The CD94/NKG2-A complex is the inhibitory receptor for the nonclassical MHC class I molecule HLA-E on human NK cells. Here we studied the molecular mechanisms underlying the inhibitory activity of CD94/NKG2-A on NK cell functions by analyzing its interference on CD16-initiated signaling pathways involved in the control of cytolytic activity. Both tyrosine phosphorylation and activation of Syk kinase together with tyrosine phosphorylation of CD16 receptor ζ subunit are markedly inhibited by the coengagement of CD94/NKG2-A complex. As a downstream consequence, CD94/NKG2-A cross-linking impairs the CD16-induced activation of extracellular regulated kinases (ERKs), a pathway involved in NK cytotoxic function. The block of ERK activation is exerted at an early, PTK-dependent stage in the events leading to p21ras activation, as the CD16-induced tyrosine phosphorylation of Shc adaptor protein and the formation of Shc/Grb-2 complex are abrogated by CD94/NKG2-A simultaneous engagement. Our observations indicate that CD94/NKG2-A inhibits the CD16-triggered activation of two signaling pathways involved in the cytotoxic activity of NK cells. They thus provide molecular evidence to explain the inhibitory function of CD94/NKG2-A receptor on NK effector functions.

Natural killer cells represent a small subset of cytotoxic lymphocytes able to recognize, through still poorly defined receptors, and lyse tumor, virus-infected, and immature hemopoietic cells without prior sensitization (1). Beside their natural cytotoxic activity, the expression of CD16 (the low affinity receptor for IgG Fc fragment, FcγRIIIA) on the vast majority of NK cells renders them the major effector for Ab-dependent cellular cytotoxicity (ADCC)3 against IgG-coated target cells (2).

The signaling pathways leading to the activation of NK lytic function have been only partially elucidated. Both CD16 receptor complex and ITIM-less, activatory counterparts of MHC-I inhibitory receptors (i.e., KAR and CD94/NKG2-C) or other NK-triggering receptors activate PTK-dependent signaling pathways (3, 4, 5, 6, 7, 8, 9, 10), and PTK activity has been shown to constitute a mandatory step in the initiation of both CD16-mediated target cell killing and natural cytotoxic activity. More recently, the activities of ERK enzymes belonging to the MAPK family and of Syk, but not the closely related PTK Zap-70, have been shown to be required for the development of both natural and CD16-mediated cytotoxic activity and for the exocytosis of cytotoxic granules (11, 12, 13).

NK cell functions are negatively regulated by MHC-I specific inhibitory receptors, so that target cell lysis represents the final balance between positive signals, which are provided by triggering receptors with the possible intervention of a wide array of costimulatory molecules, counteracted by signals initiated by inhibitory receptors (3, 4, 14).

Two classes of inhibitory receptors have been described on human NK cells to date: the killer Ig-like receptor (KIR) family, composed of HLA-A-, B-, or C-specific receptors, structurally belonging to the Ig superfamily (3, 15), and the CD94/NKG2-A heterodimer, composed of two type II proteins, both bearing a C lectin-type extracellular domain and coded for by two close genes located in the NK gene complex (3, 16, 17, 18, 19). It has been recently shown that the CD94/NKG2-A complex recognizes the nonclassical MHC-I molecule HLA-E (20, 21, 22), whose antigenic groove is preferentially loaded with peptides deriving from MHC-I signal sequences and whose membrane expression thus depends on the coincidental presence of many, but not all, classical HLA class I molecules (23, 24).

As for KIR, the inhibitory activity of CD94/NKG2-A mainly depends on the ITIM sequences located in the cytoplasmic portion of the NKG2-A chain. We have previously demonstrated that following engagement by natural MHC-I ligand-bearing cells or specific Abs, the CD94/NKG2-A inhibitory complex undergoes phosphorylation on the tyrosine residues located in the ITIM; this event induces the recruitment of SHP-1 (25), which is thought to be the main effector for inhibitory function. In fact, both the integrity of ITIM sequence and the enzymatic activity of SHP-1 seem to be crucial for the inhibitory activity of KIR and CD94/NKG2-A (3, 26, 27).

The signal transduction pathways affected by SHP-1 tyrosine phosphatase activity, and accounting for the inhibition of cytolytic activity exerted by MHC-I-specific inhibitory receptors, are still largely unknown: Ab-mediated engagement of CD94/NKG2-A has been shown to interfere with CD16-induced inositol phosphate generation and tyrosine phosphorylation of phospholipase-C and Zap-70 (6); similarly, KIR engagement by MHC-I ligand-bearing cells has been shown to inhibit intracellular calcium flux and inositol phosphate generation (28, 29, 30). Evidence that KIR-activated SHP-1 can directly dephosphorylate two adaptor proteins, such as pp36 and SLP-76, has been recently provided (29, 31).

The impact of MHC-I inhibitory receptor-activated signaling pathways on other crucial molecular intermediates of the programming for lysis has not been explored to date.

The present work investigates the interference of CD94/NKG2-A inhibitory receptor on two important pathways leading to CD16-triggered NK cytotoxicity, namely the activation of Syk and ERKs. The inhibition of these two pathways by CD94/NKG2-A coengagement provides a novel finding about the molecular mechanisms initiated by MHC-I-specific inhibitory receptors; it also contributes to a better understanding of the molecular cross-talk between activatory and inhibitory receptors in the control of NK cell functions.

Murine FcγR+ P815 mastocytoma cell line was used for reverse ADCC (rADCC).

The following mouse mAbs were used: anti-CD3 (Leu 4), anti-CD16 (Leu 11c), and anti-CD56 (Leu 19) were purchased from Becton Dickinson (Milpitas, CA); anti-CD16 (B73.1) and anti-CD56 (B159.5.2) mAbs were kindly provided by Dr. G. Trinchieri (Wistar Institute, Philadelphia, PA) and Dr. B. Perussia (Jefferson Cancer Institute, Philadelphia), respectively; Z199 mAb was kindly provided by Dr. A. Moretta (Genoa, Italy) and has been previously shown to react specifically with CD94/NKG2-A,B inhibitory heterodimers (25, 32). Goat anti-mouse IgG F(ab′)2 (GAM) was purchased from Cappel Laboratories (Cooper Biomedical, Malvern, PA). Rabbit antisera specific for Syk, Grb2, and Erk2 as well as anti-Shc and anti-ζ mAbs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Shc rabbit antiserum and anti-phosphotyrosine (pTyr) mAb were purchased from Upstate Biotechnology (Lake Placid, NY); anti-Grb2 and anti-Shc mAbs and the affinity-purified rabbit anti-Shc antiserum were obtained from Transduction Laboratories (Lexington, KY).

Polyclonal NK cell cultures were obtained by coculturing nylon nonadherent PBMC (4 × 105/ml) with irradiated (3000 rad) RPMI 8866 cells (1 × 105/ml) for 10–12 days at 37°C in a humidified 5% CO2 atmosphere as previously described (33). The cell populations used in the experiments were routinely ≥90% CD56+, CD16+, CD3 and >80% Z199+, as assessed by immunofluorescence and cytofluorometric analysis with a FACScan (Becton Dickinson, Mountain View, CA); 10–20% of the donors gave origin to NK cell cultures bearing this phenotype and were therefore selected for this study. For immunofluorescence staining, cells were incubated with saturating concentrations of the different mAbs followed by washing and labeling with FITC-conjugated goat anti-mouse F(ab′)2 (Zymed Laboratories, San Francisco, CA).

Highly purified human polyclonal NK cell pellets were incubated with primary mAbs for 30 min on ice. After washing off unbound Ab, cells were stimulated with GAM (1.5 μg/106 cells) for different time periods at 37°C.

