The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 178: 5606-5611.
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Endt, J.
Right arrow Articles by Watzl, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Endt, J.
Right arrow Articles by Watzl, C.

Inhibitory Receptor Signals Suppress Ligation-Induced Recruitment of NKG2D to GM1-Rich Membrane Domains at the Human NK Cell Immune Synapse1

Johanna Endt2,*, Fiona E. McCann2,{dagger}, Catarina R. Almeida2,{dagger}, Doris Urlaub*, Rufina Leung{dagger}, Daniela Pende{ddagger}, Daniel M. Davis3,{dagger} and Carsten Watzl3,*

* Institute for Immunology, University Heidelberg, Heidelberg, Germany; {dagger} Division of Cell and Molecular Biology, Imperial College London, London, United Kingdom; and {ddagger} Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NKG2D is an activating receptor expressed on all human NK cells and a subset of T cells. In cytolytic conjugates between NK cells and target cells expressing its ligand MHC class I chain-related gene A, NKG2D accumulates at the immunological synapse with GM1-rich microdomains. Furthermore, NKG2D is specifically recruited to detergent-resistant membrane fractions upon ligation. However, in the presence of a strong inhibitory stimulus, NKG2D-mediated cytotoxicity can be intercepted, and recruitment of NKG2D to the immunological synapse and detergent-resistant membrane fractions is blocked. Also, downstream phosphorylation of Vav-1 triggered by NKG2D ligation is circumvented by coengaging inhibitory receptors. Thus, we propose that one way in which inhibitory signaling can control NKG2D-mediated activation is by blocking its recruitment to GM1-rich membrane domains. The accumulation of activating NK cell receptors in GM1-rich microdomains may provide the necessary platform from which stimulatory signals can proceed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer cells recognize virus-infected and tumor cells through a balance of inhibitory and activating signals (1). These complex signaling events are triggered when a NK cell comes in contact with a target cell, forming an immunological synapse (IS)4 (2). NK cells can be stimulated through the engagement of different activating receptors by target cell ligands. NK cell activity is regulated by inhibitory receptors, principally recognizing self-MHC I molecules, that can block NK cell activation by dephosphorylating Vav-1 (3), thereby inhibiting actin reorganization at the NK cell IS and possibly affecting the clustering of lipid rafts (4, 5, 6).

Although several studies have addressed the organization of the inhibitory NK cell IS (7, 8, 9), less is understood about the organization of NK cell-activating receptors and ligands and, more importantly, whether or not inhibitory signals influence the organization of activating receptors and ligands at the IS. The activating receptor NKG2D and its ligands on target cells have been shown to accumulate at NK cell synapses (10, 11). NKG2D is expressed on all NK cells and a subset of T cells and recognizes stress-inducible ligands including MHC class I chain-related gene A (MICA), MICB, and UL-16-binding proteins in humans (12). NKG2D ligands are found on many tumors and are up-regulated upon infection (13, 14). Several studies have demonstrated that the enhanced cell surface expression of NKG2D ligands results in increased susceptibility of MHC I expressing tumors to NK cell cytotoxicity (15, 16, 17). This suggests that NKG2D activation can override inhibitory signaling, allowing target cell lysis to proceed in the presence of MHC I protein expression. However, a contrasting study shows that engagement of inhibitory receptors can efficiently block NKG2D-mediated cytotoxicity (18).

In this study, we imaged the distribution of NKG2D and GM1-rich microdomains at activating and inhibitory conjugates with human NK clones and biochemically determined the association of NKG2D with detergent-resistant membrane (DRM) domains in the presence of inhibitory signaling. We report that inhibitory receptor signaling can override NKG2D-mediated activation by a mechanism involving blocking the accumulation of NKG2D in GM1-rich membrane domains at the NK cell immune synapse.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and reagents

In this study, 721.221, an EBV-transformed human B cell line selected to lack endogenous expression of HLA-A, -B, or -C, was transfected to express MICA and HLA-Cw6 tagged at the C terminus with yellow fluorescent protein (MICA-YFP) or cyan fluorescent protein (HLA-Cw6-CFP). Transfectants were generated that expressed MICA alone or along with HLA-Cw6, resulting in the following target cells: 721.221 untransfected (221), 221/MICA-YFP (MICA), and 221/MICA-YFP/Cw6-CFP (MICA/Cw6). The expression of MICA was comparable between MICA and MICA/Cw6 target cells (data not shown). Untransfected 221 cells were found not to express NKG2D ligands, UL-16-binding protein 1, -2, -3, or MICA (data not shown and Ref. 19). The 221 cells expressing low and high amounts of HLA-Cw6-GFP have been described previously (20). Additionally, we generated 221 cells expressing MICA-YFP with low or high amounts of HLA-Cw6-CFP as described above. The human NK cell lines NKL, YTS-2DL1, and mouse P815 cells were cultured as described previously (4).

