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


     
 

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
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McCann, F. E.
Right arrow Articles by Davis, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McCann, F. E.
Right arrow Articles by Davis, D. M.
The Journal of Immunology, 2003, 170: 2862-2870.
Copyright © 2003 by The American Association of Immunologists

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 1,2

Fiona E. McCann*, Bruno Vanherberghen*, Konstantina Eleme*, Leo M. Carlin*, Ray J. Newsam{ddagger}, David Goulding{dagger} and Daniel M. Davis3,*

* Department of Biological Sciences and {dagger} Center for Molecular Microbiology and Infection, Imperial College, London, United Kingdom; and {ddagger} Research School of Biosciences, University of Kent, Canterbury, Kent, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, we report the organization of cytoskeletal and large transmembrane proteins at the inhibitory and activating NK cell immunological or immune synapse (IS). Filamentous actin accumulates at the activating, but not the inhibitory, NK cell IS. However, surprisingly, ezrin and the associated protein CD43 are excluded from the inhibitory, but not the activating, NK cell IS. This distribution of ezrin and CD43 at the inhibitory NK cell IS is similar to that previously seen at the activating T cell IS. CD45 is also excluded from the inhibitory, but not activating, NK cell IS. In addition, electron microscopy reveals wide and narrow domains across the synaptic cleft. Target cell HLA-C, located by immunogold labeling, clusters where the synaptic cleft spans the size of HLA-C bound to the inhibitory killer Ig-like receptor. These data are consistent with assembly of the NK cell IS involving a combination of cytoskeletal-driven mechanisms and thermodynamics favoring the organization of receptor/ligand pairs according to the size of their extracellular domains.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer cells are large granular lymphocytes that are cytotoxic to some tumors and virus-infected cells (1). The outcome of NK cell immune surveillance is the result of a complex balance between inhibitory and activating signaling (2, 3, 4, 5, 6, 7, 8, 9). Killer Ig-like receptors (KIR)4 with two Ig domains, KIR2DL1 and KIR2DL2, trigger inhibitory signaling upon recognition of the class I MHC proteins HLA-Cw4 or -Cw6, and HLA-Cw3 or -Cw7, respectively (10, 11). Immunological intercellular contacts between effector and target cells or APCs can involve the organization of proteins into micrometer scale domains at intercellular contacts (12, 13, 14, 15, 16) creating an immunological or immune synapse (IS) (17, 18, 19). This has been most extensively studied in the T cell/APC conjugate, (12, 13, 15, 20, 21). The balance between activating and inhibitory signaling also controls the supramolecular organization of proteins at the NK cell IS (14, 22, 23, 24, 25). Molecular mechanisms for the assembly of the IS include a role for the cytoskeleton (20, 21, 26, 27, 28, 29, 30), segregation of proteins according to the size of their extracellular domains (28, 31, 32, 33, 34, 35, 36), and association of proteins with lipid rafts (37, 38, 39, 40, 41).

Previously, it has been shown that HLA-C still clusters at the inhibitory NK cell IS in the presence of drugs that deplete ATP or disrupt actin, myosin motors, or microtubules, suggesting that the cytoskeleton is not necessary for accumulation of at least some proteins at the inhibitory NK cell IS (14, 24). However, within the first minute following intercellular contact, talin clusters at both the activating and inhibitory NK cell IS, though at the inhibitory NK cell IS talin then redistributes away from the IS in the ensuing five minutes (22). The speed of the redistribution of talin compares with the speed that phosphorylated lck accumulates at the activating T cell IS just two minutes after intercellular contact, closely followed by recruitment of {zeta}-associated protein of 70 kDa (42).

A role for the ezrin-radixin-moesin (ERM) proteins, linking the actin cytoskeleton to certain transmembrane proteins such as CD43, (reviewed in Refs.43, 44, 45), has been proposed for the organization of the activating T cell IS. Ezrin was either excluded from (46), or clustered at the peripheral edges of (47), the activating T cell IS (48, 49). A dominant-negative ezrin, lacking the actin-binding domain, failed to redistribute CD43 away from the activating T cell IS (46). The phosphorylation status of moesin was also observed to regulate the exclusion of CD43 from the activating T cell IS (50). Furthermore, a mutant of CD43 unable to bind ERM proteins was no longer excluded from the activating T cell IS, resulting in reduced IL-2 production (50). In contrast, another study reported that CD43 mutated to either lack the full cytoplasmic domain or just the ERM binding motif can occupy the center of the activating T cell IS, without subsequently disrupting T cell proliferation in response to Ag (51). Significantly, the exclusion of CD43 from the activating T cell/dendritic cell IS has been demonstrated in vivo within a mouse lymph node (52). Ezrin has been shown to be capable of redistributing ICAM-2 and sensitizing target cells to NK cytotoxicity (53). In this study, the distribution of filamentous actin (f-actin), ezrin, CD43, and CD45 at the inhibitory and activating NK cell IS is reported.

In addition to cytoskeletal-mediated movement, another mechanism that may contribute to the formation of the IS involves the segregation of proteins according to the size of their extracellular domains (28, 31, 32, 33, 34, 35). Pairs of proteins with longer extracellular domains such as the integrins, spanning ~40 nm (31), would be separated from protein pairs with shorter extracellular domains, such as KIR or TCR with MHC protein which span ~15 nm (54, 55, 56, 57, 58, 59). In this study, the distance across the synaptic cleft at the activating and inhibitory NK cell IS was measured and immunogold labeling was used to measure the size of the synaptic cleft where HLA-C accumulates at the inhibitory NK cell IS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Abs

NK cell transfectants, YTS/Mock (YTS, mock-transfected), and YTS/KIR2DL1 (YTS transfected to express KIR2DL1) were previously described (60). YTS is a subclone of the human NK tumor line YT (61). An EBV-transformed B cell line 721.221 (hereafter referred to as 221), selected to lack surface expression of endogenous class I MHC protein (62), was transfected to express enhanced green fluorescent protein (GFP)-tagged HLA-Cw3, -Cw4, or -Cw6 (i.e., 221/Cw6-GFP, etc.), as previously described (14). Cytotoxicity assays show that YTS/KIR2DL1 is effectively inhibited from killing 221 transfected with HLA-Cw6 (i.e., 221/Cw6) or 221/Cw4, but not 221/Cw3 or 221/Cw7, target cells (60). An activating NK cell IS is created when the intercellular contact between the NK and target cell results in lysis of the target cell. In contrast, an inhibitory NK cell IS is created at the intercellular contact of noncytolytic conjugates between the NK cell and target cell. Thus, conjugates between YTS/KIR2DL1 and 221/Cw6-GFP create an inhibitory NK cell IS and conjugates of YTS/Mock and 221/Cw6-GFP or YTS/KIR2DL1 and 221/Cw3-GFP create an activating NK cell IS.

Mouse anti-human ezrin mAb (clone 18, IgG1; BD Transduction Laboratories, Lexington, KY), rabbit polyclonal anti-human ezrin Ab (Upstate Biotechnology, Lake Placid, NY), mouse anti-human CD43 mAb (IG10, IgG1; BD PharMingen, San Diego, CA), and mouse anti-human CD45 (HI30, IgG1; BD PharMingen) were used at 5 µg/ml. Mouse anti-CD158a (EB6, IgG1; Serotec, Oxford, U.K.) was used at 10 µg/ml. Alexa Fluor 568 goat anti-mouse IgG, Alexa Fluor 633 goat anti-mouse IgG, and Alexa Fluor 568 goat anti-rabbit IgG (Molecular Probes, Eugene, OR) were used at a concentration of 2 µg/ml. To phenotype cells for expression of the various CD45 isoforms, the following FITC-conjugated mAbs (Caltag Laboratories, Burlingame, CA) were used: UCHLI (IgG2a) for CD45RO, MEM56 (IgG2b) for CD45RA, and MEM55 (IgG1) for CD45RB. For immunogold labeling, anti-GFP mAb (JL-8, IgG2a; Clontech Laboratories, Palo Alto, CA) was used at 20 µg/ml and detected with 12 µg/ml goat anti-mouse IgG conjugated with 10 nm gold (TAAB Laboratories, Berkshire, U.K.). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise.

Generation of NK clones

PBMCs were isolated from the buffy coat residues of healthy donors by centrifugation on Ficoll-Paque plus gradient (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions. Freshly isolated PBMCs and RPMI 8866 cells were irradiated (6,000 and 12,000 rad, respectively) and mixed in a ratio of 20:1 in DMEM supplemented with 10% human serum (type AB; Sigma-Aldrich), 30% nutrient mixture F-12 (Ham), 2 mM L-glutamine, 1x nonessential amino acids, 1 mM sodium pyruvate, 50 µM 2-ME, 50 U/ml penicillin-streptomycin, and 100 U/ml human recombinant IL-2 (Roche, Basel, Switzerland), referred to hereafter as DMEM plus supplements, (all from Invitrogen, Carlsbad, CA), with 1 µg/ml PHA (Sigma-Aldrich). The irradiated cells were plated into the wells of U-bottom 96-well plates (105 PBMC and 5 x 103 RPMI 8866 cells per well) and incubated overnight at 37°C/5% CO2. CD3 PBL derived from human buffy coat residues that were cultured for 1 wk with irradiated 721.221 cells (20,000 rad), in a 4:3 ratio in RPMI 1640 medium containing with 10% human serum (type AB; Sigma-Aldrich) with supplements, were sorted by FACS into the 96-well plates containing the irradiated PBMCs and RPMI 8866 cells at 1 cell per well. After sorting, 100 µl of DMEM plus supplements was added to each well. Cells in wells that had undergone expansion were split every 4 to 5 days in a 1:1 ratio in DMEM plus supplements. NK clones were periodically monitored to be CD3- and CD56+ by mAb staining and their phenotype was determined by mAb staining and cytotoxicity assays against a panel of target cells. Cytotoxicity assays and laser scanning confocal microscopy were performed at least 4 days after restimulation with fresh human recombinant IL-2.

Phenotyping of NK clones

Abs used for flow cytometry were CyChrome-conjugated anti-human CD56 (B159), CyChrome-conjugated anti-human CD3 (UCHT1), CyChrome-conjugated IgG1 control (MOPC-21), anti-human CD94 (HP-3D9), and PE-conjugated anti-KIR2DL2 (DX27), all purchased from BD PharMingen. Anti-KIR2DL1 (HP3E4) was purchased from the American Type Culture Collection (Manassas, VA) and anti-Ig-like transcript-2 (HP-F1) was a kind gift from M. López-Botet (Universitat Pompeu Fabra, Barcelona, Spain). All of these Abs were used at a concentration of 1 µg/ml. FITC-conjugated rabbit anti-mouse IgG (Sigma-Aldrich) and FITC-conjugated rat anti-mouse IgM (R6-60.2; BD PharMingen) were used at 2 µg/ml. The cytolytic activity of NK clones against various target cells was assessed in 5-h 35S-release assays as described previously (63). Assays were performed in triplicate and data values differed by ~5% of the mean. Spontaneous release of 35S was <25% of the maximal release.

Imaging the IS

221 and YTS cell transfectants (106 of each) were coincubated for 45 min at 37°C/5% CO2 in conical end tubes containing 1 ml of culture medium comprising RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1x nonessential amino acids, 50 µM 2-ME, 50 U/ml penicillin-streptomycin, (all purchased from Invitrogen), after which the cells had fallen under gravity. Excess medium was removed and the sedimented cells were fixed in Cytofix/Cytoperm (BD PharMingen) for 12 min at 4°C. The fixed cells were then washed twice in 0.1% Tween 20/PBS before cell staining. For NK clones, the same method was used except that 2 x 105 cells of each type were used and the conjugates were briefly centrifuged before fixation to ensure that the cells had sedimented before decanting excess medium. Following cell fixation, f-actin was labeled using Alexa Fluor 633-conjugated phalloidin (Molecular Probes) at 5 U/ml in 1% BSA/PBS, for 1 h at 4°C. Cells were washed three times in 0.1% Tween 20/PBS before imaging.

For imaging the NK cell IS after different times of coincubation, 106 of both YTS and 221 transfectants were incubated in a V-bottom 96-well culture plate at 37°C/5% CO2 in 100 µl of prewarmed culture medium. The cells were then fixed after 2, 10, 20, or 45 min of coincubation by adding 200 µl of Cytofix/Cytoperm directly to the well at 4°C for 15 min.

For Ab staining, fixed cells were initially incubated in a blocking solution comprising 5% horse serum (Sigma-Aldrich) and 3% BSA in Perm/Wash buffer (BD PharMingen) for 1 h at 4°C. Next, cells were incubated with primary Ab diluted in blocking solution for 90 min at 4°C. Following three washes in 0.1% Tween 20/PBS, secondary Ab, also diluted in blocking solution, was added to the cells for 1 h at 4°C. The stained cells were then washed three times in 0.1% Tween 20/PBS and 7 µl of the resuspended cell pellet was placed between a microscope slide and a 24 x 24 mm coverslip (thickness no. 1) for microscopy. Staining procedures were altered to incorporate the use of buffers containing alternative detergents such as 0.05% saponin though no effect on staining was observed (data not shown). For double immunofluorescence labeling, both primary Abs followed by both secondary Abs were added. It was confirmed that there was no species cross-reactivity between Alexa Fluor-conjugated goat anti-mouse IgG and goat anti-rabbit IgG and also that none of the secondary Abs used showed any nonspecific binding to the cells in the absence of primary Ab (data not shown).

Confocal microscopy image analysis

Cell conjugates were imaged under a 63x oil immersion objective using a confocal laser scanning microscope (TCS SP2; Leica, Deerfield, IL) equipped with argon/krypton and helium/neon lasers using excitation wavelengths of 488, 568, and 633 nm. Conjugates were scanned in the xy-direction every 0.3 µm throughout the z-plane. The face of the IS was then reconstructed using a maximum intensity projection (Confocal Software; Leica). To quantify the relative amounts of HLA-C-GFP, ezrin, and CD43 at the inhibitory IS within a single optical slice, their mean fluorescence intensities were obtained from a 15-µm2 area at the intercellular contact. The mean intensities were then measured from the same area marked onto the unconjugated membranes of both the NK and B cell and a percentage of mean fluorescence intensity at the inhibitory IS relative to the sum of the unconjugated membranes was calculated.

Electron microscopy

Live YTS and 221 transfectants were coincubated for 45 min at 37°C/5% CO2 as previously described for IS formation. For negative staining, cells were fixed in 2.5% glutaraldehyde/2.5% paraformaldehyde in PBS for 2 h at 4°C after which the cells were washed three times in PBS. Cells were then postfixed in 1% osmium tetroxide/0.5 M veronal acetate, pH 7.2, for 1 h at room temperature, then taken through a graded series of alcohols and embedded in Spurr’s resin. Ultrathin sections were cut using a diamond knife and stained with 5% uranyl acetate at 60°C for 30 min followed by Reynolds lead citrate for 10 min at room temperature. Sections were examined with a transmission electron microscope (Philips 410; Endoven, The Netherlands) operating at 80 kV and images were recorded on Kodak 4489 film (Rochester, NY). Distances across the synapse were measured from the negatives between the outer edges of the outer membranes at the intercellular contact between YTS and 221 transfectants, using image analysis software (Leica Q500 MC).

Immunogold labeling

Cell conjugates were fixed in 4% paraformaldehyde/0.2% glutaraldehyde in PBS for 10 min on ice, followed by fresh 4% paraformaldehyde/0.2% glutaraldehyde in PBS on ice for 1 h. The fixed cells were then washed three times in PBS and dehydrated through a series of alcohols as the temperature was progressively lowered to -30°C in a freeze-substitution unit (EM AFS; Leica). Next, the cells were processed in Lowicryl K4 M embedding medium (TAAB Laboratories) infiltrated overnight in 1:1 ethanol/K4 M, followed by incubation with K4 M for 24 h and finally a further incubation with fresh K4 M that was polymerized under UV light for 8 h. Ultrathin sections were cut using an ultramicrotome (UCT; Leica) and collected on Formvar support grids. The grids were blocked before labeling with 0.02 M glycine for 15 min followed by 10% FCS in PBS for 30 min at room temperature. The grids were then incubated with 20 µg/ml mouse anti-GFP (JL-8; Clontech Laboratories) in 10% FCS in PBS for 1 h at room temperature. After washing three times in PBS, the grids were incubated for 1 h in 12 µg/ml goat anti-mouse IgG conjugated with 10 nm of gold (TAAB Laboratories) in the same diluent as before. Following a further 6 washes in PBS and 10 washes in ddH2O, the grids were contrasted with uranyl acetate and lead citrate before examination with a transmission electron microscope (Philips CM100).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
F-actin clusters at the activating, but not the inhibitory, NK cell IS

Conjugates of YTS/KIR2DL1 (YTS transfected to express KIR2DL1) or YTS/MOCK (mock-transfected) and 221/Cw6-GFP, 221/Cw4-GFP, or 221/Cw3-GFP were fixed and labeled with Alexa Fluor 633-conjugated phalloidin, a stain for f-actin, and were imaged by laser scanning confocal microscopy. At the intercellular contact between conjugates lacking either KIR2DL1 or its cognate MHC ligand, thus forming an activating NK cell IS, f-actin clearly clustered (Fig. 1b). Throughout this study, clustering at the IS was confirmed by projecting a single optical slice through the IS with fluorescence intensity plotted on a third axis. Where the fluorescence intensity at the IS was more than the sum of the intensity of the unconjugated membranes, that label was defined as being clustered at the IS. The fluorescence intensity of phalloidin at the activating IS (Fig. 1b) is clearly more than the sum of unconjugated membranes, whereas at the inhibitory IS (Fig. 1a) no increase in fluorescence intensity of phalloidin above the sum of unconjugated membranes was observed. At the inhibitory IS, HLA-C clustered ~70% of the time whereas f-actin accumulated only 30% of the time. In contrast, in activating synapses, f-actin was found to accumulate 70–80% of the time, while HLA-C-GFP clustered <15% of the time (Fig. 1c).



View larger version (58K):
[in this window]
[in a new window]
  		FIGURE 1. F-actin clusters at the activating, but not inhibitory, NK cell IS. Conjugates of 221 and YTS transfectants were fixed and labeled with Alexa Fluor 633 phalloidin to stain for f-actin. In both a and b, bright-field images of conjugates followed by HLA-C-GFP and phalloidin staining with the corresponding plots of fluorescence intensity are shown. Higher peaks represent areas of higher fluorescence intensity. a, An inhibitory NK cell IS between 221/Cw6-GFP and YTS/KIR2DL1 where HLA-Cw6-GFP, but not Alexa Fluor 633 phalloidin, clusters at the intercellular contact. b, YTS/KIR2DL1 forming an activating NK cell IS with a 221/Cw3-GFP cell. In contrast to the inhibitory NK cell IS, Alexa Fluor 633 phalloidin, but not HLA-C-GFP, accumulates. Scale bars represent 10 µm. c, The distribution of HLA-C-GFP and Alexa Fluor 633 phalloidin in activating and inhibitory conjugates was assessed and the mean percentages from six independent experiments are shown. Error bars are the SEM and n is the total number of conjugates scored.

 
Ezrin and CD43 are excluded from the inhibitory, but not activating, NK cell IS

Conjugates between 221/Cw6-GFP or 221/Cw3-GFP and YTS/KIR2DL1 or YTS/Mock were fixed and either stained for ezrin and f-actin (Fig. 2) or ezrin and CD43 (Fig. 3). Consistent with analysis by flow cytometry (data not shown), the 221 transfectant often stained brighter for ezrin than the YTS cell inferring a higher level of expression in 221 cells. At the inhibitory NK cell IS, a zone devoid of ezrin and CD43 staining corresponding to the region where HLA-C clustered at the IS was evident (Figs. 2 and 3). In contrast, at the activating IS, ezrin and CD43 were evenly distributed across the membranes of apposing cells within the conjugate (Figs. 2 and 3). At the inhibitory NK cell IS, ezrin and CD43 was excluded in 50–60% of inhibitory conjugates whereas at the activating NK cell IS, exclusion of either protein occurred <10% of the time (Figs. 2d and 3f). CD43 and ezrin were equally likely to be excluded from an inhibitory NK cell IS whether each NK cell simultaneously maintains one or several synapses (data not shown). Clustering of ezrin and CD43 at either the inhibitory or activating NK cell IS was observed in <10% of all conjugates assessed. In contrast to anti-CD43 staining, an isotype-matched mAb against KIR2DL1, EB6, was observed to brightly stain the NK cell juxtaposed to where HLA-C clusters on the target cell (Fig. 3, c and e). This indicated that the staining procedure used does permit access of Abs to proteins within the inhibitory NK cell IS.



View larger version (53K):
[in this window]
[in a new window]
  		FIGURE 2. Ezrin is excluded from the inhibitory, but not activating, NK cell IS. Conjugates of 221 transfectants and YTS/KIR2DL1 cells were fixed and labeled with anti-ezrin mAb followed by Alexa Fluor 633 phalloidin to stain for f-actin. The distribution of HLA-C-GFP, ezrin, and f-actin at (a) the inhibitory NK cell IS formed between 221/Cw6-GFP and YTS/KIR2DL1 and (b) the activating NK cell IS formed between 221/Cw3-GFP and YTS/KIR2DL1 cells is demonstrated. c, A reconstruction of the face of an inhibitory IS is shown. Ezrin, but not f-actin, is excluded from the region where HLA-C-GFP clusters. Scale bars in a and b represent 10 µm, and 5 µm in c. d, Each inhibitory and activating NK cell IS was assessed for ezrin being clustered, excluded, or distributed homogeneously around the membranes of apposing cells. The mean percentages of conjugates exhibiting each ezrin distribution are shown. Error bars are the SEM and n is the total number of conjugates assessed.

 


View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 3. CD43 is excluded from the inhibitory, but not the activating, NK cell IS. Conjugates of 221 transfectants and YTS/KIR2DL1 were labeled with rabbit polyclonal anti-ezrin and mouse monoclonal anti-CD43 Abs. Columns a and b show the distribution of HLA-C-GFP, ezrin, and CD43 at the inhibitory and activating NK cell IS between (a) 221/Cw6-GFP and YTS/KIR2DL1 and (b) 221/Cw3-GFP and YTS/KIR2DL1. c, EB6 (anti-CD158a/KIR2DL1) staining at an inhibitory NK cell IS between 221/Cw6-GFP and YTS/KIR2DL1 is shown. d, Reconstruction of the face of the inhibitory NK cell IS shows that ezrin and CD43 are excluded from the inhibitory NK cell IS where HLA-C-GFP clusters. e, Reconstruction of the face of the IS demonstrates that KIR2DL1 clusters at the inhibitory NK cell IS, opposing HLA-C-GFP on the target cell. Scale bars represent 10 µm in a–c and 5 µm in d and e. f, Cells were fixed after coincubation for the times shown and the mean percentages of conjugates where ezrin or CD43 were clustered or excluded from the intercellular contact is shown. Error bars are SEM from at least three independent experiments. At least 98 conjugates were counted at each time point for both activating and inhibitory conjugates.

 
The time taken for the accumulation of HLA-C and the exclusion of ezrin and CD43 at the inhibitory NK cell IS was assessed by imaging cells that were fixed after different times of being incubated together (Fig. 3f). Interestingly, after 10 min of coincubation of cells, HLA-C is clustered at ~60% of inhibitory NK synapses, whereas ezrin and CD43 are excluded from only ~20% of these synapses. This suggests that accumulation of HLA-C precedes exclusion of ezrin and CD43 at the inhibitory NK cell IS.

The extent to which ezrin and CD43 are excluded and HLA-C-GFP is clustered at the inhibitory IS was quantified using software to analyze mean fluorescence intensities from defined regions at the inhibitory NK cell IS relative to regions on the unconjugated cell membranes, as described in Materials and Methods. From a total of 40 conjugates counted, the fluorescence intensity of ezrin and CD43 was reduced by 68% ± 12.5 and 56.7% ± 10.2, respectively, at the inhibitory NK cell IS relative to the amount on the two unconjugated membranes. Interestingly, there was no apparent trend between the extent of HLA-C-GFP clustering and the fluorescence intensity of ezrin or CD43 at the IS (Table I). Comparison of the amount of ezrin and CD43 staining on the NK cell and target cell with the amount at the inhibitory IS suggests that neither cell alone can account for the amount of ezrin and CD43 excluded from the IS. Thus, ezrin and CD43 from both the NK and target cell are excluded from the inhibitory NK cell IS.


View this table:
[in this window]
[in a new window]
  		Table I. Mean fluorescence intensity of ezrin, CD43, and HLA-C-GFP at the inhibitory NK cell ISa

 
Exclusion of ezrin and CD43 from inhibitory, but not activating, NK cell IS in conjugates involving human NK clones

Two human NK clones, 5H4 (KIR2DL1+KIR2DL2-) and 6H5 (KIR2DL1-KIR2DL2+), were selected for microscopy. By flow cytometry, both clones were found to be CD56+CD3-Ig-like transcript-2-CD94+. In agreement with the specificity of KIR for HLA-C alleles, lysis by NK clone 5H4, expressing KIR2DL1, was effectively inhibited by HLA-Cw6 while NK clone 6H5, expressing KIR2DL2, was inhibited by HLA-Cw3 expressed on 221 transfectants (Fig. 4a).



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 4. Exclusion of ezrin and CD43 from the inhibitory, but not activating, NK cell IS in conjugates involving human NK clones. a, The cytotoxicity of two human clones, 5H4 (KIR2DL1+KIR2DL2-) and 6H5 (KIR2DL1-KIR2DL2+), was assessed in a 5-h 35S release cytotoxicity assay. b, Inhibitory conjugates of 5H4 and 221/Cw6-GFP and (c) activating conjugates of 5H4 and 221/Cw3-GFP were stained for ezrin and CD43. Ezrin and CD43 are excluded from the inhibitory IS where HLA-C clusters but not the activating IS. d, Reconstructions of the face of two inhibitory NK cell IS, stained for ezrin or CD43, are shown. A region devoid of mAb staining for ezrin and CD43 corresponds to the region where HLA-C clusters. Scale bars represent 10 µm in b and c and 5 µm in d. e, The distribution of CD43 and ezrin at the inhibitory and activating NK cell IS was assessed in n conjugates.

 
Conjugates of NK clones 5H4 or 6H5 with 221/Cw6-GFP or 221/Cw3-GFP targets were fixed and stained for CD43 and ezrin using mAbs. Both CD43 and ezrin were excluded from the inhibitory IS between clone 5H4 (KIR2DL1+KIR2DL2-) and 221/Cw6-GFP (Fig. 4b). At the activating IS, ezrin and CD43 were homogeneously distributed, shown in conjugates between 5H4 and 221/Cw3-GFP (Fig. 4c). A reconstruction of the face of the inhibitory NK cell IS shows exclusion of CD43 and ezrin where HLA-Cw6-GFP clusters (Fig. 4d). For both NK clones, ezrin and CD43 were excluded from the inhibitory NK cell IS 60–80% of the time (Fig. 4e). This demonstrates that exclusion of ezrin and CD43 is a general property of the inhibitory NK cell IS rather than specific to an IS involving one type of KIR/MHC protein. At the activating NK cell IS, ezrin or CD43 was homogeneously distributed at the intercellular contact in ~80% of conjugates (Fig. 4e). Thus, the distribution of ezrin and CD43 at the inhibitory and activating IS in conjugates involving human NK clones derived from peripheral blood corresponds to that observed in conjugates of YTS and 221 transfectants (Figs. 2 and 3).

CD45 is excluded from the inhibitory, but not the activating, NK cell IS

With a view to further assessing the role of protein size in assembly of the NK cell IS, we imaged the distribution of CD45 in inhibitory and activating conjugates. Because alternative splicing produces various CD45 isoforms with extracellular domains ranging from 20 to 50 nm, we first established their respective distributions on the cells used for imaging. Abs specific for CD45RO (20–28 nm), CD45RA, and CD45RB (each ~50 nm) were used to identify the expression of the various isoforms on the 221 and YTS transfectants as well as NK clones derived from peripheral blood. By flow cytometry, it was determined that YTS/KIR2DL1 expressed only CD45RO, 221/Cw6-GFP expressed only CD45RA, and the NK clones analyzed expressed CD45RO, CD45RA, and CD45RB (data not shown). This distribution of CD45 isoforms on human NK clones is consistent with previous observations (64). Conjugates involving effectors YTS/KIR2DL1 or NK clone 5H4 (KIR2DL1+KIR2DL2-) were fixed and stained with anti-CD45 mAb (HI30), which recognizes CD45RO and CD45RB. This mAb did not stain 221 cells, inferring that it does not detect CD45RA (our unpublished observations). At inhibitory conjugates, CD45RO expressed on YTS/KIR2DL1 (Fig. 5, a and c) or CD45RO and CD45RB expressed on the NK clone 5H4 (Fig. 5d) are clearly excluded from the region where HLA-C clusters (Fig. 5, c and d). At the activating IS between YTS/KIR2DL1 and HLA-Cw3-GFP (Fig. 5b), CD45RO was evenly distributed across the face of the IS in 87% of conjugates. At the inhibitory NK cell IS between YTS/KIR2DL1 and 221/Cw6-GFP, CD45RO was found to be excluded from the IS in 67% of conjugates (Fig. 5e).



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 5. Exclusion of CD45 from the inhibitory, but not activating, NK cell IS. Inhibitory and activating conjugates of YTS/KIR2DL1 and 221/Cw6-GFP or 221/Cw3-GFP were fixed, stained with anti-CD45 mAb (HI30), and the distribution at the IS was determined. Inhibitory conjugates where CD45 is excluded from the IS are shown in a and c. b, At the activating IS, between YTS/KIR2DL1 and 221/Cw3-GFP, CD45 is evenly distributed across the intercellular contact. c, Reconstruction of the face of the inhibitory IS formed between YTS/KIR2DL1 and 221/Cw6-GFP demonstrates a region devoid of CD45 staining corresponding to that where HLA-C clusters. Scale bars represent 10 µm in a and b, and 5 µm in c. d, Reconstruction of the inhibitory IS formed between human NK clone 5H4 and 221/Cw6-GFP. Exclusion of CD45 from the region where HLA-C-GFP clusters is apparent. e, The distribution of CD45 at the inhibitory and activating IS was assessed in numerous conjugates involving YTS/KIR2DL1. n, The number of conjugates analyzed.

 
The size of the synaptic cleft at the inhibitory and activating NK cell IS

If proteins are segregated at the NK cell IS according to their size, it is necessary that the size of the synaptic cleft, defined in this study as the distance between the outer membranes of apposing effector and target cells at the IS, would vary accordingly. Thus, we assessed the ultrastructure of the activating and inhibitory NK cell IS in conjugates of 221 transfectants and YTS/KIR2DL1 cells by electron microscopy. NK cells were easily distinguished from target cells by the presence of large granules within their cytoplasm. The distance across the synaptic cleft in both activating and inhibitory conjugates varied such that distinct wide and narrow domains were evident (Fig. 6a). Distances across the synaptic cleft of conjugates between 221/Cw6-GFP and YTS/KIR2DL1 and conjugates of 221/Cw3-GFP and YTS/KIR2DL1 cells were measured at 300-nm intervals along each intercellular contact. Although the size of the synaptic cleft in activating and inhibitory conjugates was found to range from 10 to 55 nm and 10 to 42 nm, respectively, the majority of regions measured between 10 and 30 nm at both the activating and inhibitory IS (Fig. 6, c and d).



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 6. The size of the synaptic cleft at the inhibitory and activating NK cell IS. Inhibitory and activating conjugates between 221 transfectants and YTS/KIR2DL1 cells were processed for electron microscopy and the distance across the synaptic cleft was measured. a, Negative staining of intercellular contacts between NK cells and 221 transfectants reveals how the distance across the synaptic cleft varies along the length of the IS. b, Immunogold labeling of HLA-Cw6-GFP at the inhibitory NK cell IS demonstrates 10-nm gold beads clustering at regions along the IS. All scale bars represent 0.5 µm. The distance across the synaptic cleft was measured along (c) the activating and (d) inhibitory NK cell IS and the percentage of measurements occurring within 5-nm intervals between 0 and 55 nm are shown. e, Following localization of HLA-Cw6-GFP at the inhibitory IS by immunogold labeling, the distance across the synaptic cleft at each gold particle was measured and the percentage occurrence of distances between 0 and 55 nm in 5-nm intervals is shown. Data was obtained from over 100 measurements across 20 synapses in each of c and d, and 30 measurements across 3 synapses in e.

 
To characterize the distribution of HLA-C at the inhibitory NK cell IS, immunogold labeling was used to locate HLA-C-GFP. HLA-C-GFP was detected on the surface, and to a lesser extent within the cytoplasm, of the target cell. Staining was occasionally observed on the apposing NK cell, which could reflect the intercellular transfer of HLA-C-GFP from the target cell to the NK cell (24). Strikingly, accumulation of the gold beads, marking the location of HLA-C-GFP, was observed only at the tighter intercellular contacts (Fig. 6b). Although, it may be important to note that a few gold beads were located where the distance between the cells was ~100 nm, which may or may not be part of the intercellular contact. The distance across the synaptic cleft at each gold bead that was clearly at the IS i.e., HLA-C-GFP at the IS, was measured and found to be 14.5 ± 2.2 nm (Fig. 6e), which is the size of the extracellular domains of HLA-C bound to KIR (54, 55).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By laser scanning confocal microscopy, we show that, similar to the activating T cell IS, f-actin accumulates at the activating NK cell IS. Consistent with a role for f-actin in the activating NK cell IS, inhibitors of actin polymerization significantly reduced NK cytotoxicity (65, 66). Conversely, a homogeneous distribution of f-actin around the cell membranes of apposing cells was seen at the inhibitory NK cell IS. Unexpectedly, however, ezrin and one of its associated transmembrane proteins CD43 are excluded from the inhibitory, i.e., noncytolytic, NK cell IS, while both proteins are homogeneously distributed across the IS of activating conjugates. Surprisingly, the exclusion of ezrin and CD43 from the inhibitory NK cell IS is reminiscent of the exclusion of these proteins from the activating T cell IS (46, 47, 50, 51, 67). The large size of CD43 could be responsible for its exclusion from the inhibitory NK cell IS. However, both inhibitory and activating NK cell synapses contain large synaptic clefts that could accommodate CD43 (Fig. 6), and both activating and inhibitory NK cell synapses involve interactions with smaller receptor/ligand pairs (68). Also, the size of CD43 cannot be sufficient for its exclusion from the activating T cell IS, because CD43 mutants unable to bind ERM proteins can persist at the activating T cell IS with no detriment to T cell proliferation (51).

Reasons why the exclusion of CD43 from the inhibitory, but not the activating, NK cell IS may be functionally important are not obvious. The binding of CD43 to its ligands, e.g., ICAM-1, could promote adhesion (69, 70). Therefore, inclusion of CD43 in the activating NK cell IS may be necessary to maintain effector/target contact during effector functions such as secretion. Also, CD43 has been shown to associate with the signaling molecules fyn and lck involved in T cell activation (71, 72). These molecules accumulate at the activating NK cell IS (22, 23). Consistent with a role in NK cell activation, engagement of CD43 has been shown to increase NK cell proliferation and NK cytotoxicity by redirected lysis (73, 74). Thus, the exclusion of CD43 from the inhibitory NK cell IS could assist inhibition by sequestering signaling molecules such as fyn and lck away from the site of intercellular communication. Similar to CD43, we have demonstrated that CD45 is excluded from the inhibitory, but not the activating, NK cell IS. Interestingly, some anti-CD45 mAbs selectively inhibited LFA-1-mediated NK cytotoxicity, suggesting a role for CD45 in regulating NK cell killing (64). Thus, at the inhibitory NK cell IS the redistribution of CD45 away from intercellular contact may favor inhibitory effector functions.

The proposal that proteins are segregated according to the size of their extracellular domains predicts that the distance across the synaptic cleft, i.e., extracellular space between apposing cells at the interface, is determined by the size of local receptor/ligand interactions (28, 31, 32, 33, 34, 35, 36). In this study, we measured the size of the synaptic cleft between inhibitory and activating NK cell conjugates by transmission electron microscopy. We found that rather than being equidistant across the IS, the membranes of apposing cells create alternate wide and narrow regions across the synaptic cleft. Although at the activating NK cell IS the frequency of distances >30 nm remains low, perhaps these wider regions are sufficient to allow the observed prevalence of CD43 and CD45 there. Importantly, where HLA-C-GFP was localized, the synaptic cleft measured 14.5 ± 2.2 nm, which corresponds to the size of the extracellular portions of KIR/MHC, as determined by x-ray crystallography (54, 55). These findings are consistent with the hypothesis that in addition to cytoskeletal-mediated movements, proteins at the NK cell IS can redistribute due to the size of their extracellular domains. One important next goal is to locate HLA-C/KIR and ICAM-1/LFA-1 within the synaptic cleft, using electron microscopy of peripheral blood NK cells and targets.

Finally, we note that during preparation of the revised version of this manuscript, another laboratory concluded that ezrin clustered at the activating NK cell IS (75). This different conclusion may be due to the different target and effector cells used in each study, and/or the vastly different protocol by which each analysis was made. This emphasizes that the organization of proteins at an IS is sensitive to many factors, such as environmental stimuli, that are not yet understood.


   Acknowledgments
 
We are grateful to B. Askonas, K. Suhling, L. Bugeon, S. B. Taner, and K. Yanagi for critical reading of this manuscript.


   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 is supported by grants from the Medical Research Council (U.K.) and The Royal Society. Back

2 A preliminary report of this research was presented by Daniel N. Davis at the XIIIth International Congress of Histocompatibility and Immunogenetics, Seattle, WA, May 18–22, 2002. Back

3 Address correspondence and reprint requests to Dr. Daniel M. Davis, Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, London SW7 2AZ, U.K. E-mail address: d.davis{at}ic.ac.uk Back

4 Abbreviations used in this paper: KIR, killer Ig-like receptor; IS, immunological or immune synapse; ERM, ezrin-radixin-moesin; f-actin, filamentous actin; GFP, green fluorescent protein. Back

Received for publication August 12, 2002. Accepted for publication January 8, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Trinchieri, G.. 1989. Biology of natural killer cells. Adv. Immunol. 47:187.[Medline]
  2. Ljunggren, H. G., K. Karre. 1990. In search of the "missing self": MHC molecules and NK cell recognition. Immunol. Today 11:237.[Medline]
  3. Long, E. O.. 1999. Regulation of immune responses through inhibitory receptors. Annu. Rev. Immunol. 17:875.[Medline]
  4. Long, E. O., S. Rajagopalan. 2000. HLA class I recognition by killer cell Ig-like receptors. Semin. Immunol. 12:101.[Medline]
  5. Moretta, A., S. Sivori, M. Vitale, D. Pende, L. Morelli, R. Augugliaro, C. Bottino, L. Moretta. 1995. Existence of both inhibitory (p58) and activatory (p50) receptors for HLA-C molecules in human natural killer cells. J. Exp. Med. 182:875.[Abstract/Free Full Text]
  6. Pende, D., S. Parolini, A. Pessino, S. Sivori, R. Augugliaro, L. Morelli, E. Marcenaro, L. Accame, A. Malaspina, R. Biassoni, et al 1999. Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J. Exp. Med. 190:1505.[Abstract/Free Full Text]
  7. Vitale, M., C. Bottino, S. Sivori, L. Sanseverino, R. Castriconi, E. Marcenaro, R. Augugliaro, L. Moretta, A. Moretta. 1998. NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis. J. Exp. Med. 187:2065.[Abstract/Free Full Text]
  8. Sivori, S., M. Vitale, L. Morelli, L. Sanseverino, R. Augugliaro, C. Bottino, L. Moretta, A. Moretta. 1997. p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J. Exp. Med. 186:1129.[Abstract/Free Full Text]
  9. Moretta, A., R. Biassoni, C. Bottino, M. C. Mingari, L. Moretta. 2000. Natural cytotoxicity receptors that trigger human NK-cell-mediated cytolysis. Immunol. Today 21:228.[Medline]
  10. Colonna, M., G. Borsellino, M. Falco, G. B. Ferrara, J. L. Strominger. 1993. HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer cells. Proc. Natl. Acad. Sci. USA 90:12000.[Abstract/Free Full Text]
  11. Colonna, M., E. G. Brooks, M. Falco, G. B. Ferrara, J. L. Strominger. 1993. Generation of allospecific natural killer cells by stimulation across a polymorphism of HLA-C. Science 260:1121.[Abstract/Free Full Text]
  12. Monks, C. R., B. A. Freiberg, H. Kupfer, N. Sciaky, A. Kupfer. 1998. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395:82.[Medline]
  13. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221.[Abstract/Free Full Text]
  14. 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.[Abstract/Free Full Text]
  15. Bromley, S. K., W. R. Burack, K. G. Johnson, K. Somersalo, T. N. Sims, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 2001. The immunological synapse. Annu. Rev. Immunol. 19:375.[Medline]
  16. Dustin, M. L., M. W. Olszowy, A. D. Holdorf, J. Li, S. Bromley, N. Desai, P. Widder, F. Rosenberger, P. A. van der Merwe, P. M. Allen, A. S. Shaw. 1998. A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell 94:667.[Medline]
  17. Norcross, M. A.. 1984. A synaptic basis for T-lymphocyte activation. Ann. Immunol. 135D:113.
  18. Paul, W. E., R. A. Seder. 1994. Lymphocyte responses and cytokines. Cell 76:241.[Medline]
  19. Davis, D. M.. 2002. Assembly of the immunological synapse for T cells and NK cells. Trends Immunol. 23:356.[Medline]
  20. Wulfing, C., M. M. Davis. 1998. A receptor/cytoskeletal movement triggered by costimulation during T cell activation. Science 282:2266.[Abstract/Free Full Text]
  21. Wulfing, C., M. D. Sjaastad, M. M. Davis. 1998. Visualizing the dynamics of T cell activation: intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels. Proc. Natl. Acad. Sci. USA 95:6302.[Abstract/Free Full Text]
  22. Vyas, Y. M., H. Maniar, B. Dupont. 2002. Cutting edge: differential segregation of the SRC homology 2-containing protein tyrosine phosphatase-1 within the early NK cell immune synapse distinguishes noncytolytic from cytolytic interactions. J. Immunol. 168:3150.[Abstract/Free Full Text]
  23. Vyas, Y. M., K. M. Mehta, M. Morgan, H. Maniar, L. Butros, S. Jung, J. K. Burkhardt, B. Dupont. 2001. Spatial organization of signal transduction molecules in the NK cell immune synapses during MHC class I-regulated noncytolytic and cytolytic interactions. J. Immunol. 167:4358.[Abstract/Free Full Text]
  24. 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.[Abstract/Free Full Text]
  25. 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.[Abstract/Free Full Text]
  26. Valitutti, S., M. Dessing, K. Aktories, H. Gallati, A. Lanzavecchia. 1995. Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy. Role of T cell actin cytoskeleton. J. Exp. Med. 181:577.[Abstract/Free Full Text]
  27. Wulfing, C., A. Bauch, G. R. Crabtree, M. M. Davis. 2000. The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte-antigen-presenting cell interface. Proc. Natl. Acad. Sci. USA 97:10150.[Abstract/Free Full Text]
  28. van der Merwe, P. A., S. J. Davis, A. S. Shaw, M. L. Dustin. 2000. Cytoskeletal polarization and redistribution of cell-surface molecules during T cell antigen recognition. Semin. Immunol. 12:5.[Medline]
  29. Dustin, M. L., J. A. Cooper. 2000. The immunological synapse and the actin cytoskeleton: molecular hardware for T cell signaling. Nat. Immunol. 1:23.[Medline]
  30. Krummel, M. F., M. M. Davis. 2002. Dynamics of the immunological synapse: finding, establishing and solidifying a connection. Curr. Opin. Immunol. 14:66.[Medline]
  31. Springer, T. A.. 1990. Adhesion receptors of the immune system. Nature 346:425.[Medline]
  32. Shaw, A. S., M. L. Dustin. 1997. Making the T cell receptor go the distance: a topological view of T cell activation. Immunity 6:361.[Medline]
  33. Wild, M. K., A. Cambiaggi, M. H. Brown, E. A. Davies, H. Ohno, T. Saito, P. A. van der Merwe. 1999. Dependence of T cell antigen recognition on the dimensions of an accessory receptor-ligand complex. J. Exp. Med. 190:31.[Abstract/Free Full Text]
  34. Qi, S. Y., J. T. Groves, A. K. Chakraborty. 2001. Synaptic pattern formation during cellular recognition. Proc. Natl. Acad. Sci. USA 98:6548.[Abstract/Free Full Text]
  35. Chakraborty, A. K.. 2002. How and why does the immunological synapse form: physical chemistry meets cell biology. Sci. STKE 2002:PE10.
  36. Burroughs, N. J., C. Wulfing. 2002. Differential segregation in a cell-cell contact interface: the dynamics of the immunological synapse. Biophys. J. 83:1784.[Medline]
  37. Simons, K., E. Ikonen. 1997. Functional rafts in cell membranes. Nature 387:569.[Medline]
  38. Brown, D. A., E. London. 2000. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J. Biol. Chem. 275:17221.[Free Full Text]
  39. Janes, P. W., S. C. Ley, A. I. Magee, P. S. Kabouridis. 2000. The role of lipid rafts in T cell antigen receptor (TCR) signalling. Semin. Immunol. 12:23.[Medline]
  40. Simons, K., D. Toomre. 2000. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell. Biol. 1:31.[Medline]
  41. Dykstra, M., A. Cherukuri, S. K. Pierce. 2001. Rafts and synapses in the spatial organization of immune cell signaling receptors. J. Leukocyte Biol. 70:699.[Abstract/Free Full Text]
  42. Lee, K. H., A. D. Holdorf, M. L. Dustin, A. C. Chan, P. M. Allen, A. S. Shaw. 2002. T cell receptor signaling precedes immunological synapse formation. Science 295:1539.[Abstract/Free Full Text]
  43. Bretscher, A., D. Reczek, M. Berryman. 1997. Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures. J. Cell Sci. 110:3011.[Abstract]
  44. Bretscher, A.. 1999. Regulation of cortical structure by the ezrin-radixin-moesin protein family. Curr. Opin. Cell Biol. 11:109.[Medline]
  45. Tsukita, S., S. Yonemura. 1999. Cortical actin organization: lessons from ERM (ezrin/radixin/moesin) proteins. J. Biol. Chem. 274:34507.[Free Full Text]
  46. Allenspach, E. J., P. Cullinan, J. Tong, Q. Tang, A. G. Tesciuba, J. L. Cannon, S. M. Takahashi, R. Morgan, J. K. Burkhardt, A. I. Sperling. 2001. ERM-dependent movement of CD43 defines a novel protein complex distal to the immunological synapse. Immunity 15:739.[Medline]
  47. Roumier, A., J. C. Olivo-Marin, M. Arpin, F. Michel, M. Martin, P. Mangeat, O. Acuto, A. Dautry-Varsat, A. Alcover. 2001. The membrane-microfilament linker ezrin is involved in the formation of the immunological synapse and in T cell activation. Immunity 15:715.[Medline]
  48. Delon, J., S. Stoll, R. N. Germain. 2002. Imaging of T-cell interactions with antigen presenting cells in culture and in intact lymphoid tissue. Immunol. Rev. 189:51.[Medline]
  49. Das, V., B. Nal, A. Roumier, V. Meas-Yedid, C. Zimmer, J. C. Olivo-Marin, P. Roux, P. Ferrier, A. Dautry-Varsat, A. Alcover. 2002. Membrane-cytoskeleton interactions during the formation of the immunological synapse and subsequent T-cell activation. Immunol. Rev. 189:123.[Medline]
  50. Delon, J., K. Kaibuchi, R. N. Germain. 2001. Exclusion of CD43 from the immunological synapse is mediated by phosphorylation-regulated relocation of the cytoskeletal adaptor moesin. Immunity 15:691.[Medline]
  51. Savage, N. D., S. L. Kimzey, S. K. Bromley, K. G. Johnson, M. L. Dustin, J. M. Green. 2002. Polar redistribution of the sialoglycoprotein CD43: implications for T cell function. J. Immunol. 168:3740.[Abstract/Free Full Text]
  52. Stoll, S., J. Delon, T. M. Brotz, R. N. Germain. 2002. Dynamic imaging of T cell-dendritic cell interactions in lymph nodes. Science 296:1873.[Abstract/Free Full Text]
  53. Helander, T. S., O. Carpen, O. Turunen, P. E. Kovanen, A. Vaheri, T. Timonen. 1996. ICAM-2 redistributed by ezrin as a target for killer cells. Nature 382:265.[Medline]
  54. Boyington, J. C., S. A. Motyka, P. Schuck, A. G. Brooks, P. D. Sun. 2000. Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand. Nature 405:537.[Medline]
  55. Fan, Q. R., E. O. Long, D. C. Wiley. 2001. Crystal structure of the human natural killer cell inhibitory receptor KIR2DL1-HLA-Cw4 complex. Nat. Immunol. 2:452.[Medline]
  56. Garcia, K. C., M. Degano, R. L. Stanfield, A. Brunmark, M. R. Jackson, P. A. Peterson, L. Teyton, I. A. Wilson. 1996. An {alpha}{beta} T cell receptor structure at 2.5 A and its orientation in the TCR-MHC complex. Science 274:209.[Abstract/Free Full Text]
  57. Garboczi, D. N., P. Ghosh, U. Utz, Q. R. Fan, W. E. Biddison, D. C. Wiley. 1996. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 384:134.[Medline]
  58. Natarajan, K., N. Dimasi, J. Wang, R. A. Mariuzza, D. H. Margulies. 2002. Structure and function of natural killer cell receptors: multiple molecular solutions to self, nonself discrimination. Annu. Rev. Immunol. 20:853.[Medline]
  59. Rudolph, M. G., J. G. Luz, I. A. Wilson. 2002. Structural and thermodynamic correlates of T cell signaling. Annu. Rev. Biophys. Biomol. Struct. 31:121.[Medline]
  60. Cohen, G. B., R. T. Gandhi, D. M. Davis, O. Mandelboim, B. K. Chen, J. L. Strominger, D. Baltimore. 1999. The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10:661.[Medline]
  61. Yoneda, N., E. Tatsumi, S. Kawano, K. Teshigawara, T. Oka, M. Fukuda, N. Yamaguchi. 1992. Detection of Epstein-Barr virus genome in natural-killer-like cell line, YT. Leukemia 6:136.[Medline]
  62. Shimizu, Y., R. DeMars. 1989. Production of human cells expressing individual transferred HLA-A, -B, -C genes using an HLA-A, -B, -C null human cell line. J. Immunol. 142:3320.[Abstract]
  63. 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.[Abstract/Free Full Text]
  64. Poggi, A., R. Pardi, N. Pella, L. Morelli, S. Sivori, M. Vitale, V. Revello, A. Moretta, L. Moretta. 1993. CD45-mediated regulation of LFA1 function in human natural killer cells: anti-CD45 monoclonal antibodies inhibit the calcium mobilization induced via LFA1 molecules. Eur. J. Immunol. 23:2454.[Medline]
  65. Katz, P., A. M. Zaytoun, J. H. Lee, Jr.. 1982. Mechanisms of human cell-mediated cytotoxicity. III. Dependence of natural killing on microtubule and microfilament integrity. J. Immunol. 129:2816.[Abstract]
  66. Quan, P. C., T. Ishizaka, B. R. Bloom. 1982. Studies on the mechanism of NK cell lysis. J. Immunol. 128:1786.[Abstract]
  67. Sperling, A. I., J. R. Sedy, N. Manjunath, A. Kupfer, B. Ardman, J. K. Burkhardt. 1998. TCR signaling induces selective exclusion of CD43 from the T cell-antigen-presenting cell contact site. J. Immunol. 161:6459.[Abstract/Free Full Text]
  68. McCann, F. E., K. Suhling, L. M. Carlin, K. Eleme, S. B. Taner, K. Yanagi, B. Vanherberghen, P. M. French, D. M. Davis. 2002. Imaging immune surveillance by T cells and NK cells. Immunol. Rev. 189:179.[Medline]
  69. Rosenstein, Y., J. K. Park, W. C. Hahn, F. S. Rosen, B. E. Bierer, S. J. Burakoff. 1991. CD43, a molecule defective in Wiskott-Aldrich syndrome, binds ICAM-1. Nature 354:233.[Medline]
  70. Ostberg, J. R., R. K. Barth, J. G. Frelinger. 1998. The Roman god Janus: a paradigm for the function of CD43. Immunol. Today 19:546.[Medline]
  71. Pedraza-Alva, G., L. B. Merida, S. J. Burakoff, Y. Rosenstein. 1996. CD43-specific activation of T cells induces association of CD43 to Fyn kinase. J. Biol. Chem. 271:27564.[Abstract/Free Full Text]
  72. Pedraza-Alva, G., L. B. Merida, S. J. Burakoff, Y. Rosenstein. 1998. T cell activation through the CD43 molecule leads to Vav tyrosine phosphorylation and mitogen-activated protein kinase pathway activation. J. Biol. Chem. 273:14218.[Abstract/Free Full Text]
  73. Aguado, E., M. Santamaria, M. D. Gallego, J. Pena, I. J. Molina. 1999. Functional expression of CD43 on human natural killer cells. J. Leukocyte Biol. 66:923.[Abstract]
  74. Nieto, M., J. L. Rodriguez-Fernandez, F. Navarro, D. Sancho, J. M. Frade, M. Mellado, A. C. Martinez, C. Cabanas, F. Sanchez-Madrid. 1999. Signaling through CD43 induces natural killer cell activation, chemokine release, and PYK-2 activation. Blood 94:2767.[Abstract/Free Full Text]
  75. Ramoni, C., F. Luciani, F. Spadaro, L. Lugini, F. Lozupone, S. Fais. 2002. Differential expression and distribution of ezrin, radixin and moesin in human natural killer cells. Eur. J. Immunol. 32:3059.[Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
V. L. Hegde, N. P. Singh, P. S. Nagarkatti, and M. Nagarkatti
CD44 mobilization in allogeneic dendritic cell-T cell immunological synapse plays a key role in T cell activation
J. Leukoc. Biol., July 1, 2008; 84(1): 134 - 142.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. Endt, F. E. McCann, C. R. Almeida, D. Urlaub, R. Leung, D. Pende, D. M. Davis, and C. Watzl
Inhibitory Receptor Signals Suppress Ligation-Induced Recruitment of NKG2D to GM1-Rich Membrane Domains at the Human NK Cell Immune Synapse
J. Immunol., May 1, 2007; 178(9): 5606 - 5611.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Nedvetzki, S. Sowinski, R. A. Eagle, J. Harris, F. Vely, D. Pende, J. Trowsdale, E. Vivier, S. Gordon, and D. M. Davis
Reciprocal regulation of human natural killer cells and macrophages associated with distinct immune synapses
Blood, May 1, 2007; 109(9): 3776 - 3785.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. E. McCann, P. Eissmann, B. Onfelt, R. Leung, and D. M. Davis
The Activating NKG2D Ligand MHC Class I-Related Chain A Transfers from Target Cells to NK Cells in a Manner That Allows Functional Consequences
J. Immunol., March 15, 2007; 178(6): 3418 - 3426.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
E. N. M. Nolte-'t Hoen, C. R. Almeida, N. R. Cohen, S. Nedvetzki, H. Yarwood, and D. M. Davis
Increased surveillance of cells in mitosis by human NK cells suggests a novel strategy for limiting tumor growth and viral replication
Blood, January 15, 2007; 109(2): 670 - 673.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. R. Almeida and D. M. Davis
Segregation of HLA-C from ICAM-1 at NK Cell Immune Synapses Is Controlled by Its Cell Surface Density
J. Immunol., November 15, 2006; 177(10): 6904 - 6910.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Masilamani, C. Nguyen, J. Kabat, F. Borrego, and J. E. Coligan
CD94/NKG2A Inhibits NK Cell Activation by Disrupting the Actin Network at the Immunological Synapse
J. Immunol., September 15, 2006; 177(6): 3590 - 3596.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. B. Graham, M. Cella, E. Giurisato, K. Fujikawa, A. V. Miletic, T. Kloeppel, K. Brim, T. Takai, A. S. Shaw, M. Colonna, et al.
Vav1 Controls DAP10-Mediated Natural Cytotoxicity by Regulating Actin and Microtubule Dynamics
J. Immunol., August 15, 2006; 177(4): 2349 - 2355.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. S. Korbel, K. C. Newman, C. R. Almeida, D. M. Davis, and E. M. Riley
Heterogeneous Human NK Cell Responses to Plasmodium falciparum-Infected Erythrocytes
J. Immunol., December 1, 2005; 175(11): 7466 - 7473.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H. D. Kopcow, D. S. J. Allan, X. Chen, B. Rybalov, M. M. Andzelm, B. Ge, and J. L. Strominger
Human decidual NK cells form immature activating synapses and are not cytotoxic
PNAS, October 25, 2005; 102(43): 15563 - 15568.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Jansson, E. Barnes, P. Klenerman, M. Harlen, P. Sorensen, S. J. Davis, and P. Nilsson
A Theoretical Framework for Quantitative Analysis of the Molecular Basis of Costimulation
J. Immunol., August 1, 2005; 175(3): 1575 - 1585.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
E.-K. Jo, H. Wang, and C. E. Rudd
An essential role for SKAP-55 in LFA-1 clustering on T cells that cannot be substituted by SKAP-55R
J. Exp. Med., June 6, 2005; 201(11): 1733 - 1739.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Poupot, F. Pont, and J.-J. Fournie
Profiling Blood Lymphocyte Interactions with Cancer Cells Uncovers the Innate Reactivity of Human {gamma}{delta} T Cells to Anaplastic Large Cell Lymphoma
J. Immunol., February 1, 2005; 174(3): 1717 - 1722.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
B. Vanherberghen, K. Andersson, L. M. Carlin, E. N. M. Nolte-`t Hoen, G. S. Williams, P. Hoglund, and D. M. Davis
Human and murine inhibitory natural killer cell receptors transfer from natural killer cells to target cells
PNAS, November 30, 2004; 101(48): 16873 - 16878.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. J. Standeven, L. M. Carlin, P. Borszcz, D. M. Davis, and D. N. Burshtyn
The Actin Cytoskeleton Controls the Efficiency of Killer Ig-Like Receptor Accumulation at Inhibitory NK Cell Immune Synapses
J. Immunol., November 1, 2004; 173(9): 5617 - 5625.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
H. Wang, F. E. McCann, J. D. Gordan, X. Wu, M. Raab, T. H. Malik, D. M. Davis, and C. E. Rudd
ADAP-SLP-76 Binding Differentially Regulates Supramolecular Activation Cluster (SMAC) Formation Relative to T Cell-APC Conjugation
J. Exp. Med., October 18, 2004; 200(8): 1063 - 1074.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Roda-Navarro, M. Mittelbrunn, M. Ortega, D. Howie, C. Terhorst, F. Sanchez-Madrid, and E. Fernandez-Ruiz
Dynamic Redistribution of the Activating 2B4/SAP Complex at the Cytotoxic NK Cell Immune Synapse
J. Immunol., September 15, 2004; 173(6): 3640 - 3646.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
K. Eleme, S. B. Taner, B. Onfelt, L. M. Collinson, F. E. McCann, N. J. Chalupny, D. Cosman, C. Hopkins, A. I. Magee, and D. M. Davis
Cell Surface Organization of Stress-inducible Proteins ULBP and MICA That Stimulate Human NK Cells and T Cells via NKG2D
J. Exp. Med., April 5, 2004; 199(7): 1005 - 1010.
[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
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McCann, F. E.
Right arrow Articles by Davis, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McCann, F. E.
Right arrow Articles by Davis, D. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS