally infected cells, (SHP)3 1 and/or SHP-2. These phosphatases can dephosphorylate parasites and certain tumor cells (1–3). NK cells play an several substrates within the activating signaling cascade or a dis- N important role by their ability to target MHC class I tal activating receptor and inhibit NK cell activation (14, 15). negative cells that escape recognition by cytotoxic T cells (4, 5). In Although there has been great progress in understanding the addition to cytolytic activity, NK cells also produce several regu- inhibitory mechanisms that regulate NK cell function, our knowl- latory cytokines including IFN-␥, TGF␤, IL-1␤, IL-10 and GM- edge of the activating signaling pathways is slowly emerging. Sev- CSF as well as matrix metalloproteinases (MMPs) (6–9). The eral surface molecules have been identiﬁed that can activate nat-

mechanisms that control NK cell activation and cytotoxicity are ural cytotoxicity. Activating receptors such as 2B4, CD2, CD16, by guest on October 2, 2021 believed to be determined by a delicate balance between stimula- KIR3DS1-5, NKp30, NKp44, and NKp46 are members of the Ig tory and inhibitory signals received from surface receptors (10– superfamily (16–18). These molecules bear high homology on 12). NK cells also mediate the rejection of MHC mismatched bone their extracellular domains while lacking the immunoreceptor ty- marrow stem cells (13). Engagement of cytolytic activity can be rosine-based inhibitory motifs on their cytoplasmic tails. There are inhibited by MHC class I molecules on target cells interacting with also members of the lectin gene superfamily that can transduce an MHC class I receptors expressed on the surface of NK cells. NK activating signal. Members of the lectin superfamily, such as cell inhibitory receptors belong to the Ig and lectin gene super- NKR-P1A and NKR-P1C, NKG2 family, and the LY49 family can families. One common feature of the killer cell inhibitory receptors form homo- and heterodimer complexes that can be inhibitory or is the presence of immunoreceptor tyrosine-based inhibitory mo- stimulatory. Although many of the activating surface molecules tifs (ITIMs) in their cytoplasmic domains (3, 10). The ITIM motifs have been identiﬁed, information on the signaling cascade from the become phosphorylated upon receptor binding which results in the cell surface to within is fragmentary. recruitment of protein SH2 domain bearing tyrosine phosphatase 2B4 was originally identiﬁed on mouse NK cells and the subset of T cells that mediate non-MHC-restricted killing (18–21). It is a novel member of the CD2 subfamily which includes signaling lymphocyte-activation molecule, CD48, CD58, CD84, CS1, and Department of Molecular Biology and Immunology and Institute for Cancer Re- Ly9, and is expressed on all NK cells, a subset of CD8ϩ T cells, search, University of North Texas Health Science Center, Fort Worth, TX 76107 basophils, and monophils (22–24). The cytoplasmic domain of Received for publication March 28, 2001. Accepted for publication September 26, 2001. 2B4 contains novel tyrosine motifs (TxYxxV/I) that associate with signaling adaptor molecule, signaling lymphocyte-activation mol- 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 ecule-associated protein (SAP) whose defect forms the basis for with 18 U.S.C. Section 1734 solely to indicate this fact. X-linked lymphoproliferative syndrome (XLP) (25–28). It is 1 This work was supported in part by National Institutes of Health Grants AI 38938 and CA 85753. This work was supported in part by a grant from the University of North Texas Health Science Center Institute for Cancer Research. We appreciate 3 Abbreviations used in this paper: SHP, SH2 domain bearing tyrosine phosphatase; support of our work from Bank One through the University of North Texas Health SAP, signaling lympocyte-activation molecule-associated protein; XLP, X-linked Science Center Institute for Cancer Research. lymphoproliferative syndrome; MAPK, mitogen-activated protein kinase; ADCC, 2 Address correspondence and reprint requests to Dr. Porunelloor A. Mathew, De- Ab-dependent cellular cytotoxicity; ERK, extracellular signal-regulated kinase; LAT, partment of Molecular Biology and Immunology, University of North Texas Health linker for activation of T cells; h2B4, human 2B4; MEK1, MAPK kinase 1; rADCC, Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699. E-mail redirected ADCC; GEM, glycolipid-enriched microdomains; MMP, metalloprotein- address: [email protected] ase; PLC, phospholipase C.