Human NK cells and clones were isolated as previously described (21). Abs against human lymphocyte proteins were used as follows: anti-NKG2D mAbs (BAT221 supernatant for imaging (18); 149810 at 5 µg/ml for flow cytometry; and 0.5 µg/ml for redirected-lysis assay (R&D Systems)), anti-2B4 (C1.7) and anti-CD94 (HP-3B1) (both Beckman Coulter), anti-CD56 (My31) and anti-CD45 (clone 69) (both BD Biosciences), anti-CD158a (EB6, 20 µg/ml; Serotec), and anti-MHC class I (W6/32, 10 µg/ml). The anti-NKG2D Ab used for Western blotting (mouse IgG1, clone 3.1.1.1) was generated by immunizing mice with a recombinant bacterial-expressed protein containing the extracellular domain of human NKG2D. Other Abs used for Western blotting were anti-phospho-Vav-1 (pY160; Invitrogen Life Technologies), anti-Vav (V6512), and HRP-conjugated cholera toxin subunit B (CTx) (both Sigma-Aldrich). Cy5-conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) was used to detect MHC class I by flow cytometry. Alexa Fluor 568-conjugated goat anti-mouse IgG was used to detect NKG2D. GM1 ganglioside was labeled with Alexa Fluor 647-conjugated CTx, used at 20 µg/ml (Molecular Probes). For quantification of surface receptors, we used the QIFIKIT (DakoCytomation) and the Quantum Simply Cellular kit (Bangs Laboratories), which gave comparable results.

Conjugate formation and cell staining

For imaging, 2 x 105 NK cells and target cells were placed in 50 µl of prewarmed culture medium and incubated at 37°C/5% CO2 for 10 min. Conjugates were then fixed and stained as previously described (21). Alexa Fluor 647 CTx was used to stain fixed conjugates after anti-NKG2D mAb and Alexa Fluor 568 goat anti-mouse IgG. Fixed conjugates were imaged by confocal microscopy (TCS SP2; Leica). Proteins were defined as clustered at the IS if the intensity at the intercellular contact was twice that of the unconjugated membrane in an optical slice approximately in the middle of the conjugate. Data were analyzed by multinomial logistic regression analysis with fixed model design (SPSS version 14 for Windows).

Cytotoxicity assays

The susceptibility of targets to NK cytotoxicity was assessed in 5-h [35S]methionine or 4-h 51Cr release assays performed in triplicate as described previously (4, 22). Spontaneous release was <20% of the maximal release.

Ab cross-linking and preparation of DRM fractions

NKL cells expressing NKG2D were stimulated with 10 µg/ml Abs in medium for 10 min on ice. After addition of 15 µg/ml goat anti-mouse Abs, cells were transferred to 37°C for 5 min. Cells were then chilled on ice and pelleted by centrifugation, and lysates were analyzed for Vav-1 phosphorylation by Western blot. For the isolation of DRM, 1 x 108 stimulated cells were lysed in 1 ml of ice-cold TNEV buffer (10 mM Tris/Cl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, and 1 mM N3VO4) containing 0.5% Triton X-100 for 30 min on ice. Lysates were homogenized with 10 strokes of a loose fit Dounce homogenizer and mixed with 1 ml of 85% w/v sucrose in TNEV. Samples were transferred to an ultracentrifuge tube and overlaid with 6 ml of 35% followed by 3.5 ml of 5% w/v sucrose in TNEV. Samples were centrifuged at 200,000 x g for 16 h at 4°C. One-milliliter fractions were collected from the top of the tube and neighboring fractions were combined to reduce the number of samples during analysis. Fractions were then analyzed by Western blotting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NKG2D-mediated NK cell activation can be blocked in KIR2DL1+ NK clones

NK clones from several donors were isolated and analyzed for the expression of the inhibitory receptor KIR2DL1. The cytolytic activity of the NK clones was then assessed in cytotoxicity assays against 221 target cells which were transfected with MICA alone (221-MICA) or MICA and HLA-Cw6 (MICA/Cw6), a ligand for KIR2DL1 (Fig. 1A). Although all clones efficiently killed the MICA-expressing targets, lysis of MICA/Cw6 targets was clearly reduced in clones expressing KIR2DL1, but not in clones lacking this inhibitory receptor for HLA-Cw6. Similar results were obtained when we investigated the release of IFN-{gamma}, which was also inhibited by incubating KIR2DL1+ clones with MICA/Cw6 targets (data not shown). There was no difference in expression of the natural cytotoxicity receptors between the KIR2DL1-positive and -negative clones (data not shown). This suggests that the interaction between KIR2DL1 and Cw6 can overcome NKG2D-mediated NK cell activation. To get a better understanding about the strength of individual inhibitory and activating signals, we quantified activating and inhibitory receptor-ligand pairs on the NK clones and the different 221 targets. NK clones from different donors possessed surprisingly consistent numbers of NKG2D (mean of 1 x 104 receptors per NK cell) and ~10 times more KIR2DL1 (mean of 1 x 105 receptors per NK cell; Fig. 1A). The 221 transfectants expressed ~25 times more activating (MICA) and ~4 times more inhibitory (HLA-Cw6) ligands compared with the corresponding receptors on the NK clones (mean of 2.5 x 105 molecules of MICA and 4 x 105 molecules of HLA-Cw6; Fig. 1A). This suggests that during the encounter of a 221-MICA/Cw6 target by a single NK cell, NKG2D and KIR2DL1 are likely to be maximally engaged and that under these conditions the inhibitory signal can overcome NK cell activation.


