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The Journal of Immunology, 2006, 177: 3590-3596.
Copyright © 2006 by The American Association of Immunologists, Inc.

CD94/NKG2A Inhibits NK Cell Activation by Disrupting the Actin Network at the Immunological Synapse1

Madhan Masilamani*, Connie Nguyen*, Juraj Kabat{dagger}, Francisco Borrego* and John E. Coligan2,*

* Receptor Cell Biology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852; and {dagger} Biological Imaging Facility, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
An adequate immune response is the result of the fine balance between activation and inhibitory signals. The exact means by which inhibitory signals obviate activation signals in immune cells are not totally elucidated. Human CD94/NKG2A is an ITIM-containing inhibitory receptor expressed by NK cells and some CD8+ T cells that recognize HLA-E. We show that the engagement of this receptor prevents NK cell activation by disruption of the actin network and exclusion of lipid rafts at the point of contact with its ligand (inhibitory NK cell immunological synapse, iNKIS). CD94/NKG2A engagement leads to recruitment and activation of src homology 2 domain-bearing tyrosine phosphatase 1. This likely explains the observed dephosphorylation of guanine nucleotide exchange factor and regulator of actin, Vav1, as well as ezrin-radixin-moesin proteins that connect actin filaments to membrane structures. In contrast, NK cell activation by NKG2D induced Vav1 and ezrin-radixin-moesin phosphorylation. Thus, CD94/NKG2A prevents actin-dependent recruitment of raft-associated activation receptors complexes to the activating synapse. This was further substantiated by showing that inhibition of actin polymerization abolished lipid rafts exclusion at the iNKIS, whereas cholesterol depletion had no effect on actin disruption at the iNKIS. These data indicate that the lipid rafts exclusion at the iNKIS is an active process which requires an intact cytoskeleton to maintain lipid rafts outside the inhibitory synapse. The net effect is to maintain an inhibitory state in the proximity of the iNKIS, while allowing the formation of activation synapse at distal points within the same NK cell.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer cells are poised to recognize abnormal cells through a large variety of activation receptors (1). NK cells form two distinct immunological synapses: an activating synapse (aNKIS)3 (3), directing cytokine secretion and granule release toward the target cell leading to cytotoxicity, and an inhibitory synapse (iNKIS) that prevents the initiation of these processes (2, 3). An NK cell can form both activating and inhibitory synapses with susceptible and resistant targets respectively at the same time, yet, kill only the cells that are susceptible (4).

Many of the ligands recognized by activation receptors are expressed on normal cells (1, 5). To prevent killing of normal cells, NK cells express an array of inhibitory receptors, many (6) but not all (7) of which recognize MHC class I molecules. During viral infection or tumorigenesis, MHC class I molecules are often down-regulated by a variety of mechanisms, supposedly a trait selected for avoiding CTL recognition (8). This leads to a reduction or absence of inhibitory signals leading to NK cell-mediated killing of target cells. The signaling thresholds for NK cell activation vs inhibition are finely regulated, because an abnormal deviation of signaling in either direction would result in inadvertent cell killing leading to autoimmunity or impaired control of diseased cells (1). The exact means by which inhibitory signals obviate activation signals has not been defined and remains a subject of intense investigation. In this report, using the CD94/NKG2A inhibitory receptor, we study the mechanism of inhibition of activation signals in NK cells and also in CD94/NKG2A-EGFP-transfected rat basophilic leukemia –2H3 (RBL-2H3) cells. Depending on the cell type, we have used NKG2D, CD16, and Fc{epsilon}RI receptors as representative activation receptors.

CD94/NKG2A is an inhibitory receptor expressed by most human NK cells and a subset of CD8+ T cells that recognizes HLA-E on target cells (9, 10, 11). Upon ligand binding, the two ITIM motifs of CD94/NKG2A are phosphorylated by a putative src kinase and provide docking sites for src homology 2 domain-bearing tyrosine phosphatase (SHP)-1 or SHP-2 (12). The activated phosphatases then initiate dephosphorylation reactions, which ultimately lead to the suppression of NK cell activation. Beyond SHP-1 and SHP-2, the exact mechanism by which NK cell function is controlled by CD94/NKG2A is not known.