thought that NK cells in XLP patients are defective in their acti- 51Cr release cytotoxicity assay vation through 2B4 signaling (29–31). In addition to modulating K562 cells and P815 cells, where indicated, were used as target cells and cytolytic function, 2B4 activation of NK cells induces cytokine ϫ 6 51 labeled by incubating 1 10 cells with 2 MBq of Na2 CrO4 (NEN production as well as invasiveness (9, 19, 32). CD48 was recently Research Products, Boston, MA) for 90 min at 37°C under 5% CO2 in air. identified as the high-affinity counterreceptor of 2B4 in both mice The target cells were then washed three times in culture medium. Ten ␮ and humans (33, 34). CD48–2B4 interactions are physiologically thousand labeled target cells (100 l) were incubated with the effector YT cell suspension (100 ␮l), with and without mAb C1.7 (200 ng/ml). Effector important since they enhance the lytic function of human NK cells YT cells were resuspended and added at 1, 2, 5, 10, and 20 times the (23). It has been reported that 2B4 may function as a coreceptor in number of labeled target cells. After incubation for4hat37°C under 5% ϫ ␮ human NK cell activation (35). CO2 in air, the cells were pelleted at 250 g for 5 min, 100 lofthe In this paper, we investigated whether the AP-1 pathway is ac- supernatants was removed, and their radioactivity was measured. The per- centage of specific lysis was calculated by the following equation: (a Ϫ tivated in human NK cells upon 2B4 stimulation. AP-1 is an im- b/c Ϫ b) ϫ 100, where a is the radioactivity of the supernatant of target portant regulator of nuclear gene expression in leukocytes, having cells mixed with effector cells, b is that in the supernatant of target cells been found responsive to a wide range of stimuli and regulating a incubated alone, and c is that in the supernatant after lysis of target cells large number of genes (36, 37). We observed that AP-1 DNA- with 1% Nonidet P-40. All data points in each graph represent the average binding activity increased in response to 2B4 stimulation, in par- of four independent trials with similar results. Determination of statistical significance was determined on each data point representing 2B4-mediated ticular AP-1 complexes containing JunB. To dissect the mecha- cytotoxicity assays performed with inhibitor-treated effector cells or target nisms involved in 2B4-mediated cytotoxicity, we set out to cells compared with assays conducted with nontreated effector and target examine whether 2B4 stimulation of YT cells utilized the Ras- cells with Student’s t test. Data groups were considered significantly dif- dependent mitogen activated protein kinase (MAPK) pathway in ferent when p Ͻ 0.05. Downloaded from natural cytotoxicity and Ab-dependant cellular cytotoxicity (ADCC). Our results show that 2B4-induced NK cell cytotoxicity Inhibitor treatment of cells is dependent on the extracellular signal-regulated kinase (ERK) In assays using inhibitors, both effector YT cells and target K562 and P815 and p38 MAPK pathways and can be inhibited by inhibitors of Ras cells were subjected to treatment in culture medium as indicated. Cells and Raf. Examination of 2B4-stimulated release of IFN-␥ revealed were incubated in actinomycin D (20 ␮g/ml) for2htoinhibit RNA poly- the role of the p38 MAPK but not the ERK1/2 pathway in IFN-␥ merase. To inhibit p38, cells were incubated in SB203580 at 10 and 50 ␮M

concentrations for 1 h. Cells were incubated in PD089059 (100 ␮M) for 1 h http://www.jimmunol.org/ production. Immunoprecipitations revealed the constitutive asso- to inhibit MAPK kinase 1 (MEK1). Cells were incubated in FTI-277 at 7.5 ciation of linker for activation of T cells (LAT) with 2B4. and 30 ␮M for 16 h to inhibit H-Ras processing. All incubations with

inhibitors were conducted at 37°C under 5% CO2 in air. At the end of the incubation period, the cells were used in 51Cr release cytotoxicity assays. Materials and Methods Cell lines, Abs, and chemicals Immunoblot analysis YT (human NK cell line), K562 (human erythroleukemia cell line), and YT cells were incubated (1 ϫ 107/100 ␮l; 37°C) for the indicated times P815 (mouse lymphoma cell line) were cultured in culture medium (RPMI with the different stimuli. These were C1.7 mAb (10 ␮g/ml) and PMA (50 1640 supplemented with 10% FBS (HyClone, Logan, UT), 2 mM glu- ␮

