IQ Domain-Containing GTPase-Activating Protein 1 Regulates Cytoskeletal Reorganization and Facilitates NKG2D-Mediated Mechanistic Target of Rapamycin Complex 1 Activation and Cytokine Gene Translation in Natural Killer Cells

Natural killer (NK) cells are innate lymphocytes that play essential roles in mediating antitumor immunity. NK cells respond to various inflammatory stimuli including cytokines and stress-induced cellular ligands which activate germline-encoded activation receptors (NKRs), such as NKG2D. The signaling molecules activated downstream of NKRs are well defined; however, the mechanisms that regulate these pathways are not fully understood. IQ domain-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffold protein. It regulates diverse cellular signaling programs in various physiological contexts, including immune cell activation and function. Therefore, we sought to investigate the role of IQGAP1 in NK cells. Development and maturation of NK cells from mice lacking IQGAP1 (Iqgap1−/−) were mostly intact; however, the absolute number of splenic NK cells was significantly reduced. Phenotypic and functional characterization revealed a significant reduction in the egression of NK cells from the bone marrow of Iqagp1−/− mice altering their peripheral homeostasis. Lack of IQGAP1 resulted in reduced NK cell motility and their ability to mediate antitumor immunity in vivo. Activation of Iqgap1−/− NK cells via NKRs, including NKG2D, resulted in significantly reduced levels of inflammatory cytokines compared with wild-type (WT). This reduction in Iqgap1−/− NK cells is neither due to an impaired membrane proximal signaling nor a defect in gene transcription. The levels of Ifng transcripts were comparable between WT and Iqgap1−/−, suggesting that IQGAP1-dependent regulation of cytokine production is regulated by a post-transcriptional mechanism. To this end, Iqgap1−/− NK cells failed to fully induce S6 phosphorylation and showed significantly reduced protein translation following NKG2D-mediated activation, revealing a previously undefined regulatory function of IQGAP1 via the mechanistic target of rapamycin complex 1. Together, these results implicate IQGAP1 as an essential scaffold for NK cell homeostasis and function and provide novel mechanistic insights to the post-transcriptional regulation of inflammatory cytokine production.

Natural killer (NK) cells are innate lymphocytes that play essential roles in mediating antitumor immunity. NK cells respond to various inflammatory stimuli including cytokines and stress-induced cellular ligands which activate germline-encoded activation receptors (NKRs), such as NKG2D. The signaling molecules activated downstream of NKRs are well defined; however, the mechanisms that regulate these pathways are not fully understood. IQ domain-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffold protein. It regulates diverse cellular signaling programs in various physiological contexts, including immune cell activation and function. Therefore, we sought to investigate the role of IQGAP1 in NK cells. Development and maturation of NK cells from mice lacking IQGAP1 (Iqgap1 −/− ) were mostly intact; however, the absolute number of splenic NK cells was significantly reduced. Phenotypic and functional characterization revealed a significant reduction in the egression of NK cells from the bone marrow of Iqagp1 −/− mice altering their peripheral homeostasis. Lack of IQGAP1 resulted in reduced NK cell motility and their ability to mediate antitumor immunity in vivo. Activation of Iqgap1 −/− NK cells via NKRs, including NKG2D, resulted in significantly reduced levels of inflammatory cytokines compared with wild-type (WT). This reduction in Iqgap1 −/− NK cells is neither due to an impaired membrane proximal signaling nor a defect in gene transcription. The levels of Ifng transcripts were comparable between WT and Iqgap1 −/− , suggesting that IQGAP1-dependent regulation of cytokine production is regulated by a post-transcriptional mechanism. To this end, Iqgap1 −/− NK cells failed to fully induce S6 phosphorylation and showed significantly reduced protein translation following NKG2D-mediated activation, revealing a previously undefined regulatory function of IQGAP1 via the mechanistic target of rapamycin complex 1. Together, these results implicate IQGAP1 as an essential scaffold for NK cell homeostasis and function and provide novel mechanistic insights to the post-transcriptional regulation of inflammatory cytokine production.
Therefore, it is not surprising that IQGAP1 regulates immune cell function through diverse mechanisms. For example, IQGAP1 facilitates phagocytosis (27,28) and caspase-1 activation (29) in macrophages. In neutrophils, IQGAP1 mediates cell migration by promoting CXCR2 signaling processes (30). In B cells, IQGAP1 participates in B cell receptor signaling at the immunological synapse by regulating microtubule organizing center (MTOC) polarization (31); By contrast, IQGAP1 acts as a negative regulator of T cell receptor signaling (32) and nuclear factor of activated T cell-mediated transcription in T cells (33). Given the multitude of IQGAP1-regulated functions, it is expected that IQGAP1 facilitates multiple signaling processes within a specific population of immune cells and, although one study has described a role for IQGAP1 in regulating NK cell cytotoxicity (12), it is likely that IQGAP1 facilitates other NK cell functions.