We have shown previously that the formation of CD94/NKG2A iNKIS leads to exclusion of lipid rafts at the site of contact, and that ligation of both CD94/NKG2A and an activation receptor prevents the accumulation of lipid raft patches induced by cross-linking of the activation receptor (13). In this study, we demonstrate that the CD94/NKG2A iNKIS prevents formation of activation receptor synapses by disrupting the polymerized actin network necessary for the formation of a viable aNKIS. This can be explained by the observed inhibition of activation induced phosphorylation of the guanine nucleotide exchange factor Vav1 that regulates actin reorganization, and likewise of the ezrin-radixin-moesin (ERM) proteins that connect actin filaments to membrane structures. The end result is the suppression of NK cell activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and cell lines

Abs and reagents used in this study were obtained from the following vendors: anti-NKG2A (Z199, IgG2b), anti-CD94, and anti-CD16 mAbs from Beckman Coulter; anti-mouse IgG secondary Ab from Jackson ImmunoResearch Laboratories; anti-SHP-1, anti-Fc{epsilon}R1{alpha}, and anti-Vav1 from Upstate Biotechnology; phosphospecific anti-Vav1 (phosphotyrosine (pY)160) (or pVav1) from BioSource International; anti-pY from Zymed (Invitrogen Life Technologies); rabbit anti-ERM and rabbit anti-phospho-ERM from Cell Signaling Technology; anti-ezrin, anti-flotillin2, anti-CD45, anti-SHP-1, and isotype controls from BD Biosciences. The NKG2A-specific mAb 8E4 was derived by Dr. J. P. Houchins (14). Streptavidin- and protein A-coated beads were purchased from Bangs Laboratories, AlexaFluor 594-phalloidin and AlexaFluor 647- cholera toxin-B (Ctx-B) were obtained from Molecular Probes; and latrunculin A and methyl-beta-cyclodextrin (MbetaCD) were obtained from Sigma-Aldrich.

Peripheral human blood NK cells were obtained from National Institutes of Health blood bank. Polyclonal NK cells were isolated by negative selection using the NK cell isolation kit (Miltenyi Biotec). The purity of the cells was confirmed by flow cytometry. NK cells were cultured in IMDM (BioWhittaker) supplemented with 500 U/ml rIL-2 (National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD), 10% human AB serum (BioWhittaker), and glutamine (BioSource International) at 37°C under an atmosphere of 5% CO2. The NKL cell line was grown in RPMI 1640 medium (BioSource International) supplemented with L-glutamine, sodium pyruvate (BioSource International), 200 U/ml rIL-2, and 10% heat-inactivated FBS (HyClone). The NKG2A-EGFP fusion protein was expressed by cloning NKG2A cDNA into EcoRI/BglII sites of pEGFP-C1 (Clontech Laboratories). The EGFP domain is attached to the amino terminus of NKG2A, which is in the intracellular domain of the receptor. The RBL-2H3 cells are adherent, fibroblast-like cell line established by subcloning RBL cells as described previously (15, 16). The RBL-2H3 cell line was stably transfected with CD94 plus NKG2A-EGFP cDNAs and grown in complete RPMI 1640 medium supplemented with 10% heat-inactivated FBS, L-glutamine, nonessential amino acids (BioSource International), and the antibiotics neomycin and hygromycin B (Invitrogen Life Technologies). The cells were maintained in a humidified atmosphere of 5% CO2 at 37°C. The cells grow adherent and are recovered by treatment with trypsin-EDTA (Invitrogen Life Technologies) or by using a cell scraper (Corning) (13, 17).

Coating latex microspheres

Anti-human NKG2A, anti-human CD16, or anti-rat Fc{epsilon}R1{alpha} mAbs were coated on protein A latex microspheres (beads) (diameter, ~5 µm) as described previously (13). Ten microliters of streptavidin-coated beads was incubated with 1 µM biotinylated HLA-E monomer stabilized with the leader sequence peptide VMAPRTVLL from HLA-B7 or with 1 µM biotinylated MICA*004 monomer (National Institute of Allergy and Infectious Diseases Tetramer Core Facility, Atlanta, GA). The mixture was incubated at room temperature for 45 min in the dark. The mixture was then washed twice in PBS and added to CD94/NKG2A-EGFP RBL-2H3, NKL, or NK cells, followed by analyses of conjugates.