ng/ml). After stimulation, the cells were lysed with 900 l of lysis buffer by guest on October 2, 2021 tamine, 100 U/ml penicillin, 100 U/ml streptomycin, 10 mM HEPES, and (1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 10 mM HEPES (pH 10 mM nonessential amino acids). Cells were maintained at 37°Cina 7.5), 0.15 M NaCl, 10% glycerol, 1 mM PMSF, 1 mM Na VO ,50mM humidified 5% CO /95% air incubator. Cell culture reagents were obtained 3 4 2 NaF, 1 mM EDTA, and 10 ␮g/ml each of aprotinin and leupeptin). Forty from Life Technologies (Gaithersburg, MD) unless otherwise noted. C1.7 micrograms of protein lysate was analyzed in 8% SDS-PAGE (reducing Ab, which recognizes human 2B4 (25), was purchased from Coulter (Or- conditions). Western blots were performed according to the manufacturer’s lando, FL). Mouse Abs against c-Jun (catalog no. SC-822X), JunB (catalog chemiluminescence detection system instructions (Kirkegaard & Perry no. SC-8051X), anti-phospho-p38 (catalog no. SC-7973), anti-phospho- Laboratories, Gaithersburg, MD). Western blots were hybridized with anti- ERK (catalog no. SC-7383), polyclonal goat Abs anti-p38 (catalog no. phospho-p38 and anti-phospho-ERK mAbs to detect phosphorylated forms SC-535), and anti-ERK (catalog no. SC-94) and protein A/G PLUS-agar- of p38 and ERK, respectively. The Western blots were then stripped and ose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). reprobed with anti-p38 and anti-ERK Abs to detect total amounts of p38 Anti-LAT rabbit polyclonal Ab was purchased from Upstate Biotechnol- and ERK, respectively. ogy (Lake Placid, NY). All enzymes were purchased from New England Biolabs (Beverly, MA) unless otherwise stated. Poly(dI-dC) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All custom-synthe- IFN-␥ release assay sized oligonucleotides used in this study were supplied by Integrated DNA Technologies (Coralville, IA). All inhibitors used in this study were pur- Inhibitor-treated or untreated YT cells (500,000) were stimulated or un- chased from Calbiochem (San Diego, CA). stimulated with C1.7 mAb (400 ng/ml) in flat-bottom 24-well plates for 1 h at 37°C under 5% CO2 in air. Target K562 cells (50,000) were then added. ␮ After incubation for 16 h at 37°C under 5% CO2 in air, 100 l of cell-free Nuclear extraction and EMSA supernatant was collected. IFN-␥ concentration was then quantitated with Nuclear extracts were isolated from YT cells and NK-92 cells and YT cells an ELISA kit according to the manufacturer’s instructions (CLB, Amster- incubated with mAb C1.7 (200 ng/ml concentration) (38) and protein dam, The Netherlands). Each condition was tested in at least four inde- DNA-binding reactions were conducted in EMSAs (39). One million YT pendent trials. cells were incubated with C1.7 mAb for 2, 4, 6, and8hin12-well plates at 37°C in a humidified 5% CO2/95% air incubator. After the stimulation Immunoprecipitations period, the cells were collected, washed in ice-cold PBS, and lysed as described elsewhwere (38). A typical binding reaction mixture contained 2 Approximately 1 ϫ 108 YT cells were either left untreated or treated with ␮g of nuclear protein, 1 ␮g of poly(dI-dC), and radiolabeled oligonucle- 1 ␮g/ml C1.7 mAb for2hat37°C. Cells were then lysed with 1% digi- otide (20,000 cpm, ϳ0.2 ng) in 10 ␮l of reaction volume. The double- tonin, 10 mM Tris (pH 7.4), 150 mM NaCl, 100 ␮g/ml PMSF, and protease stranded radiolabeled oligonucleotide probe codes for the human 2B4 inhibitor mixture (Sigma-Aldrich, St. Louis, MO) for1honice. The ly- (h2B4) promoter sequence lies (Ϫ111 to Ϫ89) relative to the start of tran- sates were precleared with the addition of control mouse IgG Ab (Upstate scription and contains a functional AP-1 site (40). The mixture was incu- Biotechnology) for 3 h followed by protein G/A plus agarose and rotated bated on ice for 30 min and then electrophoresed through a 4% polyacryl- for3hat4°C. The precleared supernatant were used to immunoprecipitate amide gel under nondenaturing conditions in 0.25ϫ Tris-borate-EDTA at 2B4 with C1.7 mAb (5 ␮g/ml) overnight with slow shaking followed by pro- 200 V for 70 min. The gel was dried and then exposed to film. The bands tein G/A plus agarose mix for3hat4°C. Isotype control IgG (22b5) was used were visualized by autoradiography and quantified using the image quan- as negative control immunoprecipitations. Immunoprecipitates were separated titation program AlphaEase (Alpha Innotech, San Leandro, CA). by 10% SDS-PAGE and detected as described in Immunoblot analysis. 6212 2B4 ACTIVATION OF NK CELLS INVOLVES MULTIPLE MAPK PATHWAYS

