Elucidation of the integrin LFA-1–mediated signaling pathway of actin polarization in natural killer cells

Natural killer (NK) cells are innate lymphocytes that have the capacity to kill virally infected or transformed cells without prior sensitization. When stimulated to kill by activating receptors, NK cells undergo tightly regulated steps leading to target cell lysis. The first step is adhesion to the target, followed by polarization of the actin cytoskeleton. Subsequently, cytotoxic granules and the microtubule organizing center are mobilized toward the bound target cell and granules fuse with the plasma membrane. Perforin and granzymes are then exocytosed, resulting in apoptosis of the target cell. The leukocyte integrin LFA-1 is known to be important for NK-cell function as LFA-1–deficient NK cells have defective cytotoxicity.¹  In addition to mediating the adhesion of NK cells to target cells, LFA-1 is an essential component of the immunologic synapse formed by NK cells as they bind to target cells. Moreover, recent studies show that LFA-1 also functions as a costimulatory receptor on NK cells.²  The binding of LFA-1 on NK cells to its ligand intercellular adhesion molecule-1 (ICAM-1) on targets is sufficient for granule polarization in human NK cells.³  Ligation of LFA-1 on NK cells also leads to rapid phosphorylation and activation of the guanine nucleotide exchange factor Vav1 and the kinase Pyk2, which both play a role in actin polymerization signaling.^4,5  We have shown that binding of LFA-1 on NK cells to ICAM-1, in the absence of other activation signals, results in localized actin accumulation and that the cytoskeletal adaptor protein talin is required for this process.⁶  However, the precise mechanism by which LFA-1 ligation results in the reorganization of the actin cytoskeleton and how talin plays a critical role in the process remains unclear.

Talin is a large adaptor protein with a 50-kDa head and a 200-kDa rod domain. Talin head contains a FERM domain that binds the β subunit of integrin cytoplasmic tails and activates integrin functions. The FERM domain also contains binding sites for F-actin, phosphatidylinositol 4-phosphate 5-kinase type I γ (PIPKIγ)⁷  and the PIPKIγ product phosphatidylinositol-4,5-bisphosphate (PIP₂). The rod domain contains at least 2 actin binding sites and multiple binding sites for the actin cross-linking protein vinculin.⁸  Talin may be in an autoinhibited conformation in resting cells, mediated by interactions between its head and rod domains. It is unclear what relieves this autoinhibition, although binding of PIP₂ is proposed to play a role.⁸  Human NK cells express PIPKIγ, and PIP₂ is enriched at the interface between an NK cell and a sensitive, but not resistant, target cell. Moreover, disruption of this PIP₂ enrichment impairs cytotoxicity.⁹ 

The importance of the actin cytoskeleton in immune cell function has been well documented.¹⁰  The formation of branched filaments by the actin nucleating Arp2/3 complex is crucial for immune synapse formation.¹¹  An important link between integrins and Arp2/3 is vinculin. In adherent cells forming nascent focal adhesions, vinculin interacts with Arp2/3 and localizes it to the leading edge of stimulated cells.¹²  Vinculin also interacts directly with actin and talin through multiple vinculin binding sites in the talin rod. Thus, vinculin appears to play a role in Arp2/3 regulation in addition to its well-studied role as an actin cross-linking protein. Arp2/3 itself possesses little biochemical activity and must be activated by a nucleation promoting factor. The primary nucleation promoting factor at work in hematopoeitic cells is Wiskott-Aldrich Syndrome protein (WASP), the product of the gene mutated in Wiskott-Aldrich Syndrome (WAS).¹³  A hallmark of WAS is immune dysfunction, and NK cells from WAS patients have decreased cellular cytotoxicity due to impaired conjugate formation.¹⁴  WASP is found at the immune synapse and, upon activation, promotes Arp2/3-mediated actin polymerization. WASP is held in an autoinhibited conformation, and concurrent binding of PIP₂ and active Cdc42 GTPase relieves this autoinhibition.¹³  How WASP is recruited to the immune synapse is unclear. In T cells, recruitment of WASP is independent of Cdc42 but antigen-dependent.¹⁵  As WASP also binds SH3 domain-containing proteins, it has been proposed that WASP is recruited after T-cell receptor (TCR) signaling via adaptors such as CrkL and LAT.¹³  However, in cytotoxic T cells WASP localizes after LFA-1 ligation to ICAM-1, indicating a TCR-independent mechanism.¹⁶  How WASP is recruited in NK cells is unknown.