Figure 1
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 1. NKG2D signals can be blocked by KIR2DL1. A, A total of 34 NK clones from three donors was analyzed for their expression of KIR2DL1. The cytotoxicity of KIR2DL1-positive and -negative NK clones was assayed for 221-MICA and 221-MICA/Cw6 target cells at the indicated E:T ratios. A representative result is shown. Right, Average number of molecules expressed per cell. B, KIR2DL1-positive and -negative NK clones were coincubated with MICA/Cw6 target cells for 10 min, then fixed and stained with anti-NKG2D mAb and Alexa Fluor 647-conjugated CTx subunit to identify the GM1-rich lipid rafts. A glow scale is used to highlight regions of highest intensity, shown in yellow. Scale bars, 10 µm. C, The percentage of cytolytic and noncytolytic conjugates exhibiting clustering of NKG2D or CTx at the IS was assessed using two KIR2DL1– and four KIR2DL1+ NK clones with MICA/Cw6 target cells with at least 40 conjugates each. Errors are SE (***, p < 0.001).

 
Reduced frequency of clustering of NKG2D and GM1-rich microdomains at the IS of noncytolytic NK clones with MICA/Cw6 target cells

To study the distribution of NKG2D and GM1-rich microdomains in inhibitory and activating conjugates, we coincubated KIR2DL1-positive and -negative NK clones with MICA/Cw6 targets. Conjugates were imaged by confocal microscopy and the distribution of NKG2D was scored. Representative images of cytolytic and noncytolytic conjugates with MICA/Cw6 are shown in Fig. 1B. NKG2D clustered at ~40% of cytolytic synapses between KIR2DL1– NK clones and MICA/Cw6 and at only 10% of synapses of KIR2DL1+ NK clones that were inhibited by MICA/Cw6 (Fig. 1C). This demonstrates that the engagement of KIR2DL1 by Cw6 correlates with a reduced clustering of NKG2D and suggests that inhibitory receptors can block NKG2D-mediated NK cell activation by controlling the clustering of NKG2D at the IS.

The involvement of lipid rafts in NK cell activation and inhibition has been previously reported (4, 5, 6). We therefore wanted to determine whether the differential clustering of NKG2D correlated with the distribution of GM1-rich microdomains at the IS detected by staining with CTx. CTx clustered at ~65% of the cytolytic synapses between KIR2DL1– NK clones and MICA/Cw6, but at only 35% of synapses with KIR2DL1+ NK clones that were inhibited by MICA/Cw6. In 82% of conjugates where NKG2D was observed to cluster at the IS, CTx also clustered. This suggests that, after 10 min of coincubation, both NKG2D and CTx-stained GM1-rich lipid rafts cluster at cytolytic but not noncytolytic synapses.

NKG2D associates with DRMs after activation

The colocalization of NKG2D and CTx at the cytolytic IS suggests that there might be a specific association of NKG2D with GM1-rich microdomains. To directly test whether NKG2D is recruited into GM1-rich microdomains after activation, we assessed the contents of DRM fractions isolated by sucrose gradient centrifugation. Because this assay requires large cell numbers, we used the NKG2D+ NK cell line NKL. We also generated a new mAb against NKG2D (clone 3.1.1.1), which efficiently detects NKG2D by Western blot. Stimulation of NKG2D by Ab-mediated cross-linking resulted in a recruitment of the receptor to DRMs (Fig. 2). CTx staining identified the GM1-rich DRMs as pooled fractions 4 and 5. The non-raft protein CD45 marks the detergent soluble fractions 10 and 11. This demonstrates that NKG2D-mediated NK cell activation is accompanied by the recruitment of the receptor to DRMs. The presence of surface membrane cholesterol, and likely the integrity of lipid rafts, is vital for NKG2D-mediated NK cell activation because NKG2D-mediated NK cell cytotoxicity was completely abrogated in methyl-beta-cyclodextrin-treated NKL cells (data not shown).