Results with 20 ␮g/ml actinomycin D. Actinomycin D inhibits RNA poly- 2B4 stimulation induces AP-1 activation merases by complexing with the DNA preventing transcription. Pretreatment of YT cells with actinomycin D also inhibited natural Our studies on the transcriptional regulation of the mouse and cytotoxicity against target K562 cells (data not shown). Pretreat- human 2B4 gene have revealed the presence of functional AP-1 ment of YT effector cells resulted in signiﬁcantly lower levels of sites in both promoters (40, 41). The AP-1 site that resides Ϫ106 2B4-mediated cytotoxicity of K562 cells ( p Ͻ 0.05) and rADCC to Ϫ100 relative to the start of transcription is necessary for human ( p Ͻ 0.02) against P815 cells than those mediated by control non- 2B4 gene expression (40). Recently, it has been found that en- treated effector cells (Fig. 2). However, pretreatment of target cells gagement of NK cell cytotoxicity regulates AP-1 expression (42). with actinomycin D did not alter their susceptibility to NK cell To determine whether 2B4 stimulation of YT cells, a human NK cytotoxicity. Thus, these results show that critical transcriptional cell line, results in activation of AP-1, we isolated nuclear protein events are involved in 2B4-mediated cytotoxicity. after 2B4 stimulation. We then performed EMSAs with double- stranded radiolabeled probes coding for the promoter sequence Role of p38 and MEK1/ERK signaling in 2B4 stimulation (Ϫ111 to Ϫ89) of the human 2B4 promoter that contains a func- of NK cells tional AP-1 site (Fig. 1). Examination of the levels of AP-1 DNA- Several transcription factors including AP-1, NF-␬B, and Ets can binding activity through densitometric analysis revealed that AP-1 be activated by the MAPK signaling pathways. We undertook the DNA-binding activity increased in response to 2B4 cell stimula- task of examining the roles of the p38 and MEK1/ERK signaling tion, reaching maximal stimulation 6 h after 2B4 stimulation of the pathways in the 2B4 signal cascade. To accomplish this, we used cells (Fig. 1, B and C). Supershift analysis using Ab speciﬁc for

the selective inhibitor of p38, SB203580, in pretreatments of YT Downloaded from c-Jun and JunB found AP-1 factors that contained either protein cells and target K562 cells before their use in 51Cr release cyto- component increased (Fig. 1, D–F). However, the presence of the toxicity assays. YT cells pretreated with increasing concentrations supershifted band of JunB increased proportionately as shown in of SB203580 and then stimulated with anti-2B4 mAb resulted in Fig. 1B, indicating that 2B4 stimulation of NK cells results in progressively lower spontaneous cytotoxicity against K562 cells selective activation of AP-1 heterodimers involving JunB. ( p Ͻ 0.05; Fig. 3A). SB203580-pretreated NK cells’ ability for rADCC against P815 cells was also diminished. Pretreatment of