After stimulation, cells were lysed (lysis buffer containing 1% (v/v) Triton X-100, 50 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM EGTA (pH 8), 100 μg/ml PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μM Na3VO4, and 50 mM NaF (pH 8)), and lysates were cleared of debris by centrifugation at 14,000 × g for 15 min and immunoprecipitated with different Abs conjugated to protein G- or protein A-Sepharose beads. Immunocomplexes were washed six times with 50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.25% Triton-X-100, 0.1% SDS, and 0.1% sodium deoxycholate and eluted with SDS-PAGE sample buffer.

Immunoprecipitates were resolved by SDS-PAGE and transferred to Immobilon-P nitrocellulose membranes (Millipore, Bedford MA). After blocking nonspecific reactivity, filters were probed with specific Abs diluted in TBS-T (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween-20). After extensive washing, immunoreactivity was detected using an enhanced chemiluminescence kit (Amersham, Aylesbury, U.K.).

Anti-Syk or anti-Erk2 immunoprecipitates were washed four times with 50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.25% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate and twice with specific kinase assay buffer, which consisted of 20 mM Tris (pH 7.6), 10 mM MnCl2 (for Syk) (34) or 30 mM Tris (pH 8), 1 mM MnCl2, and 10 mM MgCl2 (for Erk2) (13); the kinase reaction was initiated by adding 50 μl of kinase buffer containing 10 μCi of [32P]ATP (4500 Ci/mmol; Amersham), 1 μM (Syk) or 10 μM (Erk2) ATP, and 10 μg of myelin basic protein (MBP; Sigma-Aldrich, Milan, Italy). After 30 min of incubation at 30°C, samples were boiled in sample buffer and separated by SDS-PAGE. The gels were dried and subjected to autoradiography. The rehydrated gels were then transferred to nitrocellulose membranes and subjected to Western blot analysis.

The 51Cr release assay was used to measure rADCC against P815 target cells in the presence of saturating amounts of different mAbs as previously described (33). In some experiments cells were pretreated with piceatannol (Biomol, Plymouth Meeting, PA) for 15 min at 37°C or with PD 098059 (Calbiochem-Novabiochem, La Jolla, CA) for 1 h at 37°C before target addition.

CD94/NKG2-A receptor has been shown to inhibit both natural and CD16-mediated cytotoxicity of NK clones and the NKL cell line when cross-linked by HLA-E-bearing target cells or Z199 mAb, which is uniquely able to recognize the CD94/NKG2-A heterodimer (16, 21, 22, 32, 35). Short term human NK cultures (>90% pure and >80% Z199+) were challenged in an rADCC assay using P815 FcγR+ target cells in the presence of anti-CD16 mAb. The simultaneous presence of Z199 (anti-CD94/NKG2-A) mAb suppressed the CD16-triggered killing of P815 cells, while the presence of anti-CD56 mAb, used as control, had no effect (Fig. 1). Thus, cross-linking of the CD94/NKG2-A inhibitory complex by Z199 mAb efficiently suppresses the CD16-triggered programming for lysis in normal polyclonal NK cell populations. CD94/NKG2-A engagement also affects the basal, natural cytotoxic activity of polyclonal NK cell cultures against P815 target cells in the absence of anti-CD16 Ab as previously reported (16).

A recent report has shown Syk, and not the closely related PTK Zap-70, to be a crucial intermediate in the programming of both natural and CD16-triggered cytotoxic activity of NK cell clones (12). We observed that pretreatment with piceatannol, a selective inhibitor of Syk activity (36), also inhibits CD16-directed killing of normal human NK cell populations in a dose-dependent manner (data not shown).

We then sought whether CD94/NKG2-A perturbation could affect the CD16-induced activity of Syk. To directly assess the activation state of Syk, we evaluated the in vitro kinase activity of Syk immunoprecipitates on MBP exogenous substrate. As shown in Fig. 2, simultaneous treatment with anti-CD94/NKG2-A mAb inhibited the CD16-stimulated enzymatic activity of Syk, while the presence of an anti-CD56 mAb had no effect. Western blot analysis of the same samples with anti-Syk Ab is shown in the lower panel of Fig. 2.

The SH2-mediated recruitment to immunoreceptor tyrosine-based activatory motif-containing receptor subunits together with the phosphorylation of regulatory tyrosine residues are both necessary for and parallel the enzymatic activation of Syk (37). The experiment reported in Fig. 3 shows that the coengagement of CD94/NKG2-A significantly inhibits the CD16-triggered tyrosine phosphorylation of Syk (A); the inhibitory effect is already evident at 1 min of treatment and is maintained over all the time points at which CD16 stimulation is observed under these experimental conditions (B). No such effect was observed when cells were treated with anti-CD56 in combination with anti-CD16 mAb as a control. The lower part of both panels reports the anti-Syk blot of the same membranes to show the comparable amounts of Syk protein in each lane. In A, the thicker band in stimulated samples accounts for the abundant presence of the slower migrating, tyrosine-phosphorylated form of Syk.

The recruitment of Syk on CD16/ζ-chain was then analyzed. Fig. 4 shows that CD16 cross-linking induces the appearance of Syk in anti-ζ immunoprecipitates, and that the coengagement of CD94/NKG2-A strongly and specifically affects the CD16-induced association between Syk and ζ-chain. The Western blot of the same membrane with anti-ζ mAb shows that equivalent amounts of ζ protein have been immunoprecipitated in all samples (lower panel).

These data indicate that CD94/NKG2-A engagement abrogates the CD16-induced activity of Syk by interfering with the molecular events required for its enzymatic activation (i.e., the recruitment to activated CD16 complex and its tyrosine phosphorylation).

The observation that CD94/NKG2-A abolished the ability of CD16 ζ-chain of recruiting Syk kinase following CD16 stimulation led us to investigate the effect of coengaging CD94/NKG2-A and CD16 on the tyrosine phosphorylation status of ζ-subunit.

As shown in Fig. 5 A, treatment with both anti-CD16 and Z199 mAbs inhibited the CD16-induced tyrosine phosphorylation of ζ-chain, which leads to the appearance of two major groups of bands (centered around 21 and 30 kDa, respectively), corresponding to the increasingly phosphorylated and multiubiquitinated species of the ζ molecule (38) (R. Paolini et al., manuscript in preparation). The inhibitory effect was specifically induced by engaging the CD94/NKG2-A complex, as the use of anti-CD56 mAb, as a control, did not alter the CD16-induced tyrosine phosphorylation of ζ-chain. This inhibition was already observed after 1 min of stimulation and was maintained until 10 min (B), after which time the CD16-induced signal tended to decay (data not shown). The lower region of both panels reports the immunoblotting of the same membranes with anti-ζ mAb to verify that equivalent amounts of ζ protein were immunoprecipitated in all samples.

The abrogation of CD16-associated ζ subunit tyrosine phosphorylation by CD94/NKG2-A engagement suggests that this event can be upstream of the observed effect on Syk recruitment and subsequent activation.

ERK activity has been shown to be crucially involved in NK cell functions triggered through CD16 receptor, such as cytoplasmic granule exocytosis and cytokine gene activation (13, 39). More recently, the role of ERK in the natural cytotoxic activity of NK cells has been pointed out (11). We showed that the activity of ERK is also required for the CD16-triggered cytotoxic activity of human NK cells, as the treatment with the specific MEK inhibitor PD 098059, which abrogates ERK activation, inhibits rADCC of human polyclonal NK cells in a dose-dependent fashion (Fig. 6).

We then investigated whether CD94/NKG2-A engagement could affect the CD16-induced activation of ERK. The results reported in Fig. 7 show that CD16-induced activation of ERK enzyme (evaluated as in vitro kinase activity of specific immunoprecipitates on MBP) is markedly inhibited when cells are simultaneously treated with anti-CD94/NKG2-A mAb. This effect is specific, as the use of anti-CD56 Ab does not alter CD16-triggered ERK activity. The anti-ERK immunoblot of the same samples confirms that comparable amounts of ERK protein have been loaded in each lane (data not shown).

ERK kinases are placed downstream an enzymatic cascade, which in many systems is initiated by activation of the small G protein p21ras (40). We have previously shown that CD16 stimulation induces the activation of p21ras in human NK cells, and that this event correlates with the ability of CD16 engagement of inducing the tyrosine phosphorylation of Shc adaptor protein and its association with the Grb2/Sos complex (41).

We investigated whether the events leading to ERK activation were affected by CD94/NKG2-A by analyzing the tyrosine phosphorylation of Shc, a PTK-dependent step in the activation of p21ras.

CD16 receptor and CD94/NKG2-A complex were coengaged on human polyclonal NK cells by using B73.1 (anti-CD16) and Z199 mAbs plus GAM, and the phosphorylation status of Shc in specific immunoprecipitates was analyzed by Western blot with anti-pTyr mAb. As shown in Fig. 8,A, the simultaneous coengagement of CD94/NKG2-A markedly inhibited the CD16-induced tyrosine phosphorylation of both p52 and p46 isoforms of Shc. It is worth noting that the cross-linking of CD94/NKG2-A complex per se did not appreciably affect the basal phosphorylation status of the Shc adaptor protein. The inhibition of Shc tyrosine phosphorylation was quite rapid; it was observed after 1 min of stimulation and persisted until 10 min, suggesting that CD94/NKG2-A does not merely induce a shift in the kinetics of CD16-induced Shc tyrosine phosphorylation (Fig. 8 B). This inhibition was specific, as an anti-CD56 mAb did not alter the CD16-triggered tyrosine phosphorylation of Shc. The anti-Shc blot shows that equivalent amounts of Shc protein were immunoprecipitated in all samples (A and B, lower panels).

As shown in Fig. 8, CD16 stimulation induces the association between Shc and a p145 phosphoprotein, as previously described (41). It is of interest that a diminished association of p145 with Shc parallels the inhibition of Shc tyrosine phosphorylation induced by CD94/NKG2-A. Preliminary observations indicate that this protein probably represents the SH2-containing inositol phosphatase SHIP, which has been reported to undergo tyrosine phosphorylation and association with Shc following stimulation by a variety of immune and cytokine receptors (42) (G. Palmieri, unpublished observations).

The phosphorylation-dependent association between Shc and Grb2/Sos complex constitutes an important step in the activation of the small G protein p21ras. The experiment reported in Fig. 9 indicates that in accordance with the interference on the tyrosine phosphorylation of Shc, engagement of CD94/NKG2-A complex also inhibits the CD16-induced formation of the Shc/Grb2 complex. Fig. 9 A shows that following co-cross-linking with anti-CD16 and anti-CD94/NKG2-A mAbs, a diminished amount of Grb2 is found in Shc immunoprecipitates (middle panel). The same information is confirmed by immunoprecipitating Grb2 and blotting the membrane with anti-pTyr mAb, which shows the p52- and p46-phosphorylated forms of Shc (B, upper panel). Normalization was performed by blotting with anti-Shc and anti-Grb2 (A and B, respectively).

Collectively, these results indicate that the coengagement of CD94/NKG2-A markedly inhibits the CD16-mediated activation of ERK on human NK cells, one important mechanism leading to NK cytotoxic activity. They also suggest that the block is exerted at an early step along the Ras/MAPK cascade by interfering with the tyrosine phosphorylation of Shc and its association with the Grb-2/Sos complex.

CD94/NKG2-A is the HLA-E-specific inhibitory receptor on human NK cells. Similarly to KIR, its engagement by natural ligand or specific mAb induces the tyrosine phosphorylation of ITIM sequences located in the NKG2-A chain and the recruitment of SHP-1 tyrosine phosphatase (25, 35). While a clear role for SHP-1 in the inhibitory function of MHC-I receptors has been established, controversial evidence has been provided on the involvement of SHP-2, which is structurally homologous to SHP-1, in this activity (43).

The signaling pathways regulating cytotoxic function that are affected by MHC-I-specific receptors are mostly unknown. KIR engagement has been reported to inhibit the target-induced intracellular calcium rise and inositol phosphate generation (28, 29, 30), while Ab-mediated engagement of CD94/NKG2-A has been shown to interfere with CD16-induced inositol phosphate generation, and tyrosine phosphorylation of phospholipase C-γ and Zap-70 (6). Evidence that KIR-activated SHP-1 can directly dephosphorylate two adaptor proteins, such as pp36 and SLP-76, has been provided recently (29, 31).

The present study has analyzed the mechanisms involved in the inhibitory function of CD94/NKG2-A receptor on human NK cell cytotoxic function. Our attention has focused on the effect of CD94/NKG2-A engagement on two enzymatic pathways, namely the activation of the PTK Syk and ERK kinase, which play an important role in lymphocyte cytotoxicity. Here we show that engagement of the CD94/NKG2-A complex inhibits the CD16-triggered activity of Syk by interfering with the molecular events required for its enzymatic activation. Our study also constitutes the first evidence of ERK activity inhibition by an MHC-I-specific receptor and shows that CD94/NKG2-A affects the early, PTK-dependent events in the activation of p21ras.

The direct evidence that CD94/NKG2-A cross-linking specifically hampers the CD16-mediated activation of Syk was evaluated as in vitro kinase activity on an exogenous substrate. Interestingly, we also found that Syk PTK is recruited to activated CD16 receptor complex, and that both the CD16-triggered tyrosine phosphorylation of Syk and its activation-induced association with CD16 complex ζ-chain are inhibited by the simultaneous engagement of CD94/NKG2-A receptor. Whether the lack of recruitment to the activated CD16 receptor complex is responsible for the impairment of Syk tyrosine phosphorylation and consequent activation remains to be assessed. Similarly, anti-CD94 mAb treatment of NK clones has been previously shown to affect CD16-induced Zap-70 association with phospho-ζ (6).

Here we also show that the simultaneous cross-linking of CD94/NKG2-A heterodimer markedly inhibits the CD16-induced tyrosine phosphorylation of ζ-chain; this observation provides a molecular explanation for the observed lack of recruitment (and activation) of Syk following simultaneous coengagement of CD94/NKG2-A complex and confirms a previous report in which CD94 cross-linking was found to inhibit CD16-induced tyrosine phosphorylation of ζ-chain in selected NK clones (6).

Whether the observed effect is imputable to the direct dephosphorylation of ζ-chain by CD94/NKG2-A-associated SHP-1 or, indirectly, through the inhibition of the PTK responsible for ζ phosphorylation remains to be ascertained and is presently under investigation.

The inhibition of Syk activity could represent an important mechanism for the CD94/NKG2-A-operated interference on CD16-triggered NK lytic function. Moreover, as the activity of Syk is required for both natural and CD16-mediated killing, and its tyrosine phosphorylation is not induced during KIR-mediated interaction of NK cells with MHC-bearing targets (12), it could also constitute a critical event in the CD94/NKG2-A-induced inhibition of cytotoxic activity against HLA-E-bearing cells.

Our data also suggest that CD94/NKG2-A engagement, by abrogating the tyrosine phosphorylation of CD16-associated ζ-chain, a very proximal event in the propagation of the activatory signal, can affect multiple biochemical pathways (2, 5, 37, 38).

The activity of the ERK enzymes belonging to the MAPK family has been recently shown to play a crucial role in the development of natural NK-mediated lytic function (11). Here we extend this finding by showing that the MEK-specific inhibitor, PD098059, inhibits CD16-mediated cytotoxicity as well. This observation is in agreement with our previous data on the ability of CD16 stimulation to activate ERKs in human NK cells and their involvement in the CD16-mediated exocytosis of cytotoxic granules (13). Thus, ERK activity represents another common intermediate in both natural and CD16-mediated modalities of NK cell killing.

In the present work we provide the first evidence that CD94/NKG2-A efficiently counteracts the CD16-mediated activation of ERK, and that it is capable of inhibiting the tyrosine phosphorylation of Shc and its association with Grb-2, the molecular events involved in the activation of p21ras through many receptors and by CD16 stimulation as well (41). The mechanisms underlying the diminished tyrosine phosphorylation of Shc are currently unknown. Our preliminary observations suggest that Syk could be involved in the CD16-induced phosphorylation of Shc in human NK cells, as the selective inhibitor for Syk piceatannol blocks the CD16-induced tyrosine phosphorylation of Shc and its association with Grb-2 at the same doses that effectively abrogate CD16-triggered cytotoxic activity (G. Palmieri, data not shown). The impairment of Syk activity observed upon CD94/NKG2-A cross-linking could thus be responsible for the inhibition of Shc tyrosine phosphorylation.

The interference of CD94/NKG2-A on Ras/MAPK cascade activation can be relevant for the inhibition of both the lytic function and the cytokine production of NK cells, as ERK activity is involved in CD16-triggered cytokine gene activation in human NK cells (39). In this regard, the CD94/NKG2-A complex is able to suppress FcεRI-triggered TNF-α secretion in a heterologous cell system (25), while KIR engagement inhibits target-induced IFN-γ production in human NK clones (44); similarly, Ly-49 inhibitory receptors have been shown to affect cytokine production by murine NK cells (45, 46). The ability of KIR-activated SHP-1 to directly dephosphorylate SLP-76, an important intermediate in NF-AT activation, can be also relevant in this context (31).

Collectively taken, our results show that CD94/NKG2-A engagement markedly inhibits the tyrosine phosphorylation of both proximal and distal participants along the CD16-induced signal transduction pathways. SHP-1 activity is involved in regulation of the phosphorylation status of several signal transduction molecules; in fact, higher levels of TCR-induced tyrosine phosphorylation of ζ and ε components of the TCR/CD3 complex and hyperactivation of ERK activity are observed in SHP-1-deficient me/me mice (47). Whether CD94/NKG2-A-activated SHP-1 directly dephosphorylates the involved molecules (CD16-associated ζ, Syk, and Shc) or indirectly acts by negatively regulating the PTK(s) responsible for these events is unclear as yet and will be object of future studies. The ability of KIR-activated SHP-1 to directly dephosphorylate p36 and SLP-76 has been reported (29, 31); moreover, SHP-1 overexpression has been found to decrease the tyrosine phosphorylation of a chimeric ζ molecule in a heterologous cell system, and the direct in vitro interaction with SHP-1 leads to the diminution of Zap-70 activation and tyrosine phosphorylation in T cells (48).

The observed abrogation of CD16 subunit phosphorylation leads to the speculation that CD94/NKG2-A-dependent interference in the biochemical pathways leading to the development of cytotoxic activity could be placed at a very proximal step.

In summary, our results have shown that the CD94/NKG2-A inhibitory receptor affects two key biochemical pathways involved in the development of both natural cytotoxicity and CD16-mediated killing. These data contribute to a better understanding of the mechanisms activated by inhibitory receptors and of the molecular cross-talk between signaling pathways initiated by activatory and inhibitory receptors. These information will be instrumental for a better comprehension of the regulation of NK activities.

We thank Drs. G. Trinchieri, B. Perussia, and A. Moretta for kindly providing mAbs. The expert technical assistance of Dina Milana, Anna Maria Bressan, Alessandro Procaccini, Antonio Sabatucci, and Patrizia Birarelli is gratefully acknowledged.

1

This work was supported in part by grants from Italian Association for Cancer Research, the Istituto Superiore di Sanità Italy-USA Therapy of Tumors Program, the Ministero dell’Università e della Ricerca Scientifica e Tecnologica 40% and 60%, and European Union Contract PL950062.

3

Abbreviations used in this paper: ADCC, Ab-dependent cellular cytotoxicity; ITIM, immunoreceptor tyrosine-based inhibitory motif; PTK, protein tyrosine kinase; ERK, extracellular regulated kinase; MAPK, mitogen-activated protein kinase; KIR, killer Ig-like receptor; SHP, SH2-containing tyrosine phosphatase; rADCC, reverse ADCC; GAM, goat anti-mouse IgG F(ab′)2; pTyr, phosphotyrosine; MBP, myelin basic protein.

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