Immunological synapse/receptor polarization

Approximately 106 CD94/NKG2A-EGFP-transfected cells were cultured for 24 h on sterile cover slips (Thomas Scientific) in 12-well tissue culture plates (Corning Costar). This fresh seeding of RBL-2H3 cells before the experiment allowed us to visualize individual cells adhered to the cover slip. Cells were then washed twice with PBS, and 1 ml of prewarmed medium (RPMI 1640 without phenol red) was added to the wells, with or without either 2 µg/ml latrunculin A for 2 h or 10 mM MbetaCD for 30 min. Latrunculin A inhibits actin polymerization by binding to G-actin. MbetaCD treatment leads to depletion of cellular cholesterol thereby disrupting lipid rafts. The slides were washed again, and ~0.1 mg of either HLA-E-coated microspheres or anti-Fc{epsilon}R1{alpha}-coated microspheres was added. Conjugates were allowed to form at 37°C for 20 min in the presence of AlexaFluor 647–Ctx-B to surface-stain GM1+ lipid rafts, fixed in 4% paraformaldehyde, permeabilized with 0.01% Triton X-100 in PBS, and stained with AlexaFluor 594-phalloidin to stain actin. The stained cells were washed three times with 0.01% Triton X-100 and mounted on microscopic slides with mowiol (EMD Biosciences) containing p-phenylenediamine dihydrochloride (Sigma-Aldrich). For NK cell synapse study, 106 primary NK cells were mixed with 10 µl of anti-NKG2A- or anti-CD16 mAb-coated microspheres, incubated on ice for 10–15 min and allowed to adhere to poly-L-lysine (Sigma-Aldrich)-coated cover slips for 20 min at 37°C and processed as above.

For Ab staining, the fixed and permeabilized cells were incubated in blocking buffer containing 2% rat serum (for RBL-2H3 cells) or 2% human serum (NKL cells) and 1% BSA in PBS for 30 min at room temperature. The cells were then incubated with primary Ab diluted in blocking buffer for 30 min at room temperature, washed three times, and stained with AlexaFluor-conjugated secondary Abs diluted in blocking buffer for 30 min at room temperature. The stained cells were washed three times and processed for microscopy as above.

Confocal microscopy, image acquisition, and analysis

All images were collected on a TCS SP2 AOBS microscope (Leica) at the Biological Imaging Facility (Research Technologies Branch, National Institute of Allergy and Infectious Diseases, Bethesda, MD). All images were acquired with an oil immersion 63x objective, NA 1.32. Image analysis was done using Leica confocal software (version 2.5, build 1104), Imaris (version 4.2.0; Bitplane), and Adobe Photoshop (version 7.0; Adobe Systems). Images were visually scored for the presence/absence of NKG2A, actin, and lipid rafts at the bead conjugation sites by three investigators independently and the mean data used for plotting the graph.

Pervanadate treatment, lipid rafts isolation, SDS-PAGE, and immunoblotting

NKL cells (106 per ml) were either incubated with anti-NKG2A mAb and cross-linked or incubated with freshly prepared sodium pervanadate (0.1 mM sodium orthovanadate and 10 mM hydrogen peroxide; Sigma-Aldrich) in PBS at 37°C for 20 min. Pervanadate treatment inhibits tyrosine phosphatases and thereby enables proteins to remain in phosphorylated state. The cells were washed in PBS and processed for lipid rafts isolation as described previously (18). For SDS-PAGE analyses, lipid raft fractions and soluble fractions were pooled and separated on a 4–12% gradient NuPAGE polyacrylamide gel (Invitrogen Life Technologies). The resolved proteins were transferred to nitrocellulose membranes (Invitrogen Life Technologies) and immunoblotted with anti-NKG2A (mAb, 8E4), anti-SHP-1, anti-CD45, and anti-flotillin 2 Abs.

Receptor cross-linking

Before cross-linking, NKL cells were incubated with 2% human serum for 30 min on ice to block nonspecific binding of Ab to FcR. The cells were then washed and incubated with anti-CD94, anti-NKG2D, or both together (5 µg each/100 µl) on ice for 30 min. After two washes with PBS, the cells were cross-linked with anti-mouse IgG (5 µg/100 µl) at 37°C for the indicated time. For ERM studies, cells were washed after cross-linking, fixed, permeabilized, and stained with anti-phospho ERM Ab (pERM) for flow cytometry. The anti-phospho ERM Ab was validated for FACS (19), but because of high background, it was found unsuitable for Western blot experiments. For Vav studies, after cross-linking, the cells were lysed in lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, pH 8) containing 1% Triton X-100 and subjected to immunoblot analysis with anti-pVav1 Ab. This Ab was produced against a chemically synthesized phosphopeptide derived from a region of human Vav1 that contains tyrosine 160 and identifies a 95-kDa phosphorylated Vav1 protein in Western blots. After probing with anti-pVav1, the membrane was stripped and reprobed with anti-Vav Ab that recognizes total Vav1 protein in cell lysates. Band intensity was quantified using UN-SCAN-IT gel software (Silk Scientific). The anti-pVav1 (pY160) was unsuitable for immunostaining experiments (M. Masilamani and J. E. Coligan, unpublished observations).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lipid rafts and actin cytoskeleton are disrupted at the CD94/NKG2A inhibitory but not activation NK cell synapse