2B4-mediated cytotoxicity is transcription dependent http://www.jimmunol.org/ either target cell failed to inhibit 2B4-stimulated cytotoxicity sig- Previously, we and others have established that 2B4 stimulation of niﬁcantly. These results show that p38 plays a role in 2B4-stimu- NK cell lines results in an increase in spontaneous cytotoxic and lated YT cells in ADCC as well as spontaneous cytotoxicity. redirected ADCC (rADCC) activity (9, 22, 23, 35). AP-1 induction We next examined whether the MEK1/ERK signaling pathway and other critical transcriptional events may be involved in 2B4 is part of the signaling cascade that is activated by 2B4 ligation. signaling in NK cells. To determine whether 2B4-mediated cyto- We used the MEK1 inhibitor PD089059 to determine the impor- toxicity is transcription dependent, 51Cr release cytotoxicity assays tance of MEK1 during 2B4 activation of NK cells (43). PD089059 were performed using effector and target cells pretreated for 2 h pretreatment of YT cells before their use in 51Cr release cytotox-

icity assays resulted in a signiﬁcant reduction in cytolytic activity by guest on October 2, 2021 from 2B4-stimulated YT cells against both K562 ( p Ͻ 0.02) and P815 cells ( p Ͻ 0.05 for 20:1, 10:1, and 5:1 E:T ratios; Fig. 3B). This indicates the that MEK1/ERK signaling pathway regulates 2B4 activation of the lytic function of NK cells. It appears that 2B4 signaling utilizes multiple MAPK pathways in activating cytolytic function. We have examined the effect of pretreatment with PD089059 and SB203580 on YT cells. We found that inhibition

FIGURE 1. AP-1 DNA-binding activities are increased by 2B4 stimulation. A, An EMSA experiment was performed with a radiolabeled double-stranded oligonucleotide coding for the h2B4 promoter sequence spanning (Ϫ111 to Ϫ89) relative to the start of transcription. Ϫve lane is a binding reaction done in the absence of YT cell nuclear extract. ϩve lane is a binding reaction done in the presence of 2 ␮g pf YT cell nuclear extract. Unlabeled competitor DNAs were included in the binding reactions containing YT nuclear extract at 100 and 200 molar excess, respectively. The competitor DNAs used were wild-type (wt) cold probe; AP-1, double- stranded oligonucleotides coding for AP-1 consensus binding site. B,YT cells were treated with C1.7 mAb for the indicated time intervals at 37°C. Thereafter, nuclear protein extracts were prepared from the treated cells and analyzed for AP-1 DNA-binding activity by EMSA. DNA-binding reactions were performed with 2 ␮g of nuclear protein extract and radiolabeled double-stranded oligonucleotides coding for the h2B4 promoter FIGURE 2. Effect of actinomycin D on 2B4-stimulated YT cell cyto- sequence spanning (Ϫ111 to Ϫ89) relative to the start of transcription as toxicity. YT and target K562 and P815 cells were pretreated for2hat37°C outlined in Materials and Methods. C, Graphical representation of AP-1 with actinomycin D (20 ␮g/ml) as described in Materials and Methods.YT activation observed in B. Nuclear protein extracts incubated with anti-c-Jun cells were incubated (1 h, 37°C) without (f) or with C1.7 mAb at 200 Ab (D) and anti-JunB Ab (E). F, Graphical representation of c-Jun (o) and ng/ml concentrations (Ⅺ). Pretreated effector cells (ϫ) were then incubated JunB (u) activation observed in C and D, respectively. Arrow, AP-1-DNA (1 h, 37°C) with C1.7 mAb (200 ng/ml). Pretreated target cells (ࡗ) were complex. Bracket, Ab supershifted DNA-protein complex. N.S. are non- incubated with 2B4-stimulated effector cells. All data points in each graph speciﬁc bands. Free represents unbound probe. represents the average of four independent trials with similar results. The Journal of Immunology 6213

FIGURE 4. 2B4 stimulation results in phosphorylation of ERK and p38. YT cells were stimulated with anti-2B4 mAb and PMA for the indicated FIGURE 3. Effect of MAPK inhibitors in YT lysis of tumor cells. A, YT times at 37°C. A, Cell lysates were then prepared and analyzed by Western and target K562 and P815 cells were pretreated for1hat37°C with 10 and blotting with anti-phospho-ERK (upper panel). The same membrane was ␮ 50 M with SB203580. B, Cells were pretreated for1hat37°C with 100 stripped and reprobed with anti-ERK to check for total protein amounts ␮ f M PD089059. YT cells were incubated (1 h, 37°C) without ( ) or with (lower panel). B, Cell lysates were then prepared and analyzed by Western Ⅺ C1.7 mAb at 200 ng/ml concentrations ( ). Effector cells pretreated with blotting with anti-phospho-p38 (upper panel). The same membrane was ϫ F low ( ) or high ( ) concentrations of inhibitors were then incubated (1 h, stripped and reprobed with anti-p38 to check for total protein amounts Downloaded from ࡗ 37°C) with C1.7 mAb (200 ng/ml). Target cells pretreated with low ( )or (lower panel). high (Œ) concentrations of inhibitors were then incubated with 2B4-stimulated effector cells All data points in each graph represent the average of activity (data not shown). Thus, it appears Ras and Raf are essen- four independent trials with similar results. tial for 2B4-stimulated NK cell cytotoxicity in YT cells.

