Reconstituted Killer Cell Inhibitory Receptors for Major Histocompatibility Complex Class I Molecules Control Mast Cell Activation Induced via Immunoreceptor Tyrosine-based Activation Motifs*

(Received for publication, October 8, 1996, and in revised form, December 2, 1996)

Mathieu Bléry Dagger , Jérome Delon §, Alain Trautmann §, Anna Cambiaggi Dagger , Lucia Olcese Dagger , Roberto Biassoni , Lorenzo Moretta , Philippe Chavrier Dagger , Alessandro Moretta par , Marc Daëron ** and Eric Vivier Dagger Dagger Dagger

From the Dagger  Centre d'Immunologie INSERM/CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 09, France, ** Laboratoire d'Immunologie Cellulaire et Clinique, INSERM U255, Institut Curie, Paris, 75 231 France, § Laboratoire d'Immunologie Cellulaire, CNRS URA625, CERVI, Groupe hospitalier Pitié-Salpêtrière, 75013 Paris, France,  Istituto Nazionale per la Ricerca sul Cancro, Via R. Benzi, 10, 16132 Genova, Italy, and par  Istituto di Istologia ed Embriologia Generale, University of Genova, Genova, 16 132 Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Natural killer and T cells express at their surface, members of a multigenic family of killer cell inhibitory receptors (KIR) for major histocompatibility complex Class I molecules. KIR engagement leads to the inhibition of natural killer and T cell activation programs. We investigated here the functional reconstitution of KIR in a non-lymphoid cell type. Using stable transfection in the RBL-2H3 mast cell line, we demonstrated that (i) KIR can inhibit signals induced by Fcepsilon RIgamma or CD3zeta polypeptides that bear immunoreceptor tyrosine-based activation motifs; (ii) two distinct immunoreceptor tyrosine-based inhibition motifs-bearing receptors, i.e. KIR and Fcgamma RIIB, use distinct inhibitory pathways since KIR engagement inhibits the intracellular Ca2+ release from endoplasmic reticulum stores, in contrast to Fcgamma RIIB, which only inhibits extracellular Ca2+ entry; (iii) KIR require co-ligation with an immunoreceptor tyrosine-based activation motif-dependent receptor to mediate their inhibitory function. This latter finding is central to the mechanism by which KIR selectively inhibit only the activatory receptors in close vicinity. Taken together our observations also contribute to define and extend the family of immunoreceptor tyrosine-based inhibition motif-bearing receptors involved in the negative control of cell activation.


INTRODUCTION

NK1 cells are cytotoxic lymphocytes that can induce the lysis of target cells by two mechanisms (1-3). Antibody-dependent cell cytotoxicity leads to the lysis of antibody-coated target cells, whereas natural cytotoxicity leads to the antibody-independent lysis of a variety of cell targets, including primarily virus-infected cells and tumor cells. Induction of both natural cytotoxicity and antibody-dependent cell cytotoxicity programs is controlled by the engagement of killer cell inhibitory receptors (KIR) for MHC Class I molecules; recognition of MHC Class I molecules expressed on target cell surface by KIR induces a general inhibition of both NK cell cytotoxic programs (4-8). When expressed on T cells, KIR also inhibits CD3/TCR-dependent activation programs (9, 10).

Similarly, a class of low affinity Fc receptors, Fcgamma RIIB, has been shown to inhibit B cells and T cells as well as mast cell activation (11-15). The molecular dissection of Fcgamma RIIB intracytoplasmic domain revealed the inhibitory function of a 13 amino acid ITIM (11-15). Within this sequence, we and others have reported the conservation of a (I/V)XYXX(L/V) stretch in human, as well as mouse, KIR and Fcgamma RIIB (16, 17). Upon tyrosine phosphorylation of the common (I/V)XYXX(L/V) motif, both KIR and Fcgamma RIIB bind the SH2-tandem protein-tyrosine phosphatases SHP-1 and SHP-2, which are reported to be involved in the inhibitory pathways used by KIR and Fcgamma RIIB (15-19). These data prompted us to further investigate the mechanisms by which KIR exert their inhibitory function and may thus be defined as members of the family of ITIM-bearing receptors involved in the attenuation of hematopoietic cell activation.

In this attempt, we reconstituted an HLA-Cw3-specific KIR (p58.2) in a transfected mast cell/basophil-like RBL-2H3 cell line (RTIIB). RTIIB cells express two distinct ITAM-dependent receptors: the endogenous Fcepsilon RI antigen receptor and a transfected CD25/CD3zeta chimeric molecule, as well as the murine Fcgamma RIIb2 ITIM-bearing receptor (12, 13). We also reconstituted a naturally occurring activatory form of p58.2, the p50.2 KAR (killer cell activatory receptor), in RTIIB cells. The p50.2 KAR expresses a shorter intracytoplasmic domain, which does not contain any (I/V)XYXX(L/V) stretch. This activatory receptor has been reported to trigger T and NK cell activation programs (20, 21).