Regulation of cellular metabolism has been widely studied in lymphocytes (34) and is often directly regulated by the kinase, mTOR (35). The role of mTOR has recently been appreciated in regulating IL-15 signaling, and it is essential for the development and function of NK cells (36,37). Specifically, mechanistic target of rapamycin complex 1 (mTORC1)-induced metabolic reprogramming is prerequisite for NK cell activation (37,38), and mTORC1 activation is required for NK cell effector functions in response to NKR stimulation (39). The components of mTORC1 include the central kinase, mTOR, proline-rich Akt substrate 40, mammalian lethal with Sec13 protein 8 (mLST8), DEPdomain-containing mTOR-interacting protein (Deptor), and, the defining component of mTORC1, a regulatory-associated protein of mTOR (Raptor) (40). Many cellular processes, such as cell growth, cell division, autophagy, and protein translation, are regulated by mTORC1 (41), which reflects its role as a critical regulator of cellular physiology. IQGAP1 is among many proteins that interact with mTORC1 (42). Of note, this interaction is highly conserved as mTORC1 controls cell division in yeast via Iqg1p, the yeast homolog of IQGAP1 (43). In mammalian cells, the interaction between IQGAP1 and mTORC1 promotes proliferation of fibroblasts (44) and hepatocellular carcinoma cell lines (45). Furthermore, the bacterial effector protein, OspB, regulates inTrODUcTiOn Natural killer (NK) cells are innate lymphocytes that mediate many immune functions including antiviral immunity and tumor clearance (1). NK cell activation occurs in response to cytokines as well as stress-induced ligands present on transformed or infected cells (2). These ligands activate NK cells through germline-encoded receptors (NKRs) and, along with inhibitory receptors that primarily interact with MHC class I, specify the NK cell activation repertoire (3). NK cells acquire these receptors at various stages throughout their development (4); however, unlike lymphocytes that mediate adaptive immunity, NK cells do not require either a clonotypic receptor or prior antigen-specific sensitization to become fully activated (5). In response to tumor ligands, the NK cell response can be separated into two primary effector functions. First, NK cells are cytotoxic lymphocytes and can directly lyse transformed cells. Second, NK cells promote the antitumor immune response through the secretion of inflammatory cytokines.
Although these functions often occur concurrently in response to NKR-mediated stimulation, they are regulated through distinct signaling mechanisms, and the molecules involved in these processes are subject to regulation at multiple levels (6)(7)(8). Scaffold proteins are well-known molecular rheostats capable of fine-tuning NK cell signaling in various circumstances (9). For instance, the cytohesin-associated scaffolding protein (CASP) regulates intracellular trafficking and facilitates cell migration as well as IFN-γ secretion in NK cells (10). Another example includes kinase suppressor of Ras (KSR1), a mitogen-activated protein kinase (MAPK) scaffold, which is required for NK cell lytic activity (11). Similarly, a study using the NK-like cell line, YTS, demonstrated a role for IQ domain-containing GTPase-activating protein 1 (IQGAP1) in mediating antitumor cytotoxicity (12). Given the complex nature and incomplete understanding of how scaffold proteins regulate NK cell biology, we seek to understand further the molecular mechanisms that govern NK cell function through further investigation of the multifunctional scaffold protein, IQGAP1.
IQ domain-containing GTPase-activating protein 1 is a ubiquitously expressed, 190-kDa scaffold protein (13) that regulates cellular signaling in multiple contexts (9,14,15). The scaffold function of IQGAP1 is mediated through six functional domains that facilitate its interaction with various proteins and signaling molecules. These interactions enable IQGAP1 to regulate and myriad of signaling pathways critical for diverse cellular functions. Specifically, IQGAP1 has been shown to regulate cytoskeletal reorganization (16,17) as well as multiple signaling mTORC1 activity in an IQGAP1-dependent manner to promote cell proliferation during Shigella infection (46). IQ domain-containing GTPase-activating protein 1 regulates wide range of cellular processes critical for lymphocyte function; therefore, we sought to (1) investigate the role of IQGAP1 in mediating NK cell homeostasis and effector functions and (2) describe potential mechanisms by which IQGAP1 facilitates cytoskeletal reorganization and NKR activation in the context of NK cell signaling and function. Using a global IQGAP1 knockout mouse (Iqgap1 −/− ), we observed defects in cytoskeletal reorganization in NK cells lacking IQGAP1. These alterations were associated with reduced bone marrow (BM) egress and in vitro motility as well as decreased tumor clearance in vivo. Iqgap1 −/− NK cells also showed impaired post-transcriptional cytokine production in response to NKG2D stimulation. This observation was associated with decreased NKG2D-induced mTORC1 activation and global protein synthesis. Our results demonstrate multiple roles for IQGAP1 in facilitating NK cell function and define a novel mechanism in which IQGAP1 positively regulates mTORC1 activation to facilitate cytokine translation in NK cells.

eXPeriMenTal PrOceDUres
Mice and Tumor cell lines Iqgap1 −/− mice were generously provided by our collaborator, André Bernards (Massachusetts General Hospital, Center for Cancer Research, Charlestown, MA, USA). These mice were of a mixed genetic background consisting of C57BL/6 and 129/SJL. Therefore, we bred these mice back to C57BL/6 mice for 11 generations resulting in C57BL/6 Iqgap1 −/− mice. The wild-type (WT) (control mice) used in this study were generated from the same Iqgap1 +/− × Iqgap1 +/− breeding used to generate the F11-C57BL6 Iqgap1 −/− mice. All mice were maintained in pathogen-free conditions at the Biological Resource Center at the Medical College of Wisconsin (MCW), Milwaukee, WI, USA. All animal protocols were approved by the institutional IACUC committee. EL4, RMA, RMA/S, and YAC-1 cell lines were purchased from ATCC (Rockville, MD, USA) and maintained in RPMI-1640 medium containing 10% heat-inactivated FBS (Life Technologies, Grand Island, NY, USA). Generation of H60-expressing EL4 stable cell lines has been described (47).