In this study we investigated the mechanism of LFA-1 mediated outside-in signaling that induces reorganization of the actin cytoskeleton using talin- and WASP-knockout (KO) NK cells. Our results show that talin, through its association with vinculin and PIPKIγ, mediates 2 signaling pathways that recruit Arp2/3 and WASP to the site of LFA-1 ligation and induce localized actin polymerization.

C57BL/6 mice were bred in the British Columbia Cancer Research Center Animal Resource Center. The animal care committee of the University of British Columbia approved all animal use, and animals were maintained and killed under humane conditions in accordance with the guidelines of the Canadian Council on Animal Care. C57BL/6 Was^−/− mice,¹⁷  129-derived Tln1^−/− embryonic stem (ES) cells and 129-derived wild-type (WT) ES cells have been described.¹⁸  OP9 stromal cells were from RIKEN. Anti-WASP (B9), -talin (C-20), and -PIPKIγ¹²  were from Santa Cruz Biotechnology. Purified rat cerebrum tissue extract (SC-2392) was from Santa Cruz Biotechnology. Anti-vinculin (V284) was from Millipore. Anti-PIP₂ was from Stressgen. Anti-Arp2 was from Abcam. Rhodamine-conjugated and Alexa 647–conjugated phalloidin, Alexa 488–conjugated donkey anti–mouse IgG, and Alexa 568–conjugated donkey anti–rabbit IgG were from Invitrogen Molecular Probes. Murine recombinant soluble ICAM-1 was from StemCell Technologies. Polyinosinic-polycytodylic acid (poly I:C), m-3M3FBS and poly-L-lysine (PLL) were from Sigma-Aldrich. Fluorescence staining of cells and flow cytometry were done as described.⁶ 

NK cells were isolated from 6–8 week old C57Bl/6 mice using EasySep negative selection kit (StemCell Technologies). Purified NK cells were cultured for 7 days in RPMI 1640 media supplemented with 10% fetal calf serum, penicillin, streptomycin, 5 × 10⁻⁵M 2-mercaptoethanol (StemCell Technologies) with 1000 U/mL interleukin-2 (IL-2; Peprotech) or on OP9 stromal cells with 25 ng/mL IL-15 (StemCell Technologies). Generation of NK cells from ES cells has been described.⁶  Poly I:C (200 μg) was intraperitoneally injected into 6- to 8-week-old C57Bl/6 mice and NK cells were isolated from spleen 18 to 24 hours after the injection. Where indicated, WT and WASP-KO NK cells were incubated with 4 ng/mL recombinant mouse IL15/IL15Rα complex (eBioscience) for 5 to 7 days.

Cultured NK cells were harvested and 5 × 10⁶ cells were lysed in 1 mL of ice cold lysis buffer (10mM Tris- (tris(hydroxymethyl)aminomethane) HCl, pH 8.0, 150mM KCl, 1mM EDTA [ethylenediaminetetraacetic acid] pH 8.0, 1% Triton X-100, 0.5% bovine serum albumin [BSA]) in the presence of protease inhibitors (Roche). After centrifugation at 16 060g for 20 minutes at 4°C, supernatants were taken as cell lysates. Immunoprecipitations were performed by incubating lysates with 5 μg of antibody on Protein G Dynabeads (Invitrogen) for 10 minutes at room temperature. The beads were washed 3 times with wash buffer (lysis buffer without BSA) and the immunoprecipitated proteins were eluted using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) reducing buffer. Samples were divided into 2 equal portions, separated by SDS-PAGE gel electrophoresis, blotted to polyvinylidene fluoride membrane (Pall) and detected by primary antibodies and horseradish-peroxidase conjugated secondary antibody with a chemiluminescent system (Amersham) according to manufacturer's protocol. Immunoprecipitations after binding to immobilized ICAM-1 were done as described.¹⁶ 

