Cell Cytotoxicity LFA-1 Contributes an Early Signal for NK

LFA-1 Contributes an Early Signal for NK Cell Cytotoxicity Domingo F. Barber, Mathias Faure and Eric O. Long This information is current as J Immunol 2004; 173:3653-3659; ; of September 27, 2021. doi: 10.4049/jimmunol.173.6.3653 http://www.jimmunol.org/content/173/6/3653 Downloaded from References This article cites 64 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/173/6/3653.full#ref-list-1

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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

LFA-1 Contributes an Early Signal for NK Cell Cytotoxicity

Domingo F. Barber,1,2 Mathias Faure,1,3 and Eric O. Long4

Cytotoxicity of human NK cells is activated by receptors that bind ligands on target cells, but the relative contribution of the many different activating and inhibitory NK cell receptors is difficult to assess. In this study, we describe an experimental system that ␤ circumvents some of the difficulties. Adhesion through 2 integrin LFA-1 is a common requirement of CTLs and NK cells for efficient lysis of target cells. However, the contribution of LFA-1 to activation signals for NK cell cytotoxicity, besides its role in adhesion, is unclear. The role of LFA-1 was evaluated by exposing NK cells to human ICAM-1 that was either expressed on a Drosophila insect cell line, or directly coupled to beads. Expression of ICAM-1 on insect cells was sufficient to induce lysis by NK cells through LFA-1. Coexpression of peptide-loaded HLA-C with ICAM-1 on insect cells blocked the LFA-1-dependent cytotoxicity of NK cells that expressed HLA-C-specific inhibitory receptors. Polarization of cytotoxic granules in NK cells toward ICAM-1- and ICAM-2-coated beads showed that engagement of LFA-1 alone is sufficient to initiate activation signals in NK cells.

Thus, in contrast to T cells, in which even adhesion through LFA-1 is dependent on signals from other receptors, NK cells receive Downloaded from early activation signals directly through LFA-1. The Journal of Immunology, 2004, 173: 3653Ð3659.

ysis of virus-infected cells and of tumor cells by NK cells or CD18, or after blocking LFA-1 with mAbs, target cell lysis by is controlled by multiple receptor-ligand interactions (1, cytotoxic lymphocytes is impaired (15–21). Ligand engagement by L 2). NK cells express many different activation receptors, LFA-1 induces a complex outside-in signaling cascade in T cells which can contribute to lysis of target cells, including receptors (22–26), but it is not clear whether such signals are required during http://www.jimmunol.org/ that associate with ITAM motif-containing subunits (3, 4); recep- cellular cytotoxicity. tor NKG2D, which signals through a distinct pathway by associ- It has been difficult to evaluate the minimal requirements for NK ation with the small protein DAP10 (1, 5, 6); and a number of other cell cytotoxicity because NK-sensitive target cells express ligands receptors that have costimulatory function, such as CD2 and 2B4 for multiple different NK cell receptors. For example, it is not (CD244) (7, 8). In addition, NK cell cytotoxicity is kept under known whether any one signaling pathway is required to activate control by inhibitory receptors, which recognize MHC class I mol- cytotoxicity, and whether signaling by any one receptor is suffi- ecules on target cells, and deliver an inhibitory signal through cient. High activation thresholds set by inhibitory signals could ITIM in their cytoplasmic tail (9–11). Signals from ITAM-con- mask important activation signals required for target cell lysis. taining receptors (e.g., CD16, CD94/NKG2C, NKp46, NKp30) are Furthermore, it has not been possible to determine whether LFA-1 by guest on September 27, 2021 transmitted through recruitment of tyrosine kinases Syk or ZAP70, is required for adhesion alone or whether it serves a dual role in whereas NKG2D signals through a PI3K-binding motif. However, adhesion and signaling for cytotoxicity. To circumvent the com- lysis of NK-sensitive target cells occurs with Syk/ZAP70 double- plexity inherent to the multiple receptor-ligand interactions be- deficient NK cells, even with target cells that do not express li- tween NK cells and mammalian target cells, we reconstructed a gands of NKG2D (12). These results highlight the possible redun- target cell from an evolutionary distant cell onto which ligands for dancy among NK cell activation receptors and imply that human NK cell receptors could be expressed one at a time. We alternative activation pathways, which are independent of ITAM report in this work that expression of ICAM-1 (CD54), a ligand of and of DAP10 signals, exist in NK cells. LFA-1, on Drosophila insect cells is sufficient to induce lysis by ␤ Strong adhesion to target cells mediated by the 2 integrin human NK cells through LFA-1. In addition, ICAM-1 coupled to ␣ ␤ LFA-1 (a heterodimer of CD11a/CD18, also called L/ 2)isre- beads induced granule polarization in NK cells, implying that quired for lysis by CTL and by NK cells (13, 14). In the absence LFA-1 is sufficient to induce activation signals in NK cells. of LFA-1 engagement, as in CD18-deficient leukocyte adhesion deficiency patients, and in mice with targeted mutations in CD11a Materials and Methods Antibodies Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852 For flow cytometry analysis and cell sorting, the following directly Received for publication April 20, 2004. Accepted for publication July 13, 2004. conjugated mouse mAbs were used: R-PE-conjugated (R-PE) CD11a (clone HI111), R-PE-CD11b (clone ICRF44), R-PE-CD18 (clone 6.7), 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 R-PE-CD54 (clone HA58), CyChrome CD54 (clone HA58), and FITC with 18 U.S.C. Section 1734 solely to indicate this fact. anti-HLA-A,B,C (clone G46-2.6) were all from BD Pharmingen (San 1 Diego, CA). R-PE-CD3 (clone UCHT1), R-PE-CD56 (clone N901), R-PE- D.F.B. and M.F. contributed equally to this work and share joint first authorship. CD158a (clone EB6), and R-PE-CD158b (clone GL183) were from Beck- 2 Current address: Centro Nacional de Biotecnologia, Madrid, Spain 28049. man Coulter (Miami, FL). FITC CD58 (clone TS2/9.1.4.3) was from 3 Current address: U548 Institut National de la Santeét de la Recherche Me´dicale Ancell (Bayport, MN). Isotype-matched PE-, FITC-, or CyChrome-conju- Commissariat a`l’Energie Atomique-Grenoble, 17 rue des Martyrs, 38054 Grenoble, gated mAbs were from BD Pharmingen. For blocking experiments, NK France. cells were incubated with 10 ␮g/ml purified mAbs 30 min before and 4 Address correspondence and reprint requests to Dr. Eric O. Long, Laboratory of during killing experiments. CD2 (clone TS2/18), CD11a (clone TS1/22), Immunogenetics, National Institute of Allergy and Infectious Diseases-National In- and CD18 (clone TS1/18) mAbs were from Pierce (Woburn, MD). The stitutes of Health, 12441 Parklawn Drive, Rockville, MD 20852. E-mail address: CD11b mAb (clone LM2/1) was from BioSource International [email protected] (Camarillo, CA).