nK cell isolation and culture
Isolation of splenic NK cells was done using negative selection with the EasySep™ Mouse NK Cell Isolation Kit according to the manufacturer's instructions (STEMCELL Technologies, Vancouver, BC, Canada). Isolated NK cells were determined to be >85% (CD3ε − NK1.1 + ). Generation of IL-2-cultured NK cells was previously described (49). Briefly, single-cell suspensions from spleen and BM were passed through nylon wool columns to deplete adherent populations consisting of B cells and macrophages. Nylon wool-non-adherent cells were cultured with 1,000 U/ml of IL-2 (NCI-BRB-Preclinical Repository, Maryland, MD, USA). The purity of the NK cultures was checked, and preparations with more than 90% of NK1.1 + cells were used on day 7-10. Experiments were conducted with IL-2-cultured NK cells unless otherwise specified.

cytotoxicity assays and In Vivo Tumor clearance
Natural killer-mediated cytotoxicity was quantified using [51Cr]-labeled target cells at varied E:T ratio. Percent specific lysis was calculated using amounts of absolute, spontaneous, and experimental [51Cr]-release from target cells as previously described (50). For analysis of in vivo tumor clearance, RMA and RMA/S cells were labeled with 5 µM cell trace violet and CFSE (Thermo Fisher Scientific, Waltham, MA, USA), respectively, for 10 min and quenched with 10% FBS 1640 medium. Cells were mixed at a concentration of 10 × 10 6 cells/ml of each in PBS, and 200 µl was injected retro-orbitally into each mouse. Spleens were collected 18 h later, and the fluorescence of single-cell suspensions was analyzed by flow cytometry. The ratio of RMA/S to RMA cells was calculated. Clearance of RMA/S was compared with the NK cell-depleted and IgG control groups. For in vivo NK cell depletion, NK1.1 (PK136) and IgG (Isotype control) were purchased from BioXcell (West Lebanon, NH, USA) and 200 µg was injected intraperitoneally into each mouse 24 h before tumor challenge.

rT-qPcr and Microarray analysis
For IFN-γ-encoding mRNA quantification, NK cells were activated for 6 h and processed for RNA extraction (RNeasy Mini Kit; QIAGEN). Next, cDNA was generated using the iScript cDNA synthesis kit (Bio-Rad Laboratories) according to the manufacturer's instructions. Real-time PCR was performed by using a previously published SYBR green protocol with an ABI7900 HT thermal cycler. Transcript in each sample was assayed in triplicate, and the mean cycle threshold was used to calculate the x-fold change and control changes for each gene using ΔCt. The control (housekeeping) gene actb was used for global normalization in each experiment. Primer sequences for Ifng were 5′-GACTGTGATTGCGGGGTTGT-3′ (sense) and 5′-GGCCCGGAGTGTAGACATCT-3′ (anti-sense). For whole transcriptome analysis by microarray, NK cells were activated with 2.5 µg/ml anti-NKG2D antibody for 3 h and RNA was extracted as specified above. RNA collected from NK cells derived from four WT and four Iqgap1 −/− mice were pooled, and cDNA was generated and hybridized to the 430_2 microarray platform with the assistance of Martin Hessner's laboratory (Max McGee National Research Center for Juvenile Diabetes, MCW). Quality Control and data analysis were completed using the TAC software (Affymetrix).

Puromycin incorporation assay
IL-2-cultured NK cells were left unstimulated or activated for 4 h with 5 μg/mL anti-NKG2D mitogenic antibody (A10, eBioscience). Golgi-plug (1:1,000; BD Biosciences) and, where indicated, 50 μg/ml cycloheximide (CHX) was added to the cells for an additional hour. CHX is a potent inhibitor of cellular translation and was used as the negative control for this experiment. Next, 10 μg/ml Puromycin (MilliporeSigma), was added to the cells for 15 min, and cells were harvested, fixed, and permeabilized using the intracellular staining protocol as previously described (48). The cells were then incubated with the anti-puromycin antibody (12D10; MilliporeSigma) at a 1:1,000 dilution. After two washes, AF-488 goat-anti-mouse secondary antibody (Thermo Fisher) was added at 1/1,000 for 30 min at   (52), while Iqgap1 −/− NK cells lack the full-length protein as well as any lower molecular weight cleavage products ( Figure 1A). Although Iqgap1 −/− mice maintain normal numbers of total lymphocytes (Figure 1B), the number of NK cells in the spleen of Iqgap1 −/− mice was significantly reduced while total NK cell numbers were unaltered in the BM (Figures 1C,D). Analysis of NK cell maturation by differential surface expression of CD27 and CD11b (53,54) revealed moderate alterations of CD27 + and CD27 + CD11b + NK cell populations in the spleens of Iqgap1 −/− mice (Figures 1E,F)   antibody, sacrificed after 2 min, and their BM cells were analyzed, which allowed us to quantify the number of NK cells in the sinusoidal vs. parenchymal regions of the BM, an indicator of NK cell trafficking in vivo (Figure 2A). We found a marked reduction of CD45.2-labeled sinusoidal NK cells in Iqgap1 −/− mice, indicating an impairment in their ability to traffic ( Figure 2B). Given the well-known role of IQGAP1 in mediating cell migration and motility (57,58), we evaluated Iqgap1 −/− NK cells in vitro.
