Effector functions mediated by NK cells involve cytotoxicity and transcription-dependent production and release of cytokines and chemokines. Although the JAK/STAT pathway mediates lymphokine-induced transcriptional regulation in NK cells, very little is known about transcriptional regulation induced during cell-cell contact. We demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an important component for integration of signals leading to nuclear translocation of NFAT2 and NF-κB (RelA) during cell-cell contact and NKp46-dependent signaling. This WASp function is independent of its known role in F-actin polymerization and cytoskeletal rearrangement. Absence of WASp results in decreased accumulation of calcineurin, WASp-interacting protein, and molecules upstream of calcium mobilization, i.e., activated ZAP70 and phospholipase C-γ1, in the disorganized NK cell immune synapse. Production of GM-CSF, but not IFN-γ, is decreased, while natural cytotoxicity of Wiskott-Aldrich syndrome-NK cells is maintained. Our results indicate that WASp independently regulates its dual functions, i.e., actin cytoskeletal remodeling and transcription in NK cells.

Natural killer cells mediate their function by generating cytolytic responses to virus-infected cells and transformed tumor cells (1). Activated NK cells by producing cytokines (e.g., IFN-γ, TNF-α, GM-CSF) and chemokines (e.g., MIP-1α, MIP-1β, RANTES, lymphotactin) partake in noncytolytic control of viral infections and help shape the subsequent adaptive immune response (2). NK cells are activated by IL-12 released from mature dendritic cells (3) and by type I IFNs (IFN-α and IFN-β) released from virus-infected cells (1). Lymphokine-induced transcriptional activation of cytokine and chemokine genes in NK cell uses the JAK/STAT signaling pathways (4), but the molecular constituents of gene transcription pathway(s) during cell-cell interaction are less well understood.

A sustained cell-cell contact is required for IL-2 production in Th1 cells (5), and perturbation of actin cytoskeleton inhibits IL-2 production (6). Wiskott-Aldrich syndrome (WAS)3 protein (WASp), a regulator of actin cytoskeleton, has been implicated in the formation of a stable cell-cell contact, i.e., the immunological synapse (IS) (7, 8). WASp is a key member of the Drosophila Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 domain-containing proteins, which include Ena/VASP, Spred, and Homer family of proteins (9) and mediate actin polymerization (10). Deficiency of WASp, resulting from mutations in the WASP gene, manifests as an X-linked recessive immunodeficiency disorder (11). A major role of WASp in lymphocyte functions has been implied by the various defects observed in the WAS Th cells, e.g., decreased proliferation, decreased calcium influx, and impaired IL-2 secretion (8, 12, 13). The impairment in calcium influx is well established in WASp-deficient human and murine Th cells upon TCR ligation (7, 14) and in murine mast cells upon IgE ligation (15), yet the effect of WASp deficiency on pathways upstream of calcium mobilization is not resolved. Receptor-proximal signaling events in T cells lead to increase in intracellular calcium (16) and subsequent activation of calcineurin (CN) (17). These events lead to dephosphorylation of NFAT proteins and their translocation to the nucleus (18). The WH1 domain of WASp is important for NFAT activation (19) and suggests a role for WASp in NFAT-mediated nuclear transcription that may be independent of actin polymerization. The WH1 domain-deleted mutant maintains WASp-mediated actin polymerization, but the molecular mechanisms for the defective NFAT activation have not been elucidated. Similarly, NF-κB transcription factors, which play a critical role in triggering both innate and adaptive immune responses (20) and in NK cell differentiation (21), are activated through a variety of cell surface receptors, many of which may require an intact actin cytoskeleton to effectively engage their respective ligands. The effect of disorganized actin cytoskeleton on the activity of NF-κB transcription factors is currently unknown.

WASp plays an important role in generation and maintenance of Th IS (12). However, the role of WASp in the formation of IS in cytolytic lymphocytes (i.e., CD8+ T cells or NK cells) has not been characterized other than demonstrating decreased accumulation of F-actin and perforin in the IS of WAS NK cells (22). In fact, very little is currently known of the role of WASp in different NK cell functions. It has been suggested that NK cytotoxicity is decreased in WAS patients (22, 23). Furthermore, WASp and WASp-interacting protein (WIP) selectively impair T cell, but not B cell functions (24, 25), but the role of WASp in NK cell functions has not been resolved.