The model of KIR and KAR reconstitution in RTIIB cells presents several useful features. First, RTIIB cell activation induced by Fcepsilon RI as well as by CD25/CD3zeta receptors is inhibited by Fcgamma RIIB (12, 13). Second, RTIIB, NK, and T cells express multimeric receptor complexes, that are built according to a similar architecture. Indeed, Fcepsilon RI, the antibody-dependent cell cytotoxicity receptor complex (Fcgamma RIIIA), and the CD3·TCR complexes include associated polypeptides that are necessary and sufficient for their cell surface expression as well as their transducing properties. Human Fcgamma RIIIA includes CD16 in non-covalent association with CD3zeta and FcRgamma (Fcepsilon RIgamma ) homo- and heterodimers (22-25). On T cells, the vast majority of CD3·TCR complexes include CD3zeta homodimers, but TCRgamma delta + T cells, CD3/TCR+ large granular lymphocytes, and subsets of CD8alpha alpha intraepithelial lymphocytes express FcRgamma in homodimers or in heterodimers with CD3zeta (26-28). Therefore, the effect of KIR on CD25/CD3zeta - and Fcepsilon RI-signaling pathways in RTIIB cells can be directly compared with the effect of KIR on CD3/TCR- as well as antibody-dependent cell cytotoxicity receptor complex-mediated lymphocyte activation. Third, RTIIB, NK cells, and cytotoxic T lymphocyte express intracytoplasmic granules that undergo regulated exocytosis upon ITAM-dependent receptor aggregation. Intracytoplasmic granules expressed in NK and T lymphocytes contain an array of molecules involved in lymphocyte-mediated cell cytotoxicity, such as perforin and granzymes (29). Although lymphocyte-mediated cell cytotoxicity has not been formally proven to depend upon regulated exocytosis (30), the drastic reduction of both NK and T cell-mediated cytotoxicity in perforin-/- mice supports the fact that regulated exocytosis is involved in lymphocyte-mediated cell cytotoxicity (31). In this regard, the action of KIR on serotonin release in RTIIB cells might be relevant to the inhibition of lymphocyte-mediated cell cytotoxicity by KIR.

Our results show that (i) human KIR reconstituted in RTIIB cells inhibit CD3zeta - as well as Fcepsilon RI-induced activation; (ii) transcription-factor independent effector cell responses, i.e. intracytoplasmic calcium ([Ca2+]i) increase and serotonin release, can be inhibited by KIR in RTIIB cells, whereas (iii) KAR are not functional in the same cells, and finally (iv) KIR inhibitory function requires KIR and ITAM-containing receptor cross-linking. These results also provide new insights on the importance of the elements of the signaling cascade for the functions of a novel receptor family, characterized either by intact intracytoplasmic ITIM (Fcgamma RIIB and KIR) or by their mutated version (KAR).


EXPERIMENTAL PROCEDURES

Antibodies

The following mAb and antisera were obtained from Immunotech, Marseille, France: mouse anti-human CD25 mAb (B1.49.9, IgG2a), rat anti-human CD25 mAb (33B3.1, IgG2a), mouse anti-human p58.2 mAb (GL183, IgG1), mouse anti-rat Ig (H + L) F(ab)'2 (MAR), goat anti-mouse Ig (H + L) F(ab)'2 (GAM), donkey anti-mouse Ig (H + L) F(ab)'2 (DAM), and donkey anti-rat Ig (H + L) F(ab)'2 (DAR). DAM and DAR are mouse Ig- and rat Ig- specific, respectively. Rat IgE mAb (LO-DNP-30), mouse IgE mAb (2682-I), rat anti-Fcgamma RII/III mAb (2.4G2) and mouse anti-CD25 mAb (7G7, IgG1) have been described earlier (13). The mouse anti-rat Fcepsilon RIalpha (BC4, IgG1) was a generous gift from Drs. R. Siraganian (National Institutes of Health, Bethesda, MD) and M. Benhamou (CERVI, Paris). Mouse IgE mAb was used as a dilution of hybridoma supernatants. All other mAbs were used as protein A/G-purified mAb. GL183, 2.4G2, and 7G7 mAb were used as F(ab)'2; LO-DNP-30, 2682-I, B1.49.9, and 33.B3.1 were used as intact mAbs. LO-DNP-30 and 2682-I are directed against DNP and TNP. MAR F(ab)'2 was trinitrophenylated using trinitro-benzene sulfonic acid (Eastman Kodak Co.), and 1 mol of MAR F(ab)'2 was substituted with an average number of 10 TNP mol. This TNP-F(ab)'2 MAR was used to cross-link mouse anti-TNP IgE and rat anti-Fcgamma RII/III 2.4G2.

Cells

All cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FCS and penicillin (100 IU/ml) and streptomycin (100 µg/ml). RBL-2H3 cell transfectants expressing murine Fcgamma RIIb2 and CD25/CD3zeta chimeric molecules (RTIIB) have been previously described. The CD25/CD3zeta chimeric molecule includes the complete human CD25 ecto- and transmembrane domains linked to the complete mouse CD3zeta intracytoplasmic domain (32). RTIIB cells were transfected by electroporation using either the 183.6 cDNA-encoding p58.2 (RTIIB.p58) or the 183.Act1 cDNA-encoding p50.2 (RTIIB.p50), in the RSV-5.gpt expression vector (33). Stable transfectants were established by culture in the presence of xanthine (250 µg/ml), hypoxanthine (13.6 µg/ml), and mycophenolic acid (2 µg/ml). Two representative clones of each transfection series (RTIIB.p58A, RTIIB.p58B and RTIIB.p50A, RTIIB.p50B) were examined in parallel and gave similar results. Unless indicated, results from one clone of each transfection series are shown.

Flow Cytometric Analysis

The primary mAb was incubated with cells on ice for 20-30 min, followed by three washes with phosphate-buffered saline supplemented with 0.2% bovine serum albumin. The secondary staining was performed using fluorescein-conjugated rabbit anti-mouse IgG (Immunotech), followed by three phosphate-buffered saline/0.2% bovine serum albumin washes and resuspension in PBS containing 1% formaldehyde. Cells were analyzed on a FACS-Scan apparatus (Becton Dickinson, Mountain View, CA).

Single-cell Ca2+ Video Imaging

Cells (2 × 105/sample) were allowed to adhere overnight onto glass coverslips in culture medium. Adherent cells were washed and then incubated at 37 °C for 40 min in RPMI 1640 medium supplemented with 10% FCS with a 10-3 dilution of mouse IgE (2682-I) in the presence or absence of either 1 µg/ml GL183 or 1 µg/ml 2.4G2. 1 µM Fura-2/AM (Molecular Probes, Inc., Eugene, OR) in dimethyl sulfoxide premixed with 0.2 mg/ml pluronic acid (Molecular Probes) was then added to the medium for 20 min. Cells were washed, and then measurements of intracellular Ca2+ ([Ca2+]i) mobilization were performed at 37 °C in MS buffer (140 mM NaCl, 5 mM KCl, 10 mM HEPES, pH 7.4, 1 mM CaCl2, 1 mM MgCl2) with a Nikon Diaphot 300 microscope and an IMSTAR imaging system as described previously (34). Briefly, each [Ca2+]i image (taken every 6 s) was calculated from the ratio of the average of four fluorescence images after 340 nm excitation and four fluorescence images after 380 nm excitation. Calibrations were done as previously reported (35). [Ca2+]i values were calculated according to Grynkiewicz et al. (36). The stimulation was done by adding GAM F(ab)'2 or TNP-F(ab)'2 MAR to the MS buffer for a final concentration of 50 µg/ml and 10 µg/ml, respectively. The measurements of intracellular calcium store release were done by replacing the MS buffer with a stimulation buffer (150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM EGTA, 20 mM HEPES, pH 7.3, 50 µg/ml F(ab)'2 GAM) at the time of stimulation. For each experiment, results were shown as the average variation of [Ca2+]i (nM) as a function of time for a population of 20-30 cells.

Serotonin Release

RTIIB cell transfectants harvested using trypsin EDTA were examined for serotonin release as described (12). Briefly, cells were resuspended in RPMI 1640 medium supplemented with 10% FCS at 1 × 106 cells/ml, and incubated at 37 °C for 1 h with 2 µCi/ml [3H] serotonin (Amersham Corp., Les Ulis, France), washed, resuspended in RPMI 1640 medium, 10% FCS, incubated for another hour at 37 °C, washed again, resuspended in the same medium and distributed in 96-well microculture plates at 2 × 105 cells/well. Cells were then incubated for 1 h with mouse or rat IgE, with mouse or rat anti-CD25 mAb in the absence or in the presence of GL183 F(ab)'2, in a final volume of 50 µl. Cells were washed three times in 200 µl of phosphate-buffered saline and once in 200 µl of RPMI 1640 medium, 10% FCS, and then 25 µl of culture medium were added to each well and cells were warmed for 15 min at 37 °C before challenge. Cells were challenged for 30 min at 37 °C with 25 µl of prewarmed GAM, DAM, or DAR F(ab)'2 as indicated. Reactions were stopped by adding 50 µl of ice-cold medium and by placing plates on ice. 50 µl of supernatants were mixed with 1 ml of emulsifier safe scintillation liquid (Packard Instruments, Groningen, The Netherlands) and counted in a LS6000 Beckman counter. The percentage of serotonin release was calculated using as 100% the cpm contained in 50 µl harvested from wells containing the same number of cells and lysed in 100 µl of 0.5% SDS, 0.5% Nonidet P-40.


RESULTS

Reconstitution of Human KIR and KAR in RTIIB Cells

Stable RTIIB cell transfectants expressing murine Fcgamma RIIB as well as a CD25/CD3zeta chimeric molecule at their surface, were further transfected with two distinct NK cell MHC Class I receptor p58.2 and p50.2 cDNA constructions. In NK cells, p58.2 (KIR) exerts an inhibitory effect whereas p50.2 (KAR) is an activating molecule (20, 21). Despite this striking difference, both p58.2 and p50.2 are HLA-Cw3-specific receptors and are recognized by the GL183 mAb. Representative transfected clones used thereafter were selected for their matched cell-surface expression of p58.2 (RTIIB.p58) or p50.2 (RTIIB.p50) HLA-Cw3 receptors (Fig. 1).