The preparation of ICAM-1– and PLL-coated beads and confocal microscopy using cells bound to these beads has been described.⁶  Where indicated, cells were treated with 20μM m-3M3FBS in dimethyl sulfoxide (DMSO) for 30 minutes at 37° before addition of cells to beads. Untreated cells were incubated with DMSO as a control. All primary antibodies and Alexa 647–conjugated phalloidin were used at a 1:50 dilution at 4°. All secondary antibodies were used at 10 μg/mL. Rhodamine phalloidin was used at 2 U/mL. Coverslips were mounted using Vectashield mounting medium. Cell-bead conjugates were analyzed using a Nikon C1-si confocal microscope with a 100×/1.45 numeric aperture oil objective, zoom 4. Images were collected using sequential scanning. Images were processed and merged using Volocity software (Improvision) and exported as .TIF files. For quantification of fluorescence intensity at the site of binding, the sum intensity in the channel of interest was determined using Volocity software (Version 5.0) for the area of contact between a cell and a bead and compared with the sum fluorescence intensity of the whole same cell using the following equation: [sum intensity (contact site)/sum intensity (whole cell)] × 100. For quantification of PIP₂ accumulation, fluorescence intensity of staining at the point of contact between a cell and a bead was determined using Volocity software (Version 5.0) and compared with fluorescence intensity of an area of cell membrane of equal size opposite the contact site. Fluorescence staining at the point of contact is expressed as a ratio over the fluorescence staining at the opposite point. For membrane profiles of PIP₂ staining, fluorescence intensity along a line drawn from the point of contact between a cell and a bead to the opposite side of the cell was measured using ImageJ software (Version 1.4of; National Institutes of Health). These values were exported to GraphPad software (Version 5) and graphed accordingly.

The Student 2-tailed t test was used for comparison of sets of matched samples.

NK cells purified from normal mouse spleen did not kill the standard NK target YAC-1 cells, did not have a detectable level of granzyme B or perforin and did not adhere to immobilized ICAM-1 (data not shown). This is consistent with previous reports showing that, while resting human NK cells show some latent cytotoxicity, mouse NK cells do not express granzymes and perforin until activation, suggesting murine and human NK cells have fundamental differences in their basal levels of activation.¹⁹  Therefore, we injected poly I:C into mice to prime NK cells. NK cells purified from poly I:C–injected mouse spleen readily bound to cell size beads coated with ICAM-1. PLL-coated beads, which efficiently bind to cells but do not ligate specific receptors, were used as a control. The cell-bead conjugates were fixed, permeabilized and stained for proteins of interest. As previously reported,⁶  talin and F-actin localized to the site where ICAM-1–coated beads were bound, whereas no such localization of talin or actin was seen with PLL-coated beads (Figure 1A-B). To further investigate the signaling pathways generated by LFA-1 ligation that result in localized actin accumulation we examined the subcellular localization of proteins involved in actin polymerization. Binding of ICAM-1–coated beads, but not PLL-beads, induced significant accumulation of Arp2/3 (Figure 1C), vinculin (Figure 1D), and WASP (Figure 1E) at the binding site. To quantify the level of protein accumulation seen with confocal microscopy, we measured the fluorescence intensity of staining at the point of contact between the cell and the bead and compared it to total fluorescence intensity of the entire cell (Figure 1 bar graphs to right of each corresponding panel of microscopy images). The results confirmed that these proteins localized to the site of bead binding in ex vivo NK cells after binding to ICAM-1–coated beads. To confirm specificity of antibody staining, we probed fixed and permeabilized NK cells with isotype control antibody for each protein of interest, and found that isotype control antibodies did not result in any significant staining (supplemental Figure 3, available on the Blood Web site; see the Supplemental Materials link at the top of the online article.).