Cells ages were processed using the Leica TCS-NT/SP software (version 1.6.587), Imaris 3.0.6 (Bitplane, Zurich, Switzerland), and Adobe Photo- Culture and transfection of Drosophila SC2 cells have been described (27– shop 6 (Adobe Systems, San Jose, CA). Perforin polarization was deter- 29). Expression of transfected human cDNAs was induced for 24–48hin mined visually as the percentage of NK cell-bead conjugates with polarized 1 mM CuSO4, and was monitored by ﬂow cytometry before every exper- perforin of the total number of individual conjugates. iment. HLA-C*0304 on SC2 was loaded with 10 ␮M of the peptide GAVDPLLAL, and HLA-C*0401 with 2 ␮M of the peptide QYD- DAVYKL or peptide QYDDAVYEL during the last 20 h of a 40-h induc- Results

tion period in 1 mM CuSO4 (29). The 721.221 cells were a gift of R. ICAM-1 on insect cells induces cytotoxicity by NK cells DeMars (University of Wisconsin, Madison, WI). Human NK cells were isolated from peripheral blood using the NK cell isolation kit (Miltenyi The cDNA for human ICAM-1 was transfected into Drosophila Biotec, Auburn, CA), expanded in IL-2, and cloned, as described (30). The SC2 cells using an inducible expression system (27, 28). A high A6-TCR CD8ϩ CTL clone RS56, specific for human T cell leukemia vi- expression level of ICAM-1 on SC2 cells was selected by cell rus-1 (HTLV-1),5 Tax11–19, and the HMy2.C1R-A2 (gifts from W. Bid- sorting (Fig. 1). Lysis of SC2 cells after a 3-h incubation with dison, NINCDS, Bethesda, MD) were cultured, as described (31). For in- IL-2-activated NK cells was determined by a flow cytometry assay hibition experiments, cells were preincubated for 40 min at 37°C with inhibitors and assayed in the continuing presence of inhibitors. Inhibitors (33). Very little lysis of untransfected SC2 cells was observed, used were cytochalasin D (20 ␮M), PP1 (30 ␮M), and wortmannin (400 even at high E:T cell ratios (Fig. 2). In contrast, expression of nM), all from BIOMOL (Plymouth Meeting, PA). ICAM-1 on SC2 cells was sufficient to trigger a cytotoxic response by NK cells (Fig. 2). As NK cells express the two ICAM-1 recep- Fc fusion proteins and beads tors LFA-1 and Mac-1 (CD11b/CD18), blocking mAbs were used Human B7.1/Fc, ICAM-1/Fc, and ICAM-2/Fc fusion proteins were pur- to determine the contribution of these two ␤ integrins to lysis of 2 Downloaded from chased from R&D Systems (Minneapolis, MN). Protein A-conjugated 5.6- SC2-ICAM-1 cells. Lysis was inhibited completely by mAbs to ␮m-diameter beads were from Bangs Laboratories (Fishers, IN). A total of ϫ 6 ␮ CD11a and CD18, but not by mAbs to CD11b or to CD2 (Fig. 2b). 10 10 beads was washed twice with H2O and coated with 5 gofFc fusion proteins (at 1 ␮g/ml) for2hat4°Cin400␮l. Beads were then Therefore, recognition of ICAM-1 on SC2 cells by LFA-1 alone ϫ 6 washed three times with H2O and resuspended at 20 10 beads/ml in accounts for the induction of NK cell cytotoxicity. HBSS. Coating was Ͼ98% efficient, as judged by flow cytometry. Signals forLFA-1-dependent cytotoxicity occur downstream of

Binding assays and cytotoxicity assays http://www.jimmunol.org/ adhesion Conjugate formation between NK cells and SC2 cells was determined, as 2ϩ described (32), with the following modifications: SC2 cells were resus- Activated NK cells kill sensitive target cells by a Ca -dependent pended in HBSS medium (BioSource International) ϩ 5% FBS, and 1 ϫ degranulation and release of perforin and granzymes. As expected, 105 effector cells and 4 ϫ 105 target cells were mixed in a 200 ␮l final lysis of HLA class I-deficient human 721.221 cells was inhibited volume. For binding to beads experiments, 1 ϫ 105 NK cells were resus- by EGTA/Mg2ϩ (Fig. 3a). Likewise, lysis of SC2-ICAM-1 cells ϫ 5 ␮ pended with 6 10 beads for 20 min at 37°Cin200 l. Effector cell-bead was abrogated by EGTA/Mg2ϩ (Fig. 3b), consistent with a per- conjugates were fixed with paraformaldehyde and quantified by FACS analysis, according to the side scatter scale. For cytotoxicity assays (33), forin-dependent pathway. Lysis of 721.221 cells was very sensi- 2 ϫ 104 target cells labeled with the green fluorescent cell linker PKH67- tive to inhibition of actin polymerization by cytochalasin D, of Src ␮ GLE (Sigma-Aldrich, St. Louis, MO) in a volume of 100 l were added in family tyrosine kinases by PP1, and was partially sensitive to in- by guest on September 27, 2021 duplicate to 12 ϫ 75-mm round-bottom polystyrene tubes (BD Labware, hibition of PI3K by wortmannin (Fig. 3c). Lysis of SC2-ICAM-1 Franklin Lakes, NJ). Effector cells, in a volume of 100 ␮l, were added to yield different E:T ratios. Effector and target cells were mixed by gently cells showed similar sensitivity to these three inhibitors (Fig. 3d). tapping, centrifuged at 25°C for 4 min at 300 rpm (25 ϫ g), and incubated The similar properties of lysis of SC2-ICAM-1 and 721.221 cells