Lipid rafts and a functional cytoskeleton are absolutely necessary for NK cell activation and cytotoxicity (20, 21). To investigate the functional involvement of actin cytoskeleton and lipid rafts in downstream inhibitory receptor signaling, we used a RBL-2H3 cell line expressing CD94/NKG2A-EGFP (12, 13, 17). This cell line expresses the Fc{epsilon}RI activation receptor, whose signaling can be regulated by CD94/NKG2A. Ctx-B stained (marker for lipid rafts) CD94/NKG2A-EGFP-transfected RBL-2H3 cells were incubated with anti-NKG2A, HLA-E, or anti-Fc{epsilon}R1{alpha}-coated beads as surrogate targets, fixed, and costained with phalloidin (marker for F-actin) (Fig. 1A). CD94/NKG2A-EGFP-transfected cells formed stable conjugates both with anti-NKG2A mAb and HLA-E-coated beads. CD94/NKG2A-EGFP was enriched at the site of contact with the beads, whereas lipid rafts were excluded and the actin cytoskeleton was disrupted at these sites. On the other hand, both actin and lipid rafts were present at the conjugation sites formed with beads coated with a ligand capable of generating activation signals i.e., anti-Fc{epsilon}RI{alpha} mAb.


Figure 1
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  		FIGURE 1. Exclusion of F-actin and lipid rafts at the CD94/NKG2A iNKIS. A, CD94/NKG2A-EGFP-transfected RBL-2H3 cells (green) and human primary NK cells (B) were labeled with AlexaFluor 647 Ctx-B (magenta). Conjugates were formed with beads coated with anti-NKG2A, HLA-E, anti-Fc{epsilon}RI{alpha}, and anti-CD16 as indicated, fixed, and labeled with AlexaFluor 594 phalloidin (red). Differential interference contrast (DIC) images are shown in gray. The CD94/NKG2A iNKIS is indicated by the arrow. In all panels, the beads are indicated by an asterisk. Scale bar, 5 µm.

 
We substantiated our findings with RBL-2H3 cells using NK cells isolated from human peripheral blood to form inhibitory and activating immune synapses with beads coated with anti-NKG2A and anti-CD16 (the low-affinity IgGR), respectively (Fig. 1B). The primary NK cells formed stable conjugates both with anti-NKG2A- and anti-CD16-coated beads. The cells were stained for actin and lipid rafts. The contact sites were sharply reduced for F-actin at the CD94/NKG2A iNKIS, but not at the CD16 aNKIS. Lipid rafts were excluded from the CD94/NKG2A but not CD16 contact sites.

Role of actin cytoskeleton in maintaining lipid rafts exclusion at the CD94/NKG2A iNKIS

To investigate whether the actin cytoskeleton was involved in lipid rafts exclusion at the iNKIS, we used latrunculin A to inhibit actin polymerization and reorganization. Fig. 2A shows that treatment with latrunculin A largely abolished the lipid rafts exclusion at the contact sites between CD94/NKG2A and HLA-E beads (see arrow, compare with site designated by arrow in Fig. 1A), while having no effect on CD94/NKG2A accumulation. Quantitatively, there was about a 5-fold reduction in the percentage of contact sites that exclude lipid rafts in latrunculin A-treated cells (Fig. 2B). This result indicates that despite disruption of the actin cytoskeleton within the iNKIS, the integrity of the actin cytoskeleton outside the iNKIS is required for maintaining lipid rafts exclusion at the contact site between CD94/NKG2A and its ligand HLA-E. The fact that MbetaCD fails to prevent actin disruption at the iNKIS (Fig. 2B) indicates that the actin disruption at the iNKIS is independent of lipid rafts exclusion at the site of contact with the target. Taken together, these data show that the localized disruption of actin cytoskeleton at the CD94/NKG2A iNKIS is accompanied by the exclusion of lipid rafts due to the requirement of actin network for stabilization of lipid microdomains.


