The Size of the Synaptic Cleft and Distinct Distributions of Filamentous , Ezrin, CD43, and CD45 at Activating and Inhibitory Human NK Cell Immune Synapses This information is current as of September 24, 2021. Fiona E. McCann, Bruno Vanherberghen, Konstantina Eleme, Leo M. Carlin, Ray J. Newsam, David Goulding and Daniel M. Davis J Immunol 2003; 170:2862-2870; ; doi: 10.4049/jimmunol.170.6.2862 Downloaded from http://www.jimmunol.org/content/170/6/2862

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Size of the Synaptic Cleft and Distinct Distributions of Filamentous Actin, Ezrin, CD43, and CD45 at Activating and Inhibitory Human NK Cell Immune Synapses1,2

Fiona E. McCann,* Bruno Vanherberghen,* Konstantina Eleme,* Leo M. Carlin,* Ray J. Newsam,‡ David Goulding,† and Daniel M. Davis3*

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 IS. CD45 Downloaded from 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 accord- ing to the size of their extracellular domains. The Journal of Immunology, 2003, 170: 2862–2870. http://www.jimmunol.org/ atural killer cells are large granular that are Previously, it has been shown that HLA-C still clusters at the cytotoxic to some tumors and virus-infected cells (1). inhibitory NK cell IS in the presence of drugs that deplete ATP or N The outcome of NK cell immune surveillance is the re- disrupt actin, myosin motors, or , suggesting that the sult of a complex balance between inhibitory and activating sig- cytoskeleton is not necessary for accumulation of at least some naling (2–9). Killer Ig-like receptors (KIR)4 with two Ig domains, proteins at the inhibitory NK cell IS (14, 24). However, within the KIR2DL1 and KIR2DL2, trigger inhibitory signaling upon recog- first minute following intercellular contact, clusters at both nition of the class I MHC proteins HLA-Cw4 or -Cw6, and HLA- the activating and inhibitory NK cell IS, though at the inhibitory Cw3 or -Cw7, respectively (10, 11). Immunological intercellular NK cell IS talin then redistributes away from the IS in the ensuing by guest on September 24, 2021 contacts between effector and target cells or APCs can involve the five minutes (22). The speed of the redistribution of talin compares organization of proteins into micrometer scale domains at inter- with the speed that phosphorylated accumulates at the activat- cellular contacts (12–16) creating an immunological or immune ing T cell IS just two minutes after intercellular contact, closely synapse (IS) (17–19). This has been most extensively studied in followed by recruitment of ␨-associated protein of 70 kDa (42). the T cell/APC conjugate, (12, 13, 15, 20, 21). The balance be- A role for the ezrin-radixin-moesin (ERM) proteins, linking the tween activating and inhibitory signaling also controls the su- actin cytoskeleton to certain transmembrane proteins such as pramolecular organization of proteins at the NK cell IS (14, 22– CD43, (reviewed in Refs. 43–45), has been proposed for the or- 25). Molecular mechanisms for the assembly of the IS include a ganization of the activating T cell IS. Ezrin was either excluded role for the cytoskeleton (20, 21, 26–30), segregation of proteins from (46), or clustered at the peripheral edges of (47), the activat- according to the size of their extracellular domains (28, 31–36), ing T cell IS (48, 49). A dominant-negative ezrin, lacking the and association of proteins with lipid rafts (37–41). 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 activat- *Department of Biological Sciences and †Center for Molecular Microbiology and ing T cell IS (50). Furthermore, a mutant of CD43 unable to bind Infection, Imperial College, London, United Kingdom; and ‡Research School of Bio- sciences, University of Kent, Canterbury, Kent, United Kingdom ERM proteins was no longer excluded from the activating T cell Received for publication August 12, 2002. Accepted for publication January 8, 2003. IS, resulting in reduced IL-2 production (50). In contrast, another study reported that CD43 mutated to either lack the full cytoplas- 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 mic domain or just the ERM binding motif can occupy the center with 18 U.S.C. Section 1734 solely to indicate this fact. of the activating T cell IS, without subsequently disrupting T cell 1 This work is supported by grants from the Medical Research Council (U.K.) and The proliferation in response to Ag (51). Significantly, the exclusion of Royal Society. CD43 from the activating T cell/ IS has been dem- 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 onstrated in vivo within a mouse lymph node (52). Ezrin has been 18–22, 2002. shown to be capable of redistributing ICAM-2 and sensitizing tar- 3 Address correspondence and reprint requests to Dr. Daniel M. Davis, Department of get cells to NK cytotoxicity (53). In this study, the distribution of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, Lon- filamentous actin (f-actin), ezrin, CD43, and CD45 at the inhibi- don SW7 2AZ, U.K. E-mail address: [email protected] tory and activating NK cell IS is reported. 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 In addition to cytoskeletal-mediated movement, another mech- fluorescent protein. anism that may contribute to the formation of the IS involves the

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 2863 segregation of proteins according to the size of their extracellular Phenotyping of NK clones domains (28, 31–35). Pairs of proteins with longer extracellular Abs used for flow cytometry were CyChrome-conjugated anti-human domains such as the integrins, spanning ϳ40 nm (31), would be CD56 (B159), CyChrome-conjugated anti-human CD3 (UCHT1), Cy- separated from protein pairs with shorter extracellular domains, Chrome-conjugated IgG1 control (MOPC-21), anti-human CD94 (HP- such as KIR or TCR with MHC protein which span ϳ15 nm (54– 3D9), and PE-conjugated anti-KIR2DL2 (DX27), all purchased from BD 59). In this study, the distance across the synaptic cleft at the ac- PharMingen. Anti-KIR2DL1 (HP3E4) was purchased from the American Type Culture Collection (Manassas, VA) and anti-Ig-like transcript-2 (HP- tivating and inhibitory NK cell IS was measured and immunogold F1) was a kind gift from M. Lo´pez-Botet (Universitat Pompeu Fabra, Bar- labeling was used to measure the size of the synaptic cleft where celona, Spain). All of these Abs were used at a concentration of 1 ␮g/ml. HLA-C accumulates at the inhibitory NK cell IS. FITC-conjugated rabbit anti-mouse IgG (Sigma-Aldrich) and FITC-conju- gated rat anti-mouse IgM (R6-60.2; BD PharMingen) were used at 2 ␮g/ ml. The cytolytic activity of NK clones against various target cells was Materials and Methods assessed in 5-h 35S-release assays as described previously (63). Assays ϳ Cells and Abs were performed in triplicate and data values differed by 5% of the mean. Spontaneous release of 35S was Ͻ25% of the maximal release. NK cell transfectants, YTS/Mock (YTS, mock-transfected), and YTS/ KIR2DL1 (YTS transfected to express KIR2DL1) were previously de- Imaging the IS scribed (60). YTS is a subclone of the human NK tumor line YT (61). An 6 EBV-transformed B cell line 721.221 (hereafter referred to as 221), se- 221 and YTS cell transfectants (10 of each) were coincubated for 45 min lected to lack surface expression of endogenous class I MHC protein (62), at 37°C/5% CO2 in conical end tubes containing 1 ml of culture medium was transfected to express enhanced green fluorescent protein (GFP)- comprising RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1ϫ nonessential amino acids, 50 ␮M 2-ME, 50 tagged HLA-Cw3, -Cw4, or -Cw6 (i.e., 221/Cw6-GFP, etc.), as previously Downloaded from described (14). Cytotoxicity assays show that YTS/KIR2DL1 is effectively U/ml penicillin-streptomycin, (all purchased from Invitrogen), after which inhibited from killing 221 transfected with HLA-Cw6 (i.e., 221/Cw6) or the cells had fallen under gravity. Excess medium was removed and the 221/Cw4, but not 221/Cw3 or 221/Cw7, target cells (60). An activating NK sedimented cells were fixed in Cytofix/Cytoperm (BD PharMingen) for 12 cell IS is created when the intercellular contact between the NK and target min at 4°C. The fixed cells were then washed twice in 0.1% Tween 20/PBS cell results in lysis of the target cell. In contrast, an inhibitory NK cell IS before cell staining. For NK clones, the same method was used except that ϫ 5 is created at the intercellular contact of noncytolytic conjugates between 2 10 cells of each type were used and the conjugates were briefly the NK cell and target cell. Thus, conjugates between YTS/KIR2DL1 and centrifuged before fixation to ensure that the cells had sedimented before 221/Cw6-GFP create an inhibitory NK cell IS and conjugates of YTS/ decanting excess medium. Following cell fixation, f-actin was labeled us- http://www.jimmunol.org/ Mock and 221/Cw6-GFP or YTS/KIR2DL1 and 221/Cw3-GFP create an ing Alexa Fluor 633-conjugated phalloidin (Molecular Probes) at 5 U/ml in activating NK cell IS. 1% BSA/PBS, for1hat4°C. Cells were washed three times in 0.1% Tween 20/PBS before imaging. Mouse anti-human ezrin mAb (clone 18, IgG1; BD Transduction Lab- 6 oratories, Lexington, KY), rabbit polyclonal anti-human ezrin Ab (Upstate For imaging the NK cell IS after different times of coincubation, 10 of both YTS and 221 transfectants were incubated in a V-bottom 96-well Biotechnology, Lake Placid, NY), mouse anti-human CD43 mAb (IG10, ␮ IgG1; BD PharMingen, San Diego, CA), and mouse anti-human CD45 culture plate at 37°C/5% CO2 in 100 l of prewarmed culture medium. The ␮ cells were then fixed after 2, 10, 20, or 45 min of coincubation by adding (HI30, IgG1; BD PharMingen) were used at 5 g/ml. Mouse anti-CD158a ␮ (EB6, IgG1; Serotec, Oxford, U.K.) was used at 10 ␮g/ml. Alexa Fluor 568 200 l of Cytofix/Cytoperm directly to the well at 4°C for 15 min. goat anti-mouse IgG, Alexa Fluor 633 goat anti-mouse IgG, and Alexa For Ab staining, fixed cells were initially incubated in a blocking solu- tion comprising 5% horse serum (Sigma-Aldrich) and 3% BSA in Perm/ Fluor 568 goat anti-rabbit IgG (Molecular Probes, Eugene, OR) were used by guest on September 24, 2021 at a concentration of 2 ␮g/ml. To phenotype cells for expression of the Wash buffer (BD PharMingen) for1hat4°C. Next, cells were incubated various CD45 isoforms, the following FITC-conjugated mAbs (Caltag with primary Ab diluted in blocking solution for 90 min at 4°C. Following Laboratories, Burlingame, CA) were used: UCHLI (IgG2a) for CD45RO, three washes in 0.1% Tween 20/PBS, secondary Ab, also diluted in block- MEM56 (IgG2b) for CD45RA, and MEM55 (IgG1) for CD45RB. For ing solution, was added to the cells for1hat4°C. The stained cells were then washed three times in 0.1% Tween 20/PBS and 7 ␮l of the resus- immunogold labeling, anti-GFP mAb (JL-8, IgG2a; Clontech Laboratories, ϫ Palo Alto, CA) was used at 20 ␮g/ml and detected with 12 ␮g/ml goat pended cell pellet was placed between a microscope slide and a 24 24 anti-mouse IgG conjugated with 10 nm gold (TAAB Laboratories, Berk- mm coverslip (thickness no. 1) for microscopy. Staining procedures were shire, U.K.). All other reagents were purchased from Sigma-Aldrich (St. altered to incorporate the use of buffers containing alternative detergents Louis, MO) unless stated otherwise. such as 0.05% saponin though no effect on staining was observed (data not shown). For double immunofluorescence labeling, both primary Abs fol- lowed by both secondary Abs were added. It was confirmed that there was Generation of NK clones no species cross-reactivity between Alexa Fluor-conjugated goat anti- mouse IgG and goat anti-rabbit IgG and also that none of the secondary PBMCs were isolated from the buffy coat residues of healthy donors by Abs used showed any nonspecific binding to the cells in the absence of centrifugation on Ficoll-Paque plus gradient (Amersham Pharmacia Bio- primary Ab (data not shown). tech, 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 Confocal microscopy image analysis with 10% human serum (type AB; Sigma-Aldrich), 30% nutrient mixture Cell conjugates were imaged under a 63ϫ oil immersion objective using a F-12 (Ham), 2 mM L-glutamine, 1ϫ nonessential amino acids, 1 mM so- confocal laser scanning microscope (TCS SP2; Leica, Deerfield, IL) dium pyruvate, 50 ␮M 2-ME, 50 U/ml penicillin-streptomycin, and 100 equipped with argon/krypton and helium/neon lasers using excitation U/ml human recombinant IL-2 (Roche, Basel, Switzerland), referred to wavelengths of 488, 568, and 633 nm. Conjugates were scanned in the hereafter as DMEM plus supplements, (all from Invitrogen, Carlsbad, CA), xy-direction every 0.3 ␮m throughout the z-plane. The face of the IS was with 1 ␮g/ml PHA (Sigma-Aldrich). The irradiated cells were plated into then reconstructed using a maximum intensity projection (Confocal Soft- 5 3 the wells of U-bottom 96-well plates (10 PBMC and 5 ϫ 10 RPMI 8866 ware; Leica). To quantify the relative amounts of HLA-C-GFP, ezrin, and cells per well) and incubated overnight at 37°C/5% CO2. CD3 PBL derived CD43 at the inhibitory IS within a single optical slice, their mean fluores- from human buffy coat residues that were cultured for 1 wk with irradiated cence intensities were obtained from a 15-␮m2 area at the intercellular 721.221 cells (20,000 rad), in a 4:3 ratio in RPMI 1640 medium containing contact. The mean intensities were then measured from the same area with 10% human serum (type AB; Sigma-Aldrich) with supplements, were marked onto the unconjugated membranes of both the NK and B cell and sorted by FACS into the 96-well plates containing the irradiated PBMCs a percentage of mean fluorescence intensity at the inhibitory IS relative to and RPMI 8866 cells at 1 cell per well. After sorting, 100 ␮l of DMEM the sum of the unconjugated membranes was calculated. 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 sup- Electron microscopy plements. NK clones were periodically monitored to be CD3Ϫ and CD56ϩ by mAb staining and their phenotype was determined by mAb staining and Live YTS and 221 transfectants were coincubated for 45 min at 37°C/5% cytotoxicity assays against a panel of target cells. Cytotoxicity assays and CO2 as previously described for IS formation. For negative staining, cells laser scanning confocal microscopy were performed at least 4 days after were fixed in 2.5% glutaraldehyde/2.5% paraformaldehyde in PBS for 2 h restimulation with fresh human recombinant IL-2. at 4°C after which the cells were washed three times in PBS. Cells were 2864 F-ACTIN, EZRIN, CD43, AND CD45 AT THE NK CELL IS 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 ex- amined 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 anal- ysis 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 Ϫ30°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 eth- anol/K4 M, followed by incubation with K4 M for 24 h and finally a further Downloaded from 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 ␮g/ml mouse anti-GFP (JL-8; Clontech Laboratories) in 10% FCS in PBS for1hatroom FIGURE 1. F-actin clusters at the activating, but not inhibitory, NK cell temperature. After washing three times in PBS, the grids were incubated IS. Conjugates of 221 and YTS transfectants were fixed and labeled with for1hin12␮g/ml goat anti-mouse IgG conjugated with 10 nm of gold Alexa Fluor 633 phalloidin to stain for f-actin. In both a and b, bright-field http://www.jimmunol.org/ (TAAB Laboratories) in the same diluent as before. Following a further 6 images of conjugates followed by HLA-C-GFP and phalloidin staining washes in PBS and 10 washes in ddH2O, the grids were contrasted with with the corresponding plots of fluorescence intensity are shown. Higher uranyl acetate and lead citrate before examination with a transmission elec- peaks represent areas of higher fluorescence intensity. a, An inhibitory NK tron microscope (Philips CM100). 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, Results but not HLA-C-GFP, accumulates. Scale bars represent 10 ␮m. c, The

F-actin clusters at the activating, but not the inhibitory, NK distribution of HLA-C-GFP and Alexa Fluor 633 phalloidin in activating by guest on September 24, 2021 cell IS and inhibitory conjugates was assessed and the mean percentages from six independent experiments are shown. Error bars are the SEM and n is the Conjugates of YTS/KIR2DL1 (YTS transfected to express total number of conjugates scored. KIR2DL1) or YTS/MOCK (mock-transfected) and 221/Cw6-GFP, 221/Cw4-GFP, or 221/Cw3-GFP were fixed and labeled with Al- exa 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 often stained brighter for ezrin than the YTS cell inferring a higher clustered (Fig. 1b). Throughout this study, clustering at the IS was level of expression in 221 cells. At the inhibitory NK cell IS, a confirmed by projecting a single optical slice through the IS with zone devoid of ezrin and CD43 staining corresponding to the re- fluorescence intensity plotted on a third axis. Where the fluores- gion where HLA-C clustered at the IS was evident (Figs. 2 and 3). cence intensity at the IS was more than the sum of the intensity of In contrast, at the activating IS, ezrin and CD43 were evenly dis- the unconjugated membranes, that label was defined as being clus- tributed across the membranes of apposing cells within the conju- tered at the IS. The fluorescence intensity of phalloidin at the ac- gate (Figs. 2 and 3). At the inhibitory NK cell IS, ezrin and CD43 tivating IS (Fig. 1b) is clearly more than the sum of unconjugated membranes, whereas at the inhibitory IS (Fig. 1a) no increase in was excluded in 50–60% of inhibitory conjugates whereas at the Ͻ fluorescence intensity of phalloidin above the sum of unconjugated activating NK cell IS, exclusion of either protein occurred 10% membranes was observed. At the inhibitory IS, HLA-C clustered of the time (Figs. 2d and 3f). CD43 and ezrin were equally likely ϳ70% of the time whereas f-actin accumulated only 30% of the to be excluded from an inhibitory NK cell IS whether each NK cell time. In contrast, in activating synapses, f-actin was found to ac- simultaneously maintains one or several synapses (data not cumulate 70–80% of the time, while HLA-C-GFP clustered shown). Clustering of ezrin and CD43 at either the inhibitory or Ͻ15% of the time (Fig. 1c). activating NK cell IS was observed in Ͻ10% of all conjugates assessed. In contrast to anti-CD43 staining, an isotype-matched mAb against KIR2DL1, EB6, was observed to brightly stain the Ezrin and CD43 are excluded from the inhibitory, but not NK cell juxtaposed to where HLA-C clusters on the target cell activating, NK cell IS (Fig. 3, c and e). This indicated that the staining procedure used Conjugates between 221/Cw6-GFP or 221/Cw3-GFP and YTS/ does permit access of Abs to proteins within the inhibitory NK KIR2DL1 or YTS/Mock were fixed and either stained for ezrin cell IS. and f-actin (Fig. 2) or ezrin and CD43 (Fig. 3). Consistent with The time taken for the accumulation of HLA-C and the exclu- analysis by flow cytometry (data not shown), the 221 transfectant sion of ezrin and CD43 at the inhibitory NK cell IS was assessed The Journal of Immunology 2865 Downloaded from http://www.jimmunol.org/

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 phal- loidin to stain for f-actin. The distribution of HLA-C-GFP, ezrin, and f- by guest on September 24, 2021 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 repre- sent 10 ␮m, and 5 ␮minc. d, Each inhibitory and activating NK cell IS was assessed for ezrin being clustered, excluded, or distributed homoge- neously 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.

FIGURE 3. CD43 is excluded from the inhibitory, but not the activating, NK cell IS. Conjugates of 221 transfectants and YTS/KIR2DL1 were labeled by imaging cells that were fixed after different times of being in- with rabbit polyclonal anti-ezrin and mouse monoclonal anti-CD43 Abs. Col- cubated together (Fig. 3f). Interestingly, after 10 min of coincuba- umns a and b show the distribution of HLA-C-GFP, ezrin, and CD43 at the tion of cells, HLA-C is clustered at ϳ60% of inhibitory NK syn- inhibitory and activating NK cell IS between (a) 221/Cw6-GFP and YTS/ apses, whereas ezrin and CD43 are excluded from only ϳ20% of 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 these synapses. This suggests that accumulation of HLA-C pre- YTS/KIR2DL1 is shown. d, Reconstruction of the face of the inhibitory NK cedes exclusion of ezrin and CD43 at the inhibitory NK cell IS. cell IS shows that ezrin and CD43 are excluded from the inhibitory NK cell IS The extent to which ezrin and CD43 are excluded and HLA-C- where HLA-C-GFP clusters. e, Reconstruction of the face of the IS demon- GFP is clustered at the inhibitory IS was quantified using software strates that KIR2DL1 clusters at the inhibitory NK cell IS, opposing HLA-C- to analyze mean fluorescence intensities from defined regions at GFP on the target cell. Scale bars represent 10 ␮mina–c and 5 ␮mind and the inhibitory NK cell IS relative to regions on the unconjugated e. f, Cells were fixed after coincubation for the times shown and the mean cell membranes, as described in Materials and Methods. From a percentages of conjugates where ezrin or CD43 were clustered or excluded total of 40 conjugates counted, the fluorescence intensity of ezrin from the intercellular contact is shown. Error bars are SEM from at least three and CD43 was reduced by 68% Ϯ 12.5 and 56.7% Ϯ 10.2, re- independent experiments. At least 98 conjugates were counted at each time spectively, at the inhibitory NK cell IS relative to the amount on point for both activating and inhibitory conjugates. the two unconjugated membranes. Interestingly, there was no ap- parent trend between the extent of HLA-C-GFP clustering and the fluorescence intensity of ezrin or CD43 at the IS (Table I). Com- neither cell alone can account for the amount of ezrin and CD43 parison of the amount of ezrin and CD43 staining on the NK cell excluded from the IS. Thus, ezrin and CD43 from both the NK and and target cell with the amount at the inhibitory IS suggests that target cell are excluded from the inhibitory NK cell IS. 2866 F-ACTIN, EZRIN, CD43, AND CD45 AT THE NK CELL IS

Table I. Mean fluorescence intensity of ezrin, CD43, and HLA-C-GFP human NK clones is consistent with previous observations (64). at the inhibitory NK cell ISa Conjugates involving effectors YTS/KIR2DL1 or NK clone 5H4 (KIR2DL1ϩKIR2DL2Ϫ) were fixed and stained with anti-CD45 HLA-C-GFP (%) Ezrin (%) CD43 (%) mAb (HI30), which recognizes CD45RO and CD45RB. This mAb 124 35 52 did not stain 221 cells, inferring that it does not detect CD45RA 162 39 39 (our unpublished observations). At inhibitory conjugates, 169 11 18 CD45RO expressed on YTS/KIR2DL1 (Fig. 5, a and c)or 202 27 47 CD45RO and CD45RB expressed on the NK clone 5H4 (Fig. 5d) 220 24 30 are clearly excluded from the region where HLA-C clusters (Fig. 299 28 32 355 49 67 5, c and d). At the activating IS between YTS/KIR2DL1 and HLA- 386 9 22 Cw3-GFP (Fig. 5b), CD45RO was evenly distributed across the

a The mean fluorescence intensity at the IS, relative to the sum of the unconjugated face of the IS in 87% of conjugates. At the inhibitory NK cell IS membranes of apposing NK and target cells, was calculated. From a total of 40 between YTS/KIR2DL1 and 221/Cw6-GFP, CD45RO was found conjugates assessed, the table shows eight representative examples with varying in- to be excluded from the IS in 67% of conjugates (Fig. 5e). tensities of HLA-C-GFP at the IS. There is no apparent trend between levels of the intensity of HLA-C-GFP and corresponding intensities of ezrin and CD43 at the inhibitory NK cell IS. 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 Downloaded from Exclusion of ezrin and CD43 from inhibitory, but not activating, study as the distance between the outer membranes of apposing NK cell IS in conjugates involving human NK clones effector and target cells at the IS, would vary accordingly. Thus, ϩ Ϫ Two human NK clones, 5H4 (KIR2DL1 KIR2DL2 ) and 6H5 we assessed the ultrastructure of the activating and inhibitory NK Ϫ ϩ (KIR2DL1 KIR2DL2 ), were selected for microscopy. By flow cell IS in conjugates of 221 transfectants and YTS/KIR2DL1 cells ϩ Ϫ cytometry, both clones were found to be CD56 CD3 Ig-like tran- by electron microscopy. NK cells were easily distinguished from Ϫ ϩ script-2 CD94 . In agreement with the specificity of KIR for target cells by the presence of large granules within their cyto- http://www.jimmunol.org/ HLA-C alleles, lysis by NK clone 5H4, expressing KIR2DL1, was plasm. The distance across the synaptic cleft in both activating and effectively inhibited by HLA-Cw6 while NK clone 6H5, express- inhibitory conjugates varied such that distinct wide and narrow ing KIR2DL2, was inhibited by HLA-Cw3 expressed on 221 trans- domains were evident (Fig. 6a). Distances across the synaptic cleft fectants (Fig. 4a). of conjugates between 221/Cw6-GFP and YTS/KIR2DL1 and Conjugates of NK clones 5H4 or 6H5 with 221/Cw6-GFP or conjugates of 221/Cw3-GFP and YTS/KIR2DL1 cells were mea- 221/Cw3-GFP targets were fixed and stained for CD43 and ezrin sured at 300-nm intervals along each intercellular contact. Al- using mAbs. Both CD43 and ezrin were excluded from the inhib- though the size of the synaptic cleft in activating and inhibitory ϩ Ϫ itory IS between clone 5H4 (KIR2DL1 KIR2DL2 ) and 221/ conjugates was found to range from 10 to 55 nm and 10 to 42 nm, Cw6-GFP (Fig. 4b). At the activating IS, ezrin and CD43 were respectively, the majority of regions measured between 10 and 30 by guest on September 24, 2021 homogeneously distributed, shown in conjugates between 5H4 and nm at both the activating and inhibitory IS (Fig. 6, c and d). 221/Cw3-GFP (Fig. 4c). A reconstruction of the face of the inhib- To characterize the distribution of HLA-C at the inhibitory NK itory NK cell IS shows exclusion of CD43 and ezrin where HLA- cell IS, immunogold labeling was used to locate HLA-C-GFP. Cw6-GFP clusters (Fig. 4d). For both NK clones, ezrin and CD43 HLA-C-GFP was detected on the surface, and to a lesser extent were excluded from the inhibitory NK cell IS 60–80% of the time within the cytoplasm, of the target cell. Staining was occasionally (Fig. 4e). This demonstrates that exclusion of ezrin and CD43 is a observed on the apposing NK cell, which could reflect the inter- general property of the inhibitory NK cell IS rather than specificto cellular transfer of HLA-C-GFP from the target cell to the NK cell an IS involving one type of KIR/MHC protein. At the activating (24). Strikingly, accumulation of the gold beads, marking the lo- NK cell IS, ezrin or CD43 was homogeneously distributed at the cation of HLA-C-GFP, was observed only at the tighter intercel- intercellular contact in ϳ80% of conjugates (Fig. 4e). Thus, the lular contacts (Fig. 6b). Although, it may be important to note that distribution of ezrin and CD43 at the inhibitory and activating IS a few gold beads were located where the distance between the cells in conjugates involving human NK clones derived from peripheral was ϳ100 nm, which may or may not be part of the intercellular blood corresponds to that observed in conjugates of YTS and 221 contact. The distance across the synaptic cleft at each gold bead transfectants (Figs. 2 and 3). that was clearly at the IS i.e., HLA-C-GFP at the IS, was measured and found to be 14.5 Ϯ 2.2 nm (Fig. 6e), which is the size of the CD45 is excluded from the inhibitory, but not the activating, NK extracellular domains of HLA-C bound to KIR (54, 55). cell IS With a view to further assessing the role of protein size in assem- Discussion bly of the NK cell IS, we imaged the distribution of CD45 in By laser scanning confocal microscopy, we show that, similar to inhibitory and activating conjugates. Because alternative splicing the activating T cell IS, f-actin accumulates at the activating NK produces various CD45 isoforms with extracellular domains rang- cell IS. Consistent with a role for f-actin in the activating NK cell ing from 20 to 50 nm, we first established their respective distri- IS, inhibitors of actin polymerization significantly reduced NK cy- butions on the cells used for imaging. Abs specific for CD45RO totoxicity (65, 66). Conversely, a homogeneous distribution of f- (20–28 nm), CD45RA, and CD45RB (each ϳ50 nm) were used to actin around the cell membranes of apposing cells was seen at the identify the expression of the various isoforms on the 221 and YTS inhibitory NK cell IS. Unexpectedly, however, ezrin and one of its transfectants as well as NK clones derived from peripheral blood. associated transmembrane proteins CD43 are excluded from the By flow cytometry, it was determined that YTS/KIR2DL1 ex- inhibitory, i.e., noncytolytic, NK cell IS, while both proteins are pressed only CD45RO, 221/Cw6-GFP expressed only CD45RA, homogeneously distributed across the IS of activating conjugates. and the NK clones analyzed expressed CD45RO, CD45RA, and Surprisingly, the exclusion of ezrin and CD43 from the inhibitory CD45RB (data not shown). This distribution of CD45 isoforms on NK cell IS is reminiscent of the exclusion of these proteins from The Journal of Immunology 2867

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 con- tain large synaptic clefts that could accommodate CD43 (Fig. 6), and both activating and inhibitory NK cell synapses involve inter- actions 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 pro- liferation (51). Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 5. Exclusion of CD45 from the inhibitory, but not activating, NK cell IS. Inhibitory and activating conjugates of YTS/KIR2DL1 and FIGURE 4. Exclusion of ezrin and CD43 from the inhibitory, but not 221/Cw6-GFP or 221/Cw3-GFP were fixed, stained with anti-CD45 mAb activating, NK cell IS in conjugates involving human NK clones. a, The (HI30), and the distribution at the IS was determined. Inhibitory conjugates cytotoxicity of two human clones, 5H4 (KIR2DL1ϩKIR2DL2Ϫ) and 6H5 where CD45 is excluded from the IS are shown in a and c. b, At the activating (KIR2DL1ϪKIR2DL2ϩ), was assessed in a 5-h 35S release cytotoxicity IS, between YTS/KIR2DL1 and 221/Cw3-GFP, CD45 is evenly distributed assay. b, Inhibitory conjugates of 5H4 and 221/Cw6-GFP and (c) activating across the intercellular contact. c, Reconstruction of the face of the inhibitory conjugates of 5H4 and 221/Cw3-GFP were stained for ezrin and CD43. IS formed between YTS/KIR2DL1 and 221/Cw6-GFP demonstrates a region Ezrin and CD43 are excluded from the inhibitory IS where HLA-C clusters devoid of CD45 staining corresponding to that where HLA-C clusters. Scale but not the activating IS. d, Reconstructions of the face of two inhibitory bars represent 10 ␮mina and b, and 5 ␮minc. d, Reconstruction of the NK cell IS, stained for ezrin or CD43, are shown. A region devoid of mAb inhibitory IS formed between human NK clone 5H4 and 221/Cw6-GFP. staining for ezrin and CD43 corresponds to the region where HLA-C clus- Exclusion of CD45 from the region where HLA-C-GFP clusters is apparent. e, ters. Scale bars represent 10 ␮minb and c and 5 ␮mind. e, The distri- The distribution of CD45 at the inhibitory and activating IS was assessed in bution of CD43 and ezrin at the inhibitory and activating NK cell IS was numerous conjugates involving YTS/KIR2DL1. n, The number of conjugates assessed in n conjugates. analyzed. 2868 F-ACTIN, EZRIN, CD43, AND CD45 AT THE NK CELL IS

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. Interest- ingly, some anti-CD45 mAbs selectively inhibited LFA-1-medi- ated NK cytotoxicity, suggesting a role for CD45 in regulating NK

cell killing (64). Thus, at the inhibitory NK cell IS the redistribu- Downloaded from tion 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 http://www.jimmunol.org/ interactions (28, 31–36). In this study, we measured the size of the synaptic cleft between inhibitory and activating NK cell conju- gates 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 dis- tances Ͼ30 nm remains low, perhaps these wider regions are suf- ficient to allow the observed prevalence of CD43 and CD45 there. Importantly, where HLA-C-GFP was localized, the synaptic cleft by guest on September 24, 2021 measured 14.5 Ϯ 2.2 nm, which corresponds to the size of the extracellular portions of KIR/MHC, as determined by x-ray crys- tallography (54, 55). These findings are consistent with the hy- pothesis that in addition to cytoskeletal-mediated movements, pro- teins 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 elec- tron 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 FIGURE 6. The size of the synaptic cleft at the inhibitory and activating NK cell IS. Inhibitory and activating conjugates between 221 transfectants not yet understood. and YTS/KIR2DL1 cells were processed for electron microscopy and the distance across the synaptic cleft was measured. a, Negative staining of Acknowledgments intercellular contacts between NK cells and 221 transfectants reveals how We are grateful to B. Askonas, K. Suhling, L. Bugeon, S. B. Taner, and K. the distance across the synaptic cleft varies along the length of the IS. b, Yanagi for critical reading of this manuscript. Immunogold labeling of HLA-Cw6-GFP at the inhibitory NK cell IS dem- onstrates 10-nm gold beads clustering at regions along the IS. All scale bars represent 0.5 ␮m. The distance across the synaptic cleft was measured References along (c) the activating and (d) inhibitory NK cell IS and the percentage of 1. Trinchieri, G. 1989. Biology of natural killer cells. Adv. Immunol. 47:187. 2. Ljunggren, H. G., and K. Karre. 1990. In search of the “missing self”: MHC measurements occurring within 5-nm intervals between 0 and 55 nm are molecules and NK cell recognition. Immunol. Today 11:237. shown. e, Following localization of HLA-Cw6-GFP at the inhibitory IS by 3. Long, E. O. 1999. Regulation of immune responses through inhibitory receptors. immunogold labeling, the distance across the synaptic cleft at each gold Annu. Rev. Immunol. 17:875. particle was measured and the percentage occurrence of distances between 4. Long, E. O., and S. Rajagopalan. 2000. HLA class I recognition by killer cell Ig-like receptors. Semin. Immunol. 12:101. 0 and 55 nm in 5-nm intervals is shown. Data was obtained from over 100 5. Moretta, A., S. Sivori, M. Vitale, D. Pende, L. Morelli, R. Augugliaro, C. Bottino, measurements across 20 synapses in each of c and d, and 30 measurements and L. Moretta. 1995. Existence of both inhibitory (p58) and activatory (p50) re- across 3 synapses in e. ceptors for HLA-C molecules in human natural killer cells. J. Exp. Med. 182:875. The Journal of Immunology 2869

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