at 37°Cin5%C02 for 3 h. After incubation, the reaction was stopped by suggest that the LFA-1-dependent activation of NK cells is char- placing the samples for 2 min at 4°C. A total of 10 ␮l of propidium iodide acteristic of natural cytotoxicity. Activated NK cells form tight (PI; BD Pharmingen) was added to each tube 10–15 min before data ac- conjugates with SC2-ICAM-1 cells, as shown with a two-color quisition. Target cells were gated by side scatter and fluorescence (FL-1). PI uptake was determined within the gated cells. The percentage of cells adhesion assay (Fig. 3e). The time course and the extent of con- labeled by PI in the absence of NK cells was subtracted from the number jugate formation were very similar to those observed with a sen- obtained in the presence of NK cells. Insect cells grow at temperatures sitive human target cell (32). The LFA-1-dependent adhesion to below 30°C and undergo a heat shock response at 37°C. However, the duration (3 h) of the lysis assay at 37°C did not cause detectable increase of PI uptake in insect cells alone. Immunostaining and confocal microscopy A total of 1 ϫ 106 NK cells was resuspended with 1 ϫ 106 SC2-ICAM-1, SC2-CD48, or 4 ϫ 106 uncoated or coated beads in HBSS/3% FBS in 12 ϫ 75-mm round-bottom polystyrene tubes (BD Labware), and centrifuged at 4°C for 3 min at 300 rpm (25 ϫ g). Samples were placed at 37°C for 20 min. Cells were allowed to settle on poly(lysine) (Roche Molecular Bio- chemicals, Indianapolis, IN)-coated cover glasses for 1 h, before fixation in PBS/4% paraformaldehyde, and permeabilized with PBS/0.5% Triton X-100. Nonspecific sites were saturated for 30 min with PBS/10% normal donkey serum/0.5% Triton X-100, and cells were stained with 5 ␮g/ml anti-perforin mAb (clone ␦ G9; Pierce), and revealed with a goat anti- mouse Alexa 568 secondary Ab (Molecular Probes, Eugene, OR) in FIGURE 1. Cell surface expression of human ICAM-1 and HLA-Cw4 PBS/3% normal donkey serum/0.5% Triton X-100. After three washes on transfected Drosophila SC2 cells. The single transfectants expressing with PBS, the cells were mounted on slides using the Prolong antifade kit either ICAM-1 or HLA-Cw4, as indicated within each panel, are in the top (Molecular Probes). Images were collected on a Leica TCS-NT/SP confo- panels. The double transfectant expressing both ICAM-1 and HLA-Cw4 is cal microscope (Leica Microsystems, Exton, PA) using a ϫ40 or ϫ100 oil shown in the bottom panels. HLA-Cw4 was loaded with an exogenous immersion objective NA 1.4, zoom 2. Differential interference contrast peptide. Flow cytometry was performed with a PE-conjugated mAb for images were collected simultaneously with the fluorescence images. Im- ICAM-1 (left panels) and FITC-conjugated mAb for HLA-Cw4 (right panels). Dotted lines represent controls with isotype-matched PE- or FITC- 5 Abbreviations used in this paper: HTLV, human T cell leukemia virus; KIR, killer conjugated mAbs. The same background was obtained with the specific cell Ig-like receptor; PI, propidium iodide. mAbs and untransfected SC2 cells (data not shown). The Journal of Immunology 3655

FIGURE 2. ICAM-1 expression in SC2 cells is sufficient to induce lysis by NK cells through LFA-1. a, Specific lysis of SC2 cells (asterisks) and SC2-ICAM-1 cells (diamonds) by IL-2-activated human NK cells. b, Spe- cific lysis of SC2 cells (asterisks), and of SC2-ICAM-1 cells (diamonds) in the presence of 10 ␮g/ml mAbs specific for CD2 (squares), CD11a (cir- cles), CD11b (triangles), and CD18 (inverted triangles). Downloaded from SC2-ICAM-1 cells was fairly resistant to inhibition of actin polymerization, Src family tyrosine kinases, and PI3K (Fig. 3f). Cy- tochalasin D delayed the formation of conjugates, but the inhibitory effect was largely overcome by 20 min (Fig. 3f, and data not shown). These results confirm earlier data on NK cell binding to

