The Journal of Immunology

PI3K Links NKG2D Signaling to a CrkL Pathway Involved in Adhesion, Polarity, and Granule Secretion1

Colin M. Segovis,* Renee A. Schoon,* Christopher J. Dick,* Lucas P. Nacusi,* Paul J. Leibson,* and Daniel D. Billadeau2*†

The NK cell-activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell-target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D-coated beads is impaired in the absence of CrkL. Ab-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras family GTPase Rap1 is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner and is required for conjugate formation, MTOC (microtubule organizing center) polarization, and NKG2D-dependent cellular cytotoxicity. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization, and granule release. The Journal of Immunology, 2009, 182: 6933–6942.

ytotoxic lymphocytes, such as NK cells and CD8ϩ T gates down two distinct arms of the NKG2D signaling pathway: lymphocytes, play a critical role in our body’s immune PI3K and Grb2/Vav1 (4, 7, 8). Although a number of have C response to malignant and virally infected cells. A prin- been shown to play a role downstream of these proximal signaling ciple effector mechanism of cytotoxic lymphocytes is the process molecules, questions remain regarding the signaling pathways reg- of cell-mediated cytotoxicity, during which a target cell is de- ulating NKG2D-mediated conjugate formation, granule polariza- stroyed by a cytotoxic lymphocyte. Cell-mediated cytotoxicity oc- tion, and secretion. curs in discrete steps: formation of a cytotoxic lymphocyte-target Integrins have been established as regulators of cell adhesion cell conjugate, polarization of the cytotoxic machinery toward the and are required for proper NK cell-target conjugate formation. In effector-target cell interface known as the cytotoxic synapse (CS),3 ␤ ␤ fact, blocking 1 or 2 integrins inhibits NK cell-mediated cyto- and finally lytic granule secretion (1, 2). Although the steps of toxicity (9). Moreover, patients with leukocyte adhesion deficien- cytotoxicity have been established, many questions remain regard- cies display impaired NK cell function (10–12). ITAM-containing ing the regulation of these steps. receptors have been shown to regulate integrins in T lympho- NKG2D is a non-ITAM containing NK- and -activating cytes, neutrophils, , and NK cells (13–15). Nolz receptor that recognizes “stress” ligands, such as the tumor-asso- and colleagues established a role for WAVE2 in integrin acti- ciated MICA (in humans) and Rae-1 (in mice), which are up- vation through Abl- and CrkL-C3G-mediated activation of regulated on the surface of cells undergoing genotoxic stress (3–5). Rap1 in T cell signaling (16). Vinculin and talin have also been Upon NKG2D ligation, the transmembrane protein DAP10 is implicated in integrin affinity maturation downstream of the phosphorylated by the Src kinase Lck (6). The signal then propa- TCR (17). However, whether signaling pathways activated by NKG2D signaling affect integrin activation or NK cell adhesion is unknown. In fact, two recent reports did not observe any † *Department of Immunology and Division of Oncology Research, College of Med- increase in conjugate formation upon stimulation of NKG2D, icine, Mayo Clinic, Rochester, MN 55905 suggesting that signaling through NKG2D/DAP10 may not ac- Received for publication November 17, 2008. Accepted for publication March 31, 2009. tivate integrins (8, 18). The costs of publication of this article were defrayed in part by the payment of page The Crk family of adaptor proteins are a widely expressed fam- charges. This article must therefore be hereby marked advertisement in accordance ily of signal transduction mediators involved in normal and ma- with 18 U.S.C. Section 1734 solely to indicate this fact. lignant cell processes (19). This family contains Crk, expressed as 1 Supported by the National Institutes of Health (Grants CA47752 and AI65474 to two isoforms, CrkI and CrkII, and CrkL (Crk-like) (19). CrkI has D.D.B.). D.D.B. is a Leukemia and Lymphoma Society Scholar. an N-terminal SH2 domain followed by a SH3 domain, whereas 2 Address correspondence and reprint requests to Dr. Daniel D. Billadeau, De- partment of Immunology and Division of Oncology Research, Mayo Clinic Col- both CrkII and CrkL contain an additional C-terminal SH3 do- lege of Medicine, 200 First Street SW, Rochester, MN 55905. E-mail address: main. These domains serve as binding sites for a number of pro- [email protected] teins, allowing Crk proteins to participate in multiple signaling 3 Abbreviations used in this paper: CS, cytotoxic synapse; CMAC, 7-amino-4-chlo- pathways (19). Recent work has identified CrkL as an important romethylcoumarin; IP, immunoprecipitation; KIR, killer Ig-related receptor; MICA, MICA expressing BaF3 cells; MTOC, microtubule organizing center; SFDA, 5-sul- mediator of integrin regulation in T lymphocytes (16). Although fofluorescein diacetate; siCrkL, siRNA directed against CrkL; siNeg, negative control no functional role for CrkL has been demonstrated in NK cells, siRNA; siRNA, small interfering RNA; WR, Western Reserve. Crk has been identified as a target following NK inhibitory recep- Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 tor signaling (20). www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803840 6934 CrkL REGULATION OF NKG2D-MEDIATED KILLING

In this study, we demonstrate that NKG2D-mediated conjugate Conjugate assays formation is PI3K-dependent and involves the adaptor protein Human NK clones were stained with PKH26 per included protocol and CrkL and the small Ras family GTPase Rap1. Significantly, CrkL resuspended at 0.5 ϫ 106 cells/ml in RPMI 1640. Target cells were stained suppression results in diminished cytotoxicity, decreased conju- with either 50 ␮g/ml 5-sulfofluorescein diacetate (SFDA) (Invitrogen) in gate formation, impaired microtubule organizing center (MTOC) PBSfor1hina37°C water bath or 50 ␮M 7-amino-4-chloromethylcou- polarization, and decreased granule secretion by NK cells. We also marin (CMAC) (Invitrogen) for 30 min in serum-free media and then quenched for 15 min in RPMI 1640 plus 10% CS. Target cells were washed show that Rap1 is activated by NKG2D stimulation in a PI3K- and twice in PBS and resuspended at 0.5 ϫ 106 cells/ml in RPMI 1640. NK CrkL-dependent manner and is required for NKG2D-mediated cells (0.25 ϫ 106) were mixed with 0.25 ϫ 106 target cells and spun at 500 conjugate formation, MTOC polarization, and cytotoxicity. rpm for 5 min. The resulting pellet was placed in a 37°C water bath for the desired time. Medium (0.5 ml) was removed from each sample after all samples had been heated for the desired amount of time. Samples were then Materials and Methods vortexed at maximum speed for5stodisrupt nonspecific conjugates and Reagents, cells, and Abs then fixed with 0.5 ml of 4% paraformaldehyde. Conjugate formation was analyzed using a LSRII flow cytotometer (BD Immunocytometry Sys- Reagents were purchased from Sigma-Aldrich unless otherwise stated. Re- tems). The SFDA was excited with a 488-nm laser and the emission was combinant ICAM-1 and VCAM-1, as well as mouse monoclonal IgG1 measured through a 530/30-nm bandpass (BP) filter. CMAC was excited anti-NKG2D, were purchased from R&D Systems. Human NK cells were with a 355-nm laser and the emission was measured through a 450/65-nm cloned and passed as previously described (21). Both the wild-type BaF3 BP filter. The PKH26 was excited with a 532-nm laser and the emission myeloid cell line and the MICA-expressing variant were a gift from Dr. was measured through a 575/26-nm BP filter. Data were analyzed with the L. L. Lanier (University of California at San Francisco, San Francisco, FlowJo software package (Tree Star). CA). The anti-LFA-1 Ab, mHm23, was a gift from T. A. Springer (CBR Institute for Biomedical Research, Boston, MA). Mouse monoclonal IgG1 anti-FcR (CD16) was a gift from Dr. D. Segal (Immunology Branch, Na- Cytotoxicity assays tional Institute, National Institutes of Health, Bethesda, MD). ␤ 51Cr-release assays were performed as previously described (21). NK cell Mouse monoclonal anti- 1 integrin (Chemicon International), mouse monoclonal CrkL (Abnova), and mouse monoclonal p85, clone AB6 (Up- cytotoxicity was triggered either by coating P815 tumor targets with a state Biotechnology) were also used. Rabbit polyclonal anti-Rap1 was pur- “triggering” Ab, such as anti-NKG2D, or by the BaF3 myeloid cell line chased from Cell Signaling Technology. Rabbit polyclonal anti-SLP76 was expressing the NKG2D receptor ligand MICA. generated as previously described (22).

Adhesion assays GST protein precipitation GST-CrkL SH2, GST-CrkL SH3-N, and GST-CrkL SH3-C were con- Adhesions assays were performed as previously described (16). Briefly, a ϫ 6 96-well flat bottom plate was coated overnight with ICAM-1 or VCAM-1 structed as previously described (26). Briefly, 10 10 human NK clones per sample were stimulated with anti-NKG2D and cross-linked, and then and then blocked with 2.5% BSA in PBS. Human NK cells were stained lysed on ice for 10 min with buffer containing 50 mM Tris base, 20 mM with 5 ␮M calcein-AM (Invitrogen) for 30 min in serum-free media and EDTA, and 1% Igepal CA-630. Lysates were clarified by centrifugation washed with PBS. Cells were stained with 10 ␮g/ml IgG, anti-FcR, or and transferred to reduced glutathione-agarose coated with 40 ␮gofGST anti-NKG2D at 20 ϫ 106 cells/ml then added to wells containing goat Ј fusion protein. Lysates and beads were rotated at 4°C for 1 h. Protein anti-mouse F(ab )2. Plates were incubated on ice for 1 h and then in a 37°C ␮ water bath for 15 min. After incubation, cellular input was determined complexes were eluted with 40 l of SDS sample buffer, resolved by SDS- using a fluorescent plate reader. Plates were then washed with PBS and the PAGE, and transferred to Immobilon-P membranes (Millipore). The pres- remaining fluorescence was measured to determined cell adherence. ence of protein complexes was detected with primary Abs followed by goat anti-mouse-HRP (Santa Cruz Biotechnology) or goat anti-rabbit HRP Cell stimulation, immunoprecipitation, and immunoblot analysis (Santa Cruz Biotechnology) and visualized with the SuperSignal detection system (Pierce Biotechnology). Cell stimulation, immunoprecipitation, and immunoblot analysis were con- ducted as previously described (23). Immunofluorescence Cell transfection and protein suppression Immunofluorescence staining of NK cell-target cell and NK cell-bead con- CrkL, C3G, and Rap1 were suppressed using the nucleofection technique as jugates was performed similarly to T cell- conjugate immunofluo- previously described (7). Briefly, 300 pmol of small interfering RNA (siRNA) rescence described previously (16, 27). Briefly, BaF3 and MICA tumor ␮ per 5 ϫ 106 human NK clones were introduced in human NK clones by targets were stained with 50 M CMAC for 30 min in serum-free media Amaxa nucleofection using solution V and program O-17. Cells were then then quenched for 15 min in RPMI 1640 plus 10% CS. For NK-bead ␮ placed in RPMI 1640 containing 10% FBS, 1% sodium pyruvate, 1% L-glu- conjugates, sulfate latex beads (Invitrogen) were coated with 2 g of anti- tamine, and 20 U/ml IL-2 and allowed to recover for 72 h. The following target NKG2D or IgG for 1.5 h while rocking at 37°C. Beads were washed three sequences were used to generate siRNA duplexes against proteins in human times in PBS plus 2.5% BSA. NK-target cell conjugates were formed by NK cells: CrkL, GAGTTCTTTTGGATCATAA (targeted to the 3Ј untrans- conjugating human NK clones with either MICA expressing or control lated region, nucleotides 1634–1652 of NM_005207.3), C3G, GGGTTGTGT BaF3 target cells at an E:T ratio of 2:1. NK-bead conjugates were formed GAACTGAAAT (targeted to the 3Ј untranslated region, nucleotides 5290– by conjugating human NK clones with Ab-coated sulfate latex beads at an 5308 of NM_005207.3); Rap1A, siGENOME SMART pool M-003623-02- E:T ratio of 1:1. Cells were conjugated by spinning NK and targets or NK 005, targeted against NM_002884 (Dharmacon); Rap1B, siGENOME and beads at 500 rpm for 5 min at room temperature, incubated at 37°C for SMART pool M-010364-03-005, targeted against NM_001010942 (Dharma- 10 min, transferred to poly-L-lysine-coated coverslips, and allowed to settle con); negative control, TTCTCCGAACGTGTCACGT. on coverslips for 4 min at 37°C. Conjugates were washed, fixed with 4% paraformaldehyde, and then permeablized with 0.1% Triton X-100. Con- Recombinant vaccinia virus jugates were blocked with PBS containing 1% glycerol, 0.1% gelatin from cold water fish, 5% normal goat serum, 0.1% BSA, and 0.4% azide, and Wild-type CrkL and Rap1A cDNA were cloned from human NK cell then stained with primary Ab followed by either Alexa Fluor 568 goat mRNA by RT-PCR. W160Y and W161Y mutations were introduced into anti-mouse (Invitrogen) or Cy5-goat anti-mouse (Zymed Laboratories). the wild-type CrkL cDNA to create the N-terminal SH3 CrkL mutant. The Anti-␣-tubulin-FITC was also used for MTOC staining. Actin was stained S17A mutation was introduced into wild-type Rap1A cDNA to create a with Alexa Fluor 488 or rhodamine-phalloidin (Invitrogen). Coverslips dominant negative (24). Wild-type and mutant CrkL and Rap1A cDNA were mounted in ProLong Gold (Invitrogen). Fixed conjugates were ex- were introduced into the pSP11.FLAG vector as previously described (25). amined at room temperature on a Zeiss Axiovert 200 with an Axiocam The sequence of wild-type and mutant CrkL and Rap1A vaccinia virus HRC digital camera using a ϫ100/1.4 NA oil-immersion DIC objective or were confirmed. Human NK cells were infected at a multiplicity of infec- Zeiss LSM 510 scanning confocal microscope using a ϫ100/1.4 NA oil- tion of 20:1 for CrkL, or 5:1 and 30:1 with F.Rap1A and F.Rap1 S17A, immersion objective. Image acquisition was done with either the Zeiss respectively, with appropriate Western Reserve (WR) compensation. Cells Axiovision or LSM software packages. Figure construction of images was were infected for5hat37°C in serum-free media. performed in Adobe Photoshop and Illustrator CS3. The Journal of Immunology 6935

FIGURE 1. Integrins are activated by NKG2D ligation and regulate NK-target conjugate formation in NK cell-mediated cytotoxicity. Human NK clones were analyzed for their ability to adhere to 0.1 ␮g/well VCAM-1 (A) or ICAM-1 (B) in response to stimulation with either 10 ␮g/ml anti-FcR or anti-NKG2D. Data shown are representative of three human NK clones. C, Immunofluoresence of human NK clones demonstrating recruitment of ␤ ␤ 1 or 2 integrins to the cytotoxic synapse. NK cells were allowed to form conjugates with CMAC-stained wild-type BaF3 target cells or BaF3 target cells stably expressing MICA. The conjugates were allowed to adhere to poly-L-lysine-coated coverslips, fixed, and stained with rhodamine- ␤ ␤ phalloidin (orange) and either an anti- 1 or anti- 2 integrin Ab with an Alexa Fluor 647 goat anti-mouse secondary (red) as described in Materials ␤ ␤ and Methods. 1 or 2 integrin recruitment was determined by scoring 100 conjugates per coverslip on three coverslips for integrin recruitment to ␤ the CS. D, 2 integrins are required for NKG2D-mediated conjugate formation and cytotoxicity. Human NK clones were stained with PKH26 and then treated with 1 ␮g/ml anti-LFA-1 blocking Ab (mHm23) and allowed to form conjugates with SFDA-stained MICA or BaF3 target cells. Conjugate formation was determined by two-color flow cytometry. E, Human NK clones were treated with 1 ␮g/ml anti-LFA-1 blocking Ab (mHm23) and used in a cytotoxicity assay against two different models of NKG2D-mediated NK cell killing, P815 tumor targets coated with anti-NKG2D or MICA tumor targets. Data shown are representative of two human NK clones.

Rap1 activation assays Results The amount of GTP-bound Rap1 was determined by a RalGDS Rap-bind- NKG2D ligation leads to increased adhesion and recruitment of ing domain pulldown assay as previously described (28). Briefly, human integrins to the cytotoxic synapse NK cells were stimulated with either anti-NKG2D or anti-FcR and cross- linked, then resuspended in buffer containing 50 mM Tris (pH 7.5), 50 mM The importance of NKG2D as an activating receptor of NK cells has been clearly established (4, 26), but the interplay between in- NaCl, 5 mM MgCl2, 10% glycerol, and 0.5% Nonidet P-40 (Igepal CA- 630). Cells were immediately spun at 25,000 ϫ g for 5 min at 4°C. Lysates tegrins and NKG2D-mediated cytotoxicity is unclear, despite the were transferred to reduced glutathione-agarose coated with GST-Ral-Ras- fact that integrins are known to play a critical role in natural cy- binding domain (RBD). Lysates and beads were rotated at 4°C for 15 min. totoxicity (30). Two recent reports did not observe any increase in A portion of the lysates was reserved to determine the total amount of Rap1 conjugate formation upon stimulation of NKG2D, suggesting that in the lysate (input). Proteins were eluted and visualized as previously described (23). Densitometry was performed using ImageJ (National In- signaling through NKG2D/DAP10 may not activate integrins (8, stitutes of Health), corrected for total Rap1A loading, and then plotted as 18). To more directly examine whether NKG2D signaling might fold change relative to control baseline. regulate integrin activation, we determined whether stimulation through NKG2D activates integrins in clonally derived, nontrans- formed, IL-2-activated human NK cells. Unstimulated NK clones Secretion assays displayed limited adhesion to VCAM-1- or ICAM-1-coated sur- Granule secretion was determined in human NK cells as previously de- faces. In contrast, 30.1 Ϯ 2.5% of NK clones stimulated with anti- scribed (29). Briefly, human NK clones were stimulated by in a 96-well FcR and 27.2 Ϯ 3.9% of clones stimulated with anti-NKG2D ad- plate with wells that had been previously coated with anti-FcR, anti- hered to 0.1 ␮g/well of VCAM-1 compared with 15.8 Ϯ 3.2% of NKG2D, anti-CD56, or 12-O-tetradecanoylphorbol-13-acetate and iono- Ϯ mycin. Aftera4hincubation at 37°C, supernatants were harvested. The clones treated with an IgG control (Fig. 1A). Similarly, 21.4 granzyme A activity of the supernatants was then assessed by measuring 1.5% of NK clones stimulated with anti-FcR and 18.3 Ϯ 2.2% of N-␣-carbobenzoxy-L-lysine-thiobenzyl (BLT)-esterase activity. clones stimulated with anti-NKG2D adhered to 0.1 ␮g/well 6936 CrkL REGULATION OF NKG2D-MEDIATED KILLING

ICAM-1 compared with 8.8 Ϯ 0.4% of clones treated with an IgG control (Fig. 1B). To confirm that these effects were specific to the receptor, and not to nonspecific Ab binding to the FcR expressed on the surface of human NK clones, we examined granule secre- tion by cross-linking the NK cell surface receptor CD56, which would not be expect to trigger granule release. As expected, liga- tion of FcR or NKG2D enhanced secretion, whereas ligation of CD56 did not increase secretion, demonstrating that NK cell ac- tivation in response to anti-FcR or anti-NKG2D is not a result of nonspecific binding to the NK cell FcR (supplemental Fig. 1).4 These results demonstrate that stimulation through either FcR or ␤ ␤ NKG2D increases 1 and 2 integrin-mediated adhesion of human NK cells. Conjugate and CS formation have been established as critical steps in NK cell-mediated cytotoxicity (31). Integrin recruitment to the CS has been demonstrated in a K562 tumor target model of natural cytotoxicity, but it has not been examined in primary hu- man NK cells activated by ligation of NKG2D with its natural ligand, MICA (1). BaF3 cells stably expressing MICA were con- jugated with NK cells to determine whether integrins are recruited ␤ ␤ to the CS by NKG2D ligation (Fig. 1C). 1 and 2 integrins lo- calized diffusely within the plasma membrane of isolated NK ␤ ␤ clones. In NK cells conjugated with wild-type BaF3 cells, 1 or 2 ␤ integrins did not localize to the CS. However, 1 integrins were recruited to the NK CS in 48.7 Ϯ 5.5% of NK/MICA conjugates Ϯ ␤ vs 7.7 1.5% in NK/BaF3 conjugates. Similarly, 2 integrins were recruited to the NK CS in 51 Ϯ 6% of NK/MICA conjugates FIGURE 2. PI3K is required for NKG2D-mediated conjugate formation vs 11 Ϯ 1% in NK/BaF3 conjugates. These data demonstrate that and cytotoxicity. A, Human NK clones were treated with DMSO, 0.1 ␮M ␤ ␤ wortmannin, or 30 ␮M LY294002. NK cells were stained with PKH26 and 1 and 2 integrins are recruited to the NKG2D-mediated CS. allowed to form conjugates with SFDA-stained MICA or BaF3 target cells. LFA-1 is required for NKG2D-mediated conjugate formation Conjugate formation was determined by two-color flow cytometry. Un- and cytotoxicity stained cells from the drug treatment groups were used in a cytotoxicity The role of LFA-1 in NKG2D-mediated cytotoxicity in NK cells assay as a control. B, To confirm the effect of wortmannin treatment, hu- man NK clones were treated with either DMSO or 0.1 ␮M wortmannin, has been suggested in previous studies but has not been established stimulated with anti-NKG2D/goat anti-mouse Abs for 0, 1, 2, or 5 min. in primary human lymphocytes (32, 33). In this study, we used Cells were lysed, separated by SDS-PAGE, transferred, and blotted with human NK clones and two models of cytotoxicity, reverse-Ab- anti-phospho-Akt and anti-Akt. Data shown are representative of seven dependent cellular cytotoxicity and MICA-expressing BaF3 cells, human NK clones. ␤ to establish a role for the 2 integrin LFA-1 in NKG2D-triggered NK cell-mediated cytotoxicity. Treatment of NK clones with 1 ␮g/ml of the anti-LFA-1-blocking Ab mHm23 decreased NK cell clones, PI3K activity regulates NKG2D-mediated conjugate for- conjugate formation Ͼ50% compared with IgG control Ab (Fig. mation and subsequent cytolysis. 1D). mHm23 treatment affected cytotoxicity in our reverse-Ab- dependent cellular cytotoxicity model and caused a significant de- NK cell-mediated cytotoxicity is regulated by CrkL crease in the killing of MICA-expressing BaF3 cells (Fig. 1E). To CrkL has been shown to regulate integrins following TCR ligation show that lysis of MICA-expressing BaF3 cells is specific to the in T lymphocytes (16). We therefore examined whether CrkL reg- ligation of NKG2D, we inhibited NKG2D-mediated NK cell ac- ulates integrin function in NK cells. CrkL was clearly recruited to tivation with an anti-NKG2D blocking Ab, which confirmed that the cytotoxic synapse in NK clones conjugated with MICA targets MICA engagement by NKG2D was required for killing target cells but not in control BaF3 cells (Fig. 3A). In fact, CrkL was recruited (supplemental Fig. 2). These results establish a role for LFA-1 in to the NK CS in 33.3 Ϯ 5.0% of NK-MICA conjugates vs 3.3 Ϯ NKG2D-mediated cytotoxicity in primary human NK cells. 1.2% in NK-BaF3 conjugates (see Fig. 6A). The staining of CrkL PI3K regulates NKG2D-mediated conjugate formation at the CS was highly similar to the polarized F-actin polymeriza- tion seen in NK-MICA cell conjugates. Next, we explored phos- PI3K has been established as an important signaling mediator in phorylation of CrkL in NK cells upon stimulation of NKG2D. Crk NKG2D-stimulated NK cell cytotoxicity (4). Although Giurisato family members are initially recruited by activating receptors, but et al. reported that PI3K did not affect basal conjugate formation in then phosphorylated by Abl tyrosine kinase at an inhibitory residue the NK cell tumor line NK92 (18), this is surprisingly not the case to terminate signaling. In fact, phosphorylation of CrkL occurs in in primary human NK cells. In fact, NK clones treated with the TCR-mediated signaling regulating integrin activation (16). Inter- classic PI3K inhibitors wortmannin or LY294002 decreased con- estingly, Crk was recently shown to be phosphorylated in an Abl- Ͼ jugate formation by 50% (Fig. 2A). Cytotoxicity was also sub- dependent manner upon ligation of inhibitory killer Ig-related re- stantially decreased in NK clones treated with either PI3K inhib- ceptors (KIRs) to terminate signaling from activating receptors itor. As expected, Akt phosphorylation was decreased with (20). Significantly, cross-linking NKG2D on NK clones results in wortmannin treatment (Fig. 2B). Therefore, in primary human NK a time-dependent phosphorylation of CrkL, further confirming that CrkL is recruited to NKG2D-dependent signaling pathways (Fig. 4 The online version of this article contains supplemental material. 3B). Additionally, to determine the importance of CrkL in NK The Journal of Immunology 6937

FIGURE 4. CrkL is required for NKG2D-mediated conjugate formation but not Akt or Erk phosphorylation. A, NK clones were nucleofected with either CrkL or a negative control siRNA. Seventy-two hours after nucleo- fection, siRNA-treated cells were stained with PKH26 and then allowed to form conjugates with SFDA-stained MICA or BaF3 target cells. Conjugate formation was determined by two-color flow cytometry. Suppression of CrkL was determined by SDS-PAGE and immunoblotting of siRNA- treated cell lysates. Data are representative of three human NK clones. B, Human NK clones (1.25 ϫ 106/sample) were stimulated with anti- FIGURE 3. A CrkL regulates NK cell-mediated cytotoxicity. , Immuno- NKG2D/goat anti-mouse Abs for 0, 1, 2, or 5 min and then lysed and fluorescence of human NK clones demonstrating recruitment of CrkL to clarified, followed by SDS-PAGE and immunoblotting. the cytotoxic synapse. NK cells were allowed to form conjugates with CMAC-stained BaF3 cells or MICA-expressing BaF3 cells. The conjugates were allowed to adhere to poly-L-lysine-coated coverslips, fixed, and stained with FITC-phalloidin (green) and an anti-CrkL Ab with an Alexa Our data demonstrate that NKG2D-mediated conjugate forma- Fluor 568 goat anti-mouse secondary (red) as described in Materials and tion is both CrkL- and PI3K-dependent. However, it remained pos- Methods. Bar ϭ 5 ␮M. B, Ten ϫ 106 human NK clones/sample were sible that suppression of CrkL was affecting global signaling from stimulated with anti-NKG2D/goat anti-mouse Abs for 0, 1, 2, 5, or 10 min NKG2D through an unknown mechanism. To examine this, we and then lysed and clarified. Lysates were immunoprecipitated with anti- measured the phosphorylation kinetics of Akt and Erk, two PI3K- CrkL followed by SDS-PAGE and immunoblot. C, NK clones were regulated pathways (34), in NK cells suppressed for CrkL. Impor- nucleofected with either CrkL or a negative control siRNA. Seventy-two hours after nucleofection, siRNA-treated cells were used in a cytotoxicity tantly, treatment of NK cells with CrkL siRNA (siCrkL) did not assay with MICA or BaF3 target cells or with P815 mouse tumor targets affect Akt or Erk phosphorylation compared with siNeg-treated coated with either anti-NKG2D or anti-FcR. Suppression of CrkL was controls (Fig. 4B). Therefore, CrkL suppression does not affect determined by SDS-PAGE and immunoblotting of siRNA-treated cell ly- either of these PI3K-dependent pathways. Moreover, NKG2D-de- sates. Results are representative of Ͼ10 human NK clones. pendent tyrosine phosphorylation of Vav1, SLP76, and PLC␥2 are unaffected by suppression of CrkL (C. M. Segovis and D. D. Bil- ladeau, unpublished observation). cell-mediated cytotoxicity, we suppressed CrkL in human NK CrkL is involved in NKG2D-mediated cell polarization and clones with siRNA and used these clones in a cytotoxicity assay granule secretion against various NK-sensitive targets. CrkL suppression resulted in We next examined secretion in NK cells treated with siCrkL. Sur- a significant decrease in cytotoxicity against P815 tumor targets prisingly, siCrkL-treated NK clones displayed decreased secretion coated with either anti-NKG2D or anti-FcR, and against MICA- when stimulated by either anti-FcR or anti-NKG2D (Fig. 5A). This expressing BaF3 cells (Fig. 3C). These results indicate that CrkL effect was observed at multiple Ab concentrations: 30% and 46% mediates NK cell-mediated cytotoxicity triggered by multiple ac- decrease at 1 and 0.3 ␮g/ml anti-FcR, respectively, and 84% and tivating receptors, including NKG2D. 87% decrease at 1 and 0.3 ␮g/ml anti-NKG2D, respectively. 12- Given that integrins are critical to conjugate formation in NK O-tetradecanoylphorbol-13-acetate/ionomycin-induced secretion cell-mediated cytotoxicity and the known requirement of CrkL for was not affected by CrkL suppression, indicating that granule/ integrin function in T cells, we suppressed CrkL in human NK membrane fusion was not affected and thus suggesting a role for cells to explore the role of CrkL in NK-target cell conjugate for- CrkL in establishing polarized granule secretion in NK cells. mation. CrkL suppression in NK clones results in a Ͼ90% de- Microtubule polarization to the CS is required for localized re- crease in conjugate formation between NK cells and MICA-ex- lease of granules (35). We therefore examined MTOC polarization pressing BaF3 cells compared with NK clones treated with a in primary human NK cells treated with CrkL siRNA. CrkL sup- negative control siRNA (siNeg) (Fig. 4A), implicating CrkL in pression significantly impaired MTOC polarization to the CS of NKG2D-mediated cell-cell adhesion. NK-MICA targets compared with NK clones treated with negative 6938 CrkL REGULATION OF NKG2D-MEDIATED KILLING

FIGURE 5. CrkL regulates granule secretion and MTOC polarization. Human NK cells were nucleofected with CrkL or negative control siRNA. Seventy-two hours postnucleofection, siRNA-treated cells were used in the following assays. A, BLT-esterase assay to assess granule secretion. CrkL suppression was determined by SDS-PAGE and immunoblotting. Results are representative of three human NK clones. B, CrkL or negative control siRNA-treated cells were allowed to form conjugates with CMAC-stained BaF3 or MICA tumor targets. The conjugates were allowed to adhere to poly-L-lysine-coated coverslips, fixed, and stained with FITC-conjugated anti-␣-tubulin (green) and an anti-CrkL Ab with an Alexa Fluor 568 goat FIGURE 6. PI3K interacts with the N-terminal SH3 domain of CrkL. A, ␮ anti-mouse secondary (red) as described in Materials and Methods. Three Human NK clones were treated with 0.1 M wortmannin or DMSO and coverslips were prepared per treatment group. Fifty conjugates per cover- allowed to form conjugates with CMAC-stained MICA or BaF3 target slip were scored. CrkL suppression was confirmed by immunofluorescence cells. The conjugates were allowed to adhere to poly-L-lysine-coated cov- and Western blotting. Results are representative of three human NK clones. erslips, fixed, and stained with FITC-phalloidin (green) and an anti-CrkL Bar ϭ 5 ␮M. C, CrkL or negative control siRNA-treated cells were al- Ab with an Alexa Fluor 568 goat anti-mouse secondary (red) as described lowed to form conjugates with beads coated with 2 ␮g of IgG or anti- in Materials and Methods. Each treatment group contained three cover- NKG2D. The conjugates were allowed to adhere to poly-L-lysine-coated slips. Samples were blinded, and 50 conjugates per coverslip were scored ϭ ␮ coverslips, fixed, and stained with FITC-conjugated anti-␣-tubulin (green) for CrkL polarization to the cytotoxic synapse. Bar 5 M. B, Human NK ϫ 6 and an anti-CrkL Ab with an AlexaFluor568-Goat anti-mouse secondary clones (10 10 /sample) were stimulated with anti-NKG2D/goat anti- (red) as described in Materials and Methods. Three coverslips were pre- mouse Abs for 0, 2, or 5 min and then lysed and clarified. Lysates were pared per treatment group. One hundred conjugates per coverslip were cleared with IgG-coated agarose and then immunoprecipitated with IgG, scored for NK-bead conjugate MTOC polarization. CrkL suppression was anti-CrkL, or anti-p85 followed by SDS-PAGE and immunoblot. C, Hu- ϫ 6 confirmed by immunofluorescence and Western blotting. Results are rep- man NK clones (10 10 /sample) were stimulated with anti-NKG2D/goat resentative of three human NK clones. anti-mouse Abs for 0 or 5 min and then lysed and clarified. Lysates were immunoprecipitated with anti-reduced glutathione coated with GST or GST fusion proteins, followed by SDS-PAGE and immunoblot. D,NK clones were nucleofected with either CrkL or a negative control siRNA. control siRNA (Fig. 5B). Since the loss of MTOC polarization in Seventy-two hours after nucleofection, siRNA-treated cells were infected the siCrkL NK clones could be due to diminished conjugate for- with WR, F.CrkL, or N-terminal SH3 CrkL mutant (SH3-Nmt) vaccinia mation (Fig. 4), we used anti-NKG2D beads to explore the role of virus as described in Materials and Methods. These clones were used in a CrkL in integrin-independent MTOC polarization. A 52% decrease cytotoxicity assay with P815 mouse tumor targets coated with 10 ng/well in MTOC polarization was observed in NK clones treated with anti-NKG2D. CrkL expression was determined by SDS-PAGE and immu- siCrkL in NK-bead conjugates compared with siNeg-treated NK noblotting of siRNA-treated cell lysates. Results are representative of more than two human NK clones. clones (Fig. 5C). These data demonstrate that CrkL regulates not only conjugate formation, but also MTOC polarization and subse- quent granule secretion, all of which are required for NK cell- mediated cytotoxicity. cruitment to the CS was affected. Treatment of human NK clones with wortmannin decreased recruitment of CrkL to the CS of NK- PI3K interacts with the N-terminal SH3 domain of CrkL MICA target cell conjugates by 78% (Fig. 6A). However, we can- PI3K is a critical proximal signaling regulator of NKG2D-medi- not rule out the possibility that this defect in CrkL recruitment is ated NK cell cytotoxicity (4, 36). Our results indicate that both a result of defective conjugate formation in the wortmannin-treated PI3K activity and CrkL regulate NKG2D-mediated adhesion, sug- NK cells. Since CrkL has been observed in complex with p85 in gesting that CrkL may signal downstream of PI3K. To test this, we NK cells (37), we used immunoprecipitation (IP) to establish an treated NK clones with wortmannin and determined if CrkL re- association between CrkL and p85 in our system and to determine The Journal of Immunology 6939

whether the interaction was NKG2D induced. Indeed, CrkL coim- munoprecipitated p85 in both unstimulated and NKG2D-stimu- lated human NK clones, indicating a constitutive interaction be- tween these two molecules (Fig. 6B). Similarly, an IP of p85 indicated constitutive p85-CrkL association (Fig. 6B). The N-ter- minal SH3 domain of CrkII has been shown to interact with the proline-rich region of p85, the regulatory subunit of PI3K, upon TCR activation, and in vitro an interaction has been demonstrated between the SH3 domains of CrkL and p85 (38, 39). However, which of the two CrkL SH3 domains interacts with p85 is un- known in NK cells. Using GST fusion proteins of individual SH2 and SH3 domains of CrkL, we found that the N-terminal SH3 domain of CrkL bound p85 (Fig. 6C). We next sought to determine the functional significance of this interaction in NKG2D-mediated cellular cytotoxicity by analyzing whether disruption of this p85- CrkL complex would lead to impaired killing. To test this, we performed siRNA rescue experiments in which wild-type CrkL or an N-terminal SH3 mutant (SH3-Nmt) were overexpressed in hu- man NK clones pretreated with siCrkL. Significantly, overexpres- sion of wild-type CrkL partially rescued the decrease in NKG2D- dependent cytotoxicity seen with CrkL suppression, whereas the CrkL SH3-Nmt was unable to rescue the cytotoxic defect in the CrkL-suppressed cells (Fig. 6D). These results demonstrate a reg- ulatory role for the N-terminal SH3 domain of CrkL in NKG2D- mediated NK cell cytotoxicity. Taken together, our results dem- onstrate that p85 and CrkL are in a complex, and that this interaction regulates the development of NKG2D-mediated cyto- toxicity in human NK cells.