It can be concluded that WASp deficiency leads to multiple abnormalities in Th functions, which cannot readily be explained by its known role in polymerization of F-actin and rearrangement of actin cytoskeleton. We therefore hypothesized that some or all of these defects could be caused by the lack of WASp adaptor function, resulting in defective recruitment of signaling molecules to the Th IS during interaction with APCs expressing cognate peptide/MHC ligand. By inference, we hypothesized that WASp has a similar function in the activating NK cell IS (NKIS) during interaction with susceptible target cells.

Collectively, our studies together with previous investigations of domain deletion mutants of WASp (19) suggest a major role of WASp in formation of an organized mature NKIS, which is primarily required for initiation of signaling pathways important for activation of nuclear transcription factors.

Polyclonal NK cells and NK cell clones were generated from PBMC of healthy donors and three patients with the clinical diagnosis of WAS, as previously described (26). Samples from WAS patients were obtained pre-bone marrow transplantation from Department of Pediatrics, University of Washington, and Memorial Sloan-Kettering Cancer Center, as per the Internal Review Board guidelines. WAS patient 1 (WAS-1) has mutation 211 delT (exon 2) leading to frame-shift and stop codon at aa position 75 (patient described in Refs.11 and 27). Majority of studies presented in this study were performed on the NK cells derived from WAS-1. WAS patient 2 (WAS-2) has mutation C298A (exon 2) leading to a stop codon at aa position 88. The defect in WAS patient 3 was uncharacterized. Absence of WASp in NK cells was confirmed in all three patients by imaging analysis using anti-WASp Ab (see below) and in NK and B cells from WAS-1 by Western blot analysis on whole cell extracts. Phenotypically characterized NK cells were used for cytotoxicity and conjugation assays with the target cell 721.221 (class I-negative EBV-B lymphoblastoid cell line) and anti-NKp46-coated beads as well as for quantitative cytokine analysis using ELISA. NK cells derived from patients WAS-2 and WAS-3 were used only for cytotoxicity assays.

Primary Abs.

Anti-CD56 and anti-CD3 were purchased from BD Biosciences. Mouse monoclonal anti-human α-tubulin (identifies tubules and microtubule-organizing center (MTOC)), histone H1 (identifies nucleus), Cdc42, Nck, NF-κB p65 (F-6), goat polyclonal anti-human protein kinase C (PKC)-θ, WIP, NFAT2 (7A6), Homer-3, rabbit polyclonal p-ZAP70 (Tyr319), p-phospholipase C (phosphorylated PLC)-γ1 (Tyr1254), growth factor receptor-bound protein 2 (Grb2), Vav1, and NKp46 were purchased from Santa Cruz Biotechnology. Rabbit polyclonal p-PLC-γ1 (Tyr783) was purchased from Cell Signaling Technology. Cdc42-GTP reagent was a kind gift from M. Rosen (Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX). BODIPY FL phallacidin, purchased from Molecular Probes, was used to identify F-actin. Rabbit anti-human WASp (recognizing aa 224–238) was purchased from Upstate Biotechnology, rabbit polyclonal anti-WASp Ab (503) (recognizing aa 209–226) was obtained from H. Ochs (University of Washington, Seattle, WA), and rabbit polyclonal anti-WASp (recognizing aa 1–250) was purchased from Santa Cruz Biotechnology. All three anti-WASp Ab were used in initial studies to identify WASp with similar results. Mouse anti-CN/PP2B (VA1, β-regulatory subunit) was purchased from Upstate Biotechnology. Cholera toxin B subunit, which recognizes GM1 ganglioside, purchased from Calbiochem, was used to identify lipid rafts. Mouse anti-human lysosome-associated membrane protein-1 (H4A3; identifies lysosomes) was obtained from the Developmental Studies Hybridoma Bank, Department of Biological Sciences, University of Iowa. Isotype control IgG1 and IgG2 Ab were purchased from BD Biosciences.

Secondary Abs.

Affinity-purified second Abs and species-absorbed conjugates (FITC, Cy3, and Cy5) for multiple labeling were purchased from Chemicon International. Rabbit anti-GFP Ab (Molecular Probes) was used to identify GFP-WASp-GTPase-binding domain reagent (28).

Intracellular cytokine staining and FACS.