Figure 2
View larger version (61K):
[in this window]
[in a new window]

  		FIGURE 2. Activation-induced recruitment of the NKG2D receptor to DRMs. The NKG2D receptor on NKL cells was stimulated by Ab-mediated cross-linking for the indicated times at 37°C. Cells were then lysed and DRM fractions were isolated by sucrose density centrifugation. Neighboring fractions were combined and analyzed by Western blotting using the indicated Abs. CD45 identifies the detergent-soluble fraction (10/11), whereas CTx identifies the raft fraction (4/5). Results are representative of three independent experiments.

 
Inhibitory signaling can intercept NKG2D-dependent activation and block NKG2D recruitment to DRMs

The reduced lysis of MICA/Cw6 targets by KIR2DL1+ NK clones (Fig. 1A) suggests that inhibitory killer Ig-like receptors (KIR) can control NKG2D-mediated NK cell activation. NKL cells express the inhibitory receptor CD94/NKG2A but are negative for the activating receptor NKG2C. To test whether CD94/NKG2A can also block NKG2D-mediated NK cell cytotoxicity, we performed a redirected lysis assay. For this, we used anti-NKG2D and anti-2B4 Ab concentrations that were just sufficient to result in maximal NK cell activation and combined them with increasing concentrations of anti-CD94 mAb or an isotype-matched control (Fig. 3, A and B). Although lower concentrations of anti-CD94 mAb had no effect, increasing concentrations completely abrogated any target cell lysis, demonstrating that there is a threshold where inhibitory signaling can overcome NK cell activation. Interestingly, the amount of anti-CD94 Ab necessary for inhibition (~1 ng/ml) was 20 times lower than the amount of anti-2B4 or anti-NKG2D (20 ng/ml) used. This may in part reflect the amount of 2B4, NKG2D, and CD94 on the surface of NKL cells. Quantification of these receptors showed that NKG2D and 2B4 are expressed at similar levels (2.7 x 104 2B4 and 2.5 x 104 NKG2D receptors per NKL cell), while the expression of CD94 is ~2.5 times higher (6.5 x 104 receptors per NKL cell).


Figure 3
View larger version (59K):
[in this window]
[in a new window]

  		FIGURE 3. Inhibitory signaling via CD94/NKG2A can overcome NKG2D-dependent activation and NKG2D recruitment to DRMs. A and B, Redirected lysis of P815 cells at an E:T ratio of 5:1 was induced via just-optimal concentrations (20 ng/ml) of anti-NKG2D (A) or anti-2B4 (B) Abs combined with increasing concentrations of anti-CD94 mAb or an isotype-matched control (IgG1). C, NKL cells were stimulated by Ab-mediated cross-linking with the indicated Abs (5 µg/ml) for 5 min at 37°C. Cells were then lysed and DRM fractions were isolated by sucrose density centrifugation. Neighboring fractions were combined and analyzed by Western blotting using the indicated Abs. Simultaneous cross-linking of NKG2D with CD94 but not CD56 on NKL prevents the enrichment of NKG2D to DRM fractions (4/5). Representative anti-CD45 and CTx blots are shown. Results are representative of at least three independent experiments.

 
Next, it was important to investigate the molecular mechanism whereby inhibitory signals can interfere with NKG2D-mediated NK cell activation. The reduced clustering of NKG2D and lipid rafts at the inhibitory IS suggests that inhibitory signaling may interfere with the raft association of NKG2D. To test this, we studied the raft association of NKG2D in the absence or presence of inhibitory CD94/NKG2A signaling. When NKG2D was stimulated along with CD94 by Ab-mediated cross-linking, the recruitment of NKG2D to DRMs was blocked (Fig. 3C). This demonstrates that inhibitory signals can interfere with NKG2D-mediated NK cell activation by blocking the clustering and the raft association of the receptor, thereby counteracting NKG2D signaling.

Inhibition of NKG2D-mediated NK cell activation is dependent on the strength of the inhibitory signal

The data in Fig. 3A suggest that a certain strength of inhibitory signaling is necessary to overcome NKG2D-mediated cytotoxicity. The ligands expressed by the target cells used in Fig. 1 outnumbered the corresponding receptors on the NK clones, making it likely that the activating and inhibitory receptors on the NK clones were maximally engaged. To titrate the engagement of inhibitory receptors, we used 221 transfectants with no (221), low (221/Cw6low), or high (221/Cw6high) amounts of HLA-Cw6-GFP expression (20) and generated 221 cells expressing MICA-YFP (221/MICA) with low (221/MICA/Cw6low) or high (221/MICA/Cw6high) amounts of HLA-Cw6-CFP (Fig. 4A). MICA expression levels in these target cells was between 2 and 3 x 105 molecules per cell, which vastly outnumbered the amount of NKG2D expression by NK clones (mean of 1 x 104 receptors per NK cell) to get maximal receptor engagement (Fig. 4A). The 221 transfectants with low Cw6 expression (1.5–3 x 104 molecules per cell) should only suboptimally engage the KIR2DL1 receptors on NK cell clones (mean of 1 x 105 molecules per cell), whereas 221 cells with high Cw6 expression (mean of 2 x 105 molecules per cell) are likely to maximally engage its corresponding inhibitory receptor (Fig. 4A).


Figure 4
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 4. Inhibition of NKG2D-mediated NK cell activation is dependent on the strength of the inhibitory signal. A, FACS analysis of 221 transfectants with no (221), low (221/Cw6low), or high (221/Cw6high) amounts of HLA-Cw6-GFP (top, x-axis) expression and of 221 cells expressing MICA-YFP (bottom, y-axis; 221/MICA) in combination with low (221/MICA/Cw6low) or high (221/MICA/Cw6high) amounts of HLA-Cw6-CFP. MHC class I expression was detected by W6/32 mAb staining (y-axis). Numbers indicate the average number of HLA-Cw6 and MICA molecules expressed per cell. B, Thirteen NK clones from two donors were analyzed for their expression of KIR2DL1. The cytotoxicity of YTS cells transfected with KIR2DL1 (YTS-2DL1), KIR2DL1-positive, and -negative NK clones was assayed at an E:T ratio of 10:1 for 221, 221/Cw6low, 221/Cw6high, 221/MICA, 221/MICA/Cw6low, and 221/MICA/Cw6high target cells. E:T ratios of 5:1 and 2.5:1 showed equivalent results (data not shown). Representative results are shown. The average numbers of KIR2DL1 and NKG2D molecules per NK cell are indicated.

 
To test the capacity for NK cell inhibition by low and high levels of Cw6 expression, we tested the ability of the different transfectants to inhibit the NK cell line YTS stably expressing KIR2DL1 (YTS-2DL1), which lacks functional NKG2D expression. In the absence of Cw6 expression, YTS-2DL1 lysed 221 and 221/MICA cells to a similar extent (Fig. 4B). Expression of low amounts of Cw6 led to a significant inhibition of lysis, again independent on the expression of MICA, whereas high Cw6 expression completely abrogated the lysis by YTS-2DL1.

We then analyzed 13 clones with or without KIR2DL1 expression from two donors for their lysis of the different 221 transfectants (Fig. 4B). The presence of MICA led in most cases to an enhanced lysis of 221/MICA compared with 221 targets (Fig. 4B and data not shown). In the absence of MICA, KIR2DL1+ clones were already inhibited by low Cw6 expression levels. In transfectants expressing MICA, high levels of Cw6 expression were necessary to prevent lysis, while low amounts of Cw6 were insufficient (comparing 221/MICA/Cw6high with 221/MICA/Cw6low target cells). As expected, KIR2DL1– clones were not inhibited by the 221/Cw6low targets. Interestingly, high Cw6 expression leads to an inhibition of lysis in the absence of MICA. This was possibly mediated by the induction of HLA-E surface expression by the leader peptide of HLA-Cw6 in these transfectants (23, 24). This would then engage the CD94/NKG2A receptor, which was expressed by these clones. However, in the presence of MICA, lysis occurred independently of Cw6 expression.

NKG2D-mediated activation requires F-actin

Recent studies have demonstrated that inhibitory receptors can interfere with NK cell activation by blocking the phosphorylation of Vav-1 (3). Vav-1-regulated actin polymerization is essential for raft clustering (6, 25) and the raft recruitment and function of the activating NK cell receptor 2B4 (4). To test whether inhibitory receptors might also block NKG2D-mediated NK cell activation by targeting Vav-1, we investigated the influence of inhibitory CD94 signaling on NKG2D-mediated Vav-1 phosphorylation. Vav-1 phosphorylation was detected within 1 min of NKG2D activation by Ab-mediated cross-linking (Fig. 5A). This phosphorylation was completely blocked by the coengagement of CD94, suggesting that inhibitory receptors can prevent Vav-1 phosphorylation also during NKG2D-mediated activation.


Figure 5
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 5. Inhibitory receptors can block NKG2D-mediated Vav-1 phosphorylation. A, NKG2D on NKL was cross-linked by Abs with or without co-cross-linking CD94 for the times shown. Cells were lysed and blotted for phosho-Vav-1 (Vav-P), then reblotted for Vav to show equal loading. B, Redirected lysis of P815 cells via NKG2D on NKL is abrogated by preincubating NKL cells for 30 min with 10 µM cytochalasin D. C, NKL were mixed with 221/MICA target cells at an E:T ratio of 1:1 in the presence of cytochalasin D (10 µM) or DMSO for the indicated times. Samples were analyzed as described in A. Results are representative of three independent experiments.

 
Vav-1 is known to regulate actin polymerization. The block of Vav-1 phosphorylation by inhibitory receptors would therefore also disturb local actin reorganization. We therefore wanted to test whether NKG2D signals are dependent on the actin cytoskeleton. The redirected lysis via NKG2D on NKL was totally abrogated in the presence of cytochalasin D, which disrupts the assembly of F-actin (Fig. 5B). In addition, following contact with 221/MICA cells, Vav-1 phosphorylation was blocked by cytochalasin D (Fig. 5C), demonstrating that NKG2D activating signals are dependent on actin polymerization.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We demonstrate in this study, for the first time, an activation-induced recruitment of the NKG2D receptor to DRMs. In some assays, we found that a small amount of the receptor was already associated with GM1-rich fractions even without receptor stimulation (Fig. 2), although this effect was not observed in all experiments (Fig. 3). The reason for this discrepancy is unknown. It is interesting to note that we never observed the association of any other activating NK cell receptor with DRMs in the absence of receptor stimulation (Ref. 4 and data not shown). This could reflect a higher affinity of the NKG2D receptor for GM1-rich domains, which might give the receptor an advantage over other activating receptors.

Using NK cell clones and an NK cell line, we have demonstrated that inhibitory signals can control NKG2D-mediated NK cell activation, consistent with a previous report (18). This inhibition is dependent on the strength of the negative signal (Figs. 3 and 4), suggesting that also NKG2D-mediated NK cell activation is regulated by a balance between activating and inhibitory signals. In earlier studies, that found no or only little effect of inhibitory receptors on NKG2D-mediated NK cell activation (15, 16, 17), this balance may have tipped toward activation by a high overexpression of NKG2D ligands on target cells. Human NKG2D signals through DAP10, whereas murine NKG2D can also signal through DAP12 (26, 27), which could account for a qualitatively different NKG2D signaling in murine NK cells that might be less sensitive to inhibitory signaling. However, NKG2D-mediated NK cell activation in mice can also be controlled by inhibitory Ly-49 receptors (28).

In the redirected lysis assay (Fig. 3), we needed approximately the same amount of anti-CD94 Ab to block NKG2D- and 2B4-mediated cytotoxicity of the human NK cell line NKL. Because NKL cells possess about the same number of NKG2D and 2B4 receptors (~2.5 x 104 receptors per NKL cell), this suggests, that at least in this assay, the threshold for overcoming NKG2D- and 2B4-mediated NK cell lysis is identical, arguing against the note that human NKG2D is somewhat less sensitive to inhibitory receptors.

Importantly, this study presents an insight into a molecular mechanism for the inhibition of NKG2D-mediated NK cell activation: NKG2D can be prevented from a ligand-induced association with DRMs and from clustering at the IS. NKG2D signals are dependent on actin reorganization (Fig. 5). Vav-1 is known to regulate actin polymerization and Vav-1 phosphorylation seems to be one target of inhibitory receptor signaling (Fig. 5A and Ref. 3). This suggests a model by which early signaling events after target cell contact lead to Vav-1 phosphorylation (29), inducing actin polymerization and the clustering of NKG2D and lipid rafts at the IS. The signaling of NKG2D can then in turn enhance Vav-1 phosphorylation (Fig. 5). This would result in more actin polymerization and clustering at the IS in a positive feedback loop, eventually inducing full NK cell activation. Inhibitory receptors can interfere with the early Vav-1 phosphorylation in NK cells (3), thereby preventing or dampening the positive feedback loop and the clustering of NKG2D and lipid rafts at the IS. Interestingly, engagement of KIR2DL2 in T cells does not interfere with early activation signals and does not block the formation of a mature immune synapse and the clustering of lipid rafts (30). Instead, only late signaling events are reduced by KIR engagement in T cells, including a reduced phosphorylation of Vav-1 90 min after stimulation. This is in sharp contrast to the reduced phosphorylation of Vav-1 by engagement of inhibitory receptors in NK cells, which we observed as early as 30 s after stimulation (Fig. 5A). The interference by inhibitory receptors with early signaling events is therefore likely restricted to NK cells. This could represent the molecular basis for the regulation of NKG2D-mediated NK cell activity through inhibitory receptors. Finally, limiting the clustering and DRM association of activating receptors by blocking local actin reorganization could represent a general mechanism by which inhibitory receptors control NK cell activity.


   Acknowledgments
 
We acknowledge A. Rae for assistance with cell sorting and members of our laboratories for useful discussions.


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


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

1 This work was supported by the Fundação para a Ciência e a Tecnologia (to C.R.A.), the Medical Research Council (U.K.), the Biotechnology and Biological Sciences Research Council, and a Lister Institute Research Prize (to D.M.D.), the Deutsche Forschungsgemeinschaft (SFB 405, A13), the Deutsche Krebshilfe, and the BioFuture program by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (to C.W.). Back

2 J.E., F.E.M., and C.R.A. contributed equally. Back

3 Address correspondence to Dr. Carsten Watzl, Institute for Immunology, University Heidelberg, Im Neuenheimer Feld 305, Heidelberg, Germany and Dr. Daniel M. Davis, Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College, London, U.K. E-mail addresses: Carsten.watzl{at}urz.uni-heidelberg.de and d.davis{at}imperial.ac.uk Back

4 Abbreviations used in this paper: IS, immunological synapse; MICA, MHC class I chain-related gene A; DRM, detergent-resistant membrane; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; CTx, cholera toxin subunit B; KIR, killer Ig-like receptor. Back

Received for publication March 28, 2006. Accepted for publication February 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Lanier, L. L.. 2003. Natural killer cell receptor signaling. Curr. Opin. Immunol. 15: 308-314. [Medline]
  2. Davis, D. M.. 2002. Assembly of the immunological synapse for T cells and NK cells. Trends Immunol. 23: 356-363. [Medline]
  3. Stebbins, C. C., C. Watzl, D. D. Billadeau, P. J. Leibson, D. N. Burshtyn, E. O. Long. 2003. Vav1 dephosphorylation by the tyrosine phosphatase SHP-1 as a mechanism for inhibition of cellular cytotoxicity. Mol. Cell. Biol. 23: 6291-6299. [Abstract/Free Full Text]
  4. Watzl, C., E. O. Long. 2003. Natural killer cell inhibitory receptors block actin cytoskeleton-dependent recruitment of 2B4 (CD244) to lipid rafts. J. Exp. Med. 197: 77-85. [Abstract/Free Full Text]
  5. Fassett, M. S., D. M. Davis, M. M. Valter, G. B. Cohen, J. L. Strominger. 2001. Signaling at the inhibitory natural killer cell immune synapse regulates lipid raft polarization but not class I MHC clustering. Proc. Natl. Acad. Sci. USA 98: 14547-14552. [Abstract/Free Full Text]
  6. Lou, Z., D. Jevremovic, D. D. Billadeau, P. J. Leibson. 2000. A balance between positive and negative signals in cytotoxic lymphocytes regulates the polarization of lipid rafts during the development of cell-mediated killing. J. Exp. Med. 191: 347-354. [Abstract/Free Full Text]
  7. Davis, D. M., I. Chiu, M. Fassett, G. B. Cohen, O. Mandelboim, J. L. Strominger. 1999. The human natural killer cell immune synapse. Proc. Natl. Acad. Sci. USA 96: 15062-15067. [Abstract/Free Full Text]
  8. Carlin, L. M., K. Eleme, F. E. McCann, D. M. Davis. 2001. Intercellular transfer and supramolecular organization of human leukocyte antigen C at inhibitory natural killer cell immune synapses. J. Exp. Med. 194: 1507-1517. [Abstract/Free Full Text]
  9. Vyas, Y. M., H. Maniar, B. Dupont. 2002. Visualization of signaling pathways and cortical cytoskeleton in cytolytic and noncytolytic natural killer cell immune synapses. Immunol. Rev. 189: 161-178. [Medline]
  10. Doubrovina, E. S., M. M. Doubrovin, E. Vider, R. B. Sisson, R. J. O’Reilly, B. Dupont, Y. M. Vyas. 2003. Evasion from NK cell immunity by MHC class I chain-related molecules expressing colon adenocarcinoma. J. Immunol. 171: 6891-6899. [Abstract/Free Full Text]
  11. Eleme, K., S. B. Taner, B. Onfelt, L. M. Collinson, F. E. McCann, N. J. Chalupny, D. Cosman, C. Hopkins, A. I. Magee, D. M. Davis. 2004. Cell surface organization of stress-inducible proteins ULBP and MICA that stimulate human NK cells and T cells via NKG2D. J. Exp. Med. 199: 1005-1010. [Abstract/Free Full Text]
  12. Raulet, D. H.. 2003. Roles of the NKG2D immunoreceptor and its ligands. Nat. Rev. Immunol. 3: 781-790. [Medline]
  13. Groh, V., S. Bahram, S. Bauer, A. Herman, M. Beauchamp, T. Spies. 1996. Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium. Proc. Natl. Acad. Sci. USA 93: 12445-12450. [Abstract/Free Full Text]
  14. Rolle, A., M. Mousavi-Jazi, M. Eriksson, J. Odeberg, C. Soderberg-Naucler, D. Cosman, K. Karre, C. Cerboni. 2003. Effects of human cytomegalovirus infection on ligands for the activating NKG2D receptor of NK cells: up-regulation of UL16-binding protein (ULBP)1 and ULBP2 is counteracted by the viral UL16 protein. J. Immunol. 171: 902-908. [Abstract/Free Full Text]