NKG2D-mediated cytotoxicity is Rap1-dependent FIGURE 7. Rap1 regulates NKG2D-mediated cytotoxicity in human Rap1 has been demonstrated to be critical regulator of integrin NK cells. A, Human NK clones were stimulated with anti-FcR/goat anti- activation in T lymphocytes through a CrkL-C3G complex (16), mouse or anti-NKG2D/goat anti-mouse Abs for 0, 1, 2.5, 5, or 10 min. but no role for Rap1 has been established in human NK cells (40, Cells were lysed, and the GTP-bound Rap1 was determined by immuno- 41). Ligation of either the FcR or NKG2D results in activation of precipitation with glutathione S-transferase-RalGDS followed by SDS- Rap1 (Fig. 7A). To confirm these results, we studied the activation PAGE and immunoblotting. B, Human NK clones were treated with ␮ of MST1 upon FcR and NKG2D receptor ligation. The Rap1- DMSO or 0.1 M wortmannin and then stimulated with anti-NKG2D/goat anti-mouse Abs for 0, 1, 2, or 5 min. Cells were lysed, and the GTP-bound RAPL-MST1 signaling cascade has been shown to be required for Rap1 was determined by immunoprecipitation with glutathione S-trans- adhesion and cell polarity in lymphocytes in response to chemo- ferase-RalGDS followed by SDS-PAGE and immunoblotting. C,NK kine stimulation, and activation of Rap1 results in phosphorylation clones were nucleofected with either CrkL or a negative control siRNA. of MST1 (42). Ligation of either FcR or NKG2D increased phos- Seventy-two hours after nucleofection, siRNA-treated cells were stimu- phorylation of MST1, supporting the idea that Rap1 is coupled to lated with anti-NKG2D/goat anti-mouse Abs for 0, 1, or 5 min. Cells were these activating receptors in NK cells (supplemental Fig. 3). Pre- lysed, and the GTP-bound Rap1 was determined by immunoprecipitation vious work has demonstrated that CrkL regulates the GTP loading with glutathione S-transferase-RalGDS followed by SDS-PAGE and im- and activation of Rap1 downstream of several cell surface recep- munoblotting. Fold change relative to baseline control was calculated using tors (43–45). Since Rap1 is activated by NKG2D ligation, we densitometry and corrected for total Rap1A expression. D, NK clones were asked if either PI3K activity or CrkL regulates Rap1-GTP loading. infected with WR, F.Rap1A, or F.Rap1A S17A vaccinia virus as described in Materials and Methods. Infected cells were used in a cytotoxicity assay In fact, treatment with wortmannin (Fig. 7B) or siRNA depletion with MICA or BaF3 target cells or with P815 mouse tumor targets coated of CrkL (Fig. 7C) abrogated NKG2D-stimulated Rap1 activation. with anti-NKG2D. Overexpression of F.Rap1 and F.Rap1A S17A was de- Taken together, these data indicate that Rap1 is activated following termined by SDS-PAGE and immunoblotting. Results are representative of FcR and NKG2D ligation in a PI3K- and CrkL-dependent manner. four human NK clones. E, NK clones were nucleofected with Rap1A, To determine the role of Rap1 in NKG2D-mediated cytotoxicity Rap1B, or a negative control siRNA. Seventy-two hours after nucleofec- in human NK cells, we initially expressed a wild-type or dominant- tion, siRNA-treated cells were used in a cytotoxicity assay with MICA or negative version of Rap1 (S17A) in NK cells and examined BaF3 target cells or with P815 mouse tumor targets coated with anti- NKG2D-mediated killing (24). In contrast to expression of wild- NKG2D. Suppression of Rap1 was determined by SDS-PAGE and immu- type Rap1, which did not affect killing through NKG2D, expres- noblotting of siRNA-treated cell lysates. Results are representative of more sion of Rap1 S17A inhibited killing of either MICA expressing than three human NK clones. BaF3 cells or P815 cells coated with anti-NKG2D (Fig. 7D). Since overexpression of Rap1 S17A may have off-target effects associ- ated with its dominant-negative function, we utilized siRNA to which was further diminished by suppression of both Rap1 iso- suppress Rap1A and Rap1 B in NK clones, as both Rap1A and forms (Fig. 7E). Rap1B are expressed in human NK cells (Fig. 7E, immunoblot). Given that both PI3K and CrkL regulate NKG2D-dependent Importantly, suppression of either Rap1A or Rap1B decreased Rap1 activation and conjugate formation, we used dominant-neg- NKG2D-mediated cytotoxicity against multiple tumor targets, ative Rap1A to assess the role of Rap1 in NK-target conjugate 6940 CrkL REGULATION OF NKG2D-MEDIATED KILLING

Discussion In this paper we describe an NKG2D-mediated signaling pathway for the regulation of conjugate formation, MTOC polarization, and granule secretion. Integrins have been shown to play a critical role in lymphocyte function regulated by ITAM-containing receptors such as the TCR, but little is known about the regulation of inte- grins by non-ITAM-containing receptors such as NKG2D. We es- ␤ tablish that ligation of FcR and NKG2D increase adhesion to 1 ␤ and 2 integrin ligands VCAM-1 and ICAM-1. Using blocking ␤ Abs we have established a role for the 2 integrin in NKG2D- mediated NK-tumor target formation and cytotoxicity. This is con- sistent with the observation that patients with leukocyte adhesion deficiencies display impaired NK cell function (10–12). The results presented herein indicate that NKG2D signaling reg- ulates NK cell adhesion and that LFA-1 is required for optimal killing through NKG2D. Surprisingly, previous studies aimed at dissecting NKG2D-mediated signaling in both human and mouse NK cells suggest that neither the PI3K nor Grb2/Vav1 pathways regulate NKG2D-mediated cellular adhesion (8, 18). Taken at face value, these studies would seem to indicate that neither of the two known signaling pathways engaged by NKG2D is important in stimulating adhesion. However, the amount of conjugate formation between NK cells and NKG2D-sensitive targets in both studies is only slightly, if at all, increased over control targets (8, 18), sug- gesting the possibility that NKG2D engagement does not trigger integrin activation, similar to known ITAM-containing receptors. However, our data provide evidence that NKG2D ligation does in FIGURE 8. Rap1 regulates NKG2D-mediated conjugate formation and fact increase integrin-mediated adhesion to plate-bound substrates, MTOC polarization. A, NK clones were infected with WR, F.Rap1A, or as well as conjugate formation to NKG2D ligand-bearing target F.Rap1A S17A vaccinia virus as described in Materials and Methods. In- cells. Moreover, our data identify PI3K signaling as an important fected cells were then stained with PKH26 then allowed to form conjugates mediator of NKG2D-stimulated adhesion. Thus, our work adds to with CMAC-stained MICA or BaF3 target cells. Conjugate formation was the understanding of the NKG2D-mediated signaling pathways by determined by two-color flow cytometry. Overexpression of F.Rap1 and establishing that NKG2D-mediated conjugate formation is PI3K- F.Rap1A S17A was determined by SDS-PAGE and immunoblotting. Re- dependent. Future studies will be important to determine whether sults are representative of three human NK clones. B, NK clones were the Grb2/Vav1 pathway also participates in NKG2D-stimulated infected with WR, F.Rap1A, or F.Rap1A S17A vaccinia virus as described in Materials and Methods. Infected cells were allowed to form conjugates adhesion. with beads coated with 2 ␮g of IgG or anti-NKG2D. The conjugates were The Crk family of adaptor proteins, Crk and CrkL, are important allowed to adhere to poly-L-lysine-coated coverslips, fixed, and stained regulators of cell motility and adhesion in multiple cell types and with FITC-conjugated anti-␣-tubulin (green) as described in Materials and in the regulation of the oncogenic BCR-Abl mutation (19). The Methods. Three coverslips were prepared per treatment group. One hun- lymphocyte Crk family member, CrkL, has been clearly estab- dred conjugates per coverslip were scored for NK-bead conjugate MTOC lished as an important adaptor protein in T lymphocyte biology polarization. Overexpression of F.Rap1A and F.Rap1S17A was confirmed (16) and it has been suggested that CrkL plays a role in NK cell by immunofluorescence and Western blotting. Results are representative of activation, but it has not been established if CrkL plays a role in the two human NK clones. activation of NK cells by non-ITAM-containing receptors such as NKG2D (46, 47). In this study we establish that CrkL regulates NKG2D-mediated NK cell-mediated cytotoxicity, in part through formation. Indeed, overexpression of Rap1A had no effect on con- its effects on cell adhesion and establishing cellular polarity. These jugate formation between NK cells and MICA-expressing BaF3 results are significant since a recent study has shown that engage- cells compared with WR-infected cells (Fig. 8A). In contrast, over- ment of an inhibitory KIR on NK cells results in the inactivation expression of Rap1A S17A significantly decreased conjugate for- of Crk through Abl-mediated phosphorylation at a known inhibi- mation between NK cells and MICA-expressing targets equal to tory site (20). Interestingly, the KIR-generated inhibitory signal conjugate formation between WR-expressing NK clones and BaF3 could be overcome by expression of a Crk protein lacking the control targets (Fig. 8A). We also examined the effect of Rap1A or inhibitory phosphorylation site. Our data implicate CrkL in both dominant-negative Rap1 on integrin-independent MTOC polariza- ITAM-dependent and ITAM-independent signaling pathways tion in primary human NK cells. Rap1A overexpression did not leading to granule release and cellular cytotoxicity. Whether CrkL affect MTOC polarization to the CS of NK-NKG2D-coated bead is phosphorylated by KIR engagement and serves a functionally conjugates compared with WR-infected cells (Fig. 8B). However, redundant role with Crk in the regulation of NK cellular cytotox- overexpression of dominant-negative Rap1A decreased MTOC icity remains to be determined. polarization by 53% in NK-bead conjugates compared with WR- PI3K has been shown to regulate cellular polarity and adhesion infected cells (Fig. 8B). Overall, these results demonstrate that in multiple cell types (48, 49). Additionally, the regulatory subunit Rap1 activation by NKG2D is PI3K- and CrkL-dependent, and of PI3K, p85, has also been shown to interact with CrkL in trans- that Rap1 regulates NKG2D-mediated adhesion and MTOC po- formed cell lines (38). These observations are consistent with our larization leading to NK cell cytotoxicity. study using normal human NK cells, and we add the observation The Journal of Immunology 6941 that PI3K interacts with the N-terminal SH3 domain of CrkL in 8. Graham, D. B., M. Cella, E. Giurisato, K. Fujikawa, A. V. Miletic, T. 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