NK cells were blocked with 10 μl/ml brefeldin A (Sigma-Aldrich) before activating with 1 μg/ml ionomycin and PMA (Sigma-Aldrich) for 4 h at 37°C. NK cells were fixed and permeabilized, as per manufacturer’s protocols (FIX & PERM; Caltag Laboratories). Rat anti-human GM-CSF and mouse anti-human CD69 were purchased from BD Pharmingen. Dual-labeled NK cells were analyzed by FACS (BD Biosciences).

Conjugation assay and immunofluorescent cell imaging.

Based on the Nomarsky images, NK cells that were clearly conjugated with the target cell (and in the same focal plane) were selected for fluorescent analysis. Immunofluorescent labeled NK cell-target cell conjugates were analyzed after fixing at 10 and 30 min, as previously described (26). In all experiments, a digital imaging system with a Zeiss Axiovert 200M inverted microscope (Intelligent Imaging Innovations (3-I)) was used. Images were obtained both in two-dimension (2D) (x-y-axis) and three-dimension (3D) (x-z-axis) (26). The 2-D images were used to analyze polarization events in the conjugates. An event was considered polarized if the majority of fluorescence of the molecule/organelle was located in the area closest to the contact (i.e., proximal one-third of the NK cell area closest to the immune synapse), as described earlier (26). Carefully defined NK cell contact areas (excluding the 721.221 cell contact area) were selected for acquisition, deconvolution, and rendering using segmentation and statistics capabilities of SlideBook software (3-I). Sixty to seventy serial optical sections of 0.2 μm thickness were acquired for each label. The digital recorded data were then deconvolved using both Nearest Neighbor and Constrained Iterative Deconvolution algorithm with SlideBook software, and the images were processed using Photoshop 7.0 software (Adobe Systems). The 3D images of the NKIS shown in the figures are the rendered images projected in the optical x-z-axis after applying the mask functions on the NK side of the IS in the plane of cell:cell contact. For quantitation of molecules in the IS, only those conjugates in which a clear distinction between the NK cell- and 721.221 cell-contacting membranes could be made were selected. This was done to avoid fluorescent signals of the shared molecules coming from the 721.221 cell. For each molecule, the degree of accumulation at the NKIS was calculated using the formula: relative enrichment (RE) = integrated fluorescence intensity (IFI) per unit volume at the contact site divided by the IFI per unit volume of the entire cell (29, 30). The amount of active PLC-γ1 and ZAP70 in the unconjugated and conjugated NK cells was calculated by determining the IFI of these molecules per unit volume of the entire cell, using the segmentation capability of the SlideBook software. The values obtained in the unconjugated NK cells were considered baseline and given 1.0 unit value. Increased intensity above the baseline is presented as fractions of 1.0, e.g., RE = 1.20 (20% additional accumulation), RE = 1.35 (35% additional accumulation), etc. Wilcoxon nonparametric two-sample test was used to determine the p values.

Perturbation of actin cytoskeleton by cytochalasin D (Cyt-D).

NK cells were incubated with 10 μmol/L Cyt-D (diluted with 0.1% DMSO) or with control 0.1% DMSO for 30 min. As shown previously, the viability of NK cells is maintained at this concentration of Cyt-D (31). NK cells were then used for the cytotoxicity assay (as described below) or were stimulated with PMA/ionomycin (PMA/I) and used for immunofluorescent and Western blot analysis, as shown in Fig. 4. Additionally, Cyt-D-treated normal NK cells were conjugated with NKp46-coated beads for 10 and 30 min to analyze activity of the transcription factor, NFAT2, in the face of disrupted F-actin.

FIGURE 4.

Effect of perturbation of F-actin cytoskeleton on NK cell functions. a, Immunofluorescent images of unconjugated NK cell (top panel) and NK-721.221 conjugates (bottom panel) either treated with Cyt-D (right panel) or untreated (left panel), triple labeled with indicated reagents (green, red, and blue), are shown. Normal unconjugated NK cells stimulated with PMA/I for 30 min are shown. In the NK conjugates, arrows indicate location of the NK cell and target cell. Representative images of conjugates demonstrating polarization (p) or nonpolarization (np) of F-actin along with the location of NFAT2 (n = nuclear) are shown. Data in this panel are representative of at least 30 NK cells and NK conjugates analyzed. b, Nuclear and cytoplasmic (cyto) extracts obtained from PMA/I-stimulated and Cyt-D-treated or untreated NK cells were resolved by Western blot using NFAT2 Ab. Arrow indicates the location of phosphorylated (P) or dephosphorylated (deP) forms of NFAT2 in the gel. β-actin loading control was used for each condition shown. c, Bar graph representing quantitative analysis (by ELISA) of secreted cytokines, i.e., GM-CSF and IFN-γ for PMA/I-activated and Cyt-D-treated (for 16 h) or untreated NK cells are shown. d, Cytotoxic profile of the same NK cells either Cyt-D treated or untreated is shown. e, Cyt-D-treated NK cells conjugated with NKp46-coated beads (location denoted by ∗ in the fluorescent images of the conjugate) for the indicated duration and triple labeled with the indicated reagents are shown. The data are representative of at least 30 conjugates analyzed at each time point.