IFN-␥ release is dependent on the p38 pathway with either MEK1 or p38 decreased the natural cytotoxicity of YT http://www.jimmunol.org/ IFN-␥ production by NK cells is a major function of NK cells in cells against target K562 cells (data not shown). Previous studies response to NK cell-target cell contact (1). Our studies have re- have found that the MEK1/ERK pathway is required for NK cells vealed that binding of 2B4 by C1.7 mAb results in the increase in to acquire lymphokine-activated killer activity. However, once the cytolytic activity of YT cells and the activation of Ras, Raf, and NK cells were engaged, treatment with the inhibitor PD089059 the ERK and p38 MAPK pathways. Using similar inhibitor treat- had little effect on NK activity (44). The MEK1/ERK signaling ment protocols, we investigated whether 2B4-induced IFN-␥ pro- pathway can activate AP-1, members of the Ets-1 family, whereas duction was under the control of the same pathways. Pretreatment activation of the p38 MAPK pathway leads to NF-␬B activation of YT cells with actinomycin D and SB203580 inhibited IFN-␥

(37, 45–47). Activation of these pathways can lead to important by guest on October 2, 2021 production (Fig. 6), whereas inhibitors of Ras, Raf, and the ERK transcriptional events, such as AP-1 activation, that are essential MAPK pathway did not. The induction of IFN-␥ production re- for the engagement of NK cell cytotoxicity. quired the activity of p38 as shown by the ability of SB203580 to Signal transduction through the p38 and ERK MAPK pathways completely inhibit 2B4-induced cytokine production. requires the phosphorylation of p38 and ERK, respectively. To determine whether 2B4 ligation results in the alteration of phos- 2B4 is constitutively associated with LAT phorylation states of p38 and ERK, YT cells were stimulated with Both 2B4 and LAT are localized in the glycolipid-enriched mi- C1.7 mAb and then lysed and analyzed by Western blot. 2B4 en- crodomains (GEM) fractions of the cell membrane (50). To deter- gagement by anti-2B4 mAb resulted in tyrosine phosphorylation of mine whether LAT associates with 2B4 in NK cells, 2B4 was both p38 and ERK (Fig. 4). However, PMA stimulation of YT cells resulted in phosphorylated forms of ERK only.

2B4 stimulation of NK cells utilizes a Ras-dependent pathway In general, the ERK pathway can be activated by signaling through Ras-Raf (36, 37). It has been recently reported that Ras becomes activated after target ligation or IL-2 induction (48). To determine whether 2B4 activates NK cell function in a Ras-dependent man- ner, we performed 51Cr release assays with FTI-277-treated cells. FTI-277 is a farnesyl transferase inhibitor that prevents H-Ras from localizing to the plasma membrane and becoming functionally active both in vivo and in vitro. This induces accumulation of nonfarnesylated cytoplasmic H-Ras, which bind Raf protein to form inactive Ras-Raf complexes (49). YT cells were treated for 16hat37°C with 7.5 and 30 ␮M FTI-277, known levels that FIGURE 5. Effect of Ras inhibitor in YT lysis of tumor cells. YT and inhibit Ras activation (48). Pretreatment of YT cells with FTI-277 target K562 and P815 cells were pretreated for1hat37°C with FTI-277 at 7.5 and 30 ␮M concentrations. YT cells were incubated (1 h, 37°C) markedly diminished 2B4-stimulated NK cell cytotoxicity against without (f) or with C1.7 mAb at 200 ng/ml concentrations (Ⅺ). Effectors K562 and P815 target cells (Fig. 5). However, FTI-277 treatment cells pretreated with low (ϫ) or high (F) concentrations of inhibitors were of target cells had no effect on target lysis in the same assay (Fig. then incubated (1 h, 37°C) with C1.7 mAb (200 ng/ml). Target cells pre- 51 5). These Cr release cytotoxicity assays were also conducted treated with low (ࡗ) or high (Œ) concentrations of inhibitors were then using ZM 336372, an inhibitor of Raf. ZM336372 treatment of YT incubated with 2B4-stimulated effector cells All data points in each graph effector cells also signiﬁcantly inhibited 2B4-mediated cytolytic represent the average of four independent trials with similar results. 6214 2B4 ACTIVATION OF NK CELLS INVOLVES MULTIPLE MAPK PATHWAYS