Figure 2
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  		FIGURE 2. Lipid raft independent actin disruption at the CD94/NKG2A iNKIS. A, CD94/NKG2A-EGFP (green) transfected RBL-2H3 cells were treated with latrunculin A (upper panel) or MbetaCD (lower panel), labeled with AlexaFluor 647 Ctx-B (magenta), allowed to form conjugates with the indicated beads, fixed and labeled with AlexaFluor 594 phalloidin (red). Differential interference contrast (DIC) images are shown in gray. B, The percentage of contact sites of CD94/NKG2A-EGFP transfected RBL-2H3 cells with beads that accumulate CD94/NKG2A at the contact site, along with the percentage of these sites that exclude actin and lipid rafts at the contact site are shown as a bar graph. The data shown are representative of three independent experiments (control, n = 21; latrunculin, n = 35; MbetaCD, n = 26). The CD94/NKG2A iNKIS is indicated by the arrow. In both panels, the beads are indicated by an asterisk. Scale bar, 5 µm. N/A, not applicable.

 
CD94/NKG2A signaling is initiated in the membrane-soluble fraction

To verify that CD94/NKG2A receptors function independently of lipid rafts despite the fact that they must inhibit lipid raft dependent activation signals, immunoprecipitation of NKG2A was performed from the raft and soluble fractions isolated by sucrose density gradient centrifugation from the following detergent-lysed cells: NKL cells cross-linked with anti-NKG2A (Fig. 3, left lanes), pervanadate treated (Fig. 3, right lanes), or untreated (Fig. 3, middle lanes). The precipitates were immunoblotted with anti-NKG2A (8E4) or anti-SHP-1. NKG2A was found to reside solely in the soluble fraction of untreated cells and did not translocate to the raft fraction upon cross-linking, phosphorylation, and subsequent binding of SHP-1. Pervanadate was used to enhance SHP-1 binding to NKG2A to show that such complexes reside solely in the soluble fraction. This indicates that CD94/NKG2A is a nonlipid raft resident protein and is capable of binding to SHP-1 and initiating signal transduction from outside of the lipid rafts.


Figure 3
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  		FIGURE 3. CD94/NKG2A inhibitory signaling is initiated in the membrane soluble fraction. Immunoprecipitation with anti-NKG2A mAb (clone Z199) was performed with lipid rafts (R) and detergent soluble (S) fractions of cross-linked NKG2A (xNKG2A), pervanadate-treated (PV) or -untreated NKL cells and the precipitates were immunoblotted with anti-NKG2A (8E4) or anti-SHP-1. The presence of flotillin 2 and the absence of CD45 in the lipid raft fractions confirm the purity of the lipid raft fractions.

 
Recruitment of SHP-1 at the site of CD94/NKG2A ligation

We next investigated whether ligand binding alone can lead to CD94/NKG2A phosphorylation and subsequent recruitment of SHP-1 in the absence of signals generated by other molecular interactions at cell-cell contact sites, an issue not addressed in previous studies (12, 17). To address this issue, CD94/NKG2A-EGFP-transfected cells were allowed to form conjugates with HLA-E-coated beads, fixed, and costained with SHP-1 and anti-phosphotyrosine (pY) (Fig. 4A). The colocalization of CD94/NKG2A and pY signals was enhanced at the synapse, as well as each of these with SHP-1, compared with elsewhere in the membrane (Fig. 4B). To compare and quantify the colocalization of signals at the synapse and elsewhere in the membrane, we used contour and channel masking techniques in Imaris to create the regions of interest (ROI) in 3D reconstructed images (Fig. 4C). Correlation coefficients of colocalized channels (Fig. 4D) and the percentage of ROI material colocalized between indicated proteins (Fig. 4E) at the synapse were compared with elsewhere in the membrane and the data plotted as bar graph. These analyses showed that the percentage of NKG2A protein that colocalized with SHP-1 and pY and vice versa were increased at the synapse, compared with elsewhere in the membrane. Taken together, these data show that ligand binding alone can activate CD94/NKG2A and the activated CD94/NKG2A is capable of recruiting and activating SHP-1 at the site of contact.


