The Journal of Immunology

Dedicator of Cytokinesis 8 Interacts with Talin and Wiskott-Aldrich Syndrome To Regulate NK Cell Cytotoxicity

Hyoungjun Ham,* Sabrice Guerrier,† JungJin Kim,‡ Renee A. Schoon,‡ Erik L. Anderson,*,x Michael J. Hamann,x Zhenkun Lou,‡ and Daniel D. Billadeau*,‡

Recently, patients with mutations in DOCK8 have been reported to have a combined immunodeficiency characterized by cuta- neous viral infections and allergies. NK cells represent a first-line defense against viral infections, suggesting that DOCK8 might participate in NK cell function. In this study, we demonstrate that DOCK8-suppressed human NK cells showed defects in natural cytotoxicity as well as specific activating receptor-mediated NK cytotoxicity. Additionally, compared with control NK cells, NK cells depleted of DOCK8 showed defective conjugate formation, along with decreased polarization of LFA-1, F-, and cytolytic granules toward the cytotoxic synapse. Using a proteomic approach, we found that DOCK8 exists in a macromolecular complex with the Wiskott-Aldrich syndrome protein, an actin nucleation-promoting factor activated by CDC42, as well as talin, which is required for -mediated adhesion. Taken together, our results demonstrate an important role for DOCK8 in NK cell effector function and provide important new mechanistic insight into how DOCK8 regulates F-actin and integrin-mediated adhesion in immune cells. The Journal of Immunology, 2013, 190: 3661–3669.

he dedicator of cytokinesis (DOCK) 180 family of guanine deficient patients are susceptible to recurrent viral infections sug- nucleotide exchange factors (GEF), of which there are 11 gests the existence of a general deficiency of cytotoxic . T known genes, participates in the activation of Rho family Supporting this hypothesis, one group reported lymphopenia and contributes to multiple cellular processes, including and impaired T cell expansion in DOCK8-deficient patients (5). cell migration, phagocytosis, and immune homeostasis (1–3). In However, another clinical report found that T cell numbers in 2009, mutations in the DOCK8 gene were found to be associated patients were within normal range (4), and a recent study reported with cases of autosomal recessive hyper-IgE syndrome (4, 5). The that mouse CTLs harboring a mutation within the DOCK8 DOCK disease showed an autosomal recessive pattern of inheritance, and homology region 2 (DHR2) domain had no killing defect compared all reported patients harbored homozygous or compound hetero- with control CTLs (7). Although NK cell numbers were within the zygous mutations on both alleles, leading to either null or non- normal range, it remains possible that impaired NK cell function functional expression of DOCK8. The main clinical symptoms of might be responsible for the recurrent viral infections. Interestingly, the patients were recurrent sinopulmonary and cutaneous infec- TAP-deficient patients lack CD8+ T cells and do not show particular tions and severe allergies. Immunologically, patients showed high susceptibility to viral infections (11). It is also worthwhile to note IgE and eosinophil numbers in their sera, defects in generation of that NK cell–mediated cytotoxicity is crucial for elimination of long-lasting humoral immunity, and T cells with limited cellular HSV, the most common recurrent viral infection seen in DOCK8- proliferation following activation (4–7). deficient patients (4, 5, 12). Despite this, NK cell function has yet to Direct killing by CTL or NK cells against target cells is required be assessed in patients deficient in DOCK8. for clearance of intracellular pathogens, including virus-infected Previous studies using B cells and T cells from DOCK8 mutant or transformed cells (8–10). The observation that most DOCK8- mice have shown that DOCK8 is involved in F-actin and integrin accumulation at the immune synapse (IS) (7, 13). Recent findings *Department of Immunology, College of Medicine, Mayo Clinic, Rochester, MN that DOCK8 has GEF activity toward CDC42 (14) should also be 55905; †Department of Biology, Carleton College, Northfield, MN 55057; ‡Division noted, considering an essential role of CDC42 in reorganization of of Oncology Research, Schulze Center for Novel Therapeutics, College of Medicine, the actin as well as its regulation of cell polarity. Mayo Clinic, Rochester, MN 55905; and xBiology Department, Bemidji State Uni- versity, Bemidji, MN 56601 Because F-actin polymerization is involved in integrin clustering Received for publication October 5, 2012. Accepted for publication February 2, and cytotoxic synapse formation leading to adhesion and killing 2013. (15), we decided to investigate the role of DOCK8 in human NK This work was supported by the Mayo Foundation and National Cancer Institute cells. In this study, we show that DOCK8 knockdown results in Grant R01-CA47752 (to D.D.B.). D.D.B. is a Leukemia and Lymphoma Scholar. diminished NK cell–mediated cytotoxicity. Consistent with pre- Address correspondence and reprint requests to Dr. Daniel D. Billadeau, Division of vious studies in T cells (7), we find that suppression of DOCK8 Oncology Research, Mayo Clinic, 200 1st Street SW, 19-254 Gonda, Rochester, MN affects F-actin accumulation at the cytotoxic synapse (CS), as well 55905. E-mail address: [email protected] as diminished integrin recruitment, leading to an overall decrease The online version of this article contains supplemental material. in cell–. Using proteomics, we show that DOCK8 Abbreviations used in this article: CS, cytotoxic synapse; DHR2, dedicator of cyto- kinesis homology region 2; DOCK, dedicator of cytokinesis; GEF, guanine nucleo- interacts with the integrin regulator talin, as well as the CDC42 tide exchange factor; HA, hemagglutinin; IS, immune synapse; MTOC, effector Wiskott-Aldrich syndrome protein (WASP), and the in- organizing center; PB, pull-down buffer; PtdIns, phosphatidylinositol; siRNA, small teraction with DOCK8 is involved in their accumulation at the CS. interfering RNA; WASP, Wiskott-Aldrich syndrome protein; WT, wild type. Taken together, our results provide new mechanistic information Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 regarding DOCK8 cellular function in NK cells and contribute to www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202792 3662 DOCK8 REGULATES NK CELL KILLING the understanding of the known phenotypes observed in DOCK8 1–stained YTS-721.221 conjugates were examined with a 1003 oil- deficiency. immersion objective on an LSM-710 laser-scanning confocal micro- scope (Carl Zeiss), and z-series images acquired with a space of 0.45 mm were processed into either three-dimensional reconstruction images using Materials and Methods Zen2009 software (Carl Zeiss) or three-dimensional projection images Cells, reagents, and Abs using ImageJ software (version 1.45s; National Institutes of Health). For other cases, fixed conjugates were examined with a 1003 oil-immersion All reagents were obtained from Molecular Probes, unless stated other- objective on an Axiovert 200 microscope (Carl Zeiss), and images were wise. Primary human NK cells were cloned and passaged, as previously captured with the AxioVision software package. For scoring of granule described (16). YTS was obtained from E. Long (National Institutes of polarization (based on perforin staining), .100 conjugates between YTS Health, Rockville, MD) and NKL from M. Robertson (Indiana Univer- cells and 721.221 target cells were chosen randomly per condition, and sity Cancer Center, Indianapolis, IN). Two separate rabbit polyclonal anti- categorized as front, middle, or back according to the distance of center of sera to DOCK8 (NCBI: NP_982272.2) were obtained by immunizing rabbits granules from accumulated F-actin at YTS-721.221 interface. Quantifica- with either keyhole limpet hemocyanin–conjugated DOCK8 peptide tion of fluorescence intensity for LFA-1, F-actin, WASP, and talin at the EAVEKNKRLITADQREYQQELKKC or GST-conjugated DOCK8 aa 1– YTS-721.221 interface was performed, as previously described (22). Images 238 (Cocalico Biologicals, Reamstown, PA). Each anti-DOCK8 polyclonal containing total 150 conjugates (30 conjugates for LFA-1) were taken for rabbit serum was affinity purified using Sulfolink or Aminolink (Pierce each condition (50 conjugates per experiment [10 conjugates for LFA-1]; 3 Chemical). Polyclonal rabbit antisera for WASP and ZAP-70 were previ- independent experiments), and sum fluorescence intensity of contact area ously described (17, 18). mAb for CD16 was affinity purified from 3G8 as well as that of the whole cell was measured using ImageJ software. hybridoma. Abs for a-, talin (clone 8d4), and FLAG (clone M2) Then, percentage of fluorescence at contact site was calculated using the were purchased from Sigma-Aldrich, and anti-hemagglutinin affinity ma- following equation: (sum intensity [contact site]/sum intensity [whole trix (Roche) was used for immunoprecipitation of HA-tagged WASP. HRP- cell]) 3 100. linked anti-rabbit (Cell Signaling) and anti-mouse (Santa Cruz) IgG were used for immunoblotting. Conjugate assay Plasmids and transfection NK cells (YTS, NKL, or primary human NK clones) were labeled for 30 min at 37˚C with 2 mM CellTracker Violet BMQC (Invitrogen) (violet Full-length DOCK8 (National Center for Biotechnology Information: fluorescence), and 721.221 target cells were labeled for 30 min at 37˚C NP_982272.2, wild type (WT), and V1985A) and WASP were cloned with 1.25 mM CFSE (green fluorescence). Labeled NK cells and target into the pCI2 plasmid in-frame with FLAG.YFP and HA, respectively. cells were then washed and resuspended at 4 3 106 cells/ml (2 3 106 cells/ HEK293T cells were transfected using GeneJuice Transfection Reagent ml in case of YTS cells) and 2 3 106 cells/ml, respectively. Cells (150–250 (Millipore), according to the manufacturer’s instructions. ml each) were mixed together, centrifuged, and allowed to incubate at 37˚C Immunoprecipitation and GST pull-down assays up to 15 min before fixation by adding 4% paraformaldehyde in PBS. Conjugate formation was assessed using two-color flow cytometry and For immunoprecipitation, cells were lysed in Nonidet P-40 lysis buffer (50 is revealed by simultaneous emission of violet and green fluorescence. mM Tris [pH 7.5], 120 mM NaCl, 0.5% Nonidet P-40, 10 mM NaF, 200 mM Results were analyzed as percentage of target-bound NK cells (violet+ + + Na3VO4, 1 mM PMSF, 10 mg/ml leupeptin, and 5 mg/ml aprotinin), and green ) over total NK cells (violet ). 0.75–1.0 mg clarified whole-cell lysate was used, as previously described (19). For GST fusion protein pull-downs, GST-conjugated DOCK8 frag- Size-exclusion chromatography and mass spectrometry ments used in this study include the following: DOCK8-N9 (aa 1–753), Size-exclusion chromatography to examine DOCK8-containing fractions DOCK8-Mid (aa 754–1452), and DOCK8-C9 (aa 1453–2099). A total of 3 6 40 mg GST fusion protein was bound to glutathione-agarose slurry in pull- was performed, as described previously (19), with 50–100 10 YTS cells. down buffer (PB: 1 M HEPES [pH 7.2], 50 mM CH CO K, 1 mM EDTA, Briefly, DOCK8-containing fractions were pooled and incubated with 3 2 amino-link resin covalently attached to anti-DOCK8. Resin was washed 200 mM D-sorbitol, 0.1% Triton X-100, 1 mM PMSF, 10 mg/ml leupeptin, 3 and 5 mg/ml aprotinin) for 30–60 min at 4˚C. The resin was washed once three times with 1% Nonidet P-40 lysis buffer and boiled in 4 SDS- in PB and incubated with 1.0 mg clarified whole-cell lysate for 5 h at 4˚C. PAGE laemmli sample buffer. Proteins were separated on 8.5% SDS- The protein complexes were then washed twice with PB and eluted in 40 PAGE and silver stained as per standard protocol. ml SDS-sample buffer. Eluted proteins were resolved by SDS-PAGE and Protein identification via in-gel trypsin digest–nanoLCMS/MS with transferred to Immobilon-P membranes (Millipore). hybrid orbitrap/linear ion trap mass spectrometry was performed at the Mayo Clinic Protein Chemistry and Proteomics Core. Briefly, silver- Small interfering RNA constructs and nucleofection stained SDS-PAGE gel bands were destained, reduced, and then digested in situ with 30 ml (0.005 mg/ml) trypsin (Promega, Madison, WI) in 20 mM Small interfering RNA (siRNA) duplexes targeting DOCK8 were obtained Tris (pH 8.1)/0.0002% Zwittergent 3-16, at 37˚C overnight, followed by from Dharmacon, as follows: siRNA 1, 59-GGAAACAATTCTCCGATAA- peptide extraction with 20 ml 2% trifluoroacetic acid. Concentrated sam- 39 and siRNA 2, 59-GGAGATTATTTGTGAACTT-39. Nucleofection of ples are then brought up in 0.15% formic acid/0.05% trifluoroacetic acid siRNA oligos into YTS, NKL, and NK clones was performed, as previ- for protein identification by nano-flow liquid chromatography electrospray ously described (20, 21). tandem mass spectrometry using a ThermoFinnigan LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled Cytotoxicity assay to an Eksigent nanoLC-2D HPLC system (Eksigent, Dublin, CA). The The 51Cr-release assays were performed, as described previously (16). In digest peptide mixture is loaded onto a 250-nl OPTI-PAK trap (Optimize reverse Ab-dependent cellular cytotoxicity assays, primary NK clones Technologies, Oregon City, OR) custom packed with Michrom Magic C8 were only able to kill P815 tumor target cell in the presence of either anti- solid phase (Michrom Bioresources, Auburn, CA). Tandem mass spectra FcR (a-CD16) mAb or anti-NKG2D mAb (R&D Systems). All LU values are extracted by BioWorks version 3.2. All MS/MS samples are analyzed in this study were obtained by calculating required number of NK cells to using Mascot (Matrix Science, London, U.K.; version 2.2.04), Sequest kill 20% of target cells. (ThermoFinnigan, San Jose, CA; version 27, rev. 12), and X! Tandem (www. thegpm.org; version 2006.09.15.3). Scaffold (version Scaffold_2_00_06; Cell microscopy and analysis Proteome Software, Portland, OR) is used to validate MS/MS-based pep- tide and protein identifications. Peptide identifications are accepted if 3 6 At 72 h of siRNA transfection, 250–500 ml YTS cells (1 10 cells/ml) they can be established at .95.0% probability, as specified by the Pep- were mixed with an equal volume of 7-amino-4-chloromethylcoumarin– tide Prophet algorithm. Protein identifications are accepted if they can be 3 6 stained 721.221 target cells (1 10 cells/ml) and centrifuged together at established at .95.0% probability and contain at least three identified ∼ 500 rpm in serum-free RPMI 1640 medium. The slightly pelleted cells peptides. were then incubated at 37˚C for 15 min, resuspended, and allowed to adhere to poly-L-lysine–coated coverslips for 10 min at 37˚C. Cells were Flow cytometry surface CD18 staining fixed with 4% paraformaldehyde in PBS and stained with specific Abs (anti–LFA-1 [clone MHM23], anti-perforin [BD Biosciences], anti-WASP, Equivalent numbers of cells were stained with anti-CD18 (clone MHM23), and anti-talin [clone TD77; Millipore]), followed by FITC or tetramethyl followed by FITC-labeled goat anti-mouse IgG. Cells were run on a rhodamine–labeled goat anti-mouse IgG, or goat anti-rabbit IgG. F-actin FACSCanto II flow cytometer (BD Biosciences), and the data were an- was visualized with fluorescein-phalloidin or rhodamine-phalloidin. LFA- alyzed with FlowJo (Tree Star). The Journal of Immunology 3663

Split luciferase GEF assays level (Fig. 1A). DOCK8 expression was detected in both NK cell Purification of Luc1-PAK, Luc1-Rhophilin, and Luc2-GTPases; MBP-fu- lines as well as primary human NK cells. Unexpectedly, DOCK8 sion protein purifications; and GEF assays were conducted, as previously was not expressed in Jurkat T cells, whereas we were able to detect described (23), with the following modifications to the assay: a 10-ml al- its expression in primary human CD4+ and CD8+ T cells (Fig. 1A). iquot of a luciferin solution (25 mM Tris/HCl [pH 7.8], 5.0 mM MgSO4, To determine whether DOCK8 is involved in NK cell cyto- 2.0 mM DTT, 0.5 mM luciferin, 0.5 mM EDTA, 100 mM ATP, 50 mM toxicity, we compared cytotoxic activity of YTS cells after de- acetyl-CoA, and 1 mg/ml BSA) containing either 15 nM Luc1-PAK or Luc1-Rhophilin, 50 mM GTP, and 0.2 mM appropriate MBP fusion protein pletion of DOCK8 using two different siRNA oligonucleotide (MBP, DOCK8 DHR2, or VAV2) was added to a 1.5-ml microcentrifuge duplexes. Seventy-two hours after transfection, we examined nat- tube. GEF reactions were initiated by adding 5 ml 150 nM GDP-loaded ural cytotoxicity using the 721.221 B lymphoblastoid cell line Luc2-GTPases, and the luminescence was immediately detected using (Fig. 1B). DOCK8-suppressed YTS cells showed less cytotoxicity a Modulus Luminometer. Subsequent readings were taken every 3 min for each sample. The C-terminal region of DOCK8 (aa 1586–2072) containing compared with control siRNA-transfected cells in all examined E: DHR2 domains (WT or V1985A mutant) was amplified and cloned into T ratios, and this cytotoxicity defect was DOCK8 dosage depen- modified pMal-2c (NEB, Boston, MA) to generate MBP fusion proteins. dent (Fig. 1B, left and center). LUs, a value representing relative cytotoxic activity, were also diminished in the DOCK8-suppressed Statistical analysis cells compared with that of the control group, and again, the All statistical analysis in this study was performed using two-tailed Student impact on LU depended on the level of DOCK8 expression (Fig. t test. 1B, left and right). We also observed a similar decrease in lytic activity toward 721.221 cells by both DOCK8-depleted NKL cells Results and primary human NK clones (Fig. 1C, 1D, top). In addition to DOCK8 is expressed in NK cells, and DOCK8 mediates NK natural cytotoxicity, we examined receptor-mediated cytotoxicity cell cytotoxicity using a reverse Ab-dependent cellular cytotoxicity assay against Although DOCK8 is expressed in T and B cells (4–7), DOCK8 P815 tumor target cells labeled with anti-FcR (anti-CD16) mAb or expression has not been examined in NK cells. Therefore, we pre- anti-NKG2D mAb. Suppression of DOCK8 consistently decreased pared two human NK cell lines, NKL and YTS, and primary human redirected cytotoxicity through both the FcR and NKG2D recep- NK clones along with Jurkat T cell leukemia cell line and primary tors (Fig. 1D, bottom). Taken together, these data identify DOCK8 human T cells, and then examined DOCK8 expression at the protein as a regulator of NK cell–mediated cytotoxicity.

FIGURE 1. DOCK8 is expressed in NK cells and mediates NK cell cytotoxicity. (A) Immunoblot for DOCK8 from whole-cell lysates of human NK cell lines (NKL and YTS), primary human NK clone, primary human CD4+ and CD8+ T cells, and Jurkat T cells. (B) Immunoblot for DOCK8 from whole-cell lysates derived from YTS cells transfected with indicated siRNA oligos. The numbers beneath the blots provide densitometric ratio of DOCK8 signal to a-tubulin setting value for control group as 1 (left). At 72 h posttransfection of indicated siRNA oligos into YTS cells, these cells were incubated with 51Cr- labeled 721.221 cells, and percent specific release was measured over indicated E:T ratios (center). Cytotoxic activity of control and DOCK8-depleted YTS cells was assessed by the number of LUs (right). Data shown are representative (left and center) and average (right) of three to four independent experiments. (C) Immunoblot for DOCK8 from whole-cell lysates derived from NKL cells transfected with indicated siRNA oligos. The numbers beneath the blots provide densitometric ratio of DOCK8 signal to a-tubulin setting value for control group as 1 (top). LU values of NKL cells transfected with indicated siRNA oligos against 721.221 target cells (bottom). Presented data are representative (top) and average (bottom) of three to four independent experiments. (D) Immunoblot for DOCK8 from whole-cell lysates derived from primary human NK clones transfected with indicated siRNA oligos. The numbers beneath the blots provide densitometric ratio of DOCK8 signal to ZAP70 setting value for control group as 1 (top left). LU values of primary human NK clones transfected with siRNA oligos against 721.221 (top right) or P815 target cells coated with anti-CD16 (bottom left) or anti-NKG2D (bottom right) Abs. Presented data are representative (top left) or average of independent experiments performed with .5 NK clones. Error bars indicate SEM. *p , 0.05, ***p , 0.005 compared with control group. 3664 DOCK8 REGULATES NK CELL KILLING

DOCK8 modulates conjugate formation of NK cells to target DOCK8 is required for polarization of LFA-1, actin, cells and granules toward NK cell–target cell interface NK cell cytotoxicity is a multistep process in which each step is We next examined whether DOCK8 regulates key events during regulated by specific molecules to carry out rapid and efficient NK cell cytotoxicity: accumulation of LFA-1 and F-actin at the target recognition and killing (15). The initial step of NK cell CS, as well as granule polarization. We first assessed LFA-1 ac- cytotoxicity is binding of NK cells to target cells via adhesion cumulation at the CS in control or DOCK8-depleted YTS cells molecules. Integrin-mediated firm adhesion to target cells allows conjugated with 721.221 target cells. Whereas LFA-1 was highly NK cells to scan bound targets via various activating receptors polarized to the NK–target CS in most control-transfected cells, (15, 24, 25). Additionally, previous studies using DOCK8-mutant many DOCK8-depleted YTS cells failed to polarize LFA-1 to T and B cells indicated a role for DOCK8 in integrin clustering the interface; LFA-1 was either evenly distributed on the cell at the IS (7, 13). To test whether DOCK8 was involved in ini- surface or weakly polarized (Fig. 3A). When we examined the tial target cell binding, we assessed and compared adhesion of target-contacting region of control-transfected cells using three- control-transfected and DOCK8-suppressed NK cells. Signifi- dimensional reconstruction, we were able to observe thick clus- cantly, YTS, NKL cells, and primary human NK clones depleted tering of LFA-1 into ring-shaped peripheral supramolecular of DOCK8 showed decreased conjugate formation with target activation cluster (Supplemental Video 1). However, DOCK8- cells compared with control NK cells (Fig. 2). The defect in ad- suppressed YTS cells showed much less efficient accumulation hesion of DOCK8-suppressed NK cells was not due to reduction of LFA-1 at peripheral supramolecular activation cluster con- in surface expression of integrin, because we observed similar sistent with what has been observed in DOCK8 mutant B cells CD18 (b2 integrin) surface expression levels in both control- (13) (Supplemental Video 2). For quantification of accumulated transfected and DOCK8-depleted YTS cells and NKLs (Supple- integrin at the CS, we measured sum fluorescence intensity of mental Fig. 1). Therefore, DOCK8 is involved in conjugate for- mation between NK cells and target cells.

FIGURE 2. DOCK8 mediates NK–target binding. At 72 h post- nucleofection of indicated siRNA oligos, YTS (A), NKL (B), and primary FIGURE 3. DOCK8 mediates cytotoxic synapse formation and granule human NK clones (C) were stained with CellTracker Violet. At the same polarization toward NK–target interface. YTS cells were transfected with time, 721.221 target cells were labeled with CFSE. NK cells were incu- indicated siRNA oligos. At 72 h, these cells were conjugated with CMAC- bated for the indicated time at 37˚C with 721.221 target cells, and, using stained 721.221 target cells (blue), incubated for 15 min at 37˚C, and fixed two-color flow cytometry, percentage of conjugated NK cells was calcu- for immunofluorescence staining (A, C, E). Images are shown at 3100 lated based on simultaneous emission of both violet and green fluores- original magnification and are representative of three independent ex- cence. Presented data are average of three (YTS), four (NKL), and three periments. (B, D, F) Scoring of LFA-1 (B), F-actin (D), and perforin (F) (primary human NK clones) independent experiments performed in du- polarization to YTS-721.221 interface (see Materials and Methods for plicate or triplicate. Error bars indicate SEM. *p , 0.05, **p , 0.01 details). Data shown are collected from three independent experiments. compared with control group. Error bars indicate SEM. ***p , 0.005 compared with control group. The Journal of Immunology 3665

NK–target interface and compared it with that of entire NK cell we previously applied for identification of the WASH interactome (Fig. 3B). Fluorescence of integrin at the CS was significantly (19, 31). Interestingly, we identified WASP as a candidate DOCK8- weaker in DOCK8-depleted YTS cells compared with that ob- interacting protein by mass spectrometry (Supplemental Fig. 3B). served in control cells, and this was due to a decrease in both WASP is the founding member of the WASP superfamily of F-actin contact area and density of integrin at the interface (Supplemental nucleation-promoting factors that stimulate the activity of branched Fig. 2A). F-actin via the ubiquitously expressed Arp2/3 complex (32–35). Previous studies in DOCK8-mutant T cells have found dimin- Furthermore, this potential interaction was very intriguing, be- ished accumulation of F-actin at the IS (7). We therefore examined cause the activity of WASP requires binding of active CDC42 whether DOCK depletion in NK cells would affect F-actin ac- (CDC42-GTP) (36). cumulation at the CS. Consistent with what has been observed Recently, it was shown that DOCK8 functions as a CDC42-GEF in T cells lacking DOCK8 (7), DOCK8-suppressed YTS cells via its DHR2 domain (14). Using an in vitro split luciferase assay showed defective polarization of F-actin to the CS (Fig. 3C, 3D). (23), we also observed that the DOCK8 DHR2 domain has robust Again, this defective actin accumulation was due to smaller GEF activity toward CDC42 with limited activity toward RAC1 contact area and weaker density of F-actin at the CS (Supple- and no activity toward RHOA (Supplemental Fig. 4). To confirm mental Fig. 2B). DOCK8-depleted NKLs also showed a similar the interaction of DOCK8 with WASP, we transfected 293T hu- decrease in F-actin and LFA-1 at the CS (data not shown). We next man embryonic kidney (HEK293T) cells with plasmid-encoding examined cytolytic granule polarization, because it was previously FLAG-only/FLAG-tagged DOCK8 along with HA-only/HA-tagged determined that integrin engagement in NK cells provides signals WASP and observed that WASP coimmunoprecipitated together for granule polarization (26, 27). In this experiment, each conju- with DOCK8 (Supplemental Fig. 3C). Importantly, we were also gated YTS cell was placed into three groups depending on the able to confirm that endogenous DOCK8 and WASP coimmu- position of granules from the NK–target cell interface, as follows: noprecipitate with each other in NKLs (Fig. 4A). As expected, front, middle, and back. Cytolytic granules were clearly polarized WASP did not coprecipitate with DOCK8 in Jurkat cells, where just behind the NK–target interface (front) in almost half of DOCK8 is not expressed. conjugated YTS cells, whereas DOCK8-suppressed YTS cells In addition to WASP, we also identified talin as a candidate- showed far fewer conjugates with polarized lytic granules (Fig. interacting partner of DOCK8 (Supplemental Fig. 3B). Talin not 3E, 3F). Granules were either not clearly polarized to the front only participates in integrin affinity maturation by mediating a (middle) or found at opposite position (back) from the NK–target of the integrin after cell activation (inside- interface in DOCK8-depleted YTS cells. These data suggest that out signaling), but also transmits activation signals from DOCK8 not only regulates integrin clustering and F-actin accu- (outside-in signaling) (37–41). Again, exogenous DOCK8 copre- mulation at the CS, but also participates in the development of NK cipitated together with talin (Supplemental Fig. 3D), and we could cell-mediated killing through its effects on lytic granule polari- also confirm reciprocal coimmunoprecipitation of endogenous zation. DOCK8 and talin in NKLs, but not in Jurkat cells (Fig. 4B). Interactions of DOCK8 with each protein do not seem to be de- DOCK8 interacts with talin and WASP pendent on the GEF activity of DOCK8, as we did not observe any Eleven members of the DOCK family of GEFs (DOCK1–DOCK11) change in the interaction between WT DOCK8 and GEF-deficient can be organized into four distinct subgroups (DOCK-A–DOCK-D) mutant (V1985A) (Supplemental Fig. 3E, 3F). based on domain composition and sequence homology (1). Mem- To delineate the interacting region of DOCK8 with WASP and bers of DOCK-A and DOCK-B (DOCK1–DOCK5) are able to talin, we prepared three nonoverlapping fragments of DOCK8 interact with ELMO mainly through a highly conserved N-terminal fused with GST at the N terminus (Fig. 4C). Using lysates from SH3 domain and with Crk adaptor proteins via the proline-rich HEK293T cells and HEK293T cells expressing HA-tagged WASP, motif located at their C terminus (2, 3, 28, 29). Interactions of we mapped the interacting region of DOCK8 for each binding DOCK-A and DOCK-B members with these binding partners partner by pull-down experiments. WASP was mainly pulled down are essential for their normal cellular processes. In the case of with the middle fragment of DOCK8, and weak interaction was DOCK180 (DOCK1), interaction with ELMO has been reported also observed with the N-terminal fragment (Fig. 4D). In contrast, to be critical for regulation of the DOCK180/Rac pathway (2, talin was pulled down with the C-terminal fragment of DOCK8 3, 30). In contrast, DOCK8 and other DOCK-C subfamily members (Fig. 4D). Taken together, our results demonstrate important mo- (DOCK6 and DOCK7), although possessing the DHR1 and DHR2 lecular interactions of DOCK8 with the CDC42 effector WASP as domains, do not contain other identifiable domains and therefore well as the key integrin regulator talin. are not predicted to interact with ELMO and Crk proteins and are thus likely regulated by a different mechanism. DOCK8 mediates polarization of WASP and talin to the Whereas our preceding data provide insight into the role of cytotoxic synapse of NK cells DOCK8 in NK cell–mediated cytotoxicity, the mechanism by From previous studies, it is known that both WASP and talin are which DOCK8 contributes to cellular functions is lacking as a recruited to the CS of NK cells and T cells (42–45). To de- result of limited knowledge regarding the DOCK8 interactome. termine whether DOCK8 mediates accumulation of WASP and Therefore, we first examined whether DOCK8 exists in a multi- talin at the CS, we assessed accumulation of each protein at the endogenously. YTS cell homogenate was sepa- CS in control or DOCK8-depleted YTS cells conjugated with rated via size-exclusion chromatography, and DOCK8-containing 721.221 target cells. In the case of WASP, whereas most of con- fractions were identified by immunoblot (Supplemental Fig. 3A). trol YTS cells showed clear polarization at the CS, DOCK8 sup- Interestingly, we observed that DOCK8 eluted at a much larger pression reduced WASP accumulation at the CS (Fig. 5A, 5B). size (.400 kDa) than expected (∼240 kDa), indicating that DOCK8 In addition, we observed either much weaker accumulation or might be constitutively integrated into macromolecular complexes. nonpolarization of talin at the CS in DOCK8-depleted YTS cells To identify potential DOCK8-interacting proteins, we immuno- (Fig. 5C, 5D). The defects in accumulation of both proteins at precipitated DOCK8 from the DOCK8-enriched fractions and the CS in DOCK8-suppressed NK cells were due to a decrease analyzed associated proteins by mass spectrometry, an approach in both contact area and accumulated density of each protein 3666 DOCK8 REGULATES NK CELL KILLING

FIGURE 4. DOCK8 interacts with WASP and talin. (A and B) Immunoblot analysis of whole-cell lysate and anti-DOCK8, anti-WASP, and anti-talin immunoprecipitates (IP) derived from NKL or Jurkat T cells. Rabbit and mouse IgG (rIgG and mIgG) were used as negative controls for immunoprecipitation assay. (C) Sche- matic representation of DOCK8 fragments used for GST pull-down assays. (D) Lysates from ei- ther HEK293T cells or HEK293T cells trans- fected with HA-tagged WASP were pulled down by GST or GST-DOCK8 fragments and exam- ined for talin or WASP (HA) by immunoblot (top and middle). Coomassie blue staining shows GST fusion proteins used in the pull-down assay (bot- tom). Data shown are representative of three in- dependent experiments.

(Supplemental Fig. 2C, 2D). These data suggest that DOCK8 me- Discussion diates CS accumulation of WASP and talin, two essential regulators Susceptibility to recurrent infections, various types of allergies, as of F-actin reorganization and integrin affinity maturation, respec- well as high IgE levels in the blood are main clinical symptoms tively (Fig. 6). observed in most DOCK8-deficient patients (4–7, 46). Since initial reports of DOCK8-deficient patients in 2009, research has begun to unveil why deficiency of DOCK8 leads to combined immuno- deficiency in humans. Defects in long-lasting humoral responses as well as lack of memory B cells have been observed in both DOCK8-deficient humans and DOCK8-mutant mice (6, 13). In addition, DOCK8 has been shown to be essential for survival of peripheral T cells and memory CD8+ T cells (6, 7, 13, 46), sug- gesting a potential role of DOCK8 in generation of memory in lymphocytes. Recently, DOCK8-knockout mice have been gen- erated (14), and dendritic cells of these mice showed defective migration into lymph nodes after Ag priming, a process required for initiation of adaptive immune responses. Overall, low num- bers of circulating naive T cells, defects in their priming in the lymph nodes, and failure in qualitative enhancement of immune responses by memory B cells and T cells might collectively lead to recurrent infections seen in DOCK8-deficient patients. Importantly, our current understanding does not clearly explain recurrent viral infections, which is most frequently observed in DOCK8 deficiency (4, 5, 7). Cytotoxic lymphocytes are essential for immune protection from viral infections and tumor cells (8– 10). In this study, we have demonstrated that DOCK8 plays an FIGURE 5. DOCK8 mediates localization of WASP and talin toward essential role in mediating NK cell cytotoxicity using human NK CS of NK cells. YTS cells were transfected with indicated siRNA oligos. cell lines as well as primary human NK clones. Considering this At 72 h, these cells were conjugated with CMAC-stained 721.221 target novel role of DOCK8 in NK cells, functional defects of NK cells cells (blue), incubated for 15 min at 37˚C, and fixed for immunofluo- in DOCK8-deficient patients could be part of the cause for fre- rescence staining (A, C). Images are shown at 3100 original magnifi- cation and are representative of three independent experiments. (B and D) quent occurrence of viral infections and cancer in these patients Scoring of WASP (B) and talin (D) polarization to YTS-721.221 interface (4, 5, 7). In this regard, a recent study showing normal cytotoxic (see Materials and Methods for details). Data shown are collected from activity of CTLs from DOCK8-mutant mice is very unexpected three independent experiments. Error bars indicate SEM. ***p , 0.005 and interesting (7). However, it is worthwhile to note that viral compared with control group. infections are much less commonly observed in patients lacking The Journal of Immunology 3667

FIGURE 6. Hypothetical model for how DOCK8 regulates interaction between integrin and actin cytoskeleton networks leading to formation of CS in NK cells. See Discussion for details. PIP2, PtdIns-(4,5)-biphosphate; PIP3, PtdIns-(3,4,5)-triphosphate; PIPKIg,PtdInsphos- phatekinasetypeIg.

CD8+ T cells (11), and HSV is the most common recurrent viral toxic activity (50, 51). These observations are consistent with infection observed in DOCK8-deficient patients (4, 5, 12), because the cytotoxicity defects that we observed in DOCK8-depleted NK cell–mediated killing is essential for eradication of HSV. NK cells. As an essential regulator for cell polarity in all eukary- Therefore, future studies focusing on DOCK8-dependent NK cell otes (52), CDC42 has been implicated in microtubule organizing functions in humans or mice will be important in understanding center (MTOC) polarization in T cells, macrophages, and dendritic clinical features observed in DOCK8-deficient patients. cells (53–55). Consistent with these observations, we showed de- Our data demonstrate the cellular role of DOCK8 in NK cell– fective granule polarization toward the CS in DOCK8-suppressed mediated cytotoxicity is achieved in part through integrin-mediated YTS cells. This suggests that CDC42 activity in NK cells may also adhesion to target cells, as well as polarization of F-actin and be critical for MTOC polarization toward CS, because polarization lytic granules at the NK cell CS. In the case of DOCK8-depleted of cytolytic granules toward CS is achieved by MTOC polarization NK cells, although defects in the accumulation of F-actin and (15). In addition, CDC42 was reported to be responsible for con- polarization of lytic granules were assessed for well-conjugated jugate formation between T cells and APCs, as well as actin po- NK cells only, this does not formally rule out the possibility that larization at the contact site (55, 56). Because similar defects are observed phenotypes might be due to weak adhesion in conju- observed in DOCK8-suppressed NK cells and T cells, one function gated NK cells. Integrin activation alone in NK cells is known to of DOCK8 might be to generate a localized pool of activated mediate accumulation of F-actin at NK–target interface as well CDC42 (CDC42-GTP) at the contact site to promote conjugate as polarization of lytic granules, supporting this possibility (26, formation and F-actin generation via WASP and participate in 27, 47). Further studies focusing on roles of DOCK8 in integrin MTOC polarization (Fig. 6). regulation of NK cells as well as specific NK-activating receptor- Previous studies using B cells and CD8+ T cells from DOCK8- mediated signaling will provide clearly defined cellular roles of mutant mice have demonstrated defects in integrin clustering and DOCK8 in NK cells. F-actin accumulation at the IS (7, 13). Our findings that both talin Recently, Harada et al. (14) reported that DOCK8 is a CDC42- and WASP are DOCK8-interacting partners and that DOCK8 GEF via pull-down assay, in vitro GEF assay, and structure anal- mediates their localization to the CS provide a mechanistic link ysis of DOCK8 DHR2 domain. However, whereas they did not to begin to explain the cellular phenotypes observed in T cells, note GEF activity toward RAC1, another group reported RAC1 B cells, and NK cells, as well as provide an important missing GEF activity of DOCK8 (48). Using an in vitro split luciferase piece of information explaining remarkable similarities between assay that we have developed previously (23), we observed that WASP and DOCK8 deficiency. Significantly, both diseases share the DHR2 domain of DOCK8 was most active toward CDC42, many clinical features such as recurrent infections and high IgE showed modest activity toward RAC1, and no activity toward levels in serum as well as defective cellular immunity (33–35, 57). RHOA. In addition, a GEF-deficient mutant [DHR2-V1985A NK cells from WASP-deficient patients demonstrate defects in (49)] had no detectable GEF activity toward either CDC42 or cytolytic activity, integrin-mediated adhesion, F-actin reorga- RAC1. Therefore, the observed differences in DOCK8 GEF spec- nization, and polarization of cytolytic granules to the CS similar to ificity may be a result of the sensitivity of the experimental con- DOCK8-suppressed NK cells (43, 58, 59). Thus, the finding that ditions. Spatiotemporal activation of CDC42 in NK cells was DOCK8 is a GEF for CDC42 (14) and can interact with the CDC42 recently suggested to be important in CS formation and cyto- effector WASP suggests a putative molecular link for localizing 3668 DOCK8 REGULATES NK CELL KILLING

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