  15. Cerwenka, A., J. L. Baron, L. L. Lanier. 2001. Ectopic expression of retinoic acid early inducible-1 gene (RAE-1) permits natural killer cell-mediated rejection of a MHC class I-bearing tumor in vivo. Proc. Natl. Acad. Sci. USA 98: 11521-11526. [Abstract/Free Full Text]
  16. Bauer, S., V. Groh, J. Wu, A. Steinle, J. H. Phillips, L. L. Lanier, T. Spies. 1999. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285: 727-729. [Abstract/Free Full Text]
  17. Diefenbach, A., E. R. Jensen, A. M. Jamieson, D. H. Raulet. 2001. Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity. Nature 413: 165-171. [Medline]
  18. Pende, D., C. Cantoni, P. Rivera, M. Vitale, R. Castriconi, S. Marcenaro, M. Nanni, R. Biassoni, C. Bottino, A. Moretta, L. Moretta. 2001. Role of NKG2D in tumor cell lysis mediated by human NK cells: cooperation with natural cytotoxicity receptors and capability of recognizing tumors of nonepithelial origin. Eur. J. Immunol. 31: 1076-1086. [Medline]
  19. Pende, D., P. Rivera, S. Marcenaro, C. C. Chang, R. Biassoni, R. Conte, M. Kubin, D. Cosman, S. Ferrone, L. Moretta, A. Moretta. 2002. Major histocompatibility complex class I-related chain A and UL16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent natural killer cell cytotoxicity. Cancer Res. 62: 6178-6186. [Abstract/Free Full Text]
  20. Almeida, C. R., D. M. Davis. 2006. Segregation of HLA-C from ICAM-1 at NK cell immune synapses is controlled by its cell surface density. J. Immunol. 177: 6904-6910. [Abstract/Free Full Text]
  21. McCann, F. E., B. Vanherberghen, K. Eleme, L. M. Carlin, R. J. Newsam, D. Goulding, D. M. Davis. 2003. The size of the synaptic cleft and distinct distributions of filamentous actin, ezrin, CD43, and CD45 at activating and inhibitory human NK cell immune synapses. J. Immunol. 170: 2862-2870. [Abstract/Free Full Text]
  22. Mandelboim, O., H. T. Reyburn, M. Vales-Gomez, L. Pazmany, M. Colonna, G. Borsellino, J. L. Strominger. 1996. Protection from lysis by natural killer cells of group 1 and 2 specificity is mediated by residue 80 in human histocompatibility leukocyte antigen C alleles and also occurs with empty major histocompatibility complex molecules. J. Exp. Med. 184: 913-922. [Abstract/Free Full Text]
  23. Braud, V., E. Y. Jones, A. McMichael. 1997. The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur. J. Immunol. 27: 1164-1169. [Medline]
  24. Llano, M., N. Lee, F. Navarro, P. Garcia, J. P. Albar, D. E. Geraghty, M. Lopez-Botet. 1998. HLA-E-bound peptides influence recognition by inhibitory and triggering CD94/NKG2 receptors: preferential response to an HLA-G-derived nonamer. Eur. J. Immunol. 28: 2854-2863. [Medline]
  25. Villalba, M., K. Bi, F. Rodriguez, Y. Tanaka, S. Schoenberger, A. Altman. 2001. Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells. J. Cell Biol. 155: 331-338. [Abstract/Free Full Text]
  26. Rabinovich, B., J. Li, M. Wolfson, W. Lawrence, C. Beers, J. Chalupny, R. Hurren, B. Greenfield, R. Miller, D. Cosman. 2006. NKG2D splice variants: a reexamination of adaptor molecule associations. Immunogenetics 58: 81-88. [Medline]
  27. Diefenbach, A., E. Tomasello, M. Lucas, A. M. Jamieson, J. K. Hsia, E. Vivier, D. H. Raulet. 2002. Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D. Nat. Immunol. 3: 1142-1149. [Medline]
  28. Regunathan, J., Y. Chen, D. Wang, S. Malarkannan. 2005. NKG2D receptor-mediated NK cell function is regulated by inhibitory Ly49 receptors. Blood 105: 233-240. [Abstract/Free Full Text]
  29. Riteau, B., D. F. Barber, E. O. Long. 2003. Vav1 phosphorylation is induced by beta2 integrin engagement on natural killer cells upstream of actin cytoskeleton and lipid raft reorganization. J. Exp. Med. 198: 469-474. [Abstract/Free Full Text]
  30. Henel, G., K. Singh, D. Cui, S. Pryshchep, W. W. Lee, C. M. Weyand, J. J. Goronzy. 2006. Uncoupling of T-cell effector functions by inhibitory killer immunoglobulin-like receptors. Blood 107: 4449-4457. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
E. Giurisato, M. Cella, T. Takai, T. Kurosaki, Y. Feng, G. D. Longmore, M. Colonna, and A. S. Shaw
Phosphatidylinositol 3-Kinase Activation Is Required To Form the NKG2D Immunological Synapse
Mol. Cell. Biol., December 15, 2007; 27(24): 8583 - 8599.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Endt, J.
Right arrow Articles by Watzl, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Endt, J.
Right arrow Articles by Watzl, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS