Immune Synapse 2B4/SAP Complex at the Cytotoxic NK Cell Dynamic

Dynamic Redistribution of the Activating 2B4/SAP Complex at the Cytotoxic NK Cell Immune Synapse

This information is current as Pedro Roda-Navarro, María Mittelbrunn, Mara Ortega, of September 26, 2021. Duncan Howie, Cox Terhorst, Francisco Sánchez-Madrid and Elena Fernández-Ruiz J Immunol 2004; 173:3640-3646; ; doi: 10.4049/jimmunol.173.6.3640

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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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Dynamic Redistribution of the Activating 2B4/SAP Complex at the Cytotoxic NK Cell Immune Synapse1

Pedro Roda-Navarro,* Marõ«a Mittelbrunn,† Mara Ortega,* Duncan Howie,‡ Cox Terhorst,‡ Francisco Sa«nchez-Madrid,† and Elena Ferna«ndez-Ruiz2*

The 2B4 molecule (CD244) has been described as a coreceptor in human NK cell activation. However, the behavior of 2B4 during the cytotoxic NK cell immune synapse (NK-IS) formation remains undetermined. In this study, we demonstrate the redistribution of 2B4 and the signaling adaptor molecule, signaling lymphocyte activation molecule-associated protein (SAP), to the cytotoxic NK-IS upon formation of conjugates between resting NK cells and EBV-infected 721.221 human cells. Confocal microscopy showed that 2B4 localized at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing talin within NK cell and ICAM-1 on target cells. Videomicroscopy studies with 2B4-GFP-transfected NK cells revealed that 2B4 redistributed to cytotoxic NK-IS as soon as the cell contact occurred. Simultaneously, a SAP-GFP also clustered Downloaded from at the contact site, where it remained during the interaction period. The 2B4 molecular clusters remained bound to the target cell even after NK cell detachment. These results underscore the function of 2B4 as an adhesion molecule and suggest a relevant role in the initial binding, scanning of target cells, and formation of cytotoxic NK-IS. Finally, these findings are indicative of an important role of the activating 2B4/signaling lymphocyte activation molecule-associated protein complex during the recognition of EBV-infected cells. The Journal of Immunology, 2004, 173: 3640Ð3646. http://www.jimmunol.org/ atural killer cell-mediated cytotoxicity is initiated by The glycoprotein 2B4 (3, 4), a member of the SLAM family of specific interaction with virus-infected or neoplastic tar- receptors (5), is expressed on NK cells, monocytes, basophils, N get cells. The cytolytic activity is tightly regulated by TCR␥␦ϩ T cells, and a subset of CD8ϩ T cells (6). Engagement of dominant inhibitory receptors for MHC class I molecules (1). 2B4 leads to IFN-␥ secretion, granule exocytosis, and stimulation Down-regulation of MHC class I on target cells during viral of lytic activity by NK cells (7). The ligand of 2B4 is CD48, a infection or tumor transformation enables the engagement of GPI-anchored membrane protein also belonging to the SLAM activating receptors triggering cytotoxicity and cytokine pro- family (5, 8). An activating role in NK cell-mediated lysis has been duction. A wide repertoire of NK cell-activating receptors has demonstrated for 2B4-CD48 interaction (9). Following cross-link- by guest on September 26, 2021 been identified including the natural cytotoxicity receptors, ing with specific mAb, 2B4 becomes tyrosine phosphorylated (10). NKp46, NKp30, and NKp44; the Fc receptor CD16; and In addition, pervanadate treatment also induces phosphorylation of NKG2D. Moreover, 2B4 (CD244), NTB-A (Ly-108; signaling 2B4 and recruits SLAM-associated protein (SAP) (11, 12). SAP is 3 lymphocyte activation molecule F7 (SLAMF7)), and NKp80 a signaling adaptor molecule encoded by the SH2D1A gene, which have been described as coreceptors, as their contribution to NK is mutated in patients with the inherited immunodeficiency cell-mediated cytolysis depends on natural cytotoxicity receptor X-linked lymphoproliferative disease. X-linked lymphoprolifera- expression and function (2). tive disease patients suffer from dysgammaglobulemia and abnor- mal immune responses to EBV infection (5, 13). Due to its high affinity interactions with the cytoplasmic tail of 2B4, it has been *Unidad de Biologõ«a Molecular and †Servicio de Inmunologõ«a, Hospital Universitario hypothesized that SAP could prevent binding of Src homology de la Princesa, Universidad Auto«noma de Madrid, Madrid, Spain; and ‡Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Bos- domain-containing phosphatase-2 (SHP-2) (11, 12, 14, 15). This ton, MA 02114 possible role of SAP seems to be critical for 2B4 triggering acti- Received for publication March 5, 2004. Accepted for publication July 8, 2004. vation (16). Interestingly, SHP-1 has also been shown to bind The costs of publication of this article were defrayed in part by the payment of page phosphorylated 2B4 (17). In addition, SAP is also an adaptor mol- charges. This article must therefore be hereby marked advertisement in accordance ecule that is prerequisite for binding of the src kinase Fyn to with 18 U.S.C. Section 1734 solely to indicate this fact. SLAM receptors (18, 19). A second adaptor molecule involved in 1 This work was supported by Fondo de Investigaciones Sanitarias 02/0520 from Ministerio de Sanidad y Consumo and Comunidad Auto«noma de Madrid CAM 8.3/ 2B4-mediated activation is the linker for activation of T cells 003/2001 to E.F.-R., and BMC-2002 00563 from Ministerio de Ciencia y Tecnologõ«a (LAT) (20). and Ayuda a la Investigacio«n Ba«sica Juan March 2002 to F.S.-M. P.R.-N. was initially Upon NK cell-target cell interaction, a highly organized com- recipient of a fellowship from Comunidad Autonoma de Madrid and later funded by Fondo de Investigaciones Sanitarias 02/0520. plex of molecules is formed at the contact area of both cells, 2 Address correspondence and reprint requests to Dr. Elena Ferna«ndez-Ruiz, Unidad termed the NK cell immune synapse (NK-IS). The IS was first de Biologõ«a Molecular, Hospital Universitario de la Princesa, Diego de Leo«n 62, described on the interface of T cell-APC interaction and comprises 28006 Madrid, Spain. E-mail address: [email protected] a central supramolecular activation cluster (cSMAC) of receptor- 3 Abbreviations used in this paper: SLAM, signaling lymphocyte activation molecule; ligand pairs and signaling proteins, surrounded by a peripheral cSMAC, central supramolecular activation cluster; DIC, differential interference contrast; EGFP, enhanced GFP; FN, fibronectin; h, human; IS, immune synapse; LAT, region (pSMAC) containing LFA-1 and talin (21). The NK-IS dis- linker for activation of T cells; MTOC, microtubule-organizing center; PLL, poly(L- played two distinct molecular distributions dependent upon the lysine); pSMAC, peripheral supramolecular activation cluster; PYK, proline-rich tyrosine kinase 2; SAP, SLAM-associated protein; SHP, Src homology domain-con- nature of the interaction, i.e., cytolytic or noncytolytic. Further- taining phosphatase. more, specific killer cell Ig-like receptors induce clustering of

HLA-C at the contacting surface of resistant target cells forming Conjugate formation and immunofluorescence microscopy the so-called inhibitory NK-IS (22). In contrast, when NK cells Resting NK cells and 721.221 cell line were mixed at an E:T cell ratio interact with susceptible target cells, the redistribution of signaling of 2:1 and spun at 2500 rpm for 3 min at room temperature. After molecules at the cSMAC, as well as LFA-1 and talin at the pS- incubation at 37¡C for 5 min (or 2 min in the case of conjugates stained MAC resembles the T cell-APC IS organization (23). A differen- with LAT), cells were gently resuspended and added to PLL-coated tial distribution of actin, ezrin, CD43, and CD45 at activating and slides, briefly centrifuged, and fixed in 2% paraformaldehyde. In time course experiments, cells were mixed, spun 10 s at 2500 rpm, and added inhibitory NK-IS has also been described (24). In contrast to the to PLL-coated slides for 30 s or 5 min, briefly centrifuged, gently re- inhibitory receptors, how activating receptors redistribute at the suspended, and fixed with 2% paraformaldehyde. For immunofluores- interface of NK/target cells remains ill understood, as only polar- cence assays, samples were permeabilized with 0.1% Triton X-100 for ization of NKG2D and CD2 to the contact area of NK cell-target 5 min (SAP, LAT, and microtubule-organizing center (MTOC)) or as previously described for PYK-2 (28) and blocked with TNB (0.1 M cell conjugates has been recently described (25, 26). Tris-HCl, 0.15 M NaCl, 0.5% blocking reagent; Boehringer Mannheim, In this study, we report the results of studies of the dynamic Mannheim, Germany). FcR were blocked with human ␥-globulin (100 behavior of 2B4 molecule during the resting NK cell interaction ␮g/ml; Sigma-Aldrich). After staining with primary mAb, followed by with the susceptible EBV-infected 721.221 human B cell line. Our an Alexa 488-labeled specific secondary Ab, samples were examined data demonstrate the dynamic clustering of 2B4 and its associated with a DMR photomicroscope (Leica, Mannheim, Germany) using the Leica QFISH 1.0 software. For doubled staining, cells were incubated molecule SAP at the NK-IS, which would explain the relevant role with pp35 mAb, followed by a goat anti-mouse Rhodamine Red X, of this molecular complex during the NK cell-mediated control of saturated with mouse serum, and incubated with a biotinylated ICAM-1, EBV infection. followed by streptavidin-Alexa Fluor 488, or with talin or LAT rabbit

anti-human polyclonal Ab, followed by a goat anti-rabbit Alexa 488. Downloaded from Series of optical sections were obtained with a Leica TCS-SP confocal Materials and Methods scaning laser microscope. Three-dimensional reconstructions of confo- ␮ Cells, Abs, and reagents cal sections (distanced 0.3 m in the vertical axis) were assembled using the Leica confocal software. For 2B4/SAP double staining, cells The EBV-infected B cell line 721.221 was cultured in RPMI 1640 medium were incubated with a goat anti-h2B4 Ab, followed by donkey anti-goat with Glutamax-1 (Invitrogen Life Technologies, Grand Island, NY), sup- Rhodamine Red X, saturated with goat serum, and incubated with plemented with 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 10% 10C4.2 anti-hSAP mAb, followed by a goat anti-mouse Alexa 488. FCS (complete medium). PBMC were purified from healthy donors by http://www.jimmunol.org/ Ficoll density centrifugation (Histopaque-1077; Sigma-Aldrich, St. Louis, Live-cell time-lapse fluorescence confocal microscopy MO). After depleting monocytes by plastic adhesion, resting NK cells were The 721.221 cells resuspended in HBSS with 2% FCS were allowed to purified by negative selection with immunomagnetic beads (Dynal Biotech, adhere on FN-coated coverslips. The 2B4-GFP- or SAP-GFP-transfected Oslo, Norway) coupled to anti-CD3, anti-HLA-DR, and anti-CD4 mAbs. NK cells were resuspended in HBSS with 2% FCS and added to the cham- After a second round of selection with immunomagnetic beads coupled to ber. Cells were maintained at 37¡Cin5%CO2, and confocal images were CD3 and CD19 mAbs, the obtained cell population, analyzed by flow cy- acquired using the Leica TCS-SP confocal microscope. Confocal series of Ͼ ϩ Ϫ tometry, was 97% CD56 , CD3 . fluorescence and the differential interference contrast (DIC) images were T3b (anti-CD3), HP2/6 (anti-CD4), DR (anti-HLA-DR), MEM111/B10 simultaneously obtained. The maximal projection of the most representa- (anti-ICAM-1), and D3/9 (anti-CD45) mAbs have been previously de- tive sections of the green channel (GFP signal) and the corresponding DIC scribed (27); pp35 (anti-2B4), BAB281 (anti-NKp46), and MA152 (anti- images was overlaid in a single image. by guest on September 26, 2021 NKp80) mAbs were kindly provided by A. Moretta (Universita`degli Studi di Genova, Genova, Italy). Polyclonal anti-human SAP (hSAP) directed at the C-terminal peptide CQGTTGIREDPDV (13) and 10C4.2 anti-hSAP Results mAb directed at the same peptide were previously described (15). Goat Subcellular distribution of 2B4 and SAP in resting peripheral anti-h2B4 Ab was obtained from R&D Systems (Minneapolis, MN), and blood NK cells mouse anti-hCD48 mAb from Neomarkers (Fremont, CA). Anti-talin poly- Resting NK cells isolated from peripheral blood were phenotypi- clonal Ab was kindly provided by P. Sa«nchez-Mateos (Hospital Gregorio Ϫ Ϫ Maranõn, Madrid, Spain). Anti-proline-rich tyrosine kinase 2 (PYK-2) and cally characterized by flow cytometry as CD3 , HLA-DR , anti-␣-tubulin were purchased from Santa Cruz Biotechnology (Santa CD45ϩ, CD56ϩ, NKp46ϩ, NKp80ϩ, and 2B4ϩ (Fig. 1A). To de- Cruz, CA); secondary Alexa 488, Rhodamine Red X-labeled mAbs, and termine the subcellular distribution of 2B4 in these cells, immu- streptavidin from Molecular Probes (Eugene OR); and poly(L-lysine) (PLL) and fibronectin (FN) from Sigma-Aldrich. nofluorescence analyses were performed with NK cells adhered to FN or PLL. The 2B4 was localized in patches evenly distributed GFP constructs and transient transfections throughout the surface of the cell bound to FN, whereas other polarization markers as ICAM-3 were localized at the trailing edge HindIII-2B4-BamHI fragment was obtained by RT-PCR of human NK cell of the cell (29). Cells attached to PLL showed a homogeneous total RNA, cloned in pEGFP (enhanced GFP)-N1 vector (BD Clontech, Palo Alto, CA), and sequenced to verify frame and sequence fidelity. To distribution of 2B4 and ICAM-3 (Fig. 1B). Likewise, time-lapse test the correct membrane expression of the 2B4-GFP, the Jurkat T cell line confocal videomicroscopy showed an even redistribution of 2B4- transfected with the plasmid was stained with pp35 mAb (data not shown). GFP transfected in resting NK cells, when they were migrating on EcoRI-SAP-BamHI PCR fragment was cloned in pEGFP-N3 (BD Clon- FN (Fig. 1C). Expression and subcellular distribution of SAP in tech) and sequenced. Expression of SAP-EGFP chimeric protein was as- NK cells were analyzed by Western blotting and immunofluores- sessed by transfection of 293 cells with the SAP-EGFP plasmid and staining with anti-hSAP 10C4.2 mAb (data not shown). Resting NK cells were cence experiments by using 10C4.2 anti-hSAP mAb (Fig. 1, D and transfected with the T cell Nucleofection solution (Amaxa Biosystems, E). SAP showed a similar cytoplasmic pattern on NK cells plated Koeln, Germany) using the conditions recommended by the manufacturer. onto FN or PLL.

Immunoprecipitation and Western blotting The 2B4 and SAP localized at the contact area of resting NK cells during specific recognition of susceptible target cells Immunoprecipitation experiments were performed with Jurkat T cell lysates and the 10C4.2 anti-hSAP mAb or Ig isotype control. SAP peptide To assess whether 2B4 was translocated to the contact area (200 ␮g/ml) was used to elute bound proteins. Eluates were separated on during the specific recognition of susceptible target cells, we a4Ð15% SDS-polyacrylamide gel, followed by Western bloting with anti- determined the cellular localization of 2B4 in NK-target cell hSAP polyclonal Ab. NK cells were lysed in 0.1% Nonidet P-40, and lysates were resolved in conjugates. The EBV-infected B cell line 721.221 was used as 12% polyacrylamide gels transferred to nitrocellulose membranes and an- susceptible target, because these cells lack MHC class I mole- alyzed by Western blot with the anti-hSAP 10C4.2 mAb. cules and are therefore efficiently killed by NK cells. Moreover, 3642 THE 2B4/SAP COMPLEX AT NK-IS Downloaded from

FIGURE 1. Resting human NK cells express 2B4 and SAP. A, FACS analysis of purified NK cell population using anti-CD3, HLA-DR, CD45, CD56, NKp46, 2B4, and NKp80. CTRg and CTRr indicate the isotype control in FL1 and FL2, respectively. NK cell surface molecules were expressed in Ͼ97% of cells. B, Immunofluorescence analysis of 2B4 and ICAM-3 expressed on the surface of resting NK cells plated on PLL or FN. C, 2B4-GFP-transfected human primary NK cells migrating on FN were tracked by live-cell time-lapse confocal microscopy. Each time point shows the DIC image overlaid with http://www.jimmunol.org/ the fluorescence images of NK cells. D, Immunoprecipitation and Western blotting experiments were done with lysates isolated from Jurkat cells using anti-hSAP 10C4.2 mAb and a polyclonal anti-hSAP Ab, respectively (left). Western blotting experiments were done with NK cell lysates using 10C4.2 mAb (right). IgG CTR: isotype control. Numbers in the left of each panel indicate the molecular mass markers (kDa). E, Subcellular distribution of SAP in resting NK cells plated on FN or PLL. CTR: negative control, secondary Ab alone. these cells express high levels of CD48, the cellular ligand of adaptor molecule SAP, which is known to bind to 2B4 (11), was 2B4. When NK-721.221 cell conjugates were stained with the also localized at cell-to-cell contact areas (Fig. 2, A and B). As anti-2B4 mAb, the receptor was clustered at cell-to-cell contact controls, the signaling molecule PYK-2, as well as the MTOC by guest on September 26, 2021 area (Fig. 2, A and B). The adhesion molecule ICAM-1 ex- and talin (data not shown), were also localized at NK cell-target pressed on target cells was also accumulated at the interface of cell contact areas, as reported for activated NK cells (Fig. 2A, both cells (Fig. 2A), whereas the phosphatase CD45 was ho- see arrowheads) (28). LAT was homogeneously distributed on mogeneously distributed (Fig. 2, A and B), as previously de- the plasma membrane, but a patch was observed at the central scribed (24). CD48 on the surface of the target cells was also zone of NK cell-target cell contact, colocalizing with the 2B4 localized at NK-IS (Fig. 2C, see arrowheads). The signaling cluster (Fig. 2A). SAP also colocalized with 2B4 at the NK

FIGURE 2. The 2B4 and SAP localize at the NK-IS. A, NK-721.221 cell conjugates were allowed to adhere to PLL, fixed and stained for 2B4, ICAM-1, CD45, SAP, PYK-2, ␣-tubulin (MTOC), and LAT/2B4 or SAP/2B4. DIC images are shown. The localization of the MTOC is indicated by arrowheads. B, Quantification (%) of cell conjugates in which 2B4, SAP, or CD45 was localized at the NK cell-721.221 cell contact area. More than 100 conjugates were analyzed in three independent experiments. Histogram bars represent the arithmetic mean Ϯ SD. C, Expression of CD48 in NK cell-target cell conjugates. Accumulation of CD48 at different NK-721.221 cell synapses is indicated by arrowheads. D, Comparison of NK cell-target cell conjugate formation at two time points. Immunofluorescence and quantification (%) of 2B4 and MTOC clustering at 30 s and 5 min after conjugate formation. One hundred conjugates from three independent experiments were analyzed. Histogram bars represent the arithmetic mean Ϯ SD. NK cells are identified by their smaller size. The Journal of Immunology 3643 cell-target cell contact (Fig. 2A). These data demonstrate that Dynamic redistribution of 2B4 and SAP 2B4 and SAP are localized to the cytotoxic NK-IS and support To study the dynamic distribution of these molecules during the the relevance of 2B4-triggered signaling during the control of resting NK cell-target cell interaction, NK cells were transiently EBV infection by NK cells. transfected with 2B4- or SAP-GFP constructs. The localization of Kinetic of 2B4 redistribution was analyzed in comparison with these molecules was tracked by live-cell time-lapse fluorescence Ϯ that of MTOC. The 2B4 accumulated at NK-IS in 48% 0.3 of confocal microscopy (Figs. 4 and 5). The 2B4 clusters appeared at the Ϯ 30-s conjugates and 62% 3.2 of 5-min conjugates. By contrast, cell-to-cell contact as early as 30 s after the interaction between NK Ϯ MTOC was concentrated in only 10% 1.5 of 30-s conjugates cells and target cells (Fig. 4A, see arrowhead and Supplemental Movie Ϯ and 54% 1.2 of 5-min conjugates (Fig. 2D). These data suggest 1).4 The 2B4 clusters became well established after 3 min (Fig. 4A). that 2B4 clustering occurs very rapidly after the initial cell-to-cell When NK cells simultaneously contacted with two susceptible target contact and that this molecule could be involved in early adhesive cells, 2B4 was clustered at both sites of cell-to-cell interactions as events. soon as the contact occurred (Fig. 4B; 0.30, 2.30, and 7.00 min; see Segregation of 2B4 at the cSMAC arrowheads and Supplemental Movie 2). Interestingly, the cluster of 2B4 was stable during the whole interaction period (Fig. 4B; 20.30 To topographically map the 2B4 localization within the NK-IS, min). Likewise, SAP-GFP also clustered at the contact site, and these NK-721.221 cell conjugates were also analyzed by confocal mi- clusters were maintained during the whole interaction, similar to that croscopy. As shown in Fig. 3A, clusters of 2B4 were localized at observed with 2B4-GFP (Fig. 4C and Supplemental Movie 3). In the central zone of the contact area. Next, conjugates were double addition, the staining of SAP-GFP-transfected NK-target cell conju- stained with 2B4 and talin, a component of the cortical cytoskel- gates with 2B4 revealed a clear-cut colocalization of this molecule Downloaded from eton localized at the peripheral zone of NK-IS (23). A ring of talin with SAP-GFP at the contact zone of cell conjugates, as it was at the pSMAC surrounded 2B4 clusters at the cSMAC. Likewise, observed by confocal microscopy (Fig. 4D). when conjugates were double stained with 2B4 and ICAM-1, the Remarkably, the interaction of NK cells with 721.221 cells was cellular ligand of LFA-1 expressed on target cells, ICAM-1 was associated with the formation in NK cells of very thin and elon- also detected at the pSMAC surrounding 2B4 clusters (Fig. 3B). gated cytoplasmic protrusive structures, which connect both cells. The 2B4 distribution pattern was observed in 70% of a total of 20 This connective structure was formed and maintained after NK cell http://www.jimmunol.org/ conjugates scored from three different donors showing a peripheral detachment (Fig. 5). A very high concentration of 2B4 and SAP localization of talin. Finally, NK-target cell conjugates were was detected in this structure (Fig. 5, see arrowheads and Supple- stained with both LAT and 2B4, and a partial colocalization of 2B4 mental Movies 4 and 5). When connective structures were broken, clusters with LAT at the NK-IS was found (Fig. 3C, see arrow- a portion of 2B4-containing membrane remained attached to the head). These data show that, like other costimulatory receptors target cells (Fig. 5A; 7.45 min). The significance of this phenom- such as CD2 or CD28 in T cell-specific IS, the activating core- enon is currently unclear, but it could be due to the high affinity of ceptor 2B4 is localized at the cSMAC of cytotoxic NK-IS, and that 2B4 with its ligand CD48 (8, 9). the colocalization of 2B4 and LAT only occurs at the interface of These data show the formation of 2B4- and SAP-containing by guest on September 26, 2021 resting NK cells and 721.221 target cells. connective structures, the dynamic redistribution of 2B4 and SAP, and the association of these molecules at NK-IS throughout the interaction between resting NK cells and EBV-infected cells.

Discussion In this study, we report the subcellular localization of activating coreceptor 2B4 and signaling adaptor molecule SAP during the recognition of susceptible EBV-infected 721.221 cells by human NK cells. In this model, a cytotoxic NK-IS was established due to the absence of MHC class I molecules on target cells. We have analyzed this NK-IS by confocal microscopy and live-cell time- lapse fluorescence confocal microscopy. First, 2B4 molecule was shown to be homogeneously distributed on the surface of resting peripheral blood NK cells adhered to FN and PLL, but localized at the cytotoxic NK-IS when conjugates between NK cells and target cells were formed, which suggest that 2B4 is translocated to the NK-IS after cell contact occurs. In these experiments, we showed how SAP and PYK-2 NK cell molecules and ICAM-1 target cell molecule also localized at the NK-IS. Previous studies have demonstrated accumulation of ICAM-1 at the contact site of APCs in T cell IS (30). Moreover, the interaction of ICAM-1 with LFA-1 expressed on resting NK cells mediates the adhesion to target cells (31). The localization of LFA-1 and talin at the pSMAC and mul- FIGURE 3. The 2B4 segregates at the cSMAC of the NK-IS. timolecular signaling complex at the cSMAC of the cytotoxic NK-721.221 cell conjugates were allowed to adhere to PLL, fixed and NK-IS has recently been reported (23). Our data show the local- stained for 2B4 (red) (A), and double stained for 2B4 (red) and talin, ization of the activating coreceptor 2B4 at the cSMAC and the ICAM-1, and LAT (green) (B and C). One representative confocal section, DIC images, and three-dimensional reconstructions (A and B) or three- dimensional high magnification (B, right panels) are shown. The colocalization of 2B4 and LAT is shown by an arrowhead. 4 The on-line version of this article contains supplemental material. 3644 THE 2B4/SAP COMPLEX AT NK-IS Downloaded from http://www.jimmunol.org/

FIGURE 4. Dynamic redistribution of 2B4 and SAP at the NK-IS. The 721.221 target cells were seeded onto FN-coated coverslips, and then resting NK cells transiently transfected with 2B4-GFP (A and B) or SAP-GFP (C) were added to the chamber. Cells were monitored by time-lapse confocal microscopy at 30-s intervals, and representative images are shown. Each time point shows the DIC image overlaid with the fluorescence images of NK cells. D, Conjugates were formed with SAP-GFP-transfected NK cells and target cells, adhered to FN, and fixed and stained with 2B4. Confocal sections, DIC, and merged images are shown. Clustering of different GFP and colocalization of 2B4 and SAP-GFP are shown by arrowheads. by guest on September 26, 2021 adhesion molecule ICAM-1, expressed on target cells, at the pS- T cells and CTLs, which consists of localization of receptors in- MAC. These data suggest that the rearrangement of surface pro- volved in activating signaling pathways in a cSMAC, surrounded teins at the cytotoxic NK-IS resembles the one described for CD4ϩ by a pSMAC containing the pair LFA-1/ICAM-1 and talin (21, 32). It has been previously demonstrated that phosphorylated 2B4 is localized in lipid rafts upon activation of NK cells by specific mAbs or CD48ϩ target cells (33), and that lipid rafts are accumulated at cytotoxic NK-IS (34). In contrast, it has been described that LAT is constitutively associated with rafts in T lymphocytes (35), and with 2B4 in the NK cell line YT (7). As we found that a fraction of LAT and 2B4 molecules colocalized at cytotoxic NK-IS, we can suggest that 2B4 and LAT are in part associated within lipid rafts concentrated at the NK-IS. Accordingly, the localization of mouse 2B4 in lipid rafts was shown to be required for the association of this molecule with LAT (36). In addition, our data show that a fraction of LAT is not translocated to NK-IS and does not colocalize with 2B4. The dynamic redistribution of activating receptors or signaling molecules during the NK cell-target cell interaction has not been described to date. Then we developed chimeric 2B4- and SAP- GFP and transfected resting NK cells to study this issue by time- FIGURE 5. Formation of connective structures during the interaction of lapse videomicroscopy. Our data show a stable clustering of 2B4 resting NK cells and target cells. The 721.221 target cells were seeded onto and SAP, and demonstrate that 2B4 clusters can be simultaneously FN-coated coverslips, and then resting NK cells transiently transfected formed when one NK cell interacts with two target cells. Simul- with 2B4-GFP (A) or SAP-GFP (B) were added to the chamber. Cells were monitored by time-lapse confocal microscopy at 30-s intervals, and repre- taneous clusters have also been described for talin, but not for the sentative images are shown. Each time point shows the DIC image overlaid signaling molecules SLP-76 and protein kinase C-␪, which were with the fluorescence images of NK cells. The accumulation of GFP at the polarized in the NK cell toward only one of the two targets (23). connective structure is indicated by arrowheads. Likewise, ICAM-1 clusters were detected at the contact site of both The Journal of Immunology 3645

APCs in T cell-APC triple-cell conjugates (30). Kinetic and dynamic binding of 2B4 to CD48 could participate in a variety of cell-to- studies show that 2B4 clusters occur very rapidly, which suggests that cell interactions that are important for immune responses. it could be involved in the initial binding of resting NK cells to target cells, before the killing process. In this regard, it has been described Acknowledgments recently that coexpression of CD48 with ICAM-1 on insect cells en- We thank Dr. A. Moretta for kindly providing mAbs used in this study, and hances the adhesion of resting NK cells (31). In addition, the signaling Dr. Manuel Lo«pez-Cabrera for critical reading of the manuscript. adaptor molecule SAP has been suggested to prevent the binding of SHP-2 to the cytoplasmic tail of 2B4 (11). Moreover, it is conceivable References 1. Ravetch, J. V., and L. L. Lanier. 2000. Immune inhibitory receptors. Science that SAP might couple the src kinase fyn to 2B4, as it has been de- 290:84. scribed for SLAM (18, 19). Our data show the association of SAP 2. Biassoni, R., C. Cantoni, D. Pende, S. Sivori, S. Parolini, M. Vitale, C. Bottino, with 2B4 at cytotoxic NK-IS and strongly suggest that the perma- and A. Moretta. 2001. Human natural killer cell receptors and co-receptors. Im- munol. Rev. 181:203. nency of activating 2B4/SAP complex is necessary to block the re- 3. Valiante, N. M., and G. Trinchieri. 1993. Identiﬁcation of a novel signal trans- cruitment of phosphatases during the interaction between resting NK duction surface molecule on human cytotoxic lymphocytes. J. Exp. Med. 178:1397. cells and EBV-infected cells. In this regard, the stable localization of 4. Garni-Wagner, B. A., A. Purohit, P. A. Mathew, M. Bennett, and V. Kumar. SAP at NK-IS during the interaction period would prevent the block- 1993. A novel function-associated molecule related to non-MHC-restricted cy- ade of lipid raft polarization to NK-IS, exerted by SHP-1 associated to totoxicity mediated by activated natural killer cells and T cells. J. Immunol. 151:60. inhibitory killer cell Ig-like receptors (34). Then this fact would per- 5. Engel, P., M. J. Eck, and C. Terhorst. 2003. The SAP and SLAM families in mit the stable assembly of the multimolecular signaling complex as- immune responses and X-linked lymphoproliferative disease. Nat. Rev. Immunol. 3:813. Downloaded from sociated to glycolipid-enriched microdomains at the cSMAC (37). 6. Boles, K. S., S. E. Stepp, M. Bennett, V. Kumar, and P. A. Mathew. 2001. 2B4 This signaling complex includes PTKs as Lck, Fyn, and the trans- (CD244) and CS1: novel members of the CD2 subset of the immunoglobulin membrane LAT. Finally, these data would also explain the exclusion superfamily molecules expressed on natural killer cells and other leukocytes. Immunol. Rev. 181:234. of phosphatases, as SHP-1 from the NK-IS very early after cytotoxic 7. Chuang, S. S., P. R. Kumaresan, and P. A. Mathew. 2001. 2B4 (CD244)-medi- interactions is formed (38). ated activation of cytotoxicity and IFN-␥ release in human NK cells involves distinct pathways. J. Immunol. 167:6210. We found a very thin membrane structure, called connective 8. Brown, M. H., K. Boles, P. A. van der Merwe, V. Kumar, P. A. Mathew, and http://www.jimmunol.org/ structure, associated to the interaction formed between resting NK A. N. Barclay. 1998. 2B4, the natural killer and T cell immunoglobulin super- cells and 721.221 target cells. This structure is formed in unstable family surface protein, is a ligand for CD48. J. Exp. Med. 188:2083. 9. Nakajima, H., M. Cella, H. Langen, A. Friedlein, and M. Colonna. 1999. Acti- conjugates when NK cells are detached from target cells. Similar vating interactions in human NK cell recognition: the role of 2B4-CD48. Eur. unstable conjugates and connective structures have been previously J. Immunol. 29:1676. 10. Watzl, C., C. C. Stebbins, and E. O. Long. 2000. NK cell inhibitory receptors reported in cytotoxic contacts formed between mouse NK cells and prevent tyrosine phosphorylation of the activation receptor 2B4 (CD244). J. Im- target cells, where they have been proposed to be involved in inducing munol. 165:3545. physical damage to susceptible targets during NK cell-mediated 11. Tangye, S. G., S. Lazetic, E. Woollatt, G. R. Sutherland, L. L. Lanier, and J. H. Phillips. 1999. Human 2B4, an activating NK cell receptor, recruits the cytotoxicity (39). Interestingly, we ﬁnd a high concentration of 2B4 protein tyrosine phosphatase SHP-2 and the adaptor signalling protein SAP.

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nological synapse of CTL contains a secretory domain and membrane bridges. ICAM-3 in the initial interaction of T lymphocytes and APCs. Nat. Immunol. http://www.jimmunol.org/ Immunity 15:751. 3:159. by guest on September 26, 2021