Figure 4
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  		FIGURE 4. CD94/NKG2A phosphorylation and SHP-1 recruitment at the iNKIS. A, CD94/NKG2A-EGFP transfected RBL-2H3 cells were allowed to form conjugates with HLA-E-coated beads, fixed, and costained with anti-SHP-1 mAb (red) and rabbit anti-pY (magenta). B, The colocalization channels were generated by merging the indicated channels. White structures near the bead contact site indicate colocalization of the proteins. The bead is indicated by an asterisk. Scale bar, 5 µm. C, Contour and channel masking technique in Imaris was used to create the ROI in 3D reconstructed images. All three channels were overlaid as a 3D image (Ci). Constructed 3D surface object (gray) was used for masking of the channels inside iNKIS (ROI 1) (Cii) and the rest of the membrane (ROI 2) (Ciii). Only voxels inside of the surface object were used for colocalization analysis. The channel mask was then used in conjunction with the automatic thresholding to calculate colocalization statistics in ROI 1 and ROI 2 representing the synapse and rest of the membrane, respectively. D and E, The channel correlation in colocalized volume (correlation coefficient) between indicated proteins and the percentage of ROI material colocalized between indicated proteins are plotted. The data shown are the results from three independent experiments. The error bars indicate SEM of correlation coefficient of six synapses.

 
CD94/NKG2A cross-linking inhibits activation induced Vav1 and ERM phosphorylation

We investigated whether the CD94/NKG2A inhibitory signals could block activation signals through interference with Vav1 and ERM protein functions, known to be involved in actin reorganization (22, 23, 24). NKG2D and CD94 on NKL cells were co-cross-linked with mAbs for the indicated times. The cells were then lysed, and the lysates were analyzed by Western blotting with phosphospecific Abs against Vav1, stripped, and reblotted with anti-Vav1 Ab (Fig. 5A). The band intensities of pVav1 were quantified, normalized to total Vav, and plotted on the graph (Fig. 5B). Phosphorylation of Vav1 on tyrosine residues within its acidic domain is important for its function as a guanine nucleotide exchange factor (25). Although the total Vav1 levels were similar in all conditions, NKG2D cross-linking significantly enhanced the phosphorylation of Vav1, compared with the IgG control, and this enhanced phosphorylation was clearly inhibited by co-cross-linking with anti-CD94 Ab.


Figure 5
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  		FIGURE 5. CD94/NKG2A cross-linking induces Vav1 dephosphorylation. A, NKL cells were incubated with anti-NKG2D, anti-CD94 or both mAb on ice for 30 min and cross-linked with secondary Ab for 1, 5, and 15 min. The cells were lysed, and equal amounts of cell lysates were immunoblotted with anti-pVav1, stripped, and then reblotted with anti-Vav1. B, The band intensities of pVav1 were quantified, normalized to total Vav, and plotted in the graph. The data shown are the results from six independent experiments.

 
Because ERM proteins are shown to be involved in Vav1-Rac1 pathway (19, 26), we asked whether the observed actin depolymerization correlated with an inhibition of ERM activation by CD94/NKG2A. Flow cytometric analyses of NKL cells cross-linked as above and stained with Ab specific for phosphorylated C-terminal residues in ERM (ezrin, Thr567; radixin, Thr564; moesin, Thr558) showed an ~25% decrease in ERM phosphorylation in CD94 cross-linked cells (Fig. 6). The phosphorylation at these C-terminal threonine residues of ERM proteins plays a key role in modulating the conformation and function of ERM proteins (27). The pERM levels moderately increased within 1 min of NKG2D activation. On the other hand, co-cross-linking of cells with both anti-CD94 and anti-NKG2D resulted in decreased levels of pERM levels. These results indicate that the formation of CD94/NKG2A iNKIS prevents phosphorylation (i.e., activation) of Vav1 and ERM proteins and the predominance of inhibitory signals in the presence of both activation and inhibitory signaling.


Figure 6
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  		FIGURE 6. CD94/NKG2A cross-linking induces ERM dephosphorylation. A, NKL cells were incubated with anti-NKG2D, anti-CD94, or both mAb on ice for 30 min and cross-linked with secondary Ab for 1, 5, and 15 min, fixed, permeabilized, and stained with pERM Ab, and analyzed by flow cytometry. B, The percentage difference in mean fluorescence between the isotype Ab treated (set to 100%) and cells cross-linked with anti-NKG2D, anti-CD94, or both mAb are plotted on the graph. The graph shows the results from three independent experiments. The error bars indicate SEM.

 
ERM proteins are excluded at the CD94/NKG2A inhibitory synapse

ERM proteins act as a linker between actin cytoskeleton and the cytoplasmic portion of cell surface proteins. We reasoned that the observed absence of the actin cytoskeleton at the CD94/NKG2A iNKIS (Fig. 1) could be due to a break in this link because dephosphorylation/inactivation of ERM proteins at C-terminal threonine residues alters the conformation such that it masks F-actin binding sites (28) and leads to disruption of actin (23, 24). To evaluate this possibility, we examined the localization of ezrin at the activation and inhibitory synapse in NKL cells. HLA-E, as well as MHC class I chain-related A (MICA) protein, a ligand for the NK cell activation receptor NKG2D (29), were coated on beads and allowed to form inhibitory and activating synapses, respectively. The cells were fixed and costained with anti-human ezrin, phalloidin, and phosphospecific anti-pERM Ab (Fig. 7). Along with actin, ezrin was excluded from the site of contact with HLA-E beads but not with MICA beads. Moreover, the fact that ezrin staining colocalized with pERM and actin at the cell membrane suggests that the ERM proteins associated with actin are phosphorylated (data not shown).


Figure 7
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  		FIGURE 7. Localization of ezrin and pERM at the inhibitory and activating synapse. NKL cells were allowed to form conjugates with HLA-E or MICA*004-coated beads, immobilized on poly-L-lysine-coated cover slips, fixed, and stained with anti-ezrin mAb (green), anti-pERM (magenta), and phalloidin (red). DIC images are shown in gray. The bead is indicated by an asterisk. Scale bar, 5 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We show that formation of CD94/NKG2A iNKIS is accompanied by the depolymerization of the actin cytoskeleton and active exclusion of lipid rafts at such sites (Fig. 1). Even after ligation, CD94/NKG2A resides in the membrane-soluble fraction (Fig. 3) and yet regulates receptors that signal in a raft-dependent manner (13). The actin depolymerization at the CD94/NKG2A iNKIS is linked with the dephosphorylation (or inhibition of phosphorylation) of Vav1 and ERM proteins (Figs. 5 and 6). This is most likely accomplished by SHP-1 phosphatase recruited to the site of the iNKIS (Fig. 4), which presumably maintains Vav1 and, consequently, ERM proteins in a dephosphorylated state thereby preventing actin polymerization. ERM proteins within the iNKIS are not phosphorylated and absent, most likely as a result of dephosphorylation, and therefore cannot connect cell surface proteins to the cytoskeleton (Fig. 7). The disruption of the actin network at the iNKIS prevents the recruitment of raft associated activation receptors required to generate activation signals. On the other hand, an intact cytoskeleton outside the iNKIS is required to maintain lipid rafts exclusion at the site of CD94/NKG2A contact with ligand. Cells treated with latrunculin A no longer exclude lipid rafts from the iNKIS, indicating that rafts exclusion is an active process and not a mechanical one (Fig. 2).

We have used the natural ligand HLA-E to cross-link CD94/NKG2A instead of the Ab usually used for the study of lymphocyte synapses (13, 30, 31). The use of HLA-E-coated beads, rather than HLA-E-expressing target cells, facilitates the study of CD94/NKG2A inhibitory synapse by avoiding potential complicating signals generated by other receptor–ligand interactions present at the cell-cell contact sites. Using HLA-E beads, we could show that SHP-1 binding and activation requires only CD94/NKG2A ligation (Fig. 4) and occurs outside of lipid rafts (Fig. 3). In contrast, in T cells, only lipid raft-associated SHP-1 mediates inhibitory effects on TCR signaling (32, 33). It has been shown that inhibitory killer cell Ig-like receptors (KIR) also activate SHP-1, and several targets of SHP-1 have been proposed (34, 35). The guanine nucleotide exchange factor Vav1 has been shown to bind to a chimeric receptor that contains a substrate trapping mutant of SHP-1 in place of the KIR cytoplasmic tail (34), suggesting that Vav1 is a target for SHP-1-mediated dephosphorylation. Other than recruiting SHP-1, it is not known whether KIRs and CD94/NKG2A use identical or related inhibitory signaling pathways.