MAPK signaling pathways activates AP-1 and other transcription factors including NF-␬B and Ets proteins. IL-2 activation of human NK cells results in an increase in ERK activity but not p38 activity, and IL-2-activated NK functions are dependent on the ERK pathway (44). FcR-triggered TNF-␣ secretion and integrin- mediated IFN-␥ production also are dependent on the ERK pathway (52, 53). In our study, YT cytotoxicity in spontaneous and rADCC against target K562 and P815 cells was increased by 2B4 ligation with Ag-specific mAb. Pretreatment of YT cells with various cell-permeable specific inhibitors revealed the activation of the Ras/Raf/ERK and p38 MAPK pathways during 2B4-mediated signaling. Previously, it has been found that p38 and MEK1/ERK signaling are involved in direct tumor cell lysis by NK cells while the c-Jun N-terminal kinase MAPK pathway does not play a sig- nificant role (44, 48, 52). The p38 MAPK pathway’s role in other FIGURE 6. p38 activation controls 2B4-induced IFN-␥ production in NK cell functions include Fc␥RIIIA-induced granule exocytosis ϫ 5 YT cells. YT cells (5 10 ) were incubated with medium only or cell- and target cell-induced IFN-␥ mRNA accumulation (52). Although permeable inhibitors as described in Materials and Methods. The untreated some studies have shown that activation of natural cytotoxicity can and treated cells were then stimulated with anti-2B4 mAb for1hat37°C under 5% CO in air. Target K562 cells (5 ϫ 104) were then added. After be Ras independent, IL-2 activation of the MAPK pathway was Downloaded from 2 completely dependent on intact Ras (44, 48). It appears that the incubation for 16 h at 37°C under 5% CO2 in air, cell-free supernatants were collected. IFN-␥ concentration was quantitated by ELISA. role of these proteins in natural cytotoxicity is complex and dependent on the activation state of the NK cells. 2B4 stimulation of NK cells may utilize distinct but overlapping immunoprecipitated from the YT cells, which was either unstimu- pathways from those engaged by other receptors and these path- lated or stimulated with anti-2B4 mAb. Immunoprecipitates were

ways regulate different aspects of NK cell function. Ras inhibitor, http://www.jimmunol.org/ analyzed by Western blot using anti-2B4 mAb and subsequently FTI-277, could not interfere with perforin and granzyme B polar- after stripping the blot was reprobed with anti-LAT polyclonal Ab. ization toward the contact point with a target cell (48). 2B4 stim- As shown in Fig. 7, LAT is associated with 2B4 consitutively. ulation of NK cells revealed the activation of the ERK and p38 Densitometric analysis indicates that the level of association be- pathway as well as an integral role for Ras (Figs. 3–5). However, tween 2B4 and LAT remains unchanged after 2B4 stimulation. pretreatment of YT cells with an inhibitor of Ras did not inhibit 2B4/target cell-stimulated production of IFN-␥ (Fig. 6). Alterna- Discussion tively, treatment of YT cells using actinomycin D and the p38 Transcription factor AP-1 has been found to be an important reg- inhibitor SB203580 did inhibit IFN-␥ production. Additionally, ulator of gene expression in leukocytes involving T and B cell SB202190, a p38 inhibitor, inhibited accumulation of target by guest on October 2, 2021 activation, Ig production, cytokine expression, cell differentiation, (K562)-induced IFN-␥ mRNA in NK cells (52). This suggests that and development (36). In NK cells, AP-1 factors have been im- 2B4-mediated IFN-␥ production is Ras/Raf independent, unlike ␤ plicated in cell activation, cytolytic activity, and cytokine produc- 1 integrin- and IL-2-activated secretion of IFN-␥ (44, 53). tion (42, 44, 51). Our previous studies in understanding the tran- Our study demonstrated that 2B4 associates constitutively with scription of the 2B4 gene in both mouse and humans has revealed LAT (Fig. 7). Bottino et al. (54) have also shown that 2B4 is that AP-1 plays an important role in the regulation of expression of constitutively associated with LAT and Ab ligation of 2B4 leads to 2B4 (40, 41). We have examined the effect of 2B4 stimulation of phosphorylation of 2B4 and LAT. Furthermore, phosporylated YT cells on AP-1 DNA-binding activity and found that AP-1 LAT recruits other signaling molecules, phospholipase C (PLC) ␥ DNA-binding activity increased significantly. Supershift EMSAs and Grb2 (54). The role of PLC␥1orPLC␥2 in natural cytotox- revealed that increases in AP-1 complexes containing JunB corre- icity has not been clearly defined. Both are found in NK cells and lated strongly with increases in AP-1 DNA-binding activity. These become tyrosine phosphorylated upon Fc␥R activation (55). In results are consistent with other findings that show an increase in JunB mRNA and the increase in JunB-containing AP-1 complexes other signaling pathways, LAT is required for FcR-dependent ␥ during natural cytotoxicity (42). phosphorylation of PLC . LAT has been found to associate with Grb2, Gads, and PLC␥1 in T cell Ag receptor-mediated signaling (56, 57). In T cells, the activation pathways through LAT may be determined by the signaling molecules that it associates with (58– 60). The Ras pathway is blocked in LAT-deficient cells, resulting in inhibition of MAPK activation and transcriptional activity of AP-1 (61, 62). On the other hand, overexpression of LAT in NK cells leads to increased ADCC and spontaneous cytotoxicity (56). LAT has emerged as a major transmembrane adaptor protein in T FIGURE 7. 2B4 is constitutively associated with LAT. YT cells were cell activation after TCR engagement by Ag-MHC (58–60, 63). lysed and immunoprecipitated with anti-2B4 mAb. Immunoprecipitates TCR engagement results in the phosphorylation of LAT which were analyzed by Western blotting with anti-2B4 mAb. The same mem- then associates with other adaptor proteins including PLC␥, Grb-2, brane was stripped and reprobed with anti-LAT Ab. Lane 1, cells stimu- Grap, phosphatidylinositol 3-kinase, and Gads which can then lated with anti-2B4 mAb (200 ng/ml) for2hat37°C before lyses; lane 2, immunoprecipitation with unstimulated YT cells; lane 3, YT cells lysed form multimeric signaling complexes by the recruitment of other and immunoprecipitated with a control IgG; and lane 4, YT cells lysed and adaptor proteins including Vav, SLP-76 Sos, Shc, Pyk-2, and Graf immunoprecipitated with protein A plus G only. The migration positions of (58–60). Many of these adaptor proteins are also expressed in NK 2B4 and LAT are indicated. cells and have been found to play various roles in the signaling The Journal of Immunology 6215 cascade in natural cytotoxicity and ADCC. Grb2-Sos complex as- References sociates with LAT and activates the Ras-dependent MEK/ERK 1. Trinchieri, G. 1989. Biology of natural killer cells. Adv. Immunol. 47:187. pathway (36, 43, 64). Many other kinases have also been impli- 2. Scott, P., and G. 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Bennett, tional distinction between cytotoxicity and cytokine production V. Kumar, and P. A. Mathew. 1999. Molecular characterization of a novel human natural killer cell receptor homologous to mouse 2B4. Tissue Antigens 54:27. (79). KIR2DL4 induction of resting NK cells induced IFN-␥ pro- 23. Nakajima, H., M. Cella, H. Langen, A. Friedlein, and M. Colonna. 1999. Acti- duction but not cytotoxicity. We have observed that the LLT-1 vating interactions in human NK cell recognition: the role of 2B4-CD48. Eur. receptor (80), another NK cell molecule, induces IFN-␥ secretion J. Immunol. 29:1676. 24. Boles, K. S., and P. A. Mathew. 2001. Molecular cloning of CS1, a novel human while failing to increase cytolytic activity (H. K. Pham, S. S. natural killer cell receptor belonging to the CD2 subset of the immunoglobulin Chuang, and P. A. Mathew, unpublished observation). Cumula- superfamily. Immunogenetics 52:302. tively, these data show that NK cell functions may be differentially 25. 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