Fig. 1. Reconstitution of p58.2 and p50.2 HLA-Cw3-specific KIR and KAR in RTIIB cells. Representative transfected RTIIB clones were stained by indirect immunofluorescence using mAb directed against rat Fcepsilon RI (BC4), human CD25 (B1.49.9), as well as human p58.2 KIR and p50.2 KAR (GL183). Negative controls were only incubated with fluorescein-conjugated rabbit anti-mouse IgGs that were also used as secondary reagents.
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Inhibition of ITAM-dependent Serotonin Release by KIR Reconstituted in RTIIB Cells

RTIIB cells can be induced to release serotonin by one of two ways: aggregation of the endogenous rat Fcepsilon RI receptor complex or aggregation of the CD25/CD3zeta chimeric molecule (12, 37). Mouse IgE binding to Fcepsilon RI is not sufficient to induce RTIIB serotonin release, and aggregation of Fcepsilon RI receptors was obtained using GAM F(ab)'2. The integrity of ITAM expressed by both receptors is required for RTIIB serotonin release (38) indicating that both Fcepsilon RI- and CD25/CD3zeta -induced serotonin release utilizes ITAM-dependent signaling mechanisms. Using RTIIB cells expressing p58.2 KIR or p50.2 KAR in addition to Fcepsilon RI and CD25/CD3zeta , we investigated whether the expression or the aggregation of reconstituted HLA-Cw3 receptors modulate ITAM-dependent serotonin release.

As shown in Fig. 2, A and B, aggregation of Fcepsilon RI or CD25/CD3zeta receptors induces a dose-dependent serotonin release of RTIIB cells expressing either p58.2 KIR (RTIIB.p58) or p50.2 KAR (RTIIB.p50). The larger serotonin release elicited by anti-CD25 in RTIIB.p58 compared with RTIIB.p50 cells most likely reflects the difference in surface expression of CD25/CD3zeta in the two cell types. When p58.2 KIR were aggregated using anti-p58.2 mAb (GL183), no RTIIB serotonin release was observed. This result is consistent with the lack of detectable changes in NK and T cell function upon anti-p58.2 mAb stimulation. However, no serotonin release was detected in response to GL183 mAb in RTIIB.p50 cells, in contrast with the stimulatory effect of p50.2 KAR reported in both NK and T cells (21, 39).


Fig. 2. Surface receptor-induced serotonin release in RTIIB cells expressing human KIR. RTIIB.p58 cells (A) or RTIIB.p50 cells (B) were incubated for 1 h at 37 °C either with serial dilutions of mouse IgE (2682-I, initial concentration: straight hybridoma supernatant) (closed circles), anti-hCD25 (F(ab)'2 7G7, initial concentration: 1 mg/ml) (closed squares), or GL183 F(ab)'2 (initial concentration: 1 mg/ml) (open triangles). After being washed, cells were challenged for 30 min at 37 °C with 50 µg/ml GAM F(ab)'2. The serotonin released in supernatants was measured. The experiment shown is representative of three independent experiments. These experiments also defined the optimal concentration of mAb needed for cell activation.
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In a second set of experiments, p58.2 KIR versus p50.2 KAR and Fcepsilon RI receptors were co-aggregated using mouse IgE, mouse GL183 and GAM. Using saturating concentrations of GL183, the serotonin release induced by Fcepsilon RI was impaired in RTIIB.p58 cells (Fig. 3A). This inhibition was detectable at suboptimal (<10-3 dilution), as well as optimal concentrations of IgE (10-3 dilution). Using saturating concentrations of both GL183 (1 µg/ml) and IgE (10-3 dilution), 72.8 ± 2.2% and 55.8 ± 9.9% (mean ± S.E., n = 3) inhibition of serotonin release was observed in two distinct RTIIB.p58 clones. Similar results were obtained when serotonin release was triggered via the CD25/CD3zeta chimeric molecule (Fig. 3C). Using saturating concentrations of both GL183 (1 µg/ml) and anti-hCD25 (3 µg/ml), serotonin release was inhibited by 39.3 ± 11.9% in the representative RTIIB.p58B clone.


Fig. 3. Human KIR inhibit ITAM-dependent serotonin release in RTIIB cells. RTIIB.p58 cells (A, C) and RTIIB.p50 cells (B, D) were incubated for 1 h at 37 °C with 3 µg/ml GL183 F(ab)'2 and mouse IgE (2682-I) (A and B, open circles) or with 3 µg/ml GL183 F(ab)'2 and anti-hCD25 (F(ab)'2 7G7) (C and D, open circles). Controls were made without GL183 F(ab)'2 (closed circles). After being washed, cells were challenged for 30 min at 37 °C with 50 µg/ml GAM F(ab)'2. The serotonin released in supernatants was measured. The experiment shown is representative of three independent experiments.
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In contrast, no significant inhibition or potentiation of ITAM-dependent cell activation was detected when p50.2 KAR was co-aggregated with either Fcepsilon RI or CD25/CD3zeta surface receptors, even at suboptimal concentrations of triggering IgE or anti-hCD25, in the presence of a saturating concentration of GL183 (1 µg/ml) (Fig. 3, B and D). These results first demonstrate that p58.2 KIR reconstituted in RTIIB cells are functional and inhibit ITAM-dependent cell activation. Second, they show that the integrity of the p58.2 intracytoplasmic sequence is required for KIR-mediated inhibition of RTIIB serotonin release. Finally, these data indicate that RTIIB cells provide an appropriate cellular environment for a functional reconstitution of p58.2 KIR but not of p50.2 KAR.

KIR Inhibitory Function Requires Co-engagement of KIR and ITAM-containing Receptors

In the next set of experiments, RTIIB.p58 cells were stimulated via Fcepsilon RI in the presence of aggregated p58.2 in one of two ways: DAM was used to co-aggregate Fcepsilon RI and p58.2 KIR via mouse IgE and mouse GL183, respectively (Fig. 4C), or a combination of DAR and DAM was used to independently aggregate Fcepsilon RI and p58.2 KIR via rat IgE and mouse GL183 respectively (Fig. 4D). As shown in Fig. 4A (closed circles), Fcepsilon RI-p58.2 KIR co-aggregation induced a GL183 dose-dependent inhibition of Fcepsilon RI-induced serotonin release, consistent with our observation reported in Fig. 3A. By contrast, the independent aggregation of Fcepsilon RI and p58.2 KIR failed to inhibit Fcepsilon RI-induced serotonin release at any GL183 concentration (Fig. 4A, open circles). Similar results were obtained when RTIIB.p58 serotonin release was induced via the CD25/CD3zeta chimeric molecule (Fig. 4B). These results demonstrate that KIR require a co-aggregation with activatory receptors (Fcepsilon RI or CD25/CD3zeta ) to exert their inhibitory functions. They also suggest that KIR-mediated inhibition takes place in the immediate vicinity of KIR molecules. Consistent with this conclusion, co-aggregation of Fcepsilon RI and KIR only inhibits RTIIB cell serotonin release induced by Fcepsilon RI but not by the triggering of the CD25/CD3zeta chimeric molecule (data not shown).


Fig. 4. Inhibition of ITAM-dependent RTIIB cells serotonin release requires KIR co-aggregation. A, RTIIB.p58 cells were incubated 1 h at 37 °C with indicated concentrations of GL183 F(ab)'2 and either 10-3 murine IgE (2682-I) dilution (closed circles) or 10-3 rat IgE (LO-DNP-30, 1 mg/ml initial concentration) dilution (open circles). After being washed, cells were challenged for 30 min at 37 °C with 50 µg/ml DAM F(ab)'2 (closed circles) or with 50 µg/ml DAM F(ab)'2 plus 50 µg/ml DAR F(ab)'2 (open circles). The serotonin released in supernatants was measured. B, RTIIB.p58 cells were incubated for 1 h at 37 °C with indicated concentrations of GL183 F(ab)'2 and either 3 µg/ml anti-hCD25 mAb (mB1.49.9) (closed circles) or 3 µg/ml anti-hCD25 mAb (r33B3.1) (open circles). After being washed, cells were challenged for 30 min at 37 °C with 50 µg/ml DAM F(ab)'2 (closed circles) or with 50 µg/ml DAM F(ab)'2 plus 50 µg/ml DAR F(ab)'2 (open circles). The serotonin released in supernatants was measured. This experiment is representative of five independent experiments. Co-aggregation and independent aggregation experiments are schematized in C and D, respectively.
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From these data, one can predict that an inhibitory effect of p58.2 KIR should only be observed when the relative proportion of co-aggregated KIR-activatory receptors is high enough. To test this point, we have selected a clone expressing an unusually high level of CD25/CD3zeta chimeric molecules. In this particular clone, using saturating concentrations of GL183 F(ab)'2 (1 µg/ml), only low levels of inhibition (14.1 ± 5.0%) of CD25/CD3zeta -induced serotonin release were observed. In addition, we have observed that in all clones stimulated with supraoptimal IgE concentrations (>10-3) KIR fail to inhibit Fcepsilon RI-induced RTIIB serotonin release. These data are compatible with an inhibition requiring the co-aggregation of activatory and inhibitory receptors.

KIR-Fcepsilon RI Co-aggregation Inhibits Fcepsilon RI-induced Intracytoplasmic Ca2+ Mobilization

Activation of RTIIB cells via ITAM triggers [Ca2+]i mobilization (40, 41). To test whether reconstituted human KIR inhibit early ITAM-dependent activatory signals, measurements of [Ca2+]i were performed using a single-cell imaging system. RTIIB.p58 and RTIIB.p50 cells were stimulated via the Fcepsilon RI receptor complex in the absence or presence of GL183 using GAM as a cross-linker. In both RTIIB.p58 and RTIIB.p50 cells, aggregation of the Fcepsilon RI receptor complex induced a large [Ca2+]i response consisting of a peak followed by a sustained plateau (Fig. 5A, dotted line). When p58.2 KIR was co-aggregated with Fcepsilon RI, the IgE-induced [Ca2+]i peak was impaired (Fig. 5A, continuous line). Using saturating concentrations of both GL183 F(ab)'2 (1 µg/ml) and IgE (10-3 dilution), the [Ca2+]i reached at the peak decreased from 1459 ± 92 nM (n = 58 cells) to 756 ± 59 nM (n = 60 cells) (mean ± S.E., p < 0.001) (Table I).