The central role of the actin cytoskeleton in lipid rafts redistribution for activation receptor signaling has been reported for T cells (36) and RBL-2H3 cells (37). Several lymphocyte signaling intermediates that regulate actin cytoskeletal dynamics have been identified, including rho-family GTPases, WASp, and Arp2/3 (22, 38). The activities of rho-family GTPases are controlled by the phosphorylation status of the bound guanine nucleotide. Vav1 is the main guanine nucleotide exchange factor activated by TCR ligation (39) and is a mediator of actin cytoskeletal reorganization in T cells (40, 41). Vav1 contains multiple protein binding motifs and plays a role in multiple pathways by serving as an adaptor protein for linking cell surface receptor ligation to downstream signaling proteins (22, 39). Vav1 and Rac1 have been shown to be the important regulators of NK cell-mediated killing (42). The acidic domain of Vav1 contains three tyrosines (Y142, Y160, and Y174) whose phosphorylation after receptor ligation are highly correlated with the guanine nucleotide exchange activity of Vav1 proteins (43, 44, 45). We were able to show by Western blotting that ligation of CD94/NKG2A leads to dephosphorylation of acidic domain of Vav1. The anti-pVav1 (pY160) was unsuitable for immunostaining experiments.

ERM belongs to a family of proteins that connect membrane proteins, such as CD43, CD44, ICAM-1 etc., to the actin cytoskeleton (46, 47, 48). ERM proteins also are involved in cytoskeletal reorganization and signaling (24) and inactivation of ezrin leads to disruption of actin and microvillar breakdown (23). ERM proteins have been shown to promote T cell–APC conjugation by regulating cytoskeletal relaxation (19). Roumier et al. (49) reported that ERM proteins are involved in IS formation and are enriched at the T cell–APC contact site. The ezrin enriched at the T cell IS was serine/threonine phosphorylated and localized within the lipid rafts, where it seems to stabilize the lipid raft-associated signaling molecules at the IS (50). ERM proteins also have been shown to complex with CD43 distal to the T cell IS (31, 51) and the exclusion of CD43 from the IS depends on the phosphorylation of ERM proteins (52). In NK cells, ezrin is known to be excluded from the inhibitory KIR2DL1 synapse, but not from the activating synapse (53). We also show that ezrin is excluded from the CD94/NKG2A iNKIS, which parallels the actin disruption at the iNKIS (Fig. 4). Small rho GTPases are known to be associated with ERM protein activation (54) and ERM function is regulated by Vav1 in T cells (19, 26).

We conclude that the CD94/NKG2A inhibitory signaling prevents the formation of NK cell activation synapse by localized disruption of actin, thereby preventing lipid raft recruitment. This agrees with the previous observation that lipid rafts are excluded from the KIR2DL1 iNKIS (55). The actin disruption can be explained by our observation that Vav1 is dephosphorylated after engagement of CD94/NKG2A. Such dephosphorylated Vav1 can no longer mediate signaling that leads to phosphorylation of ERM proteins necessary for actin reorganization. Without functional ERM proteins, actin fails to reorganize, thereby preventing the coalescence of prerequisite lipid raft regulatory platforms at target cell contact points that are required for activating receptor function. Whether the effect of Vav1 on actin cytoskeleton is only through ERM or whether other alternate routes of cytoskeletal reorganization independent of ERM are involved is not clear at this moment. Thus, by providing the localized actin depolymerization and lipid rafts exclusion at the iNKIS, while maintaining the normal status of the cytoskeleton elsewhere, the NK cell can still kill susceptible target cells simultaneous to a resistant target cell engagement. Our findings provide a better understanding of how an inhibitory receptor engagement such as CD94/NKG2A can regulate the function of activation receptors.


   Acknowledgments
 
We thank the National Institute of Allergy and Infectious Diseases Tetramer Core Facility for production of the HLA-E and MICA monomers, Dr. Owen Schwartz for his help with confocal microscopy, Robert Valas for NK cell isolations, and Ishani Pathmanathan for technical assistance. We also thank Drs. Steven Burgess, Gul’nar Fattakhova, Kerima Maasho, Alina Marusina, Sriram Narayanan, and Xiaobin Tang for their critical reading of the manuscript.


   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 funds from the intramural program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. John E. Coligan, Receptor Cell Biology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, Twinbrook II, Room 205, 12441 Parklawn Drive, Rockville, MD 20852. E-mail: JCOLIGAN{at}niaid.nih.gov Back

3 Abbreviations used in this paper: Ctx-B, cholera toxin-B; ERM, ezrin-radixin-moesin; KIR, killer cell immunoglobulin-like receptor; MbetaCD, methyl-beta-cyclodextrin; iNKIS, inhibitory NK cell immunological synapse; aNKIS, activation NK cell immunological synapse; pY, phosphotyrosine; SHP-1, src homology 2 domain-bearing tyrosine phosphatase-1; ROI, region of interest; MICA, MHC class I chain-related A. Back

Received for publication April 12, 2006. Accepted for publication June 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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