Many previous studies on LFA-1 signaling in NK cells were done using in vitro cultured cells. Therefore, we tested whether the same protein localization was seen with in vitro cultured NK cells. Purified splenic NK cells, without priming with poly I:C, were cultured with IL-15 in the presence of OP9 stromal cells or with a high dose (1000 U/mL) of IL-2. In both cultures, NK cells vigorously expanded, enlarged, contained high levels of granzyme B and were highly cytotoxic against YAC-1 cells (data not shown). IL-15 activated cells became cytotoxic during the culture and killed off all OP9 stroma cells. Flow cytometric analysis showing that all cells in the final culture expressed LFA-1, which is not expressed on stromal cell lines. Thus, NK-cell populations did not contain contaminating OP9 stromal cells (data not shown). Further, staining of NK cells cultured with recombinant IL-15/IL-15Rα complex in the absence of stromal cells showed the same patterns of talin, vinculin, Arp2/3, WASP, and actin accumulation as those seen in NK cells cultured on OP9 with IL-15 (supplemental Figure 3). The distribution of talin, actin, and Arp2/3 in the in vitro–cultured NK cells was similar to that seen with the poly I:C–primed ex vivo NK cells. Unexpectedly, binding of ICAM-1–coated beads did not induce vinculin localization in IL-2–cultured NK cells (Figure 1D). Similarly, WASP did not significantly localize to the bead binding site in IL-2–cultured NK cells (Figure 1E), which is consistent with a previously described WASP-independent pathway of actin polymerization.^17,20  As IL-2–activated NK cells appear to be different from ex vivo or IL-15–activated NK cells, we compared the expression of LFA-1, the NK-cell activating receptor NKG2D, and the NK-cell inhibitory receptors Ly49C/I and Ly49G in all 3 types of NK cells. While LFA-1, NKG2D, and Ly49G expression was similar between the ex vivo and cultured cells, Ly49C/I was significantly down modulated after culture in IL-2, but not IL-15 (data not shown).

We have previously shown that talin is required for the accumulation of F-actin in NK cells bound to ICAM-1–coated beads.⁶  Talin-KO mice are not viable.²¹  Therefore, we generated NK cells in vitro from talin-KO and WT ES cells as in our previous study⁶  and examined the role of talin in the recruitment of actin polymerization machinery. As talin-KO NK cells do not bind ICAM-1, they were incubated in the presence of 1mM Mn⁺⁺ to induce high-affinity LFA-1 and binding to ICAM-1. In WT cells bound to ICAM-1 beads, actin (Figure 2A), Arp 2/3 (Figure 2B), vinculin (Figure 2C), and WASP (Figure 2D) accumulated at the site of contact between cell and bead. Neither WT cells bound to PLL beads nor talin-KO NK cells bound to either ICAM-1 or PLL beads showed this accumulation. Quantitative analysis of the confocal images confirmed that binding of LFA-1 to ICAM-1 resulted in significant localization of actin, Arp2/3, vinculin, and WASP in WT, but not talin-KO NK cells (Figure 2 bar graphs). These results indicate that talin is required for the recruitment of Arp2/3, vinculin, and WASP to the site of LFA-1 ligation.

We isolated NK cells from WASP-KO mice primed with poly I:C as well as those cultured with IL-2 or IL-15. To confirm that NK cells from various culture conditions bound ICAM-1, we assayed adhesion to immobilized ICAM-1 as previously described.²²  While unstimulated cells showed minimal adhesion to ICAM-1, ex vivo NK cells from poly I:C–injected mice as well as IL-2 and IL-15 activated NK cells from WT and WASP-KO C57Bl/6 mice bound ICAM-1 with similar avidities (supplemental Figure 1). Similarly, talin-KO ES-derived NK cells bound ICAM-1 after treatment with Mn⁺⁺ as previously shown.⁶  Upon binding of ICAM-1–coated beads, talin (Figure 3A) and Arp2/3 (Figure 3B) accumulated at the contact site in all 3 types of WASP-KO NK cells. Vinculin (Figure 3C) also accumulated at the contact site in ex vivo and IL-15–activated WASP-KO NK cells, but unexpectedly no significant accumulation of vinculin was seen in IL-2–activated WASP-KO NK cells. Actin (Figure 3D) did not accumulate at the contact site in ex vivo and IL-15–cultured WASP-KO NK cells, whereas it accumulated at the contact site in IL-2–cultured WASP-KO NK cells. These results show that WASP is required for actin accumulation at the site of LFA-1 ligation in ex vivo NK cells and IL-15–cultured NK cells whereas IL-2–cultured NK cells use a WASP-independent pathway of actin polymerization. The results also show that WASP is not required for the recruitment of talin, vinculin, and Arp2/3.

When IL-15–cultured NK cells bound ICAM-1–coated beads, WASP and Arp2/3 as well as vinculin and Arp2/3 colocalized at the site of bead binding. IL-2–activated cells, consistent with the results in the previous paragraph showed no apparent colocalization of vinculin and Arp2/3 or WASP and Arp2/3, either before or after binding to ICAM-1–coated beads (Figure 4A). To determine whether colocalization of these proteins was due to physical association, we carried out coimmunoprecipitation experiments using IL-2– or IL-15–cultured NK cells, which express similar levels of our proteins of interest (supplemental Figure 2). Immunoprecipitation of talin from NK cells cultured with IL-15, but not IL-2, resulted in coprecipitation of vinculin (Figure 4B top panel). Moreover, immunoprecipitation of vinculin from NK cells cultured with IL-15, but not IL-2, resulted in coprecipitation of talin as well as Arp2 (Figure 4B middle panel). On the other hand, WASP was not coprecipitated with talin or vinculin. Despite apparent colocalization of WASP and Arp2/3, immunoprecipitation of WASP from both IL-2– and IL-15–cultured NK cells did not coprecipitate Arp2, talin, or vinculin (Figure 4B bottom panel). Immunoprecipitations carried out using isotype control antibody confirmed the specificity of our antibodies. We also carried out the same coimmunoprecipitation analyses using NK cells bound to immobilized ICAM-1 and obtained the same results (Figure 4C), indicating that the talin-vinculin-Arp2 association is most probably constitutive in NK cells cultured with IL-15.

The results in Figures 2 and 3 showed that talin and WASP are required for the accumulation of F-actin at the site of LFA-1 ligation in ex vivo and IL-15–cultured NK cells and that the recruitment of WASP is talin-dependent. However, no direct association was detected between WASP and talin, vinculin, or Arp2/3. WASP has a PH domain that binds PIP₂ and talin binds PIPKIγ, which generates PIP₂.²³  Therefore, we tested whether binding of ICAM-1–coated beads to NK cells induces elevated levels of PIP₂ at the contact site and whether talin is required for the process. As seen in Figure 5A (left panel), binding of WT, but not talin-KO, NK cells to ICAM-1–coated beads resulted in the accumulation of PIP₂ at the contact site. We also tested whether WASP is required for the accumulation of PIP₂ at the contact site. WASP-KO NK cells, after binding to ICAM-1–coated beads, showed the same level of PIP₂ accumulation at the contact site as WT NK cells (Figure 5A right panel). Quantification of the PIP₂ accumulation showed that the fluorescence intensity of PIP₂ staining at the contact site was approximately 2-fold higher than that at the opposite side of the same cell in WT and WASP-KO NK cells, but not talin-KO NK cells (Figure 5B). Thus, the localized increase in PIP₂ is dependent on talin, but not WASP. To test talin-PIPKIγ association, PIPKIγ was immunoprecipitated from IL-15–cultured WT NK cells and probed for talin. The results showed coprecipitation of talin and PIPKIγ (Figure 5C). We also showed, using purified rat brain extract as a positive control, that immunoprecipitated PIPKIγ from NK cells corresponds predominantly with the larger of 2 isoforms found in mouse.

To test whether the elevated level of PIP₂ at the site of LFA-1 ligation is responsible for the recruitment of WASP, we tested the effects m-3M3FBS, a compound shown to activate phospholipase-Cγ (PLCγ).²⁴  As PLCγ converts PIP₂ into diacylglycerol and inositol-1,4,5-trisphosphate, m-3M3FBS was expected to lower the level of PIP₂. Treatment of NK cells with various concentrations of m-3M3FBS showed that 20μM m-3M3FBS efficiently reduced PIP₂ staining whereas higher concentrations affected the cell viability (data not shown). NK cells treated or not with 20μM m-3M3FBS were incubated with ICAM-1–coated beads and stained for PIP₂. The treatment significantly reduced the overall level of PIP₂ and accumulation of PIP₂ at the site of bead binding (Figure 6A). The residual staining in the treated cells appeared to be mostly in the cytoplasm of the cell, suggesting that cytoplasmic staining may be nonspecific. To quantify the membrane specific reduction of PIP₂ after treatment, we measured the fluorescence intensity of staining along a line from the point of contact between a cell and a bead. With untreated NK cells bound to PLL-coated beads, 2 equal peaks of PIP₂ staining corresponding to the cell membrane at the contact site and the opposite end were detected (Figure 6B left). In untreated cells bound to ICAM-1–coated beads, a single high peak of staining at the contact site was seen (Figure 6B center). Treatment with PLCγ activator almost completely abrogated the staining of antibody, particularly at the membranes (Figure 6B right). We also tested the effects of m-3M3FBS on the localization of talin, WASP, and actin. While talin accumulation was unaffected by the treatment, actin accumulation and WASP recruitment was almost completely abrogated (Figure 6C). Quantification of these results confirmed that m-3M3FBS treatment significantly decreased the intensity of PIP₂, WASP and actin staining at the contact site after binding of a cell to an ICAM-1–coated bead while talin localization was not significantly affected (Figure 6D). These results suggest that localized production of PIP₂ is required for recruitment of WASP but not talin, and that this PIP₂ dependent WASP recruitment is required for actin polymerization after binding of LFA-1 to ICAM-1.

LFA-1 is considered to be an important stimulatory receptor on NK cells.²⁵  It is thought to be required for the formation of the immunologic synapse and spatial distribution of multiple receptors in the synapse as NK cells interact with target cells.²⁶  We have also shown previously that binding of ICAM-1 to LFA-1 on mouse NK cells induces reorganization of the actin cytoskeleton and polarization of NK cells, which is a prerequisite for subsequent redistribution of cytotoxic granules toward the bound targets. Moreover, talin is required for this LFA-1–induced NK-cell polarization.⁶  However, the precise signaling events induced by LFA-1 ligation have been difficult to discern as NK-target interaction involves multiple receptor-ligand combinations that generate complex signaling events. In this study we used ICAM-1–coated cell-sized beads as artificial targets and talin-deficient and WASP-deficient NK cells to study the effects of LFA-1 ligation independent of other receptors. Our results have revealed the mechanisms by which LFA-1 ligation results in the polarization of the actin cytoskeleton in NK cells. As previously shown,⁶  binding of ICAM-1–coated beads to NK cells induces talin redistribution from the cytosol to the site of the plasma membrane where the beads are bound. ICAM-1 binding presumably separates the 2 chains of LFA-1 and exposes the cytoplasmic tail of the β-chain, where talin binds and stabilizes the high affinity conformation of LFA-1.²⁷  Our current results have shown that talin recruited by ligand-bound LFA-1 brings and activates actin polymerization machinery by 2 separate pathways. First, talin brings the actin nucleating Arp2/3 to the site of LFA-1 ligation via association of vinculin with both talin and Arp2/3. Second, talin also binds PIPKIγ and recruits it to the site of LFA-1 ligation, where it synthesizes PIP₂. WASP, which has a PH domain, is recruited by the elevated level of PIP₂ at the site. Thus, talin brings together Arp2/3 and WASP to promote the actin nucleation activity of Arp2/3, resulting in the accumulation of F-actin at the site of LFA-1 ligation.

While the LFA-1–mediated signaling pathways described in the previous paragraph are in operation in freshly isolated NK cells from poly I:C–injected mice and those cultured with IL-15, NK cells cultured with a high dose of IL-2 seem to have distinct LFA-1–mediated signaling pathways. In IL-2–cultured NK cells, vinculin and WASP do not localize to the site of LFA-1 ligation, and no vinculin association with talin or Arp2/3 is detectable. Nevertheless, Arp2/3 and F-actin accumulate at the site of LFA-1 ligation in IL-2–cultured NK cells. Moreover, WASP-KO NK cells cultured with IL-2 accumulate F-actin at the site of LFA-1 ligation, indicating a WASP-independent pathway of actin polymerization. Consistent with our findings, NK cells from WAS patients have been shown to have profound defects in NK-cell cytotoxicity due to impaired conjugate formation with targets.¹⁴  However, IL-2 stimulation restores target binding and cytotoxicity. Currently, what recruits Arp2/3 and activates its actin nucleating function in IL-2–activated NK cells is unknown. While not investigated in this study, it would be interesting to determine whether the constitutive association of talin, vinculin and Arp2/3 is present in resting NK cells. It is likely that this complex is a result of IL-15 activation. It would also be interesting to further examine the role that vinculin plays in this system. It is possible that, while vinculin does not localize after IL-2 treatment, it is still required and simply has a lowered threshold for recruitment and activation, a hypothesis that could be tested using vinculin-deficient NK cells. Alternatively, as the talin-vinculin interaction is controlled by the physical extension of talin after substrate binding,²⁸  it is possible that IL-2 signaling affects the rigidity of the cytoskeleton and the forces exerted on talin.

Although the role of vinculin in the immune system is still unclear, it has been reported to form a complex with Arp2/3, talin, and the nucleation promoting factor WASP verprolin homologous 2 (WAVE2) in T cells.²⁹  Vinculin knock-down results in reduced localization of talin to the immunologic synapse formed between Jurkat T cells and super antigen-pulsed Raji B cells, suggesting that vinculin is required for the recruitment of talin to the synapse in T cells.²⁹  In our system, talin is required for vinculin recruitment after LFA-1 ligation. While NK cells express WAVE, we saw no WAVE localization after LFA-1 binding in IL-2– or IL-15–activated NK cells (not shown). The hematopoeitic cortactin homologue HS1 has been shown to play a role in NK-cell synapse formation,³⁰  but it is still unknown whether HS1 mediates actin polymerization after LFA-1 ligation in IL-2–activated NK cells.

Our results have shown that PIP₂ is important for the recruitment of WASP to the site of LFA-1 ligation in ex vivo NK cells and IL-15–activated NK cells. PIP₂ is on the inner leaflet of the plasma membrane and it binds to a number of PH domain-containing proteins, including WASP. Although it is quite abundant in the plasma membrane, LFA-1 ligation induces a localized elevation of PIP₂ level at the site, most likely due to recruitment of PIPKIγ by talin. Micucci et al have reported that PIPKIα and -γ are expressed in human NK cells. Moreover, interaction of a human NK cell with a sensitive, but not resistant target, results in the production and then consumption of cellular PIP₂, and quenching of PIP₂ in these cells by competitive inhibition results in impaired cytotoxicity.⁹  PIP₂ and PIPKIγ are known to regulate cytoskeletal dynamics in adherent cells. Talin recruits PIPKIγ to focal adhesions, where it is phosphorylated and activated by focal adhesion kinase, which both increases local production of PIP₂ and strengthens the association of PIPKIγ with talin.²³  PIP₂ also strengthens vinculin-talin and talin-integrin β tail interactions.^31,32 

While our results have shown that PIP₂ is required for the recruitment of WASP to the site of LFA-1 ligation, the function of WASP seems to be regulated by multiple mechanisms. In T cells, WASP is held in a complex with WIP, which prevents activation of WASP by Cdc42. After TCR signaling, a ZAP-70-CrkL-WASP-WIP complex is formed and WASP is translocated to the membrane, where phosphorylation of WIP by PKCθ causes dissociation and subsequent activation of WASP.³³  SH3 domain–containing proteins have been implicated in WASP recruitment and activation in several systems. In NK cells, LFA-1 ligation has been reported to activate Vav1,⁴  Cdc42,³⁴  and Pyk2.⁵  Although we have been unable to detect an enrichment of Cdc42 at the site of LFA-1 ligation (not shown), it is possible that a small pool of GTP-bound Cdc42 is localized to the site and activates WASP.³⁵  LFA-1 ligation also induces rapid phosphorylation and localization of Pyk2 at the site.⁶  Pyk2 thus activated may be responsible for the activation of PIPKIγ that is recruited to the site by talin.

LFA-1–mediated binding of NK cells to sensitive target cells recruits many receptors and signaling molecules that form the immunologic synapse. LFA-1 is critical for the organization of the synapse, which is dependent on the actin cytoskeleton. Our current study has revealed the mechanisms by which LFA-1 ligation induces actin reorganization and will help us understand how NK- cell cytotoxicity is initiated.

We thank Drs Susan Monkley and David Critchley for talin-KO and WT ES cell lines, and Pak Kwong for technical assistance.

This work was supported by a grant from the Canadian Institute of Health Research. Expenses for materials used in this work were partially defrayed by StemCell Technologies.

Contribution: E.M.M. designed and performed experiments, analyzed data, and wrote the manuscript; J.Z. and K.A.S. contributed vital materials; and F.T. designed experiments and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Fumio Takei, 675 West 10th Ave, Vancouver, BC V5Z 1L3; e-mail: ftakei@bccrc.ca.