Fig. 5. Human KIR inhibit ITAM-dependent intracytoplasmic Ca2+ mobilization in RTIIB cells. Dotted line, RTIIB.p58 cells were pre-incubated with mouse IgE mAb (2682-I) (10-3 dilution). Continuous lines, RTIIB.p58 cells were pre-incubated 1 h with a combination of mouse IgE mAb (10-3 dilution) and GL183 F(ab)'2 mouse mAb (1 µg/ml) (A, C) or mouse IgE mAb (10-3 dilution) and 2.4G2 F(ab)'2 rat mAb (1 µg/ml) (B, D). At a time indicated by the arrow, cells were stimulated with a GAM F(ab)'2 (50 µg/ml) (A, C), or TNP-F(ab)'2 MAR (10 µg/ml) (B, D). A and B, RTIIB.p58 cells were stimulated in the presence of extracellular calcium. C and D, RTIIB.p58 cells were stimulated in the absence of extracellular calcium. Values were obtained from 59 to 117 tested cells from 3 to 5 independent experiments.
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Table I.

Control of [Ca2+]i mobilization in RTIIB, RTIIB·p50, and RTIIB·p58 cells

IgE (2682-I), GL183 F(ab)'2, 2.4G2 F(ab)'2, GAM F(ab)'2, and TNP-MAR F(ab)'2 were used as indicated in Fig. 5. Values were obtained from 59 to 117 tested cells from 3 to 5 independent experiments.


IgE + GAM IgE + GL183 + GAM IgE + TNP-F(ab)'2MAR IgE + 2.4G2 + TNP-F(ab)'2MAR

RTIIB
  Basal levela 104  ± 5 104  ± 3
  Peak responseb 1234  ± 78 1287  ± 68 NDc ND
  Plateaud 645  ± 35 692  ± 36
RTIIB · p50
  Basal levela 145  ± 5 165  ± 6
  Peak responseb 829  ± 67 873  ± 67 ND ND
  Plateauf 519  ± 26 534  ± 25
RTIIB · p58
  Basal levela 110  ± 4 100  ± 5 102  ± 5 96  ± 4
  Peak responseb 1459  ± 92 756  ± 59 1032  ± 47 985  ± 48
  Plateaud 700  ± 35 339  ± 25 515  ± 27 253  ± 12
RTIIB · p58 without calciume
  Basal levela 99  ± 3 118  ± 5 104  ± 7 79  ± 2
  Peak responseb 625  ± 39 368  ± 32 712  ± 32 901  ± 36
  Plateaue 67  ± 3 94  ± 6 74  ± 4 66  ± 3

a [Ca2+]i at 2 min, before stimulation in nM ± S.E.
b [Ca2+]i at the peak of the response in nM ± S.E.
c ND, not done.
d [Ca2+]i at 17 min in nM ± S.E.
e [Ca2+]i at 12 min in nM ± S.E.
f The experiment was performed in the absence of extracellular calcium and in the presence of 0.5 mM EGTA as indicated under "Experimental Procedures."

In contrast, when p50.2 KAR was co-aggregated with Fcepsilon RI, no significant alteration of IgE-induced [Ca2+]i mobilization was observed. At the [Ca2+]i peak, using saturating concentrations of both GL183 (1 µg/ml) and IgE (10-3 dilution), [Ca2+]i was 829 ± 67 nM (n = 67 cells) versus 873 ± 67 nM (n = 67 cells) (mean ± S.E.) for IgE versus IgE-GL183 co-aggregation, respectively (Table I).

To evaluate whether KIR inhibited ITAM-dependent release of [Ca2+]i from intracellular stores and/or [Ca2+]i influx, further experiments were performed on RTIIB.p58 cells in the absence of extracellular Ca2+. For these conditions, only a small peak corresponding to the release of Ca2+ from intracellular stores was observed upon IgE stimulation (Fig. 5C, dotted line). Using saturating concentrations of both GL183 (1 µg/ml) and IgE (10-3 dilution), [Ca2+]i decreased from 625 ± 39 nM (n = 56 cells) to 368 ± 32 nM (n = 37 cells) (mean ± S.E., p < 0.001) at the [Ca2+]i peak (Table I, Fig. 5C). Therefore, p58.2 KIR is able to inhibit the release of Ca2+ from intracellular stores upon co-aggregation with Fcepsilon RI. In addition, no [Ca2+]i mobilization was detected when p58.2 or p50.2 was aggregated on RTIIB.p58 and RTIIB.p50 cells in the absence of IgE stimulation (data not shown).

These results show that p58.2 KIR impairs ITAM-induced Ca2+ mobilization in RTIIB cells. Furthermore, p50.2 KAR was not capable of mediating any detectable Ca2+ mobilization when expressed in RTIIB cells, in contrast to its stimulatory function reported in both T and NK cells.

Fcgamma RIIB-Fcepsilon RI Co-aggregation Only Inhibits Extracellular Ca2+ Influx in RTIIB Cells