solid-phase ICAM-1, which was also resistant to inhibition of actin http://www.jimmunol.org/ polymerization, Src family kinase, and PI3K (28). These results show that signaling for cytotoxicity occurs downstream of adhesion. Formation of conjugates between NK cells and SC2-ICAM-1 cells in the presence of cytochalasin D, which did not result in extensive lysis (see Fig. 3, d and f), suggested that SC2-ICAM-1 FIGURE 3. LFA-1-dependent signals for NK cell cytotoxicity are cells are not simply sensitive to contact with cytotoxic cells or to downstream of adhesion to SC2-ICAM-1 cells. a, Specific lysis of human 721.221 cells in the presence (‚) or absence (ࡗ) of EGTA/Mg2ϩ. b, Spe- cross-linking of ICAM-1. To confirm this point, lysis assays and cific lysis of SC2 cells (asterisks) and SC2-ICAM-1 cells in the presence by guest on September 27, 2021 conjugation assays were performed with a highly lytic CD8 T cell (Œ) or absence (diamonds) of EGTA/Mg2ϩ. Adhesion of NK cells to SC2- line, specific for the Tax11–19 peptide of HTLV-1 and restricted ICAM-1 cells was not inhibited by EGTA/Mg2ϩ (data not shown). c and by HLA-A2 (31). SC2-ICAM-1 cells and SC2 cells expressing d, NK cells preincubated for 40 min with 20 ␮M cytochalasin D, 30 ␮M both ICAM-1 and CD58 (28) were not lysed by the CD8 T cell line PP1, and 400 nM wortmannin were tested for lysis of 721.221 cells (c) and (Fig. 4a), despite ICAM-1-dependent conjugate formation (Fig. SC2-ICAM-1 cells (d). Lysis in the presence of inhibitors is shown relative 4b). Conjugate formation with SC2-ICAM-1-CD58 cells was to lysis without inhibitors. Error bars represent the SD in four independent greater than that with SC2-ICAM-1 cells (Fig. 4b). As expected, experiments. e, Adhesion of NK cells to SC2 cells (asterisks) and SC2- lysis of the HLA-A2 B cell line C1R-A2 was peptide dependent ICAM-1 cells (diamonds) was determined by flow cytometry, and is rep- (Fig. 4a). Addition of specific peptide also induced strong conju- resented as the fraction of NK cells that shifted into two-color conjugates. f, NK cells preincubated for 40 min with 20 ␮M cytochalasin D, 30 ␮M gate formation (Fig. 4b), as expected from a TCR-dependent in- PP1, and 400 nM wortmannin were tested for conjugate formation with side-out signal to LFA-1 (34). We conclude that lysis of SC2- SC2-ICAM-1 cells. Data are represented as percentage of control. Error ICAM-1 cells is not induced simply by contact of ICAM-1 with bars represent the SD in four independent experiments. LFA-1, but requires an LFA-1-dependent signal in the NK cells. ICAM-1 cells (Fig. 5a). The recognition of HLA-Cw4 by a LFA-1-dependent cytotoxicity is sensitive to inhibition by killer CD158a KIR is dependent on the side chain at position 8 in the cell Ig-like receptors (KIR) bound peptide (37). This peptide selectivity in recognition by in- The KIR, CD158a and CD158b, inhibit human NK cells upon hibitory CD158a KIR provided a perfect specificity control. SC2- binding to HLA-C allotypes on human target cells (35, 36). It was ICAM-1 ϩ HLA-Cw4 cells were loaded either with a peptide therefore of interest to test whether NK cell cytotoxicity triggered (QYDDAVYKL) that is compatible with recognition by CD158a by LFA-1 was also sensitive to inhibition by CD158a/b. SC2 cells KIR, or a peptide (QYDDAVYEL) that interferes with binding of ␤ were cotransfected with cDNAs encoding human 2-microglobu- CD158a KIR (37). Both peptides were equally effective in stabi- lin and the H chain of HLA-Cw3 or HLA-Cw4, either without or lizing cell surface HLA-Cw4 (37) (data not shown). Peptide-spe- with ICAM-1 (29). As insect cells do not load MHC class I mol- cific inhibition of lysis was observed with a CD158aϩ NK cell ecules with endogenous peptides (27), HLA-C expression at the clone incubated with SC2-ICAM-1 ϩ HLA-Cw4 (Fig. 5b). There- surface of SC2 cells was stabilized by addition of specific peptides fore, LFA-1-dependent NK cell cytotoxicity is sensitive to inhibi- (29). CD158aϩ (specific for HLA-Cw4) and CD158bϩ (specific tion by ITIM-containing KIR. for HLA-Cw3) NK clones were used to test lysis of SC2 cells coexpressing ICAM-1 and cognate HLA-C. Peptide-loaded HLA- ICAM induces granule polarization in NK cells Cw3 inhibited lysis of SC2-ICAM-1 ϩ HLA-Cw3 cells by a A contribution to the killing of SC2-ICAM-1 cells by receptors CD158bϩ clone (Fig. 5a). Inhibition was specific because the other than LFA-1 on NK cells, upon binding conserved determi- same cells not loaded with peptide were lysed comparably to SC2- nants on insect cells, cannot be ruled out. Therefore, to test 3656 LFA-1 SIGNALS FOR NK CELL CYTOTOXICITY

FIGURE 4. SC2-ICAM-1 cells are not lysed simply by contact with LFA-1. a, Lysis by an HLA-A2-restricted CD8ϩ CTL clone, specific for HTLV-1 Tax11–19, of SC2-ICAM-1 cells (diamonds), SC2-ICAM-1- CD58 cells (triangles), HMy2.C1R-A2 cells without peptide (E), and HMy2.C1R-A2 cells loaded with Tax11–19 peptide (F). b, Conjugate formation, as determined in Fig. 3e, of the HLA-A2-restricted CD8ϩ CTL clone with SC2 cells (asterisks), SC2-ICAM-1 cells (diamonds), SC2- FIGURE 6. Polarization of perforin-containing granules in NK cells to- ICAM-1-CD58 cells (triangles), HMy2.C1R-A2 cells without peptide (E), ward ICAM. NK cells incubated with SC2-CD48 cells (a), with SC2- Downloaded from and HMy2.C1R-A2 cells loaded with Tax11–19 peptide (F). ICAM-1 cells (b and c), with uncoated beads (d), with beads coated with ICAM-1 (e), and beads coated with ICAM-2 (f) were fixed, permeabilized, and stained with an anti-perforin mAb and Alexa 568 secondary Ab. NK whether engagement of LFA-1 alone was sufficient to trigger sig- cells in conjugates (a–c) are marked by a star. nals, we investigated the interaction of NK cells with purified

ICAM-1. Polarization of perforin-containing cytotoxic granules to- http://www.jimmunol.org/ ward sensitive target cells (38, 39) was used as a measure for a late Our results suggest that lysis of SC2-ICAM-1 cells occurs by tar- step in the activation of NK cell cytotoxicity. Perforin-containing geted release of granules by NK cells, and demonstrate that LFA-1 granules polarized toward NK-sensitive SC2-ICAM-1 cells, but binding to ICAM ligands is sufficient to signal for granule polar- not toward NK-resistant SC2-CD48 cells, in a majority of NK cells ization in NK cells. (Fig. 6, a–c, and data not shown). This granule polarization assay was used with beads coated with soluble rICAM. Strong polariza- Discussion tion of granules toward beads coated with ICAM-1 and ICAM-2, Expression of individual ligands of NK cell receptors on insect but not toward uncoated beads, was observed (Fig. 6, d–f). NK cells provides a very useful tool to dissect the requirements for cells bound avidly to ICAM-1- and ICAM-2-coated beads, but did target cell lysis by NK cells. Transfected insect cells have been by guest on September 27, 2021 not bind to uncoated beads nor beads coated with B7.1 (Fig. 7a). used previously to define requirements for T cell activation, and A total of 25–30% of NK cells displayed polarization of granules revealed that ICAM-1 provides unique costimulatory function (40, in the absence of target cells and beads (Fig. 7b), consistent with 41). With this approach we show that ICAM-1 expression on in- another report (39). The frequency of granule polarization did not sect cells is sufficient to induce LFA-1-dependent cytotoxicity by change after incubation with uncoated beads or beads coated with IL-2-activated NK cells. Furthermore, the use of beads coated with B7.1 (Fig. 7b). In contrast, ϳ70% of NK cells that were in contact either ICAM-1 or ICAM-2 demonstrated that engagement of with ICAM-1-coated beads displayed granule polarization toward LFA-1 alone was sufficient to trigger polarization of cytotoxic the bead, which was similar to the polarization observed in NK granules toward ICAM. Earlier experiments had established an cells incubated with sensitive 721.221 human target cells (Fig. 7b).

FIGURE 7. LFA-1 is sufficient to induce perforin polarization in NK FIGURE 5. LFA-1-induced cytotoxicity of NK cells is inhibited by cells. a, Human NK cells were mixed with uncoated beads (Ϫ), and with KIR. a, Specific lysis by a CD158bϩ NK clone of SC2 cells (asterisks), beads coated with B7.1, ICAM-1, or ICAM-2, as indicated, for 20 min at SC2-ICAM-1 cells (diamonds), and SC2-ICAM-1/Cw3 cells without 37°C. The percentage of NK cells bound to beads was determined by flow added peptide (Ⅺ) and SC2-ICAM-1/Cw3 cells after loading HLA-Cw3 cytometry on the basis of side scatter. Error bars represent the SD in three with a peptide (f). b, Specific lysis by a CD158aϩ NK clone of SC2- independent experiments. b, NK cells with polarized perforin before (Ϫ) ICAM-1 cells (diamonds), and SC2-ICAM-1/Cw4 cells after loading HLA- and after incubation with 721.221 cells (.221), with uncoated beads (Ϫ), Cw4 with a peptide that prevents binding of KIR2DL1 (Ⅺ) and after load- and beads coated with ICAM-1 or B7.1, as indicated. Data are expressed ing HLA-Cw4 with a peptide that promotes binding of KIR2DL1 (f). as percentage of NK cells that display polarized perforin. The number of Similar results were obtained with other NK clones. NK cells scored is given in parentheses above each bar. The Journal of Immunology 3657 important role of LFA-1 in target cell killing by CTLs and NK in this work, we have shown that incubation of NK cells with cells, but could not distinguish between a role in adhesion alone SC2-ICAM-1 cells resulted in a Src family kinase-dependent ac- and a dual role in adhesion and in signaling for cytotoxicity. The tivation of the guanine exchange factor Vav1, upstream of actin signals for LFA-1-dependent NK cell cytotoxicity are downstream polymerization (50). In contrast, the enhancement of Vav1 phos- of adhesion because inhibition of actin polymerization, of Src fam- phorylation induced by SC2 cells coexpressing ICAM-1 and ily kinase, and of PI3K inhibited lysis of target cells much more so CD48, the ligand of receptor 2B4, was downstream of actin poly- than adhesion to target cells or binding to ICAM-1 on plates (28). merization (50). Thus, our data suggest a model for regulation of The use of a Drosophila insect cell to reconstitute a target cell for NK cell cytotoxicity whereby LFA-1 provides an early signal for NK cells was further validated by functional recognition of actin polymerization, through activation of the GTPase Rac1 by HLA-C by inhibitory KIR on NK cells. Vav1, which is necessary for clustering and signaling by other It is possible that NK cell receptors recognize structures on in- receptors such as 2B4 (53). LFA-1 engagement is sufficient to sect cells once adhesion through LFA-1 occurs. If such high evo- initiate signaling because ICAM-1 on beads induced a Pyk2-de- lutionary conservation exists, it would more likely occur with non- pendent activation of Rac1 in NK cells (51). Furthermore, Ab- protein ligands. NK cells express an inhibitory member of the mediated cross-linking of LFA-1 on human NK cells induced sialic acid-binding Siglec receptor family, Siglec-7 (or AIRM1) phosphorylation of Vav and of the MAPK ERK1/2 (52). In T cells, (42), and the hyaluronic acid-binding receptor CD44, which pro- binding of the active form of LFA-1 to ICAM-1 promotes actin vides costimulation in NK cells (43, 44). Furthermore, the recent polymerization (49). Binding of human T lymphoblasts to ICAM-1 structure of activation receptor NKp44 suggests the possibility of on plates and concomitant activation of LFA-1 resulted in transient a carbohydrate binding site (45). Using heat-inactivated influenza stimulation of Rac1 and in PI3K-dependent activation of Pyk2 Downloaded from virus, which binds sialic acid via its hemagglutinin, as a probe for (26). Furthermore, binding of soluble, dimeric ICAM-2 to LFA-1 sialic acid, we found that SC2 cells do not express sialylated pro- on Jurkat cells leads to a cytohesin-dependent activation of teins at their surface (our unpublished observation), consistent with ERK1/2 (25). This signaling pathway induced by LFA-1, although other data on Drosophila cells (46). In addition, treatment of SC2- not sufficient to activate cytotoxicity in T cells, could be important ICAM-1 cells with neuraminidase did not reduce lysis by NK cells. in triggering NK cell cytotoxicity (52). The LFA-1 signaling path-

␤ http://www.jimmunol.org/ Likewise, enzymatic removal of cell surface hyaluronic acid, way in NK cells may be similar to a novel pathway attributed to 3 ␤ which was monitored with a hyaluronic acid-binding protein, had integrin in transfected Chinese hamster ovary cells: binding of 3 no effect on lysis by NK cells (our unpublished observation). integrin to fibrinogen resulted in a Syk-dependent phosphorylation Therefore, sialic acid and hyaluronic acid are not required for lysis of Vav1, and activation of ERK2 independently of actin polymer- of SC2-ICAM-1 cells by NK cells. ization (54). Early LFA-1 signals in NK cells, upstream of signal- To date, no receptor has been shown to be either necessary or ing by other receptors that are dependent on actin polymerization, sufficient in signaling for NK cell cytotoxicity. The importance of may be a common requirement of otherwise redundant activation certain receptors, such as NKG2D and the natural cytotoxicity re- pathways for natural cytotoxicity. ceptor NKp46, in lysis of tumor cells and virus-infected cells has Signaling events triggered by ligation of LFA-1 in NK cells, been well documented with specific combinations of target cells such as costimulation of CD16 signals for TNF-␣ secretion (55), by guest on September 27, 2021 and NK cells, indicating a possible redundancy of activation re- and induction of NK cell migration (56) and NK cell adhesion (28, ceptors and activation pathways in NK cells (1, 4). Our data sug- 57), have been reported, but not in the context of target cell lysis. gest that LFA-1 may be an important signaling receptor for cyto- Association of LFA-1 with other cell surface molecules, such as toxicity by NK cells. ITAM- and NKG2D-independent natural CD87 and DNAM-1, has been observed (21, 58). It is possible that cytotoxicity was observed in mouse NK cells with a double Syk LFA-1 signaling for cytotoxicity is dependent on such associations. and ZAP70 deficiency and with target cells lacking NKG2D li- Coexpression of peptide-loaded HLA-C with ICAM-1 on insect gands (12). This cytotoxicity was sensitive to inhibition of Src cells resulted in inhibition by KIR of the LFA-1-dependent cyto- family kinases and PI3K, as was the LFA-1-triggered target cell toxicity. We have recently shown in a transfected cell system that killing reported in this work. Such results are consistent with a KIR engagement by HLA-C on target cells leads to dephosphor- contribution of LFA-1 signals to ITAM/NKG2D-independent nat- ylation of Vav1 by the tyrosine phosphatase Src homology region ural cytotoxicity. 2 domain-containing phosphatase-1, which is recruited by phos- The interaction of NK cells with mammalian target cells is very phorylated ITIMs in KIR. Vav1 was the only tyrosine-phosphory- complex, involving many receptor-ligand interactions, some of lated protein associated with a trapping mutant of Src homology which exert a negative effect on cytotoxicity. Recent data on region 2 domain-containing phosphatase-1 during inhibition of NK mouse NK cells have revealed that natural cytotoxicity is con- cells by HLA-C on target cells; and Vav1 trapping was indepen- trolled not only by inhibitory receptors specific for MHC class I, dent of actin polymerization (59). These results, together with the but also by inhibitory NKR-P1B/D, which bind to Clr-b/Ocil (47, actin polymerization-independent activation of Vav1 by SC2- 48). As Clr-b/Ocil molecules are widely expressed (48), activation ICAM-1 cells (50), suggest that inhibitory KIR block NK cell acti- thresholds for lysis of many target cells by NK cells may be quite vation at a very early step, upstream of signals from other receptors. high. The simplified target cell system used in this study has elim- Our results point to ICAM as important ligands for stimulation inated most of this complexity and has the potential to reveal im- of natural cytotoxicity. The redistribution of ICAM-2 by the pro- portant receptor-ligand interactions, which are not easily detected tein ezrin into uropods of target cells resulted in increased sensi- with mammalian target cells. Even though induction of NK cell tivity to lysis by NK cells, in support of an important role for cytotoxicity by LFA-1 signals alone may rarely occur in vivo, our LFA-1 ligands in natural cytotoxicity (60). Expression of ICAM-1 approach has revealed a signaling function for LFA-1, which could is enhanced by inflammatory cytokines, including IFN-␥, and by provide essential activation signals together with other cytotoxicity infections of endothelial and epithelial cells with bacteria and vi- receptors in many NK-target cell combinations. ruses (61, 62). ICAM down-regulation could be a mechanism used Several recent studies on LFA-1 in NK cells and T cells indicate by pathogens to evade lysis by NK cells. Indeed, down-regulation that binding to ICAM is sufficient to trigger signaling pathways of ICAM-1 and B7.1 by protein K5 of Kaposi sarcoma-associated (26, 49–52). Taking advantage of the insect cell system described herpesvirus reduces sensitivity to NK cells (63). Regulation of 3658 LFA-1 SIGNALS FOR NK CELL CYTOTOXICITY natural cytotoxicity, at least in part, by expression of ICAM on 20. Shier, P., K. Ngo, and W. P. Fung-Leung. 1999. Defective CD8ϩ T cell activation target cells could serve useful functions. As many different cell and cytolytic function in the absence of LFA-1 cannot be restored by increased TCR signaling. J. Immunol. 163:4826. types require protection from lysis by NK cells, low expression 21. Shibuya, K., L. L. Lanier, J. H. Phillips, H. D. Ochs, K. Shimizu, E. Nakayama, levels of ICAM could diminish sensitivity to natural cytotoxicity H. Nakauchi, and A. Shibuya. 1999. Physical and functional association of LFA-1 with DNAM-1 adhesion molecule. Immunity 11:615. as an alternative protection mechanism to the expression of ligands 22. Sims, T. N., and M. L. Dustin. 2002. The immunological synapse: integrins take for NK cell inhibitory receptors. Conversely, up-regulation of the stage. Immunol. Rev. 186:100. ICAM during inflammation or infection may serve to modulate 23. Hynes, R. O. 2002. Integrins: bidirectional, allosteric signaling machines. Cell 110:673. NK cell responses at the proper time and place. 24. Kim, M., C. V. Carman, and T. A. Springer. 2003. Bidirectional transmembrane The contribution of early signals for cytotoxicity in NK cells by signaling by cytoplasmic domain separation in integrins. Science 301:1720. LFA-1 is very different from integrin-mediated activation of T 25. Perez, O. D., D. Mitchell, G. C. Jager, S. South, C. Murriel, J. McBride, L. A. Herzenberg, S. Kinoshita, and G. P. Nolan. 2003. Leukocyte functional cells. T cells do not adhere to ICAM-bearing cells unless an inside- antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 out signal has been received, which up-regulates the avidity of and Jun-activating binding protein 1. Nat. Immunol. 4:1083. adhesion through LFA-1 (34, 64). In contrast to T cells, NK cells 26. Sańchez-Martıń, L., N. Sańchez-Sańchez, M. D. Gutie´rrez-Lo´pez, A. I. Rojo, M. Vicente-Manzanares, M. J. Pe´rez-Alvarez, P. Sańchez-Mateos, X. R. Bustelo, can use LFA-1 directly to bind to target cells (28) and to transmit A. Cuadrado, F. Sańchez-Madrid, et al. 2004. Signaling through the leukocyte signals for cytotoxicity. In this respect, triggering of NK cell cy- integrin LFA-1 in T cells induces a transient activation of Rac-1 that is regulated by Vav and PI3K/Akt-1. J. Biol. 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