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The Journal of Immunology, 2006, 177: 3170-3177.
Copyright © 2006 by The American Association of Immunologists, Inc.

Molecular Analysis of NTB-A Signaling: A Role for EAT-2 in NTB-A-Mediated Activation of Human NK Cells1

Philipp Eissmann2 and Carsten Watzl3

Institute for Immunology, University Heidelberg, Heidelberg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Engagement of NTB-A on human NK cells by homophilic interaction with NTB-A-expressing target cells can trigger NK cell cytotoxicity, cytokine production, and proliferation. To better understand how NTB-A can activate NK cells, we analyzed the molecular mechanisms of NTB-A signaling. We show that NTB-A is tyrosine phosphorylated in unstimulated human NK cells and associates with SLAM-associated protein (SAP) and EAT-2. This phosphorylation of NTB-A is mediated by Src family kinases and is most likely a result of the homophilic interaction of NTB-A among neighboring NK cells. Stimulation of NK cells by NTB-A-positive targets results in increased NTB-A phosphorylation. The cytoplasmic tail of NTB-A contains three tyrosines, two of which are embedded within an immunoreceptor tyrosine-based switch motif. We generated a NTB-A-negative NK cell line, in which we expressed different mutants of NTB-A. Functional studies showed that the second tyrosine is sufficient and essential for NTB-A-mediated cytotoxicity. EAT-2, but not SAP, is recruited to this second tyrosine, indicating that SAP may be dispensable for this NTB-A function. To further investigate this, we silenced SAP expression in NK cell lines. Functional analysis of these cells showed that NTB-A can mediate NK cell cytotoxicity in the absence of SAP, probably via EAT-2. In contrast, NTB-A-mediated IFN-{gamma} production was greatly reduced in the absence of SAP, demonstrating that cytokine production and cytotoxicity are differentially dependent on SAP and possibly EAT-2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer cells are a subset of lymphocytes belonging to the innate arm of the immune system. They represent a first line of defense against transformed and virally infected cells (1). NK cell activity is regulated by a fine balance of signals from activating and inhibitory receptors. Inhibitory NK cell receptors recognize self-MHC class I molecules and thereby ensure the tolerance toward normal cells (2). Upon triggering of activating receptors by their respective ligands, NK cells can respond by secreting cytokines and killing of susceptible cells. NK cells express a variety of activating receptors, including NKp30, NKp44, NKp46, NKp80, NKG2D, DNAX accessory molecule-1, CD96, 2B4 (CD244), CRACC (CS-1), and NTB-A (3).

2B4, CRACC, and NTB-A belong to the recently defined family of signaling lymphocytic activation molecule (SLAM)4-related receptors (SRR) (4, 5). One characteristic of SRR is the presence of an immunoreceptor tyrosine-based switch motif (ITSM) in their cytoplasmic tail. The consensus sequence of an ITSM is TxYxxV/I (single amino acid code, in which x represents any amino acid) (6). Phosphorylated ITSM can recruit several signaling molecules such as SLAM-associated protein (SAP) (SH2D1A), EAT-2 (SH2D1B), Src homology (SH) region 2 domain-containing phosphatase (SHP)-2, SHP-1, SHIP, Csk, and 3BP2 (4, 5). SAP is a small adapter molecule almost entirely composed of an SH2 domain. The importance of SAP for lymphocyte function is well established, as it is mutated in patients suffering from X-linked lymphoproliferative syndrome (XLP) (7). XLP is characterized by the inability of the immune system to control EBV infections, which is at least in part a result of a defect in lymphocyte fine-tuning through SRR (8). SAP mediates the activation of lymphocytes through SRR by recruiting the Src family kinase FynT through an untypical SH2-SH3 domain interaction (9, 10). Furthermore, SAP can act as a natural blocker of SH2-mediated interactions, thereby preventing the association of phosphatases such as SHP-1 and SHP-2 with the ITSM of several SRR (11, 12, 13). EAT-2 is a second member of the family of SAP-related adapters. Similar to SAP, EAT-2 is almost entirely composed of an SH2 domain (14), and it was also proposed that EAT-2 might function as a blocker of SH2 domain interactions (15). The study of mice deficient for EAT-2 suggests that EAT-2 is a negative regulator of NK cell function (16). How EAT-2 mediates these inhibitory signals is not known, but inhibition strongly depends on tyrosine residues within a short C-terminal part of the molecule (16). Interestingly, a recent report suggests that human EAT-2 can transmit activating signals and mediate CRACC-dependent NK cell activation (17).

The SRR family member NTB-A is expressed on human NK, T, and B cells, as well as on human eosinophils (18, 19, 20). Similar to other SRR members, NTB-A represents its own ligand through homophilic binding (21, 22, 23). Engagement of NTB-A on human T cells can substitute the CD28 costimulatory pathway and induces polarization toward a Th1 phenotype (23). However, CD4-positive T cells from NTB-A (Ly-108) knockout mice show an impairment in IL-4 production, suggesting a role of NTB-A in Th2 polarization (24). The reason for this discrepancy is unknown to date. Stimulation of NTB-A on human NK cells by Ab-mediated cross-linking, NTB-A fusion proteins, or homophilic interaction with NTB-A-expressing cells stimulates cytotoxicity and proliferation, as well as IFN-{gamma} and TNF-{alpha} production (18, 21, 22, 25). The cytoplasmic tail of NTB-A contains three tyrosines, two of which are embedded within an ITSM (18, 19). Upon phosphorylation, NTB-A is able to associate with SAP, EAT-2, SHP-1, and SHP-2 (18, 19, 23). The importance of SAP for NTB-A function is supported by the finding that NK cells from XLP patients cannot be activated via NTB-A and that NTB-A might even become inhibitory in the absence of SAP (18). Nonetheless, the initial molecular mechanisms leading to NTB-A-mediated activation of human NK cells are poorly understood.

In this study, we analyze the molecular mechanisms of early NTB-A signaling in human NK cells. We show that NTB-A associates with SAP and EAT-2 after stimulation by NTB-A-expressing target cells. Using receptor point mutants, we demonstrate that the second tyrosine of NTB-A is sufficient and essential for NTB-A-mediated cytotoxicity. EAT-2, but not SAP, can associate with this second tyrosine, suggesting that EAT-2 can mediate NTB-A-dependent NK cell cytotoxicity. This is confirmed by down-regulating SAP expression in human NK cell lines, which does not affect NTB-A-mediated NK cell cytotoxicity, but impairs IFN-{gamma} production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells, Abs, and inhibitors

Human polyclonal NK cells were isolated from PBLs using the NK cell negative isolation kit (Dynal Biotech). NK cells were between 95 and 99% CD3–, CD56+. Cells were grown in IMDM with 10% human serum, penicillin/streptomycin, and 100 U/ml IL-2 (National Institutes of Health cytokine repository). Cell lines used in this study were 721.221, P815 (both grown in IMDM, 10% FCS, penicillin/streptomycin), Baf/3 (grown in RPMI 1640, 10% FCS, penicillin/streptomycin), and the human NK cell lines NKL (grown in RPMI 1640, 10% FCS, penicillin/streptomycin, 100 U/ml IL-2) and NK92 (grown in {alpha} medium with 2 g/L NaHCO3, 12.5% FCS, 12.5% horse serum, penicillin/streptomycin, 1% L-glutamine, and 50 µM 2-ME). The NTB-A-positive and -negative sublines of NKL were obtained by repeated FACS sorting of the parental NKL cell line.

The following Abs were used: IgG1 control (MOPC-21; Sigma-Aldrich), biotinylated anti-phosphotyrosine 4G10 (Upstate Cell Signaling Solutions), anti-2B4 (C1.7; a gift from G. Trinchieri, The Wistar Institute, Philadelphia, PA), anti-Vav (Sigma-Aldrich), anti-phospho-Vav-1 (pY160) (BioSource International), HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology), and HRP-conjugated streptavidin (Amersham). The mouse monoclonal anti-SAP, the mouse monoclonal anti-NTB-A, and the rabbit polyclonal anti-2B4 Abs have been described (13, 21, 26). The rabbit anti-EAT-2 Ab was raised against the peptide DLPYYHGRLTKQDCETLC (single amino acid code), and the rabbit anti-NTB-A Ab against the peptide CNHSKESKPTFSRATA (single amino acid code). Both Abs were affinity purified.

The following inhibitors were used: piceatannol, PP1, wortmannin, cytochalasin D, latrunculin, PD-98059, SB-202190, U73343, and U73122 (all obtained from BIOMOL).

cDNA constructs, short hairpin RNA (shRNA) constructs, and retroviral transduction

NTB-A point mutants were generated by replacing tyrosines in the cytoplasmic tail of the wild-type receptor with phenylalanine by site-directed mutagenesis exchanging adenosine with thymidine at the following positions (base pair number of the wild-type receptor in which the A of the start codon is 1: Y1 (818), Y2 (851), Y3 (923). All mutations were confirmed by DNA sequencing. NTB-A mutants were cloned in the pBABE retroviral expression vector carrying a puromycin resistance gene. For the stable silencing of SAP, we used a shRNA retroviral expression vector (pSHAG-MAGIC2; Biocat). Retroviral transduction was done using the packaging cell lines Phoenix ampho or Phoenix eco (American Type Culture Collection). Infected NK92, NKL, or Baf/3 cells were selected in puromycin (Sigma-Aldrich) and, if necessary, further enriched by FACS.

Cytotoxicity assays

Target cells were grown to mid-log phase, and 5 x 105 cells were labeled in 100 µl of CTL medium (IMDM with 10% FCS and penicillin/streptomycin) with 100 µCi of 51Cr for 1 h at 37°C. Cells were washed twice in CTL medium and resuspended at 5 x 104 cells/ml in CTL medium. Five thousand target cells/well were used in the assay. Effector cells were resuspended in CTL medium and, where applicable, preincubated with Abs (0.5 µg/ml final concentration) and/or pharmacological inhibitors. After preincubation, effector cells were mixed with labeled target cells in a V-bottom 96-well plate. Maximum release was determined by incubation in 1% Triton X-100. For spontaneous release, targets were incubated without effectors in CTL medium alone. All samples were done in triplicates. After a 1-min centrifugation at 1000 rpm, plates were incubated for 4 h at 37°C. Supernatant was harvested, and 51Cr release was measured in a gamma counter. Percentage of specific release was calculated as ((experimental release – spontaneous release)/(maximum release – spontaneous release)) x 100. The ratio between maximum and spontaneous release was at least 4 in all experiments. Pharmacological inhibitors were titrated by serial dilution and preincubated with the effector cells for 30 min at 37°C. Percentage of inhibition was calculated as 100 – ((percentage of lysis of Baf-NTB-A Y123F after pretreatment with the respective inhibitor – percentage of lysis of Baf-GFP after pretreatment with the respective inhibitor)/(percentage of lysis of Baf-NTB-A Y123F after pretreatment with DMSO – percentage of lysis of Baf-GFP after pretreatment with DMSO)) x 100.

Cell mixing, immunoprecipitation, and Western blotting

Cell mixing was essentially done as described (27). For immunoprecipitation, precleared lysates were first incubated for 1 h at 4°C with 2 µg of control IgG1, followed by 2 µg of specific Ab, both coupled to recombinant protein G agarose (Invitrogen Life Technologies). Beads were washed three times in ice-cold lysis buffer and boiled in 2.5x reducing sample buffer (5% SDS, 25% glycerol, 12.5% 2-ME, 0.156 M Tris/Cl (pH 6.8), and 0.01% bromphenol blue). For Western blotting, samples were separated on either 10 or 12% SDS NuPAGE gels (Invitrogen Life Technologies) and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% milk powder in PBST (0.05% Tween 20 in PBS) for 1 h at room temperature and incubated with the indicated Ab overnight at 4°C. After washing, the membrane was incubated with the respective HRP-conjugated secondary Ab for 1 h at room temperature and developed using SuperSignal West Pico or Dura (Pierce).

IFN-{gamma} release assay

A total of 1 x 105 NK cells and 1 x 105 target cells was incubated for 20 h at 37°C in a 96-well plate. All samples were done in triplicates. Supernatants were harvested, and quantification of IFN-{gamma} was performed using a quantitative sandwich ELISA (Quantikine kit; R&D Systems), according to the manufacturer’s instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Signaling pathways activated by NTB-A

The molecular mechanisms and signaling pathways that lead to NTB-A-mediated activation of human NK cells are poorly defined. To analyze which signaling pathways are activated by NTB-A, we used a variety of pharmacological inhibitors and tested their effect on NTB-A-mediated NK cell cytotoxicity (Fig. 1). For this, the mouse B cell line Baf/3, stably transfected with human NTB-A, served as a target for primary human NK cells. Blocking actin polymerization with either cytochalasin D or latrunculin completely inhibited NTB-A-mediated killing, as did the Src kinase inhibitor PP1. Similarly, blocking PI3K with wortmannin or phospholipase C (PLC) with the inhibitor U73122 inhibited NTB-A function. The use of the MEK kinase inhibitor PD98059 resulted only in a partial reduction of NTB-A-mediated killing. In contrast, inhibition of Syk kinase by piceatannol or p38 MAPK by the inhibitor SB202190 did not affect NTB-A function. This demonstrates that NK cell activation via NTB-A is dependent on actin reorganization and the activity of Src kinases, PLC, and PI3K and to a lesser extent on the MEK kinase pathway. Syk family kinases and the p38 MAPK pathway do not seem to be involved in NTB-A-mediated NK cell cytotoxicity.


Figure 1
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  		FIGURE 1. Signaling pathways involved in NTB-A-mediated NK cell activation. NTB-A-specific killing of IL-2-activated human NK cells in the presence of three decreasing concentrations of the following inhibitors: cytochalasin D (5, 2.5, and 1.25 µM), latrunculin (1, 0.5, and 0.25 µM), PP1 (10, 5, and 2.5 µM), piceatannol (100, 50, and 25 µM), PD98059 (20, 10, and 5 µM), SB202190 (100, 50, and 25 nM), wortmannin (100, 50, and 25 nM), U73122 (2.5, 1.25, and 0.6 µM), U73343 (an inactive analog to U73122, which serves as a control; 2.5, 1.25, and 0.6 µM). The data are from an E:T ratio of 5. Similar results were obtained at an E:T ratio of 10. Percentage of inhibition was calculated as 100 – ((percentage of lysis of Baf-NTB-A Y123F after pretreatment with the respective inhibitor – percentage of lysis of Baf-GFP after pretreatment with the respective inhibitor)/(percentage of lysis of Baf-NTB-A Y123F after pretreatment with DMSO – percentage of lysis of Baf-GFP after pretreatment with DMSO)) x 100. The data represent mean and deviation of at least three independent experiments using NK cells from different donors.

 
NTB-A associates with SAP and EAT-2 upon phosphorylation

After determining the signaling pathways involved in NTB-A-mediated NK cell cytotoxicity, we wanted to investigate the molecular mechanisms that initiate these pathways. Previous reports that found an association of NTB-A with SAP, EAT-2, SHP-1, and SHP-2 used the yeast two-hybrid system, pervanadate treatment of NK cells, or Ab-mediated cross-linking of NTB-A on T cells (18, 19, 23). To investigate the receptor-proximal signaling of NTB-A in human NK cells, we first used a mAb to cross-link NTB-A on IL-2-activated human NK cells (Fig. 2). We observed that NTB-A is already phosphorylated in untreated cells, and to our surprise phosphorylation of the receptor decreased after cross-linking. This effect was specific for Ab-mediated cross-linking of NTB-A as neither cross linking of CD56 nor incubation of NK cells on 37°C without the addition of any Ab reduced NTB-A phosphorylation. In a time course, the phosphorylation of NTB-A started to decrease as early as 1 min and was almost undetectable after 5 and 10 min of cross-linking (data not shown). To investigate whether receptor engagement indeed reduces NTB-A phosphorylation, we mixed primary human NK cells with the NTB-A-expressing target cell line 721.221, which resembles a more physiological way of receptor stimulation (Fig. 3A). In this assay, we also observed NTB-A phosphorylation in untreated NK cells. However, the phosphorylation of NTB-A increased after target cell contact, reaching its maximum ~3 min after stimulation (Fig. 3A). To exclude that the phosphorylation we detected after cell mixing results from NTB-A phosphorylation in the target cells, we generated a mutant NTB-A receptor in which all three tyrosines were replaced by phenylalanine. Such a mutant receptor cannot be phosphorylated anymore (data not shown). We stably expressed this mutant receptor in Baf/3 cells and used these as targets for the stimulation of NTB-A in NK cells. In this experiment, we observed a similar increase in NTB-A phosphorylation (Fig. 3B), demonstrating that NTB-A phosphorylation was induced in the NK cells. As a control, we did not observe any increase in NTB-A phosphorylation after incubating NK cells with control-transfected Baf-GFP cells, demonstrating that the increase in NTB-A phosphorylation in the NK cells was not induced by homophilic interactions between NK cells (data not shown). We also observed an induction of Vav-1 phosphorylation when incubating NK cells with Baf-NTB-A, but not Baf-GFP targets (Fig. 3C). This demonstrates that engagement of NTB-A on NK cells by NTB-A-expressing target cells induces receptor phosphorylation, leading to the induction of downstream signal transduction as evident by the Vav-1 phosphorylation.


Figure 2
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  		FIGURE 2. NTB-A phosphorylation decreases after Ab-mediated receptor cross-linking. NTB-A was cross-linked on IL-2-activated human NK cells using the NT-7 mouse mAb, followed by a secondary goat anti-mouse Ab. CD56 cross-linking serves as a control. Samples were either left on ice (0') or stimulated for 5 min at 37°C. An additional sample was handled the same way without the prior addition of any Ab (–). NTB-A or CD56 (control) was immunoprecipitated from cell lysates, and samples were analyzed for tyrosine phosphorylation (pY). To confirm equal immunoprecipitation, membranes were reblotted with Abs against NTB-A.

 

Figure 3
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  		FIGURE 3. NTB-A phosphorylation increases after target cell contact. A, A total of 10 x 106 IL-2-activated human NK cells was mixed with the NTB-A+ target cell line 721.221 at an E:T ratio of 2 for the indicated time points. Target cells were also added to time zero samples of all experiments. Cells were lysed and immunoprecipitated with a control Ab (IgG), followed by an Ab against NTB-A. Samples were analyzed by Western blotting for tyrosine phosphorylation (pY) and coimmunoprecipitation of SAP and EAT-2. To confirm equal immunoprecipitation, membranes were reblotted with Abs against NTB-A. B, A total of 10 x 106 IL-2-activated human NK cells was mixed with the mouse cell line Baf/3 expressing a triple mutant (Y123F) of the NTB-A receptor at an E:T ratio of 2. Cells were lysed and immunoprecipitated with a control Ab (IgG), followed by an Ab against NTB-A. Samples were analyzed as described in A. Longer exposures also revealed a phosphorylation of NTB-A in unstimulated NK cells. No increase in NTB-A phosphorylation was detected after incubating IL-2-activated human NK cells with Baf-GFP (data not shown). C, A total of 5 x 106 IL-2-activated human NK cells was mixed with Baf-NTB-A-Y123F or Baf-GFP targets as a control at an E:T ratio of 2 for the indicated time points. Cells were lysed, and the lysate was analyzed for Vav phosphorylation using an anti-phospho-Vav-1 Ab. Vav levels confirm equal loading of the gel.

 
We detected an association of SAP and EAT-2 with NTB-A after mixing primary human NK cells with 721.221 targets (Fig. 3A). This coimmunoprecipitation must result from a recruitment of both molecules to the NTB-A receptor in NK cells, as the 721.221 cells do not express either SAP or EAT-2 (Fig. 4Aii). Although a change in the amount of coimmunoprecipitated SAP or EAT-2 after target cell contact was hard to detect and differed between various experiments, the overall pattern suggests that the association of both molecules with NTB-A follows the time course of the phosphorylation (Fig. 3A and data not shown). However, NK cell stimulation was not necessary to detect NTB-A phosphorylation and association of SAP and EAT-2, as it was also detected after immunoprecipitation of NTB-A from NK cells without any pretreatment (Fig. 4Ai). This background phosphorylation is not unique to NTB-A as it can also be detected for 2B4 (Fig. 4Ai) and is most likely a result from an engagement of the receptors between neighboring NK cells. In comparison, the 2B4 phosphorylation in unstimulated NK cells was even slightly higher than that of NTB-A. However, while the association of SAP and EAT-2 with NTB-A was easily detectable, hardly any SAP and EAT-2 were associated with 2B4 under identical conditions (Fig. 4Ai). This suggests that in unstimulated cells, NTB-A might have a higher affinity for the recruitment of SAP and EAT-2 than 2B4. We detected no NTB-A phosphorylation when we immunoprecipitated the receptor from 721.221 cells, which do not express SAP or EAT-2 (Fig. 4A). This suggests that at least one of the adapters may be essential for NTB-A phosphorylation.


Figure 4
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  		FIGURE 4. Phosphorylation-dependent association of SAP and EAT-2 with NTB-A. A, A total of 10 x 106 untreated IL-2-activated human NK cells or 5 x 106 721.221 cells was lysed and immunoprecipitated with a control Ab (IgG), followed by an Ab against NTB-A or 2B4. Immunoprecipitates (i) were analyzed by Western blotting for tyrosine phosphorylation (pY) and coimmunoprecipitation of SAP and EAT-2. To confirm equal immunoprecipitation, membranes were reblotted with Abs against NTB-A and 2B4. Lysates (ii) were analyzed for SAP and EAT-2 expression. B, A total of 10 x 106 IL-2-activated human NK cells was pretreated with either 10 µM PP1 or DMSO for 30 min at 37°C and analyzed, as described in A. Data are representative of at least three independent experiments.

 
To test whether the association of SAP and EAT-2 with NTB-A is dependent on receptor phosphorylation, we incubated NK cells with the Src kinase inhibitor PP1 (Fig. 4B). This treatment completely blocked the phosphorylation of NTB-A, demonstrating that Src kinases are essential for this event. More importantly, SAP and EAT-2 coimmunoprecipitated with phosphorylated NTB-A from control (DMSO)-treated NK cells, but not with unphosphorylated NTB-A from PP1-treated samples. This clearly demonstrates that the association of NTB-A with SAP and EAT-2 is dependent on tyrosine phosphorylation.

SAP and EAT-2 associate with the same NTB-A molecule at the same time

When we immunoprecipitated SAP from unstimulated NK cells, we detected a strong association with NTB-A, but only a very weak assocciation with 2B4 (Fig. 5A), confirming the results in Fig. 4A. The strong band seen on the antiphosphotyrosine blot comigrates with NTB-A, again suggesting that SAP associates with phosphorylated NTB-A. To our surprise, we also detected a coimmunoprecipitation of EAT-2 with SAP (Fig. 5A). This could either be the result of a direct interaction between SAP and EAT-2, or it could be an indirect association mediated via NTB-A. To investigate these possibilities, we made use of the human NK cell line NKL. The parental cell line contained two subpopulations, with and without NTB-A expression. Using four rounds of FACS sorting, we generated two stable sublines that either do or do not express NTB-A (Fig. 5B). SAP and EAT-2 expression was identical between the two sublines (data not shown). Functional data confirmed that Baf-NTB-A targets were lysed only by the NTB-A-positive, but not by the negative NKL line, whereas 721.221 were lysed only slightly less by the NTB-A-negative NKL line, demonstrating that while NTB-A is involved in the lysis of 721.221 cells, other receptor/ligand interactions play a major role in the recognition of these targets (data not shown). We immunoprecipitated SAP from either the NTB-A-positive or the NTB-A-negative NKL line and analyzed the association of NTB-A and EAT-2 (Fig. 5C). We coimmunoprecipitated EAT-2 only from the NTB-A-positive NKL line, in which we also detected the association between SAP and NTB-A. This demonstrates that the association of EAT-2 with SAP is most likely indirect and may be mediated via NTB-A. It also indicates that SAP and EAT-2 associate with the same NTB-A molecule at the same time and that this association is most likely independent of either molecule.


Figure 5
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  		FIGURE 5. SAP and EAT-2 associate with one NTB-A molecule at the same time. A, SAP was immunoprecipitated from 30 x 106 IL-2-activated primary human NK cells. Samples were analyzed by Western blotting for SAP; for coimmunoprecipitation of EAT-2, NTB-A, and 2B4; and for tyrosine phosphorylation (pY) of coimmunoprecipitated molecules. B, NTB-A-negative and -positive NKL lines were analyzed for NTB-A expression via surface staining with either an isotype control Ab (gray curve) or an anti-NTB-A Ab (NT-7, open curve). C, SAP was immunoprecipitated from 20 x 106 NTB-A-negative or NTB-A-positive NKL cells. Samples were analyzed as described in A. Equal immunoprecipitation of SAP from each sample was confirmed by reblotting the membrane with an anti-SAP mAb. Specificity of immunoprecipitations was controlled with an isotype control Ab (IgG). Data are representative of at least three independent experiments.

 
The second ITSM is essential for NTB-A function

The cytoplasmic tail of NTB-A contains three tyrosine residues. The first tyrosine (Y1) at position 273 of the amino acid sequence is not embedded within the consensus sequence of an ITSM. The second (Y2, aa 284) and the third (Y3, aa 319) tyrosines are surrounded by amino acids that form an ITSM (19). To determine the impact of the tyrosines on NTB-A-mediated NK cell activation, we mutated the different tyrosines of NTB-A to phenylalanine (Y->F mutations) either alone or in combination. The wild-type receptor and the different mutants were then stably expressed in the NTB-A-negative NKL cell line. Equal expression levels of the transfected receptors were confirmed by anti-NTB-A surface staining (Fig. 6). These mutants were then tested in redirected lysis assays against the Fc{gamma}R-positive mouse cell line P815 (Fig. 6). A single mutation of the first tyrosine (Y1F) did not result in a reduced killing compared with the wild-type receptor (Fig. 6A). In contrast, the triple mutant (Y123F) of NTB-A had completely lost the ability to mediate killing of the P815 cell line (Fig. 6A). This demonstrates that the tyrosines in the cytoplasmic tail of NTB-A are essential for the function of this receptor and that Y1 is dispensable for full NTB-A function. Next, we analyzed the ITSM tyrosine mutants (Fig. 6B). A mutation of the second tyrosine (Y2F) resulted in a complete loss of NTB-A-mediated killing, whereas a mutation of the third tyrosine (Y3F) only reduced NTB-A-mediated killing compared with the wild-type receptor. This demonstrates that Y2 is essential for NTB-A function and Y3 is needed for the full activity of the receptor. Double mutants lacking the second tyrosine (Y12F and Y23F) also did not display any killing, underlining the essential role of Y2 for NTB-A function. In contrast, the Y13F mutant, only containing a functional Y2, was still active, although to a lesser extent than the wild-type receptor. This demonstrates that Y2 is not only essential, but also sufficient for NTB-A-mediated cytotoxicity. As a control, all mutants showed similar killing when triggered via the 2B4 receptor (data not shown). These data could also be confirmed using Baf-NTB-A or Baf-GFP cells as targets. Although NKL cells expressing the wild-type receptor showed enhanced lysis of Baf-NTB-A cells, double mutants lacking the second tyrosine (Y12F and Y23F) did not show any NTB-A-specific lysis. Again, the Y13F mutant, only containing a functional Y2, was almost as active as the wild-type receptor (data not shown).


Figure 6
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  		FIGURE 6. Importance of cytoplasmic tyrosines for NTB-A function. A and B, The tyrosines in the cytoplasmic tail of NTB-A were mutated to phenylalanine (Y->F) either alone or in combination. The different mutants and the wild-type receptor were expressed in the NTB-A-negative NKL line by retroviral gene transfer. Expression was monitored by surface staining with an isotype control Ab (gray curve) or an anti-NTB-A Ab (NT-7, open curve) and is shown in the right corner of each graph. Transfectants were tested in 51Cr release assays against P815 target cells in the presence of a mouse IgG mAb (control) or an anti-NTB-A (NT-7) mAb at different E:T ratios. Error bars represent SD of triplicates. Data are representative of at least three independent experiments.

 
The second tyrosine specifically binds EAT-2

As SAP and EAT-2 can both associate with NTB-A, we were interested to determine which molecule is recruited to the second tyrosine. We therefore immunoprecipitated NTB-A from NKL cells expressing either the wild-type, the Y2F, or the Y13F NTB-A receptor (Fig. 7A). SAP and EAT-2 coimmunoprecipitated with the wild-type receptor, which is consistent with the data in Fig. 4. When the second tyrosine was mutated (Y2F), the association of EAT-2 with NTB-A was lost, while SAP still associated. The Y13F mutant, only containing functional Y2, coimmunoprecipitated EAT-2, but not SAP. The second tyrosine therefore specifically recruits EAT-2, whereas SAP associates with Y3 and possibly with Y1. As Y2 is essential, but Y1 and Y3 are dispensable for full NTB-A-mediated NK cell cytotoxicity, this suggests that NTB-A function may be dependent on EAT-2, but not on SAP.


Figure 7
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  		FIGURE 7. SAP and EAT-2 associate with different ITSM. A, NTB-A was immunoprecipitated from NKL transfectants (75–100 x 106 cells per sample) expressing either the wild-type receptor or the Y2F or Y13F mutant. Samples were analyzed by Western blotting for the coimmunoprecipitation of SAP and EAT-2. Equal immunoprecipitation of NTB-A was confirmed by immunoblotting for NTB-A. Immunoprecipitation with an isotype control Ab (IgG) confirms specificity. B, Lysates of 0.4 x 106 NKL cells or NKL cells obtained from two independent SAP shRNA transfections (T1 and T2) were analyzed for SAP expression by immunoblotting. Actin levels confirm equal loading of the gel. EAT-2 expression is shown to confirm specificity of SAP down-regulation. C, NKL cells or the transfectants T1 and T2 were tested in a 51Cr release assay against the target cell line P815 at different E:T ratios in the presence of a mouse IgG Ab (control), an anti-NKG2D Ab as a positive control, or an anti-NTB-A Ab. Error bars represent SD of triplicates. D and E, Analysis of NK92 cells with down-modulated SAP expression, as described in B and C.

 
To investigate whether SAP could be dispensable for NTB-A-mediated NK cell activation, we generated NKL cells with stably down-regulated SAP protein expression via retroviral shRNA delivery. The down-regulation of SAP and the presence of EAT-2 were confirmed by immunoblotting (Fig. 7B). T1 and T2 represent NKL subclones derived from two independent transfections. These transfectants were then tested in redirected lysis assays for their ability to mediate killing of P815 cells via NTB-A (Fig. 7C). Although the extent of killing differed between the two transfectants, down-regulation of SAP did not result in a loss of NTB-A function. This demonstrates that SAP can be dispensable for NTB-A-mediated cytotoxicity, although it is formally possible that the residual levels of SAP expression are still sufficient for NTB-A function. To confirm these results with another NK cell line, we also down-regulated SAP expression in NK92 cells (Fig. 7D). Also in these cells, the down-regulation of SAP did not impair NTB-A-mediated cytotoxicity (Fig. 7E).

NTB-A can also stimulate the release of cytokines such as IFN-{gamma}. To test whether SAP is involved in this function of NTB-A, we tested the IFN-{gamma} production of the NK92 cells with and without SAP expression after incubation with either Baf-GFP or Baf-NTB-A target cells (Fig. 8). The incubation of control-transfected NK92 cells with Baf-NTB-A targets induced enhanced IFN-{gamma} production compared with the incubation with Baf-GFP targets. This NTB-A-mediated IFN-{gamma} production was significantly reduced in the NK92 cells with down-modulated SAP expression, demonstrating that SAP is involved in NTB-A-mediated cytokine production.


Figure 8
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  		FIGURE 8. Defective NTB-A-mediated IFN-{gamma} release in the absence of SAP. NK92 cells transfected with a shRNA against SAP or CD4 as a control were incubated with Baf-GFP or Baf-NTB-A-Y123F targets for 20 h. Supernatants were harvested, and IFN-{gamma} concentrations were determined by ELISA. Results are mean and deviation of four independent experiments with each sample performed in triplicates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
NTB-A belongs to the family of SRRs who play an important role in the fine-tuning of lymphocyte activity (5). The molecular mechanisms that regulate activation of lymphocytes via NTB-A are poorly defined. We therefore investigated NTB-A signaling in human NK cells. We show that NTB-A-mediated NK cell cytotoxicity is mainly dependent on the PLC and PI3K and to a lesser extent on the MEK kinase signaling pathways. Not surprisingly, inhibiting actin polymerization or Src kinases also disrupted activation of NK cells via NTB-A. The role of PLC and PI3K in the generation of NK effector functions is well established (28). PI3K has been shown to regulate NK cytotoxicity via a pathway that involves the activation of MEK1/2 and subsequently ERK1/2 (29). The activation of NK cells through the engagement of 2B4 also results in phosphorylation of PLC and requires PI3K and ERK (27, 30, 31, 32). Additionally, and in contrast to our findings for NTB-A, 2B4 also signals via the p38 MAPK pathway (31). Recently, it was shown that 2B4-mediated ERK1/2 activation through a pathway involving Vav-1, Rac-1, Raf-1, and MEK is defective in NK cells from XLP patients (33), suggesting that this pathway is dependent on SAP. NTB-A function was only partially affected by MEK inhibition, which is consistent with our finding that SAP can be dispensable for NK cell cytotoxicity mediated by NTB-A. Although our inhibitor studies provide only a rough overview of NTB-A-activated signaling pathways, our results demonstrate that SRR use only in part overlapping pathways and further studies will be needed to understand the complexity and integration of SRR signaling.

NTB-A-mediated NK cell activation is dependent on tyrosine-based motifs (ITSM) within its cytoplasmic tail, as evident from our studies using mutant receptors. We detected tyrosine phosphorylation of NTB-A already in unstimulated NK cells. This phosphorylation is dependent on Src kinases, as the inhibition of these kinases resulted in a loss of NTB-A phosphorylation. Furthermore, NTB-A baseline phosphorylation is independent of IL-2 stimulation of cultured NK cells, as we also observed phosphorylation of NTB-A in freshly isolated NK cells (data not shown). Surprisingly, our approach to study NTB-A phosphorylation and association with signaling molecules by Ab-mediated receptor cross-linking resulted in a decrease of NTB-A phosphorylation. In contrast, when we stimulated NK cells with NTB-A-expressing target cells, we observed an increase in NTB-A phosphorylation. NTB-A is a homophilic receptor, and the interaction of NTB-A between neighboring NK cells could already induce phosphorylation. We therefore propose that by cross-linking NTB-A with Abs, we block this interaction and hence phosphorylation of NTB-A. It is interesting to note that adding the anti-NTB-A Ab in the absence of a cross-linking Ab did not reduce phosphorylation (data not shown). This suggests that somehow cross-linking enhances the block of NTB-A homophilic interaction.

NTB-A has been shown to bind to SAP and EAT-2 in a yeast two-hybrid system (19). In this study, we confirm this interaction of SAP and EAT-2 with NTB-A in primary human NK cells expressing endogenous levels of these molecules. Additionally, we show that SAP and EAT-2 can associate with the same NTB-A molecule at the same time. The association of both molecules to the receptor is dependent on tyrosine phosphorylation. It was difficult to assess whether SAP and EAT-2 recruitment increases with increased receptor phosphorylation. This might result from the fact that NTB-A association with SAP and EAT-2 is already detectable under steady state conditions. Interestingly, we hardly detected an association of either molecule with phosphorylated 2B4 under the same conditions, suggesting that SAP and EAT-2 bind to NTB-A with a higher affinity.

Our functional analysis of NTB-A point mutants demonstrates that only the two ITSM tyrosines are important for NTB-A-mediated NK cell activation. Among these, the second tyrosine (Y284), which specifically binds the EAT-2 molecule, is essential and sufficient for NTB-A-mediated cytotoxicity. The third tyrosine (Y319) contributed to full NTB-A-mediated NK cell activation and most likely binds SAP, but no EAT-2. This demonstrates that SAP and EAT-2 are both necessary for full NTB-A function. However, SAP was dispensable for NTB-A-mediated cytotoxicity in two independent NK cell lines in which we knocked down SAP by shRNA. This is in contrast to a previous study, which demonstrated that NTB-A-mediated NK cell activation is defective in XLP patients (18). SLAM, 2B4, and CD48 expression was identified on hemopoietic stem cells and nonself-renewing multipotent hemopoietic progenitors (34). Although identified as markers to distinguish between two differentiation stages, it is possible that these receptors also serve a functional role during the development of lymphocytes. One reason for the discrepancy could be that the absence of SAP during the development of NK cells may be responsible for the defective NTB-A function in XLP patients, whereas silencing of SAP in mature NK cells, as presented in this study, does not result in a loss of NTB-A-mediated cytotoxicity. However, we did detect a defect in NTB-A-mediated IFN-{gamma} release upon down-modulation of SAP. This demonstrates that the pathways mediating cytotoxicity and cytokine production may already diverge at the level of NTB-A-associated adapter molecules, with EAT-2 mediating cytotoxicity and SAP mediating IFN-{gamma} release.

SAP can block the binding of phosphatases such as SHP-1 and SHP-2 to ITSM of SRR (11, 12, 13), and may therefore also prevent the binding of SHP-1 or SHP-2 to the third tyrosine in the cytoplasmic tail of NTB-A. In XLP patients, the binding of those phosphatases in the absence of SAP could counteract the positive signaling events initiated by EAT-2, thereby reducing or even abolishing NTB-A-mediated NK cell activation. Depending on the strength of the positive signal mediated by EAT-2 and the negative signal by SHP-1 or SHP-2, the outcome of NTB-A triggering in the absence of SAP could differ. This could be another explanation for the discrepancy of NTB-A activity in XLP patients and SAP-silenced NK cells.

There are several other examples of SRR signaling pathways that are independent of SAP. CD84, which also associates with SAP, was shown to be functional and phosphorylated in T cells derived from XLP patients (35, 36). Phosphorylated SLAM was still detectable in platelets derived from SAP–/– mice (37), although the dependence on SAP for SLAM phosphorylation is well documented in lymphoid cells (38). Additionally, the activation of NK cells through CRACC is completely independent of SAP (39) and seems to be mediated via EAT-2 (17).

Our results suggest an important role of EAT-2 for NTB-A-mediated NK cell cytotoxicity. This is in contrast to a recent report that established a negative regulatory role of mouse EAT-2 (16). This inhibitory function was dependent on the phosphorylation of two tyrosines located within a short C-terminal extension of EAT-2 (16). The C-terminal tail of human EAT-2 contains only one of these tyrosines. Although we could detect phosphorylation of human EAT-2 after pervanadate treatment when we overexpressed the molecule in 293T cells, we were unable to detect EAT-2 phosphorylation in primary human NK cells or NK cell lines (data not shown). It is therefore possible that human EAT-2 does not possess the inhibitory properties of its murine counterpart. EAT-2-mediated activation of human NK cells was also suggested in a recent report investigating CRACC signaling (17). To date, we were unable to silence EAT-2 in human NK cells, which could proof the importance of EAT-2 for the function of NTB-A. Furthermore, it will be necessary to silence SAP and probably EAT-2 in primary human NK cells in the future to also investigate the influence of these molecules on NTB-A-mediated NK cell proliferation.

How can EAT-2 initiate a signaling pathway leading to human NK cell activation? It was shown that EAT-2 can function as a blocker of SH2 domain interactions, thereby preventing the association of phosphatases with SRR (15). However, an activating function of EAT-2 cannot just be explained by blocking the association of other molecules with the ITSM of SRR. Future studies will be necessary to identify molecules that are recruited to EAT-2 and to investigate whether the C-terminal tyrosine does play any role for EAT-2 function.

Our analysis of NTB-A-mediated activation of human NK cells provides further evidence for SAP-independent pathways in the activation of human NK cells through SRR. These signals are probably delivered by EAT-2. Our observations will help to further understand the complexity of lymphocyte fine-tuning by SRR and the phenotype of XLP.


   Acknowledgments
 
We gratefully thank Mina Sandusky for DNA sequencing and Birgitta Messmer and Sabine Wingert for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Deutsche Forschungsgemeinschaft (SFB 405, A9). Back

2 Current address: Division of Cell and Molecular Biology, Imperial College London, London, U.K. Back

3 Address correspondence and reprint requests to Dr. Carsten Watzl, Institute for Immunology, University Heidelberg, INF 305, 69120 Heidelberg, Germany. E-mail address: carsten.watzl{at}urz.uni-heidelberg.de Back

4 Abbreviations used in this paper: SLAM, signaling lymphocytic activation molecule; ITSM, immunoreceptor tyrosine-based switch motif; PLC, phospholipase C; SAP, SLAM-associated protein; SH, Src homology; SHP, SH region 2 domain-containing phosphatase; shRNA, short hairpin RNA; SRR, SLAM-related receptor; XLP, X-linked lymphoproliferative syndrome. Back

Received for publication February 1, 2006. Accepted for publication June 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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