Contractile actomyosin complex helps to form blebs, and actin polymerization is required for lamellipodia expansion to facilitate coordinated cellular migration (59). To define the role of IQGAP1 in the directional movement of NK cells, we quantified the number of leading edges per NK in IL-2 culture. The majority of the NK cells from WT mice exhibited directional movement with a single-leading edge ( Figure 2C). However, NK cells from Iqgap1 −/− mice displayed multiple blebs and quantification of the number of lamellipodia suggested impaired directional motility ( Figure 2D). To quantify the motility, we cultured the NK cells in IL-2 and, using a multiphoton confocal microscope, performed live cell imaging in real-time ( Figure 2E). As expected, we observed a significant reduction in overall motility of Iqgap1 −/− NK cells in IL-2 culture (Figure 2F).

lack of iQgaP1 results in impaired MTOc Morphology
To further assess cytoskeletal components involved in cellular motility and migration, we utilized confocal microscopy to evaluate the formation and morphology of the MTOC. Repositioning of the MTOC is an essential component of cellular polarity and is essential for directional cell movement (60). Similarly, the directional translocation of MTOC from the perinuclear region to the proximity of the plasma membrane is central to the formation of the immunological synapse and directed degranulation in NK cells. Lack of IQGAP1 resulted in considerable changes to the structure and appearance of the MTOC. NK cells from the WT mice contained well-defined MTOC located above the perinuclear region (Figure 2G, left panel). However, NK cells from Iqgap1 −/− mice showed an increase in MTOC length associated with an overall increase in cell diameter ( Figure 2G, right panel). We then quantified the size of the MTOC using a reference line across the longitudinal length of individual NK cells. Figure 2H shows representative NK cells from WT and Iqgap1 −/− mice. Quantification of MTOC size in 100 individual NK cells from each strain revealed that the lack of IQGAP1 did not affect the formation of the MTOC; however, the overall size and morphology of the MTOC were significantly increased in Iqgap1 −/− NK cells (Figure 2I). Interestingly, the significant increase in MTOC diameter observed in NK cells lacking IQGAP1 was also associated with morphologically irregular microtubule filaments and an overall increase in the fluorescent intensity of α-tubulin suggesting altered microtubule dynamics in Iqgap1 −/− NK cells. Therefore, these data demonstrate a critical role for IQGAP1 in maintaining proper cytoskeletal structures and subsequent motility in NK cells.

iQgaP1 is required for Optimal nK cell-Mediated antitumor responses In Vivo
To ascertain the role of IQGAP1 in antitumor immunity, we employed the RMA-RMA/S tumor clearance model as an in vivo evaluation of NK cell function. Mice were depleted of NK cells by intra-peritoneal injection of the PK136 monoclonal antibody, 24 h before intravenous tumor inoculation, serve as the control for NK cell-mediated tumor clearance. Using this "self" vs. "missing-self" model ( Figure 3A), in vivo cytotoxic potential of Iqgap1 −/− NK cells was determined through changes in the ratio of RMA/S to RMA tumor cells recovered from the spleens of tumorbearing mice when compared with WT mice as well as those sufficiently depleted of NK cells (Figures 3B,C). The ratio RMA/S to RMA cells recovered from the spleen of Iqgap1 −/− mice was similar to that observed in NK cell-depleted mice, demonstrating an inability of Iqgap1 −/− NK cells to mediate antitumor immunity ( Figure 3D). Using the NK cell-depleted condition as a standard for no tumor clearance, we calculated the percent cytotoxicity of Iqgap1 −/− NK cells and found that it was significantly reduced when compared with the IgG (WT) control ( Figure 3E). To determine if the inability of Iqgap1 −/− mice to clear RMA/S tumors was due to the reduction in peripheral NK cells or if the deficiency was on a per cell basis, we isolated splenic NK cells from WT and Iqgap1 −/− mice and evaluated the cytotoxic potential of these cells against various targets using the standard 4-h 51 Cr-release assay. We found that Iqgap1 −/− NK cells were unable to mediate tumor killing to the level of WT controls ( Figure 3F). This observation was consistent among multiple models that utilize distinct mechanisms of NK cell activation, implying a deficiency in the activation of critical molecular determinants of NK cell cytotoxic function in Iqgap1 −/− NK cells.  hypothesized that the reduced cytotoxic potential of IQGAP1deficient NK cells was due to disrupted actin polymerization. Using fluorescent phalloidin as an indicator of F-actin, we evaluated actin polymerization in WT and Iqgap1 −/− NK cells. A significant reduction in the fluorescent intensity of phalloidin in Iqgap1 −/− NK cells imaged directly after isolation indicated impaired F-actin accumulation in these cells (Figures 4A-C). To determine if reduced actin polymerization in IQGAP1-deficient NK cells was the reason for the observed reduction in cytotoxic activity, we attempted to rescue NK cell-mediated cytotoxicity in  Iqgap1 −/− NK cells with the addition of recombinant IL-2 which is known to bypass WASp-dependent actin polymerization in NK cells (63). Interestingly, the addition of recombinant IL-2 during the 51 Cr-release assay was able to rescue the ability of Iqgap1 −/− NK cells to lyse YAC-1 targets in a dose-dependent manner ( Figure 4D). IL-2-cultured Iqgap1 −/− NK cells were also able to kill various tumor cells in vitro ( Figure 4E). Importantly, this IL-2-mediated rescue in cytotoxic potential was associated with restored F-actin accumulation in Iqgap1 −/− NK cells (Figures 4F-H). Therefore, these data demonstrate the ability of IQGAP1 to mediate actin polymerization required for optimal NK cell antitumor activity.

lack of iQgaP1 impairs cytokine Production in nKr-stimulated nK cells
In addition to facilitating the functional reorganization of cytoskeletal components, IQGAP1 mediates multiple signaling pathways involved in a wide variety of cellular processes.
Therefore, we hypothesized that loss of IQGAP1 would result in additional defects in NK cell function beyond cell-mediated cytotoxicity. To test this hypothesis, we investigated the ability of Iqgap1 −/− NK cells to produce inflammatory cytokines. Evaluation of cytokine production in response to NKR activation revealed significant impairment in the ability of Iqgap1 −/− NK cells to produce cytokines, such as IFN-γ and GM-CSF, as well as chemokines, such as CCL3, CCL4, and CCL5 ( Figure 5A). Cytokines present in the supernatants of activated Iqgap1 −/− NK cells were significantly reduced after stimulation through various NKRs including NKG2D, CD137, NK1.1, Ly49D, and CD244. Since IQGAP1 has previously been shown to regulate intracellular protein trafficking (64), we performed intracellular cytokine anal ysis by flow cytometry to determine if the defect in cytokine production was due to deficiencies in the exocytosis of cytokine-containing vesicles. To this end, we found a significant reduction in Iqgap1 −/− NK cells containing IFN-γ (Figures 5B,C) and CCL3 (Figures 5D,E) in response to NKG2D stimulation. However, Iqgap1 −/− NK cells were able to produce similar amounts of cytokines when stimulated  with IL-12 and IL-18 when compared with WT controls (Figure 5F). These data suggest an NKR-specific role for IQGAP1 with regards to cytokine generation; therefore, we utilized NKG2D as a model receptor to further investigate this phenomenon.

iQgaP1 regulates inflammatory cytokine Production Through a Post-Transcriptional Mechanism
NKG2D stimulation results in the activation of critical signaling pathways that lead to cytokine gene transcription in NK cells (3). IQGAP1 regulates multiple kinase signaling programs known to be activated in response to NKG2D ligation, including Erk1/2 and Akt (14,65). Therefore, we examined the requirement of IQGAP1 in NKG2D-mediated signaling in the context of cytokine production. Western blot analysis showed similar induction of Akt phosphorylation at both the Ser 473 and Thr 308 sites in Iqgap1 −/− NK cells ( Figure 6A). Furthermore, degradation of IκBα ( Figure 6B) and the phosphorylation of MAP kinases, Erk1/2, and Jnk1/2 ( Figure 6C) were also unaltered. Consistent with these observations, we found no defect in the induction of Ifng mRNA in Iqgap1 −/− NK cells in response to NKG2D stimulation ( Figure 6D).
To determine if IQGAP1 has a global effect on mRNA expression, we performed a genome-wide transcriptome analysis by microarray. Comparison of NKG2D-activated WT and Iqgap1 −/− NK cells showed similar induction of many cytokine mRNAs including Ifng, csf1, ccl3, and ccl4 (Figure 6E). Thus, the reduction  Interestingly, Iqgap1 −/− NK cells showed notable deregulation in the mRNA expression of several solute carrier proteins ( Figure 6F). These included amino acid transporters, such as the glutamine transporters, SLC1A2 and SLC1A7. Emerging evidence suggests a role for amino acid transporters as amino acid sensors capable of monitoring nutrient availability to control the activation of mTORC1, a master regulator of protein synthesis (66,67). Although IQGAP1 has not been directly implicated in amino acid sensing, previous reports have demonstrated a functional interaction between IQGAP1 and mTORC1 (22,46). Therefore, we next evaluated the contribution of mTORC1 activation to IFN-γ production and addressed mTORC1 activation in Iqgap1 −/− NK cells.
iQgaP1 Promotes nKg2D-induced mTOrc1 activation in nK cells To address the requirement of IQGAP1-mediated mTORC1 activation for IFN-γ production in NK cells, we inhibited mTORC1 with rapamycin ( Figure 7A). Rapamycin treatment resulted in a significant decrease in IFN-γ production in NKG2D-stimulated WT NK cells; however, while rapamycin treatment also reduced IFN-γ production in Iqgap1 −/− NK cells, the change was not statistically significant ( Figure 7A). These data are consistent with a previous report demonstrating that mTOR is dispensable for IFN-γ production in response to IL-12 and IL-18, while IFN-γ production in response to NKR stimulation requires mTOR signaling (36). Since the reduction in IFN-γ generation in Iqgap1 −/− NK cells mirrors that of rapamycin-treated WT NK cells when stimulated via NKG2D and Iqgap1 −/− NK cells have no apparent defect in IFN-γ production in response to IL-12-and IL-18mediated stimulation, we sought to further evaluate mTORC1 activation and signaling in response to NKG2D stimulation in Iqgap1 −/− NK cells. The surface expression of the amino acid transporter CD98 is positively regulated by mTORC1 activation (68,69), and in NK cells, its induced expression is impaired by rapamycin treatment (38). As shown in Figures 7B,C, we found that NKG2Dinduced surface expression of CD98 was significantly reduced in Iqgap1 −/− NK cells. To further examine this defect, we investigated the mTORC1-mediated activation of specific signaling molecules in response to NKG2D stimulation. The ribosomal protein S6 is a component of the 40S ribosomal subunit and is phosphorylated at Ser 240/244 by p70S6 kinase (S6K1), a direct target of mTORC1 (41). Using intracellular flow cytometry, we found a significant reduction in the phosphorylation of S6 at Ser 240/244 following NKG2D stimulation in Iqgap1 −/− NK cells (Figures 7D,E). Taken together, these results suggest a role for IQGAP1 in promoting NKG2D-mediated mTORC1 activation in NK cells.

Phosphorylation of s6 and global Protein synthesis are reduced in Iqgap1 −/− nK cells
To further investigate the activation of the mTORC1 → S6K1 → S6 signaling axis in Iqgap1 −/− NK cells, we conducted a time course experiment to examine S6 phosphorylation in response to NKG2D stimulation by western blot. As shown in Figures 8A,B, Iqgap1 −/− NK cells were unable to sufficiently phosphorylate S6 at Ser 235/236 as well as Ser 240/244 . Upon mitogenic stimulation, S6 is phosphorylated at Ser 235/236 by P90S6K (RSK) through activation of the MAPK pathway; however, rapamycin-mediated inhibition of mTORC1 decreases these phosphorylation events (70)(71)(72), which suggests an exclusive role for mTORC1 in regulating S6 phosphorylation at multiple residues. Phosphorylation of S6 promotes its recruitment to the mRNA cap-binding complex and may play a key role in facilitating translation initiation (71), which suggests a potential role for IQGAP1 in mediating translation in activated NK cells. To evaluate global protein synthesis, we utilized a puromycin incorporation assay. Puromycin is a structural analog of aminoacyl tRNAs and is incorporated into the nascent polypeptide chain of an actively translating ribosome (73,74). Thus, the amount of puromycin retained within a cell directly relates to the amount of protein synthesis occurring within that cell. Analysis of puromycin incorporation in Iqgap1 −/− NK cells revealed a significant decrease in global protein synthesis in response to NKG2D stimulation (Figures 8C,D). Along with protein translation, mTORC1 also regulates metabolic reprogramming; therefore, we assessed the metabolism of Iqgap1 −/− NK cells using the Seahorse stress test system ( Figures S2A-D in Supplementary Material). The rates of extracellular acidification (ECAR) and oxygen consumption (OCR) were unaltered in Iqgap1 −/− NK cells, suggesting that mTORC1 activity and the metabolic processes regulated by this complex are maintained in Iqgap1 −/− NK cells in the absence of NKR stimulation. Since Iqgap1 −/− NK cells were unable to sufficiently translate proteins in response to activation via NKG2D, our data suggest a role for IQGAP1 in regulating NKR-mediated cytokine translation through the activation of mTORC1.

DiscUssiOn
In this study, we define multiple roles for IQGAP1 in regulating NK cell physiology. Despite having no apparent defects in development or maturation, Iqgap1 −/− mice have significantly less splenic NK cells and altered NK cell distribution within the BM. This apparent deficiency in BM egress is associated with altered cell polarization and motility. However, it is possible that IQGAP1 plays a highly specific role in NK cell homeostasis within the BM. NK cell development occurs within the BM, and their movement between specific niches is a highly regulated process controlled by chemokine gradients (55). Furthermore, retention of NK cells within the BM is controlled by CXCL12-CXCR4 signaling. Indeed, IQGAP1 has been shown to regulate chemokine receptor signaling in immune cells. For instance, IQGAP1 interacts with CXCR2 in neutrophils (30) and promotes CXCR4 function and trafficking in Jurkat T cells (75). The role of IQGAP1 in these interactions may be of importance with regard to BM egression of NK cells; however, IQGAP1 can also directly regulate cell migration by regulating phosphoinositol signaling at the leading edge of migrating fibroblasts (57). Therefore, given the marked decrease in leading edge formation and cell motility in vitro, our data suggest that the alterations within the BM compartment are due to defects in conserved signaling mechanisms that are critical for leading-edge formation and cell migration.
We also observed a significant deficiency in the antitumor response of Iqgap1 −/− NK cells. The inability of Iqgap1 −/− mice to sufficiently clear RMA/S tumors in vivo demonstrates an apparent defect in the ability of Iqgap1 −/− NK cells to mediate antitumor immunity in response to this "missing-self" tumor. The RMA tumor cell line is of C57BL/6 origin and maintains normal levels of MHC Class I surface expression. Therefore, RMA cells are recognized by NK cells as immunological "self" and are not targeted for lysis due to interactions between MHC Class I expressed on the target cell and inhibitory receptors on NK cells (76). Conversely, the RMA/S tumor line, derived from RMA, harbors a nonsense mutation in Tap2, a key peptide transporter and regulator of MHC Class I surface expression (77,78). This mutation results in a marked reduction of MHC Class I surface expression and represents the immunological "missing-self. " In the absence of inhibitory signaling induced by MHC Class I expressed on the target cell, NK cells are able to efficiently lyse RMA/S tumors through the targeted release and delivery of granules containing the pore-forming enzyme, perforin, along with a specific class of serine proteases known as granzymes (79). Moreover, when isolated and co-cultured with various tumor cells, Iqgap1 −/− NK cells have significantly reduced cytotoxic potential, which suggests a role for IQGAP1 in mediating NK cell cytotoxicity through a cell-intrinsic mechanism.
Specific F-actin reorganization mediated by actin-related protein-2/3 branching is required for the cytotoxic function of NK cells (80). Given that F-actin in isolated Iqgap1 −/− NK cells is compromised, it is likely that IQGAP1 regulates NK cell cytotoxicity via actin polymerization. In line with this idea, the addition of IL-2 did indeed rescue the cytotoxic potential as well as the ability of Iqgap1 −/− NK cells to accumulate F-actin. It is known that IQGAP1 stimulates actin polymerization through WASp (17) and WASp is required for cytotoxicity in uncultured NK cells (62). However, in a study using NK cells isolated from patients with Wiskott-Aldrich syndrome, Orange et al. demonstrated the ability of IL-2 to rescue both cytotoxicity and actin polymerization in WASp-deficient NK cells (63). Furthermore, this group showed that IL-2 was able to bypass WASp-dependent actin polymerization by activating the WASp-related actin reorganizer, WASp-family verprolin-homologous protein (WAVE) (63), and IQGAP1 does not mediate actin polymerization through WAVE (17). Therefore, it appears that IL-2 renders IQGAP1 dispensable for actin polymerization and, by extension, cytotoxicity in IL-2conditioned NK cells.
Given the role of IQGAP1 in regulating multiple signaling pathways necessary for NK cell function, we investigated the ability of Iqgap1 −/− NK cells to produce inflammatory cytokines. Our data show that Iqgap1 −/− NK cells produce significantly less cytokines in response to NKR activation; however, Iqgap1 −/− NK cells have no defect in cytokine production when activated with IL-12 and IL-18. Activation of NK cells via NKRs, such as NKG2D, and cytokines, such as IL-12 and IL-18, initiate distinct signaling programs in NK cells. For instance, IL-18 alone is not sufficient to stimulate IFN-γ production in NK cells but synergizes with IL-12 signaling (81). Therefore, a marked increase of IFN-γ production is expected when IL-18 is added in combination with IL-12 to activate NK cells. IL-12 signaling is propagated by the Janus-activated kinase/signal transducer and activator of transcription (STAT) pathway (81) and IL-18, a member of the IL-1 cytokine family, signals via the IL-18 receptor (IL-18R) complex (82). Specifically, STAT4 induction by IL-12 promotes IFN-γ production by enhancing Ifng gene transcription (81), while IL-18R signaling activates p38 MAPK which enhances Ifng transcript stability (83).
Although stimulation of NK cells via IL-12 and IL-18 results in substantial IFN-γ production, chemokine generation via this activation mechanism is not as robust compared with activation through NKRs. Differences in signaling mechanisms may be one potential explanation for this discrepancy. Distinct from IL-12 or IL-18, signaling events initiated in response to NKG2D stimulation are mediated through the adaptor molecules DNAXactivating protein of 10 kDa (DAP10) and DNAX-activating protein of 12 kDa (DAP12) via YXXM tyrosine-based and immunoreceptor tyrosine-based activation motifs (ITAMs), respectively (3). Upon NKG2D stimulation, the YXXM motif of DAP10 recruits and activates the p85 subunit of phosphoinositide 3-kinase, while the ITAM motif of DAP12 recruits ZAP70 and Syk to mediate NK cell activation via PLC-γ (84,85). NGK2D signaling mediated through either DAP10 or DAP12 activates MAP kinases, Erk1/2 and P38, as well as Akt which results in the activation of critical transcription factors, such as AP-1 and NF-κB, that promote cytokine gene transcription (7). Therefore, IQGAP1 appears to regulate NKR-specific processes required for inflammatory cytokine generation.
Another essential difference between NKR and IL-12-and IL-18-induced IFN-γ generation is the requirement of the metabolic regulator, mTOR (36). In fact, inhibition of the critical metabolic processes, glycolysis, and oxidative phosphorylation, significantly reduces NKR-mediated IFN-γ generation, whereas IFN-γ production in response to IL-12 and IL-18 remains unaffected in NK cells (86). Therefore, we assessed these two critical metabolic processes in Iqgap1 −/− NK cells using the Seahorse stress test system. Consistent with the observation that Iqgap1 −/− NK cells have no defect in IL-2-induced proliferation in our culture system (data not shown), the rates of extracellular acidification and oxygen consumption were unaltered in Iqgap1 −/− NK cells suggesting that mTORC1 activity and the metabolic processes regulated by this complex are maintained in Iqgap1 −/− NK cells in the absence of NKR stimulation.
To further investigate this defect in IFN-γ production, we evaluated vital signaling molecules activated downstream of NKG2D and found no differences in NKG2D-induced stimulation of these pathways in Iqgap1 −/− NK cells. Importantly, we also found no difference in NKG2D-induced Ifng gene transcription in Iqgap1 −/− NK cells. We next performed a genome-wide transcriptome analysis by microarray, which showed similar induction of NKG2D-induced cytokine transcription in WT and Iqgap1 −/− NK cells. However, the mRNAs of many solute carriers were differentially expressed in Iqgap1 −/− NK cells in response to NKG2D stimulation. Solute carrier proteins play a crucial role in cellular responses to nutrient availability by regulating mTORC1 activation through amino acid sensing (67); therefore, we sought to investigate further mTORC1 activation in Iqgap1 −/− NK cells stimulated via NKG2D.
Using the mTORC1 inhibitor, rapamycin, we confirmed the requirement for mTORC1 in NKG2D-induced IFN-γ production and, as expected, IFN-γ production in response to IL-12 + IL-18 was unaffected by rapamycin. Further investigation of mTORC1 activation by CD98 upregulation and S6 phosphorylation demonstrated a deficiency of mTORC1 activation in Iqgap1 −/− NK cells stimulated via NKG2D. S6 is a well-known regulator of cellular translation (71) and, consistent with these previous observations, global protein synthesis in response to NKG2D stimulation was reduced in Iqgap1 −/− NK cells. Along with protein translation, mTORC1 also regulates metabolic reprogramming required for optimal NK cell functions (36,86), suggesting defects in cytokine production in IQGAP1-deficient NK cells could be a result of metabolic defects in addition to protein translation. Consistent with the observation that Iqgap1 −/− NK cells have no defect in IL-2-induced proliferation in our culture system (data not shown), the rates of extracellular acidification and oxygen consumption were unaltered in Iqgap1 −/− NK cells, suggesting that mTORC1 activity and the metabolic processes regulated by this complex are maintained in Iqgap1 −/− NK cells in the absence of NKR stimulation. The molecular mechanism(s) by which IQGAP1 might mediate mTORC1-driven processes remains to be shown in NK cells; however, our observations suggest an essential role for IQGAP1-mediated mTORC1 activation in regulating protein translation in NKR-stimulated NK cells.
Our results demonstrate multiple roles for IQGAP1 in regulating NK cell effector responses. Although the role of IQGAP1 in facilitating cytoskeletal reorganization and cytotoxicity has been described in the human NK-like cell line, YTS (12), whether the function of IQGAP1 in regulating mTORC1 signaling is conserved between mice and humans was not addressed in this study and remains to be determined. With 98% amino acid homology between mice and humans (9), IQGAP1 is a highly conserved scaffold protein. Therefore, investigations into how IQGAP1 modulates NK cell effector functions may have clinical applications as recent advances in cellular immunotherapy have led to the utilization of NK cells as effector lymphocytes with anti-cancer properties in patients suffering from a wide range of malignancies (87). NK cell therapy has shown great promise as an anti-cancer therapeutic in clinical trials (88). However, an in-depth understanding of how NK cells function at the molecular level is required to further advance the therapeutic potential of NK cells. Cytokine release syndrome, induced by excessive immune cell activation, has been associated with the use of cellular immunotherapy for cancer treatment and clinical intervention is required to mitigate this potentially dangerous condition (89). However, targeted genetic or pharmacological manipulation of cellular products pre-transfusion may serve as a promising approach to reduce or eliminate many of the adverse effects associated with cellular immunotherapy. Multiple studies have concluded that, in NK cells, cytokine secretion and cytotoxicity are regulated differentially downstream of ITAM-containing receptors, such as NKG2D (7,90,91). Our findings demonstrate that IQGAP1 facilitates NK cell function at multiple levels and plays distinct roles in facilitating cytotoxicity vs. cytokine production. Therefore, the molecular mechanisms by which IQGAP1 differentially regulates NK cell function is of clinical significance and warrants further investigation for the development of next-generation NK cell-based therapeutics.

eThics sTaTeMenT
All mice used in this study were utilized responsibly, and all protocols were approved by the institutional IACUC committee at the Medical College of Wisconsin (MCW), Milwaukee, WI, USA.
aUThOr cOnTriBUTiOns AA designed the study and performed experiments, data collection, analysis, and drafted the original manuscript. AT, ZG, JS, CY, NS, and KD participated in data collection and assisted in manuscript preparation. MT and SM conceived of the study, participated in its design, and performed data analysis and writing of the manuscript. All the authors have read and approved the final manuscript.    (e) Cell death was assessed using Annexin V and propidium iodide in splenic NK cells from WT and Iqgap1 −/− mice. Error bars represent SD with four mice from two independent experiments, *p < 0.05 using two-way ANOVA accounting for multiple comparisons.
FigUre s2 | Metabolic characterization of Iqgap1 −/− natural killer (NK) cells. Seahorse assay was used to measure ECAR and OCR rates in IL-2 cultured wild-type and Iqgap1 −/− NK cells. (a,B) A glycolytic stress test was used to evaluate glycolytic parameters following the addition of glucose, oligomycin, and 2-DG. (c,D) A mitochondrial stress test was used to evaluate oxidative phosphorylation following the addition of oligomycin, FCCP, and antimycin A. Error bars represent SD using three mice from two independent experiments. reFerences