NKG2A-expressing transfectant

Cellular Immunology 219 (2002) 57–70 www.academicpress.com

Expression of p58.2 or CD94/NKG2A inhibitory receptors in an NK-like cell line, YTINDY, leads to HLA Class I-mediated inhibition of cytotoxicity in the p58.2- but not the CD94/NKG2A-expressing transfectant Hui Lin Chuaa and Zacharie Brahmia,b,* a

b

Department of Microbiology/Immunology, Indiana University School of Medicine, Riley Hospital, RM 0615, 702 Barnhill Drive, Indianapolis, IN 46202-5200, USA Department of Medicine, Indiana University School of Medicine, Riley Hospital, RM 0615, 702 Barnhill Drive, Indianapolis, IN 46202-5200, USA Received 13 August 2002; accepted 17 September 2002

Abstract Natural killer cytotoxicity is down-regulated by HLA Class I-specific inhibitory receptors classified as killer inhibitory receptors (KIRs) or C-type lectins. The regulation of their inhibitory signaling pathways is not completely understood. The YTINDY NK-like cell line was transfected to express p58.2 KIR (YT/C143 transfectant) or CD94/NKG2A C-type lectin (YT/CD94 transfectant); and YT/C143, but not YT/CD94, cytotoxicity was down-regulated by Class I. YT/C143 and YT/CD94 expressed equally low p56lck levels, suggesting that p56lck is not absolutely required for p58.2 signaling but may be required for CD94/NKG2A signaling. Lower SHP-1 levels and activity were observed in YT/CD94 compared to YT/C143. However, increasing SHP-1 to equivalent levels in YT/ C143 did not restore inhibition in YT/CD94. Our results suggest that the combination of low p56lck and SHP-1 levels may be responsible for the absent inhibitory signal in YT/CD94. In addition, the possible expression of CD94/NKG2C activating receptor may override inhibitory signals transduced through CD94/NKG2A. Ó 2002 Elsevier Science (USA). All rights reserved.

1. Introduction Natural killer (NK)1 cells are an integral part of the innate immune system, able to mediate cytotoxicity of transformed and virus-infected cells without prior antigen sensitization [1]. The ‘‘missing-self’’ hypothesis describes the down-regulation of NK cytotoxicity through the binding of their inhibitory receptors to specific ‘‘self’’ HLA Class I ligands [2,3]. In this way, NK cells are able to distinguish normal autologous cells from virusinfected cells that show reduced levels of ‘‘self’’ Class I molecules. Such aberrant cells include those infected with herpes simplex virus [4,5] or human cytomegalovirus [6]. *

Corresponding author. Fax: +1-317-2741108. E-mail address: [email protected] (Z. Brahmi). 1 Abbreviations used: Ab, antibody; EBV, Epstein–Barr virus; ITIM, immunotyrosine-based inhibitory receptor; KIR, killer inhibitory receptor; NK, natural killer.

Transplanted allogeneic cells introduced during bone marrow transplantation may lack ‘‘self’’ HLA Class I molecules and are recognized by NK cells as ‘‘non-self,’’ stimulating NK cells to mediate graft-versus-host disease (GVHD) [7]. The HLA Class I-specific receptors in humans are divided into the C (calcium-dependent)-type lectin group or the killer inhibitory receptor (KIR) group belonging to the immunoglobulin (Ig) superfamily. The inhibitory receptors possess immunoreceptor tyrosine-based inhibitory motifs (ITIMs), I/VXYXXL/V, in their cytoplasmic domains, which are responsible for transducing the inhibitory signal [8,9]. This study focuses on the CD94/NKG2A and p58.2 KIR inhibitory receptors. The CD94/NKG2 C-type lectin complex consists of CD94 covalently associated by disulfide bonds with a member of the NKG2 family, NKG2A/B, C, or E. Whereas the CD94 component does not possess any characteristic signaling motifs in its short cytoplasmic tail, NKG2A

0008-8749/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 8 - 8 7 4 9 ( 0 2 ) 0 0 5 7 8 - 6

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and B possess cytoplasmic ITIMs [10,11]. The ‘‘activating’’ NKG2C, D, and E members lack cytoplasmic ITIMs but instead associate with ITAM-containing proteins, which can transduce activating signals. NKG2C and E associate with the ITAM-containing DAP12, whereas the NKG2D homodimer associates with DAP10 [12–15]. Recent findings identify the nonclassical HLA-E as the predominant ligand recognized by CD94/NKG2A, B, and C [16–19]. The KIR family of receptors is characterized according to the two (KIR2D) or three (KIR3D) Ig-like domains they possess in their extracellular region. They are further divided according to their cytoplasmic domains, which are either long (L) and possess one or two ITIMs; or short (S) and without ITIMs, but which associate with DAP-12 through a lysine residue in their transmembrane domain [13]. The ligand for p58.2 (KIR2DL) is HLA-Cw3 [20,21]. Blocking of NK activation involves simultaneous engagement of an activating receptor such as CD 16 (FccRIIIA) or 2B4 and a Class I inhibitory receptor such that components of the two opposing pathways are brought into close proximity with each other. Following receptor ligation, p56lck associated with the activating receptor [22–26] becomes activated and phosphorylates tyrosine residues in the cytoplasmic ITIMs of the inhibitory receptor. The SHP-1 phosphatase is then recruited to the phosphorylated ITIMs by its SH2 domains where it is activated to dephosphorylate components of the activating signal such as the CD3f chain, ZAP70 and PLCc1/2 proteins [27,28]. The compensatory role of SHP-2 phosphatase in cells lacking SHP-1 has been suggested [29]. The inhibitory signaling pathway has not been fully elucidated and whether pathways utilized by the two types of inhibitory receptors are distinct or differentially regulated is not known. To investigate this, we used the YTINDY NK-like leukemic cell line, which was previously derived by serial passaging of the parental YT cell line [30]. YTINDY was transfected to express CD94/ NKG2A or p58.2 and the transfectants, YT/CD94 and YT/C143, were obtained respectively. The YT/C143 transfectant showed down-regulation of cytotoxicity against target cells expressing the HLA-Cw3 ligand, whereas the YT/CD94 transfectant did not show downregulation of cytotoxicity against target cells expressing the HLA-E ligand. From our results we concluded that the absent inhibitory signal in YT/CD94 may be due to low p56lck and SHP-1 expression as well as the possible expression of the activating CD94/NKG2C.

Harlan (Indianapolis, IN). The anti-NKG2A Ab, Z199 was a gift from Dr. F. Navarro (Hospital Universitario de la Princesa, Madrid, Spain); and the anti-CD94 Ab, NKH3 from Dr. M.J. Robertson (Indiana University School of Medicine, Indianapolis, IN). Anti-phosphotyrosine (4G10), -p56lck and -SHP1 Abs were purchased from Upstate Biotechnology (Lake Placid, NY), anti-SHP2 Ab from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-HLA Class I, W6/32 from Sera Lab (Crawley Down, Sussex, UK). Anti-p58.2 (GL183 clone) and anti-2B4 (C1.7.1 clone) Abs were purchased from Immunotech (Miami, FL). The anti-p56lck Ab used in immunoprecipitation experiments was purchased from Zymed (San Francisco, CA). Isotype (IgG) control and secondary goat anti-mouse IgG Abs were purchased from BD-Pharmingen (San Diego, CA). The DMRIE-C transfection reagent was purchased from Life Technologies (Gaithersburg, MD). 2.2. Cell lines

2. Materials and methods

The 721.221 cell line and its transfectants, expressing HLA-B7 and -Cw3 molecules, as well as the vector control transfectant, 721.221/pHebo, were obtained from Dr. C. Lutz (University of Iowa, Iowa City, IA). The NKL cell line was kindly provided by Dr. M.J. Robertson (IU School of Medicine, Indianapolis, IN) and the Jurkat cell line from Dr. P.H. Krammer (German Cancer Research Center, Heidelberg, Germany). The JCaM1.6 and K562 cell lines were purchased from the American type Culture Collection (Rockville, MD). The YTINDY NK-like cell line was previously derived in our laboratory by serial passaging of the parental YT cell line [30]. The YT/CD94 clone was derived by cotransfection of YTINDY with the pSV2neo selection plasmid and the pJFE14/LL300 expression vector, containing the cDNA encoding CD94. The YT/neo vector control cell line was derived by co-transfection of YTINDY with pSV2neo and the empty pJFE14 plasmid. YTINDY was also transfected to express p58.2 KIR to obtain the YT/C143 clone as described by Tarazona et al. [31]. The YT/C143, YT/CD94, and YT/ neo transfectants were gifts from Drs. A.G. Brooks and F. Borrego (NIH, Bethesda, MD). All cell lines were HLA-typed in our laboratory using standard techniques; and routinely screened for mycoplasma contamination [32]. Cells were cultured in RPMI 1640 supplemented with 100 U/ml penicillin, 50 lg/ml streptomycin, and 2 mM L -glutamine, 10 mM non-essential amino acids, and 10% heat-inactivated fetal calf serum or cosmic calf serum.

2.1. Antibodies and reagents

2.3. Flow cytometry analysis

The anti-CD94 monoclonal Ab, HP3B1; HRP- and FITC-conjugated secondary Abs were purchased from

Cells were prepared for flow cytometry analysis by indirect immunofluorescence staining. The cells (5
105 )

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were incubated with the primary Ab for 30 min on ice, washed with PBS containing 1% FCS and incubated for a further 30 min with a secondary FITC-conjugated Ab. The cells were washed, fixed in 2% paraformaldehyde and analyzed using the FACScan flow cytometer and CellQuest software (Becton–Dickinson, San Jose, CA). Background immunofluoresence was determined by analyzing cells that had been incubated with an isotype control Ab or the secondary Ab alone. 2.4. Cytotoxicity assays Effector cell lytic activity was determined using the 4h chromium-release assay as previously described [33]. Briefly, 2
106 target cells were labeled with 100 lCi radioactive sodium [51 Cr]chromate (NEN Life Science Products, Boston, MA) for 1 h; and 1
104 (100 ll) target cells were incubated with effector cells at a ratio of 20:1 (effector-target cells) in 96-well microtiter plates (COSTAR, Corning, NY) at 37 °C for 4 h. Samples were prepared in triplicate wells. After incubation, the supernatants were collected using a harvesting system (Skatron Instruments, Lier, Norway) and the amount of radioactivity collected was measured using a Beckman gamma scintillation counter (Beckman–Coulter, Brea, CA). Lytic activity was calculated according to the formula: %51 Cr-release ¼

ðSample cpm  spontaneous cpmÞ ðMaximum cpm  spontaneous cpmÞ
100:

Maximum cpm refers to the radioactive count in 100 ll of labeled target cells, spontaneous cpm to the count released spontaneously from target cells alone and sample cpm to the count released from target cells incubated with effector cells. Spontaneous release of 51 Cr was consistently less than 20%. Alternatively, the JAM test was used to determine effector cell DNA fragmentation activity [34]. Target cells (2
106 ) were labeled with 30 lCi [3 H]thymidine (NEN Life Science Products, Boston, MA) and the assay was set up as described above. After a 4-h incubation at 37 °C, the supernatants were harvested onto glass fiber filter papers using a harvesting system (Skatron Instruments, Lier, Norway). The amount of radioactivity retained on the filter papers was determined using a Beckman scintillation counter. DNA fragmentation activity was calculated according to the formula:

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collected from wells containing target cells incubated with effector cells. Data were analyzed by the StudentÕs t test using the GraphPad software (San Diego, CA) and p values of less than 0.05 were considered statistically significant. 2.5. Cell activation To stimulate tyrosine phosphorylation of cellular proteins, cells were treated with sodium pervanadate (5 ll/ml cell suspension) at room temperature for 10 min. Sodium pervanadate was prepared by combining 50 mM hydrogen peroxide (Fisher Scientific, Pittsburg, PA) and 48 mM sodium orthovanadate (Sigma, St. Louis, MO) and incubating at room temperature for 10 min [35,36]. Alternatively, cells were treated with anti-2B4 Ab and anti-CD94 (HP3B1) or -p58.2 (GL183) Abs, in addition to a secondary cross-linking goat anti-mouse Ab for 10 min at 37 °C. The cells were then incubated in lysis buffer (30 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1% NP-40 (v/v), 50 mM NaF, 1 mM Na orthovanadate, 2 mM EDTA, 4 mM PMSF, and 100 lg/ml each of leupeptin, aprotinin, and pepstatin A) for 30 min on ice. Cell lysates were immunoprecipitated using anti-2B4 Ab, immune complexes were captured using Protein G– agarose (Roche Molecular Biochemicals, Indianapolis, IN), boiled in 2
SDS sample buffer, then resolved by 10% SDS–PAGE and transferred onto a nitrocellulose membrane for Western blot analysis. 2.6. Western blot analysis Resolved protein samples were transferred onto a nitrocellulose membrane, which was then treated with anti-p56lck , -SHP1 or -SHP2 Abs, followed by a secondary HRP-conjugated Ab and the ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ). Phosphorylated proteins were visualized by treating the nitrocellulose membrane with the anti-phosphotyrosine Ab, 4G10, and the ECL detection system. To achieve equal loading of samples, protein concentrations of samples were determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). The same amount of each sample used for Western blot analysis was resolved on a separate gel by SDS–PAGE and protein bands were visualized with either Coomassie blue (Bio-Rad, Hercules, CA) or by silver staining [37]. 2.7. p56lck in vitro tyrosine kinase assay

% DNA fragmentation ¼

4-h spontaneous cpm  sample cpm
100: 4-h spontaneous cpm

Four-hour spontaneous cpm is the amount of radioactivity collected from wells containing target cells only; and sample cpm is the amount of radioactivity

Cell lysates were pre-cleared with Protein G–agarose and then immunoprecipitated with 3 lg anti-p56lck (Zymed) Ab overnight at 4 °C. Immune complexes were captured with Protein G–agarose and washed in kinase assay buffer (30 mM Tris–HCl, pH 7.5, 10 mM MnCl2 ). The kinase reaction was carried out for 5 min at 30 °C in

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50 ll of kinase buffer containing 20 mM ATP and 20 lCi ½c-32 P]ATP with 3 lg of enolase (Sigma) as substrate. The kinase reaction was stopped by boiling in 2
SDS sample buffer. The samples were resolved by 7.5% SDS– PAGE, transferred onto a nitrocellulose membrane and kinase activity was detected by exposing the membrane to scientific imaging film (Eastman Kodak Co.). Western blot analysis was also carried out to determine the amount of p56lck from each sample that was loaded into the kinase reaction. 2.8. Transfection of YT/CD94 DMRIE-C transfection reagent (Life Technologies, Gaithersburg, MD) was used according to manufacturerÕs instructions. YT/CD94 cells were transfected with the pMEPC4 plasmid containing the cDNA for SHP-1, provided by Dr. D.L. Durden (Herman B. Wells Center, IU School of Medicine). Vector control cells were obtained by transfection with the empty pMEPC4 plasmid. Transfected cells were sub-cloned to obtain single clones.

3. Results 3.1. HLA Class I-specific inhibitory receptor expression in YTINDY transfectants The YTINDY cell line was transfected to express either CD94/NKG2A or p58.2 KIR. Surface expression of inhibitory receptors was determined by flow cytometry analysis. The YT/CD94 clone showed positive staining with the Z199 (anti-NKG2A) Ab. In contrast, the YT/neo vector control transfectant did not, whereas another NK cell line NKL, which expresses endogenous CD94/NKG2A, showed positive staining with Z199 Ab (Fig. 1A). YT/CD94 and NKL also showed positive staining with another CD94specific Ab, HP3B1 (data not shown). Therefore, transfection of YTINDY with the cDNA for CD94 led to the expression of both CD94 and NKG2A components on the cell surface. The YT/C143 clone showed positive staining with the GL183 (anti-p58.2) Ab, but not the untransfected YTINDY cell line (Fig. 1B).

Fig. 1. (A) NKL and YT/CD94 showed positive staining with anti-NKG2A (Z199) Ab but not YT/neo. Similar staining profiles were observed with the anti-CD94 Abs, HP3B1, and NKH3 (data not shown). (B) YT/C143 cells showed positive staining with the anti-p58.2 (GL183) Ab, but not YTINDY. Filled histograms represent cells stained with the primary and the secondary FITC-conjugated Abs, whereas outlined histograms represent cells stained with the secondary Ab only.

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3.2. To determine co-expression of CD94 and NKG2A in YT/CD94 The CD94/NKG2A receptor complex consists of covalently-linked CD94 and NKG2A components. The NKG2A component of 43 kDa molecular weight, possesses cytoplasmic ITIMs containing tyrosine residues, which become phosphorylated following CD94/ NKG2A ligation. We attempted to show that the CD94 and NKG2A components were co-expressed as a heterodimer in YT/CD94. YT/CD94 cells were treated with sodium pervanadate to promote the accumulation of tyrosine-phosphorylated proteins [35,36] including the ITIM-containing NKG2A. Cell lysates were immunoprecipitated with the anti-CD94 Ab, HP3B1 and immune complexes were captured and analyzed by Western blotting using the HRP-conjugated antiphosphotyrosine specific Ab (4G10). A 43 kDa phosphorylated band corresponding to the molecular weight of NKG2A was observed. In contrast, immunoprecipitation of YT/CD94 cell lysate with an IgG2a isotype control Ab for HP3B1 did not yield a 43 kDa phosphorylated band (Fig. 2A). In a similar experiment, YT/ CD94 and the vector control YT/neo cells were treated

Fig. 2. Co-expression of CD94 and NKG2A in YT/CD94 cells. YT/ CD94 cells were treated with sodium pervanadate and cell lysates were immunoprecipitated with (A) anti-CD94 (HP3B1) Ab, the isotype control (IgG) Ab; or (B) the anti-NKG2A (Z199) Ab. The samples were analyzed by Western blot analysis using the antiphosphotyrosine (4G10) Ab and a phosphorylated band of approximately 43 kDa corresponding to NKG2A was observed for YT/ CD94 immunoprecipitated with anti-CD94 Ab, but not in YT/neo. (C) NKL cells were similarly treated with sodium pervanadate and cell lysates were immunoprecipitated with HP3B1 or Z199, yielding a phosphorylated band of 43 kDa molecular weight corresponding to NKG2A.

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with sodium pervanadate and cell lysates were immunoprecipitated with the anti-NKG2A Ab, Z199. The immunoprecipitated YT/CD94 cell lysate sample showed a phosphorylated band at 43 kDa corresponding to the molecular weight of NKG2A, whereas the YT/ neo sample did not (Fig. 2B). NKL was similarly treated with sodium pervanadate and cell lysates were immunoprecipitated with HP3B1 or Z199. A 43 kDa phosphorylated band, corresponding to the molecular weight of NKG2A, was observed following immunoprecipitation with the HP3B1 and Z199 Abs, but not with the isotype control Ab (Fig. 2C). Together with the flow cytometry data (Fig. 1A), these results (Figs. 2A and B) suggest that CD94 and NKG2A may be co-expressed as a heterodimer in the YT/CD94 transfectant.

Fig. 3. 721.221 cells transfected to express the HLA-B7 and -Cw3 Class I molecules showed positive staining with the pan anti-Class I Ab, W6/ 32; but not the 721.221/pHebo vector control cell line. Solid-lined histograms represent cells stained with the primary and secondary FITC-conjugated Abs, whereas dotted histograms represent cells stained with the secondary Ab only.

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3.3. HLA Class I-mediated inhibition of cytotoxicity in YT/C143, but not in YT/CD94 The cytotoxic activity of YT/CD94 and YT/C143 was determined by the 4-h 51 Cr-release assay that measures the degree of target cell lysis [33] and by the 4-h JAM test [34] that measures the degree of target cell DNA fragmentation, which is a hallmark of apoptosis. The target cells used in these assays are derivatives of the HLA Class I-null 721.221 cell line [38], each transfected to express a specific HLA Class I molecule, namely HLA-B7 or -Cw3; as well as the vector control Class Ideficient transfectant, 721.221/pHebo. 721.221 cells express intracellular HLA-E, however, HLA-B7 or -Cw3 expression in 721.221 cells up-regulates HLA-E surface expression [18,19,39,40]. The 721.221/B7 and /Cw3 cell lines consistently showed positive staining with the pan HLA Class I-specific Ab, W6/32, when analyzed by flow cytometry. As expected, 721.221/pHebo did not show positive staining with the W6/32 Ab (Fig. 3). Results from the 51 Cr-release assay showed that YT/ CD94 did not exhibit significant down-regulation of cytotoxicity against 721.221/B7 and /Cw3 target cells, compared to 721.221/pHebo target cells (p > 0:05, not

significant). In contrast, NKL, the NK cell line expressing endogenous CD94/NKG2A, showed downregulation of cytotoxicity against 721.221/B7 and /Cw3 cells, compared to 721.221/pHebo cells (p < 0:05). As expected, the YT/neo vector control cells showed similar cytotoxic activity against the target cells tested (p > 0:05, not significant) (Fig. 4A). This suggests that 721.221/B7 and /Cw3 cells express sufficient HLA-E levels to inhibit the cytotoxicity CD94/NKG2Aexpressing NK cells. Similar results were obtained for the JAM test (Fig. 4B). YT/C143 showed significant down-regulation of cytotoxicity in response to HLA Class I, against target cells expressing the HLA-Cw3 ligand for p58.2 KIR (p < 0:05). In contrast, YTINDY showed similar cytotoxic activity against 721.221/Cw3 and /pHebo target cells (p > 0:05, not significant) using the 51 Cr-release assay (Fig. 4C) and the JAM test (Fig. 4D). 3.4. Comparative levels of p56lck , SHP-1, and SHP-2 in YTINDY and its transfectants Cellular expression levels of described components of the inhibitory signaling pathway, namely p56lck , SHP-1,

Fig. 4. YT/CD94 cells did not show HLA Class I-mediated down-regulation of cytotoxicity against the 721.221/B7 and /Cw3 TCs (N.S., not significant); whereas NKL cells showed down-regulation of cytotoxicity against 721.221/B7 and /Cw3, but not against 721.221/pHebo, as observed using the Cr-release assay (A) and the JAM test (B). YT/C143 cells, in contrast to YTINDY, showed down-regulation of cytotoxicity against 721.221/Cw3 TCs, but not against the 721.221/pHebo TCs using the Cr-release assay (C) and the JAM test (D). The results shown are representative of three experiments.

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and SHP-2 [9,27,28] were compared in YTINDY and its transfectants by Western blot analysis. An inhibitory signal is initiated following simultaneous engagement of activating and Class I inhibitory receptors. The ITIM tyrosine residues of the inhibitory receptor become phosphorylated by p56lck src tyrosine kinase. SHP-1 binds to the phosphorylated ITIM through its SH2 domains and becomes activated to dephosphorylate components of the activating signal. YTINDY and its transfectants expressed approximately 2.5-fold lower levels of p56lck compared to the NKL cell line. Jurkat cells were included as a positive control, whereas JCaM1.6 cells, which express a truncated form of p56lck , were included as a negative control (Fig. 5). The cells were also analyzed for p56lck kinase activity (Fig. 6A, upper panel); and Western blot analysis was carried out to determine the amount of p56lck loaded into the kinase reaction for each sample (Fig. 6A, lower panel). Although YT/C143 and YT/CD94 expressed similar levels of p56lck (Fig. 5), densitometry analysis of the p56lck kinase assay and the corresponding Western blot autoradiograms showed that p56lck isolated from YT/C143 had approximately 2.5-fold lower p56lck kinase activity per molecule compared to p56lck isolated from YT/ CD94 (Fig. 6B). The role of SHP-1 in the inhibitory signal has been established [9]; in addition, a possible role of SHP-2 in the inhibitory signaling pathway has been suggested [29]. The expression level of SHP-1 in YT/CD94 was approximately 2-fold lower than that of the other cell lines tested (Fig. 7A), whereas SHP-2 levels were comparable (Fig. 7B). The results suggest that the

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Fig. 6. YT/C143 expressed the lowest level of p56lck kinase activity per molecule of immunoprecipitated p56lck . (A) p56lck was immunoprecipitated from whole cell lysates, subjected to a kinase assay and the samples were resolved by 10% SDS–PAGE. Whole cell lysates were also resolved and analyzed by p56lck Western blot analysis. (B) Densitometry analysis was used to normalize kinase activity levels to the amount of p56lck loaded into the kinase assay for each sample.

absence of HLA Class I-mediated inhibition in YT/ CD94 may be in part due to its low SHP-1 expression levels; in addition, SHP-2 does not appear to play a role in inhibitory signal transduction in YT/CD94. 3.5. Reduced 2B4 receptor phosphorylation following simultaneous ligation with p58.2 KIR

Fig. 5. YTINDY and its transfectants expressed lower levels of p56lck compared to NKL. The Jurkat and JCaM1.6 cell lines were included as positive and negative controls, respectively. Whole cell lysates were resolved by 10% SDS–PAGE and Western blot analysis was carried out using the anti-p56lck and secondary HRP-conjugated Abs (upper panel). Equal protein loading was ensured by resolving an equal amount of each sample on another gel, which was then stained with Coomassie blue (lower panel).

Inhibitory signal transduction through HLA Class I-specific inhibitory receptors is initiated upon simultaneous engagement of an activating and a Class I inhibitory receptor. Following receptor co-ligation, tyrosine residues of the cytoplasmic ITIMs belonging to the inhibitory receptor become phosphorylated by p56lck . The SHP-1 phosphatase is then recruited to the phosphorylated ITIM tyrosine residues by its SH2 domains to dephosphorylate components of the activating signal. To determine if these initial events occurred in YT/CD94, the 2B4 and CD94/NKG2A receptors were simultaneously ligated in YT/CD94 cells. The 2B4 activating receptor has been shown to induce ‘‘natural

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Fig. 7. (A) YT/CD94 expressed the lowest level of SHP-1 whereas (B) SHP-2 levels were comparable among the cell lines tested. Whole cell lysates were resolved by 10% SDS–PAGE and Western blot analysis was carried out using anti-SHPl or -SHP2 Abs accordingly (upper panels). Equal protein loading was ensured by resolving an equal amount of each sample on another gel, which was then stained with Coomassie blue (lower panels).

killing’’ in NK cells; and simultaneous ligation of 2B4 and CD94/NKG2A or p58.2 leads to 2B4 dephosphorylation by SHP-1 as well as inhibition of NK cytotoxicity [41]. To determine if the early inhibitory signaling event of 2B4 dephosphorylation occurred in YT/CD94, the 2B4, and CD94/NKG2A receptors were simultaneously ligated in YT/CD94 cells. NKL, YTINDY, and its transfectants express 2B4 (Fig. 8). YT/CD94 cells were treated with anti-2B4 Ab with or without antiCD94 (HP3B1) Ab. Cell lysates were immunoprecipi-

tated with anti-2B4 Ab and samples were analyzed by Western blot analysis using the anti-phosphotyrosine 4G10 HRP-conjugated Ab. A heavily phosphorylated band of approximately 66 kDa corresponding to 2B4 was immunoprecipitated following ligation of the 2B4 receptor; however, 2B4 reduced phosphorylation was not observed following simultaneous ligation of the 2B4 and CD94/NKG2A receptors. In addition, we were unable to detect a 43 kDa band corresponding to the molecular weight of NKG2A (Fig. 9A). In YT/C143

Fig. 8. YTINDY and its transfectants showed positive staining with the anti-2B4 (C1. 7.1 clone) Ab. K562 cells were included as the negative control. Filled histograms represent cells stained with the anti-2B4 and secondary FITC-conjugated Abs; whereas outlined histograms represent cells stained with the secondary Ab only.

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Fig. 9. Reduced 2B4 phosphorylation was observed in YT/C143 cells following 2B4 and p58.2 KIR ligation, but not in YT/CD94 cells following simultaneous ligation of 2B4 and CD94/NKG2A. (A) YT/CD94 and (B) YT/C143 cells were treated with anti-2B4 Ab, with or without anti-CD94 or anti-p58.2 Abs, respectively. Immunoprecipitation was carried out and samples were analyzed by Western blotting using the 4G10 anti-phosphotyrosine Ab (upper panels). Equal protein loading was ensured by resolving an equal amount of each sample on another gel and protein bands were visualized by silver staining (lower panels).

cells, ligation of the 2B4 receptor led to the phosphorylation of 2B4; and simultaneous ligation of 2B4 and p58.2 led to reduced phosphorylation of the 2B4 receptor. We were, however, unable to detect a 58 kDa phosphorylated band corresponding to the molecular weight of p58.2 (Fig. 9B). 3.6. Increased SHP-1 expression in YT/CD94 We hypothesized that low SHP-1 levels may be responsible for the absence of HLA Class I-mediated inhibition of cytotoxicity in YT/CD94. YT/CD94 was transfected with the pMEPC4 plasmid containing the cDNA for SHP-1 to increase its SHP-1 expression. By SHP-1 Western blot analysis, four clones, DM2-8, -10, -12, and -19 were found to express approximately 2-fold higher SHP-1 levels compared to untransfected YT/ CD94. YT/CD94vec, the vector control transfectant of YT/CD94 was obtained by transfection of YT/CD94 cells with the empty pMEPC4 plasmid alone (Fig. 10A). Densitometric values of SHP-1 expression in the transfectants were normalized against that of untransfected YT/CD94 (Fig. 10B). CD94/NKG2A expression was analyzed in NKL, YT/CD94, and the four DM2 clones over a period of several weeks. Flow cytometry analysis

showed that NKL, YT/CD94 and its DM transfectants stained with the anti-NKG2A (Z199) Ab expressed varying but similar CD94/NKG2A levels (Fig. 11). The DM2 clones were tested for their cytotoxic activity against 721.221/Cw3 target cells using the JAM test, however, down-regulation of cytotoxicity in response to Class I was not observed in any of the DM2 clones. The YT/C143 cell line, which exhibits HLA Class I-mediated inhibition of cytotoxicity was included in the experiment as a positive control (Fig. 12). Therefore, increasing SHP-1 expression in YT/CD94 did not restore the inhibitory signal, suggesting that factors other than low SHP-1 are responsible for the absence of HLA Class I-mediated inhibition of cytotoxicity in YT/CD94.

4. Discussion The ‘‘missing-self’’ hypothesis describes the downregulation of NK cytotoxicity through the ligation of NK inhibitory receptors to their HLA Class I ligands [2,3]. The YTINDY NK-like leukemic cell line was previously derived by serial passaging of the parental YT cell line and was described to mediate cytotoxicity against EBV-transformed cell lines regardless of their

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Fig. 11. The DM2 clones showed similar CD94/NKG2A expression levels compared to YT/CD94 and NKL by flow cytometry analysis of cells stained with the anti-NKG2A (Z199) Ab. Change in mean channel fluorescence (D MCF) values from five experiments are shown.

Fig. 10. (A) YT/CD94 cells transfected to increase SHP-1 expression yielded four clones showing higher SHP-1 expression levels compared to YT/CD94 and the vector control transfectant. Western blot analysis was carried out using the anti-SHP1 Ab (upper panel). Equal protein loading was ensured by resolving an equal amount of each sample on another gel, which was then stained with Coomassie blue (lower panel). (B) Densitometry analysis was used to normalize SHP-1 levels of the clones against the level expressed by YT/CD94.

Fig. 12. The JAM test was used to determine the cytotoxic activity of the DM2 clones against 721.221/pHebo and /Cw3 TCs. In contrast to YT/C143, the DM2 clones did not show down-regulation of cytotoxicity in response to Class I against the 721.221/Cw3 TCs. The results shown are representative of two experiments.

HLA Class I expression [30]. In accordance with the ‘‘missing-self’’ hypothesis, the apparent unresponsiveness of YTINDY to HLA Class I expressed by the various cell lines suggests that YTINDY expresses a limited repertoire of Class I-specific inhibitory receptors, if any. Our preliminary flow cytometry analysis showed that YTINDY does not express either CD94/NKG2A, p70 KIR (data not shown) or p58.2 KIR (Fig. 1B). In this study, YTINDY was transfected to express either CD94/NKG2A or p58.2 HLA Class I inhibitory receptors and two clones, designated as YT/CD94 and YT/ C143 were obtained, respectively. Interestingly, YTINDY transfected with the cDNA encoding CD94 showed surface expression of the NKG2A component (Fig. 1A). Studies have shown that the YT cell line expresses NKG2A intracellularly as an immature form;

and that NKG2A expression on the cell surface as a mature protein requires co-expression of its CD94 counterpart [11]. Cell surface expression of both CD94 and NKG2A receptor components was detected by flow cytometry (Fig. 1A). We attempted to determine if CD94 and NKG2A were co-expressed as a heterodimer in YT/CD94 by inducing tyrosine phosphorylation of the NKG2A component and co-immunoprecipitating it using anti-CD94 Ab. Treatment of cells with sodium pervanadate promotes the accumulation of phosphotyrosine-containing proteins by stimulating tyrosine phosphorylation of cellular proteins and inhibiting tyrosine phosphatase activity [35,36]. Cell lysates from YT/CD94 cells treated with sodium pervanadate were immunoprecipitated with the anti-CD94 (HP3B1) Ab and a 43 kDa phosphorylated protein corresponding to

H. Lin Chua, Z. Brahmi / Cellular Immunology 219 (2002) 57–70

the molecular weight of NKG2A was co-immunoprecipitated (Fig. 2A). Taken together, the flow cytometry and immunoprecipitation data suggest that CD94 and NKG2A are co-expressed as a heterodimer in YT/ CD94. Transfection of 721.221 cells to express specific HLA Class I molecules such as HLA-B7 and -Cw3 up-regulates HLA-E surface expression, since the leader peptides of HLA-B7 and -Cw3 are able to bind to the peptide-binding groove of HLA-E to stabilize its expression on the cell surface [18,19,39,40]. 721.221/B7 and /Cw3, as well as the vector control transfectant 721.221/pHebo, were used as target cells in the chromium-release assay and JAM test to measure the cytotoxicity of the NK effector cells. HLA Class I-mediated inhibition of cytotoxicity was observed in YT/C143 against 721.221/Cw3 target cells expressing the HLACw3 ligand for p58.2 (Figs. 4C and D). In contrast, YT/ CD94 did not show similar inhibition against the 721.221/B7 and /Cw3 target cells. However, the NKL cell line, that expresses endogenous CD94/NKG2A, showed down-regulated cytotoxicity against 721.221/B7 and /Cw3 target cells. Besides CD94/NKG2A, NKL also expresses ILT2/LIR1 and KIR2DL4, both of which recognize HLA-G [19,42], but not HLA-B7 or -Cw3. Therefore, the down-regulated cytotoxicity observed is most likely due to inhibition through CD94/NKG2A. The YT/neo vector control did not show down-regulated cytotoxicity against the 721.221/B7 or /Cw3 cell lines tested (Figs. 4A and B). We were unable to determine HLA-E surface expression on transfected 721.221 cells due to commercial unavailability of HLAE-specific Abs, such as 3D12 [19]. However, there appears to be a sufficient level of up-regulated HLA-E expression in these cells to down-regulate the cytotoxicity of NKL cells expressing the CD94/NKG2A receptor. The absence of a functional inhibitory signal in YT/ CD94 was investigated. Transduction of the inhibitory signal through HLA Class I inhibitory receptors in NK cells is initiated upon simultaneous engagement of activating and Class I inhibitory receptors. Following receptor ligation, the p56lck src tyrosine kinase associated with the activating receptor becomes activated and phosphorylates tyrosine residues in the cytoplasmic ITIMs of the inhibitory receptor. The p59fyn src tyrosine kinase does not appear to play a similar role [27]. The SHP-1 phosphatase is then recruited to the phosphorylated ITIMs by its SH2 domains where it is activated to dephosphorylate components of the activating signal [27,28]. Thus, it is necessary for simultaneous ligation of receptors to occur such that components of the activating and inhibitory signaling pathways are brought into close proximity to each other, allowing SHP-1 phosphatase activity to block the activating signal. Although the role of SHP-1 in the inhibitory signal has been established [9], the compensatory role of another

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phosphatase, SHP-2, was suggested when NK cells from viable motheaten (mev =mev ) or SHP1-deficient motheaten mice (me/me) [29] or transgenic mice expressing a catalytically inactive dominant-negative SHP-1 mutant [43] showed residual phosphatase activity. In addition, in vitro studies showed both SHP-1 and SHP-2 capable of binding to the ITIMs of human and murine inhibitory receptors on phosphorylation of critical tyrosine residues [44,45]. Although both SHP-1 and -2 are recruited to the NKG2A component following pervanadate treatment, only SHP-1 is recruited upon crosslinking with the Z199 (anti-NKG2A) Ab [46]. Other in vitro studies only tested for SHP-1 recruitment following Ab treatment of human NK clones [27,28]. Studies comparing the two phosphatases showed that SHP-1 has a higher catalytic activity as well as a more efficient level of binding to phosphorylated tyrosine residues compared to SHP-2 [44,47]. Western blot analysis showed that YTINDY and its transfectants expressed approximately 2.5-fold lower levels of p56lck as compared to NKL, a CD94/NKG2Aexpressing NK cell line that exhibited HLA Class Imediated inhibition of cytotoxicity. Although both YT/ C143 and YT/CD94 both express similar p56lck levels (Fig. 5), YT/C143 showed approximately 2.5-fold lower p56lck kinase activity compared to YT/CD94 (Fig. 6). In addition, the expression level of SHP-1 in YT/CD94 was approximately 2-fold lower than that of the other cell lines tested (Fig. 7A), whereas SHP-2 levels were comparable (Fig. 7B). The variation observed in the levels of SHP-1 expression in YTINDY and its transfectants may be due to the post-transfection selection of the transfectant clones differing in their level of SHP-1 expression from that of YTINDY. The higher p56lck kinase activity per molecule observed in YT/CD94 is consistent with its low SHP-1 expression, since SHP-1 is able to inactivate p56lck through dephosphorylation of the tyr 394 residue [48]. Therefore, the results suggest that the absence of HLA Class I-mediated inhibition in YT/CD94 might be due to its low expression levels of SHP-1. In addition, SHP-2 does not appear to play a compensatory role in the inhibitory signaling pathway in YT/CD94. We then wanted to determine if the low levels of p56lck in YT/C143 and YT/CD94 were sufficient to phosphorylate inhibitory receptor ITIMs and to recruit SHP-1 for dephosphorylation of activating signal components following activating and inhibitory receptor coligation. The CD16 and 2B4 activating receptors induce antibody-dependent cellular cytotoxicity (ADCC) and ‘‘natural killing’’ in NK cells respectively; and simultaneous engagement of Class I inhibitory receptors with either CD16 or 2B4 leads to inhibition of NK cytotoxicity [9,41,49]. Because YTINDY expresses 2B4 (Fig. 8) but not CD16 [30], 2B4 was used as the activating receptor in the following experiments. Studies have shown that 2B4 becomes phosphorylated by p56lck following

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2B4 ligation and that during inhibitory signal transduction, SHP-1 phosphatase is recruited to the signaling complex to dephosphorylate 2B4 [41]. The importance of 2B4-induced ‘‘natural killing’’ in NK cells in clearing EBV infection is evident in the inability of X-linked proliferative disease (XLP) patients to clear EBV-infection due to a defect in 2B4 signaling [50]. Ligation of 2B4 in YT/CD94 and YT/C143 led to 2B4 phosphorylation; and simultaneous engagement of 2B4 and p58.2 KIR in YT/C143 led to reduced 2B4 phosphorylation (Fig. 9B). In contrast, reduced 2B4 phosphorylation was not observed in YT/CD94 following 2B4 and CD94/ NKG2A co-ligation (Fig. 9A). These results suggest that there is insufficient SHP-1 activity recruited to the signaling complex to dephosphorylate activating signal components such as 2B4 and is consistent with the low SHP-1 expression levels in YT/CD94 compared to YT/ C143. To investigate whether low SHP-1 expression in YT/ CD94 is responsible for the absence of an inhibitory signal, YT/CD94 was transfected with the pMEPC4 plasmid to increase its SHP-1 expression and four single clones were obtained, DM2-8, -10, -12, and -19. The SHP-1 expression levels of the DM2 clones were approximately 2-fold higher than that of YT/CD94 (Fig. 10). In addition, the DM2 clones exhibited comparable expression levels of CD94/NKG2A to YT/CD94 and NKL. It is of note that although the average CD94/ NKG2A expression level in NKL was slightly higher than YT/CD94 and the DM2 transfectants, the CD94/ NKG2A expression levels were not significantly different (Fig. 11). Using the JAM test, we found that none of the DM2 clones exhibited down-regulation of cytotoxicity in response to Class I, (Fig. 12). Therefore, our results suggest that factors besides low SHP-1 expression levels are responsible for the absence of an inhibitory signal in YT/CD94. Low p56lck levels in YT/CD94 may also be partly responsible for the absent inhibitory signal. YT/CD94 and YT/C143 both expressed a similar level of p56lck , which was 2.5-fold lower than in NKL (Fig. 5). In addition, 2B4 phosphorylation was observed in NKL, YT/ CD94, and YT/C143 cells following 2B4 ligation implying the induction of src tyrosine kinase activity. However, we were unable to discern whether NKG2A or p58.2 phosphorylation had occurred following 2B4 and Class I inhibitory receptor ligation (Fig. 10). The low level of p56lck expressed in YT/CD94 may be insufficient to phosphorylate NKG2A and the absence of NKG2A phosphorylation will thus prevent recruitment and activation of SHP-1. Since YT/C143 was able to transduce an inhibitory signal, it is possible that another tyrosine kinase may compensate for the low p56lck levels in YT/C143, but not in YT/CD94. In support of our findings, Tarazona et al. [31] recently showed that YT/ C143 does not require p56lck for transduction of the

inhibitory signal and suggested that another tyrosine kinase such as lyn may play a role here instead. Another possible factor responsible for the absent inhibitory signal may be the co-expression of CD94 with the activating NKG2C component, as well as with the inhibitory NKG2A in YT/CD94. NKG2A and C share 94% homology in their extracellular domains [51] and both CD94/NKG2A and CD94/NKG2C recognize HLA-E as their predominant ligand [16,17,19]. Whereas NKG2A possesses cytoplasmic ITIMS, NKG2C does not possess cytoplasmic signaling motifs, however it is capable of transducing an activating signal through association with the DAP12 signaling molecule [9]. Transduction of activating signals through CD94/ NKG2A may override inhibitory signals transduced through CD94/NKG2A. These possibilities are currently under investigation. Although a 39 kDa phosphorylated band corresponding to NKG2C was not observed following immunoprecipitation of YT/CD94 cell lysates with anti-CD94 Ab, HP3B1 (Fig. 2A), we cannot rule out the possibility of NKG2C expression without testing this using an anti-NKG2C specific Ab. The P25 Ab recognizes both NKG2A and NKG2C [52]. NKG2C expression could be tested by extensively preclearing YT/CD94 cell lysate with Z199 (anti-NKG2A Ab) to remove all NKG2A protein, followed by immunoprecipitation or Western blot analysis with P25. In conclusion, our results show that the absence of a functional inhibitory signal in YT/CD94 may be due to low expression levels of p56lck as well as SHP-1. Expression of CD94/NKG2C activating receptor is also a possibility. Transfection of YT/CD94 to increase expression levels may restore a functional inhibitory signal.

Acknowledgments We thank Dr. A. Bajpai for his assistance in performing the p56lck kinase assay, Drs. A.G. Brooks and F. Borrego for providing the YT/CD94, YT/neo, and YT/C143 transfectants; and Dr. R. Brutkiewicz for his critical review and comments of the manuscript.

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