RTIIB.p58 cells also express Fcgamma RIIB ITIM-bearing receptors. Similar to KIR, Fcgamma RIIB was shown to inhibit serotonin release in RTIIB cells (13). By contrast to KIR, Fcgamma RIIB has been reported in B cells to only inhibit Ca2+ entry (42). Therefore, we examined the effect of Fcgamma RIIB-Fcepsilon RI co-aggregation on Ca2+ mobilization, to document whether the differential effect of KIR and Fcgamma RIIB on Ca2+ mobilization is due to a difference between B cells and RTIIB cells or is the consequence of distinct inhibitory strategies used by these two ITIM-bearing receptors. RTIIB.p58 cells pre-incubated with murine IgE (10-3 dilution) in the presence or absence of 2.4G2 F(ab)'2 (1 µg/ml) were challenged with a TNP-F(ab)'2 MAR (10 µg/ml). Aggregation of the Fcepsilon RI receptor complex induced a large [Ca2+]i response consisting of a peak followed by a sustained plateau (Fig. 5B, dotted line). When Fcgamma RIIB was co-aggregated with Fcepsilon RI, the [Ca2+]i peak was not impaired but the plateau was not sustained (Fig. 5B, continuous line). Using saturating concentrations of both 2.4G2 F(ab)'2 (1 µg/ml) and IgE (10-3 dilution), the [Ca2+]i reached at the plateau decreased from 515 ± 27 nM (n = 60 cells) to 253 ± 12 nM (n = 59 cells) (mean ± S.E., p < 0.001) (Table I).

To dissect the effects of Fcgamma RIIB inhibition on Ca2+ mobilization, additional experiments were performed on RTIIB.p58 cells in the absence of extracellular Ca2+. In these conditions, only a peak corresponding to the release of Ca2+ from intracellular stores was observed upon IgE stimulation (Fig. 5D, dotted line). Using saturating concentrations of both 2.4G2 (1 µg/ml) and IgE (10-3 dilution), no inhibition of Ca2+ release was observed, but rather [Ca2+]i increased from 712 ± 32 nM (n = 79 cells) to 901 ± 36 nM (n = 59 cells) (mean ± S.E.) at the [Ca2+]i peak (Table I, Fig. 5D). These results indicate that Fcgamma RIIB has no effect on the release of Ca2+ from intracellular stores upon co-aggregation with Fcepsilon RI. In addition, no [Ca2+]i mobilization was detected when Fcgamma RIIB was aggregated on RTIIB.p58 cells in the absence of IgE stimulation (data not shown). Therefore p58.2 KIR and Fcgamma RIIB ITIM-bearing receptors exert distinct regulatory function on Fcepsilon RI-dependent Ca2+ mobilization.


DISCUSSION

The molecular cloning of MHC Class I-specific KIR has been reported both in human and in mouse (33, 43-45). Human KIR belong to a multigenic family of Ig-like members and are characterized, in their extracellular portions by two or three Ig-like domains, corresponding to HLA-C-specific p58, HLA-Bw4-specific p70/NKB1, and HLA-A3-specific NKAT4 receptors. By contrast, mouse KIR belong to a family of type II integral dimeric lectin proteins (7). Despite these striking differences of genetic origin, mouse and human KIR express one or two conserved intracytoplasmic (I/L)XYXX(Y/V) ITIM motifs, respectively (16-19). We and others have described that these amino acid stretches recruit upon phosphorylation of the tyrosine residue, the SH2-tandem protein-tyrosine phosphatases, SHP-1 and SHP-2 (16, 17). The implication of SHP-1 in the inhibition of ITAM-dependent activatory signals also has been suggested for B lymphocyte surface molecules expressing an intracytoplasmic (I/V)XYXX(L/V) amino acid stretch, such as Fcgamma RIIB and CD22 (15, 46, 47). In the present paper, we have investigated the influence of the cellular environment on the function of KIR, and the results are compared with those obtained with Fcgamma RIIB (11-15, 42, 48).

We report here that HLA-Cw3-specific human KIR expressed in RTIIB cells can inhibit [Ca2+]i mobilization and serotonin release induced by the Fcepsilon RI receptor as well as by a CD25/CD3zeta chimeric molecule (Figs. 3 and 5). Therefore, KIR and Fcgamma RIIB share several features. First, KIR and Fcgamma RIIB inhibit early steps of the signaling cascade, which are transcription-independent and are likely to reflect NK cell killing mechanisms, such as regulated exocytosis (49). Second, KIR and Fcgamma RIIB control the signals induced via polypeptides including only one ITAM (FcRgamma ), as well as receptors including three sequential ITAM (CD3zeta ). However KIR and Fcgamma RIIB appear to use distinct inhibitory strategies. Indeed KIR inhibits Ca2+ release from ER stores, whereas Fcgamma RIIB only inhibits influx from the extracellular compartment. In addition, upon phosphorylation of the ITIM, Fcgamma RIIB recruits preferentially the phosphatidylinositol 5-phosphatase SHIP (48),2 whereas KIR recruits the SHP-1 tyrosine phosphatase (16-19).2 It remains to be elucidated whether these two findings are related.

We also show that in the absence of intracytoplasmic (I/V)XYXX(L/V), the HLA Class I receptor, p50.2 KAR, fails to transmit an inhibitory signal. These data strongly support the involvement of (I/V)XYXX(L/V) in KIR inhibitory function. In addition, and in contrast to T and NK cells, RTIIB cells expressing KAR are not activated upon KAR aggregation (Figs. 2 and 3). KAR molecules present a charged amino acid residue in their transmembrane domains. In T and NK cells, but not in RTIIB cells, KAR are part of a multimeric complex including three KAR-associated polypeptides (KARAP).3 It is thus tempting to speculate that in NK and T cells, KAR do not function in isolation but rather associate with other proteins, including KARAP, to mediate their activatory function.

Finally, we show that the mere cell-surface expression of KIR does not modulate RTIIB cell activation (Fig. 2), just as described for endogenous KIR expressed in NK and T cells and in contrast to other regulators of lymphocyte activation, such as CD45 (50). On the contrary, co-aggregation between an activatory receptor (the Fcepsilon RI receptor complex or the CD25/CD3zeta chimeric molecule) and p58.2 KIR is required for the inhibition of serotonin release by RTIIB cells (Fig. 4). Although our data are obtained in a heterologous cell system, they imply that the KIR-inhibitory effect occurs in the immediate vicinity of the molecule, a feature shared by Fcgamma RIIB (12, 13). The requirement for a co-engagement of KIR with an activatory receptor may reflect the necessity for KIR to be phosphorylated by a non-diffusible protein-tyrosine kinase associated with the activatory receptor. Indeed, the tyrosine phosphorylation of KIR is mandatory to the recruitment of SHP-1. Thus, the same protein-tyrosine kinase might induce the tyrosine phosphorylation of both ITAM and ITIM, which is consistent with data showing that ITIM YXX(L/V) sequence is a potential substrate for the Src family member protein-tyrosine kinase used by ITAM-containing receptors, such as Lyn, Lck, and Fyn (51). An additional basis for the obligation of proximity between activatory and inhibitory molecules may be that the tyrosine-phosphorylated targets of SHP-1 belong to the ITAM-dependent activation cascade, and thus must be brought in close proximity to SHP-1.

The identification of the major targets for SHP-1 involved in the KIR inhibitory pathways is not yet achieved. Nevertheless, the recognition of target cells protected from NK cell lysis by the surface expression of HLA-B alleles leads to an inhibition of phosphatidylinositol 4,5-biphosphate hydrolysis, resulting in the prevention of [Ca2+]i mobilization in NK cells (52). Similarly, our results show that KIR inhibit ITAM-induced [Ca2+]i mobilization in RTIIB cells. Therefore, phospholipase C-gamma , and/or its potential upstream signaling effector/adaptors, such as p36-38 (53), as well as the ITAM-binding SH2-tandem protein-tyrosine kinase (ZAP-70 and p72syk) may be SHP-1 targets involved in KIR signaling pathways. This model is supported by recent data in B cells, where p72syk, phospholipase C-gamma 1, and SHP-1 are recruited to CD22 upon BCR cross-linking (54), as well as in T cells, where ZAP-70 is reported to be a direct substrate for SHP-1 (55).

In conclusion, the balance between receptor-mediated activation and inactivation is central to cell homeostasis. We propose that KIR belong to a novel family of ITIM-containing receptors possessing the intracytoplasmic (I/V)XYXX(L/V) ITIM. Members of this family are broadly expressed within the hematopoietic lineage and include receptors for Ig (Fcgamma RIIB) and MHC Class I molecules (KIR), as well as B cell molecule CD22 and a KIR homologue gp49B expressed on macrophages and mast cells (56, 57). All of these receptors act in a spatio-temporal coordinated way with ITAM-containing receptors to regulate the amplitude as well as the rates of signal initiation and termination in hematopoietic cells.


FOOTNOTES

*   This work was supported by institutional grants from INSERM, CNRS, Ministère de l'Enseignement Supérieur et de la Recherche, specific grants from Association pour la Recherche contre le Cancer and Ligue Nationale Contre le Cancer (to E. V., M. B., L. O., and A. C.), and by the Associazione Italiana per la Ricerca sul Cancro, Consiglio Nazionale delle Ricerche, Istituto Superiore di Sanità and Progetto Finalizzato ACRO (to A. M., L. M., and R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger Dagger    A member of the Institut Universitaire de France. To whom correspondence should be addressed. Tel.: 33-491269444; Fax: 33-491269430; E-mail:vivier{at}ciml.univ-mrs.fr.
1   The abbreviations used are: NK, natural killer; [Ca2+]i, intracellular Ca2+ concentration; mAb, monoclonal antibody; DAM, donkey anti-mouse Ig antiserum; DAR, donkey anti-rat Ig antiserum; GAM, goat anti-mouse Ig antiserum; ITAM, immunoreceptor tyrosine-based activation motif(s); ITIM, immunoreceptor tyrosine-based inhibition motif(s); KAR, killer cell activatory receptor(s); MAR, mouse anti-rat; FCS, fetal calf serum; KIR, killer cell inhibitory receptor(s); MHC, major histocompatibility complex; ER, endoplasmic reticulum; TCR, T cell receptor; TNP, trinitrophenol.
2   F. Vély, S. Olivero, L. Olcese, A. Moretta, J. E. Damen, L. Liu, G. Krystal, J. C. Cambier, M. Daëron, and E. Vivier, submitted for publication.
3   L. Olcese, L. Olcese, A. Cambiaggi, C. Bottino, A. Morretta, and E. Vivier, submitted for publication.

ACKNOWLEDGEMENTS

We thank Odile Malbec (INSERM U255) for help in serotonin release assay and TNP-F(ab)'2 MAR production, Corinne Béziers-Lafosse for graphic expertise, and Valéry Renard, Frédéric Vély, Bernard Malissen, Michel Fougereau, Pierre Golstein (CIML), Wolf H. Fridman (INSERM U255), and Marc Benhamou (CERVI) for helpful discussions and continuous encouragement.


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