FIGURE 4.

Effect of perturbation of F-actin cytoskeleton on NK cell functions. a, Immunofluorescent images of unconjugated NK cell (top panel) and NK-721.221 conjugates (bottom panel) either treated with Cyt-D (right panel) or untreated (left panel), triple labeled with indicated reagents (green, red, and blue), are shown. Normal unconjugated NK cells stimulated with PMA/I for 30 min are shown. In the NK conjugates, arrows indicate location of the NK cell and target cell. Representative images of conjugates demonstrating polarization (p) or nonpolarization (np) of F-actin along with the location of NFAT2 (n = nuclear) are shown. Data in this panel are representative of at least 30 NK cells and NK conjugates analyzed. b, Nuclear and cytoplasmic (cyto) extracts obtained from PMA/I-stimulated and Cyt-D-treated or untreated NK cells were resolved by Western blot using NFAT2 Ab. Arrow indicates the location of phosphorylated (P) or dephosphorylated (deP) forms of NFAT2 in the gel. β-actin loading control was used for each condition shown. c, Bar graph representing quantitative analysis (by ELISA) of secreted cytokines, i.e., GM-CSF and IFN-γ for PMA/I-activated and Cyt-D-treated (for 16 h) or untreated NK cells are shown. d, Cytotoxic profile of the same NK cells either Cyt-D treated or untreated is shown. e, Cyt-D-treated NK cells conjugated with NKp46-coated beads (location denoted by ∗ in the fluorescent images of the conjugate) for the indicated duration and triple labeled with the indicated reagents are shown. The data are representative of at least 30 conjugates analyzed at each time point.

Close modal

Treatment of NK cells with Cyt-D impaired the ability of NK cells to form conjugates, resulting in decreased number of conjugates formed at different time points, necessitating multiple experiments to accumulate a robust n value. However, the conjugates that did form remained stable for the duration of the experiment and were included in the analysis.

Cytotoxicity assay.

Polyclonal NK cells and NK clones, derived from normal and WAS patients, were used as effectors, and 51Cr-labeled HLA class I-negative EBV-B lymphoblastoid cell line, 721.221, as target. Assays were performed in triplicates for 4 h at an E:T ratio of 10:1, and the percent-specific lysis was calculated as per the standard formula.

ELISA.

IL-2-activated NK cells were stimulated with 25 ng/ml PMA and 1 μg/ml ionomycin for 48 h (or for the indicated duration) in NK cell medium. The supernatants were analyzed for IL-2, IFN-γ, GM-CSF, and TNF-α using standard protocol for quantitative ELISA as per the manufacturer’s recommendations (R&D Systems).

Extraction of nuclear and cytoplasmic fractions and Western blot analysis.

For detection of NFAT2 and NF-κB in the cell extracts, 1 × 106 NK cells were stimulated for the indicated length of time with either anti-NKp46-coated beads or PMA (25 ng/ml) and ionomycin (1 μg/ml) (Sigma-Aldrich). Epoxy beads from Dynal Biotech were washed with PBS and incubated overnight with 30 μl of anti-NKp46 Ab. Nuclear and cytoplasmic fractions were extracted, according to manufacturer’s instructions, using NE-PER kit (Pierce). For Western blotting, 10 μl of nuclear and cytoplasmic extracts was resolved on Tris-HCl Ready Gels (Bio-Rad) with anti-NFAT2, anti-NF-κB p65, anti-p-ZAP70, and anti-p-PLC-γ1 primary Abs. mAb to β-actin (Sigma-Aldrich) that specifically recognizes only β-actin isoform was used as a loading control to determine total amount of each protein in whole cell, nuclear, or cytoplasmic lysates. In our in vitro conditions, the efficiency of conjugate formation between NK cells and NKp46 beads ranged from 15 to 25% at 10 and 30 min. Therefore, Western blot analysis of the NK cell:bead mixture will reflect events occurring in ∼20% of NK conjugates and ∼80% of unconjugated NK cells.

Initially, the ability of WAS NK cells to form cytolytic conjugates with 721.221 cells was evaluated at 3, 5, 10, and 15 min. At each time point, WAS NK cells form fewer conjugates (i.e., 5–8%, n = 3) compared with normal NK cells (i.e., 15–33%, n = 3) (data not shown), as determined by FACS. As a measure of cytoskeletal remodeling, we evaluated NK conjugates at 10 min for polarization of F-actin, MTOC, and lysosomes to the cell-cell contact area (Fig. 1). The strength of fluorescent signals of F-actin in WAS and normal NK cells was similar, suggesting unimpaired polymerization of actin in WAS NK cells. However, F-actin was polarized in two-thirds of normal, but in only one-third of WAS NK conjugates, and the amount of F-actin accumulated in the WAS-NKIS was lower compared with normal NKIS (Table I). F-actin segregates in the peripheral supramolecular activation clusters (pSMAC) of normal NKIS, whereas in WAS-NKIS, F-actin filaments are randomly distributed (Fig. 1,a, 3D images). Similarly, MTOC and lysosomes are polarized in the majority of normal, but in only a minority of WAS NK conjugates (Fig. 1,a and Table I). Furthermore, in the majority of normal NKIS, lipid rafts (rafts) are polarized and cluster predominantly in the central SMAC (cSMAC) (Fig. 1,a). In the WAS-NKIS, accumulation of rafts is decreased and rafts are distributed either in the p-SMAC or randomly within the NKIS (Fig. 1 a).

FIGURE 1.

Configuration of normal and WAS cytolytic NKIS at 10 min. a, Representative 2D and 3D (i.e., NKIS) images of normal (top panel) and WAS (bottom panel) NK cell conjugates dual labeled with the indicated reagents (in green and red) analyzed at 10 min are shown. Same images with either green or red color removed are shown for clarity of individual events. In the conjugate, top cell is the NK cell and the bottom cell is 721.221. Data in the figure are representative of at least 50–60 conjugates analyzed for each combination. b, Topology of the molecular segregation within the microdomains (i.e., peripheral, p; intermediate, i; central, c) of normal and WAS-NKIS is illustrated in the diagram. Arrows indicate location of the molecules within each microdomain (i.e., pSMAC, iSMAC, or cSMAC) of the individual NKIS.

FIGURE 1.

Configuration of normal and WAS cytolytic NKIS at 10 min. a, Representative 2D and 3D (i.e., NKIS) images of normal (top panel) and WAS (bottom panel) NK cell conjugates dual labeled with the indicated reagents (in green and red) analyzed at 10 min are shown. Same images with either green or red color removed are shown for clarity of individual events. In the conjugate, top cell is the NK cell and the bottom cell is 721.221. Data in the figure are representative of at least 50–60 conjugates analyzed for each combination. b, Topology of the molecular segregation within the microdomains (i.e., peripheral, p; intermediate, i; central, c) of normal and WAS-NKIS is illustrated in the diagram. Arrows indicate location of the molecules within each microdomain (i.e., pSMAC, iSMAC, or cSMAC) of the individual NKIS.

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Table I.

Quantitative analysis of normal and WAS NK conjugatesa

ACTIN % (n =)MTOC % (n =)Lysosomes % (n =)WASP % (n =)CDC42 % (n =)CDC42-GTP % (n =)VAV % (n =)WIP % (n =)GRB2 % (n =)p-ZAP70 % (n =)NCK % (n =)RAFTS % (n =)PKC-θ % (n =)CN % (n =)p-PLC-γ1 % (n =)HOMER-3 % (n =)
2D analysisb                 
65 (232) 64 (136) 70 (60) 55 (108) 71 (100) 73 (40) 60 (52) 61 (92) 73 (40) 60 (33) 67 (57) 62 (50) 80 (25) 60 (33) 73 (103) 50 (50) 
WAS 35 (180) 33 (64) 46 (35)  67 (45) 60 (43) 62 (60) 35 (160) 75 (44) 52 (21) 54 (90) 45 (33) 77 (30) 37 (75) 36 (138) 42 (100) 
3D analysisc                 
1.36 ± 0.02 (183)   1.22± 0.02 (89) 1.27± 0.02 (53) 1.30± 0.03 (40) 1.26± 0.02 (47) 1.28± 0.02 (57) 1.27± 0.04 (35) 1.27± 0.04 (30) 1.24± 0.03 (47) 1.35± 0.04 (27) 1.27± 0.04 (25) 1.17± 0.05 (31) 1.16± 0.03 (31) 1.22± 0.03 (20) 
WAS 1.30± 0.03 (124)    1.25± 0.02 (45) 1.25± 0.05 (32) 1.23± 0.03 (49) 1.15± 0.02 (83) 1.18± 0.04 (43) 1.19± 0.04 (31) 1.20± 0.03 (53) 1.17± 0.02 (21) 1.20± 0.03 (30) 1.0± 0.03 (35) 1.0± 0.03 (35) 1.11± 0.07 (20) 
p Value 0.01    0.5 0.24 0.5 <0.001 0.1 0.01 0.4 <0.001 0.18 0.01 0.004 0.1 
ACTIN % (n =)MTOC % (n =)Lysosomes % (n =)WASP % (n =)CDC42 % (n =)CDC42-GTP % (n =)VAV % (n =)WIP % (n =)GRB2 % (n =)p-ZAP70 % (n =)NCK % (n =)RAFTS % (n =)PKC-θ % (n =)CN % (n =)p-PLC-γ1 % (n =)HOMER-3 % (n =)
2D analysisb                 
65 (232) 64 (136) 70 (60) 55 (108) 71 (100) 73 (40) 60 (52) 61 (92) 73 (40) 60 (33) 67 (57) 62 (50) 80 (25) 60 (33) 73 (103) 50 (50) 
WAS 35 (180) 33 (64) 46 (35)  67 (45) 60 (43) 62 (60) 35 (160) 75 (44) 52 (21) 54 (90) 45 (33) 77 (30) 37 (75) 36 (138) 42 (100) 
3D analysisc                 
1.36 ± 0.02 (183)   1.22± 0.02 (89) 1.27± 0.02 (53) 1.30± 0.03 (40) 1.26± 0.02 (47) 1.28± 0.02 (57) 1.27± 0.04 (35) 1.27± 0.04 (30) 1.24± 0.03 (47) 1.35± 0.04 (27) 1.27± 0.04 (25) 1.17± 0.05 (31) 1.16± 0.03 (31) 1.22± 0.03 (20) 
WAS 1.30± 0.03 (124)    1.25± 0.02 (45) 1.25± 0.05 (32) 1.23± 0.03 (49) 1.15± 0.02 (83) 1.18± 0.04 (43) 1.19± 0.04 (31) 1.20± 0.03 (53) 1.17± 0.02 (21) 1.20± 0.03 (30) 1.0± 0.03 (35) 1.0± 0.03 (35) 1.11± 0.07 (20) 
p Value 0.01    0.5 0.24 0.5 <0.001 0.1 0.01 0.4 <0.001 0.18 0.01 0.004 0.1 
a

Percentage of normal (N) and WAS NK cell conjugates showing polarization of the indicated molecules/organelles to the contact area (i.e., synapse) is shown under “2D analysis”. Bold, underlined items highlight significant change in the values in the WAS NK cells compared to normal. The degree of accumulation (i.e., RE, relative enrichment) (±SEM) at the synapse of the indicated molecules is shown under “3D analysis.” See Materials and Methods for details on quantitating RE (e.g., RE of 1.0 is the baseline concentration with no additional recruitment; RE of 1.36 is 36% additional enrichment above the baseline). The RE values are averages obtained from analyzing numerous conjugates. The number of conjugates (n) analyzed is shown under each label. Values of p comparing RE in normal vs WAS NK conjugates are also shown.

b

Polarization to the synapse. Conjugation time: 10 min.

c

Degree of accumulation at the synapse. Conjugation time: 10 min.

In summary, WASp deficiency results in disorganized F-actin and raft rearrangement within the microdomains of the cytolytic NKIS.

We then evaluated the effect of disorganized F-actin and raft rearrangement on the formation of cytolytic WAS-NKIS. Initially, 10-min conjugates were analyzed as normal NKIS matures at this time point (26). In the majority of WAS conjugates, polarization of WIP was decreased, whereas polarization of total Cdc42, Cdc42-GTP, Vav1, Nck, PKC-θ, p-ZAP70, and Grb2 occurred normally. In comparison, these molecules as well as WASp were polarized in the majority of normal NK conjugates (Fig. 1,a and Table I).

The 3D analysis of the NKIS was made to evaluate: 1) degree of accumulation (i.e., RE) and 2) pattern of segregation of the recruited molecules within the cytolytic NKIS. Consistent with the polarization data, the degree of accumulation of WIP was significantly lower in WAS-NKIS compared with normal NKIS, while total Cdc42, Cdc42-GTP, Vav1, Nck, PKC-θ, and Grb2 were accumulated to the same degree in both normal and WAS-NKIS (Table I). The amount of p-ZAP70 accumulated in the WAS-NKIS was lower than normal. More significantly, the pattern of segregation of these proteins within the two NKIS was markedly different. In normal cytolytic NKIS, F-actin accumulated in the pSMAC, forming a ring enclosing the recruited signaling molecules as well as MTOC and lysosomes, which were all predominantly segregated in the cSMAC. WASp and WIP colocalized in the microdomain of NKIS that lies between the prototypical pSMAC and cSMAC, called the intermediate SMAC (iSMAC). By contrast, in the WAS-NKIS, Nck, Vav1, p-ZAP70, and PKC-θ either segregated in the pSMAC (majority) or were distributed without any specific pattern (minority). Cdc42-GTP and Grb2 localized in the cSMAC and WIP in the iSMAC in both WAS and normal NKIS. Homer-3, included in this analysis because of its role in actin-related protein complex (Arp) 2/3-independent actin polymerization (9), was polarized to the NKIS in ∼40–50% of normal and WAS NK conjugates, and segregated in a non-cSMAC microdomain of the NKIS (i.e., p/i SMAC).

In summary, WASp deficiency results in mislocation of the recruited signaling molecules and formation of disorganized cytolytic NKIS.

Because the induction of NFAT/AP-1-mediated gene transcription in the T cells depends on WIP binding WH1 region of WASp (32), and the transcription activity requires a stable cell-cell contact (5), we initially evaluated the fate of NFAT2 (or NFATc1) nuclear translocation in the face of WASp deficiency and the resulting WIP and NKIS defects.

Under physiological conditions, normal and WAS NK cells were mixed with 721.221 cells in the presence of 0.8 mM [Ca2+]ex at 37°C for 10 min, the duration that normally leads to generation of high intracellular Ca2+ concentration levels (33). Conjugates were triple-labeled with F-actin, NFAT2, and histone (identifies the nucleus). Normal NK cell conjugates demonstrated partial (i.e., cytoplasmic plus nuclear) or complete nuclear translocation of NFAT2 in 80% compared with only 27% in WAS NK cells (Fig. 2, A1, b and g, and A2). In the majority of WAS NK cell conjugates, NFAT2 remains cytoplasmic in location. To determine whether NFAT2 remains in the cytoplasm at any length of interaction, NK conjugates were analyzed at 30 min. At 30 min, NFAT2 is translocated to the nucleus in 86% of normal (Fig. 2, A1, c and e, and A2) and 66% of WAS NK conjugates (Fig. 2, A1, i and j, and A2). Furthermore, NKp46-expressing normal and WAS NK cells were conjugated with anti-NKp46-coated beads for 10 and 30 min in the presence of 0.8 mM [Ca+2]ex. Nuclear and cytoplasmic extracts obtained from this cell:bead mixture were analyzed for the presence of NFAT2 by Western blot. At 10 min, normal NK cells demonstrated nuclear NFAT2, while WAS NK cells failed to demonstrate NFAT2 in the nucleus. At 30 min, however, the magnitude of NFAT2 nuclear translocation was similar in both WAS and normal NK cells (Fig. 3,a, left panel). It should be pointed out that this NK cell:bead mixture contains only ∼20% cell:bead conjugates and ∼80% unconjugated NK cells. Therefore, nuclear translocation in cell:bead mixtures identified by biochemistry (in Fig. 3) may appear less efficient compared with nuclear translocation identified by selective imaging of well-formed cell:cell conjugates (in Fig. 2).

FIGURE 2.

Temporal relationship between actin cytoskeleton and transcription factors in normal and WAS NK cells. NK cell conjugates analyzed at 10 min (A1, b and g; B1, b, c, f, and g) and 30 min (A1, c–e and h–j; B1, d and h) were triple labeled with the indicated reagents (in green, red, and blue), and the representative 2D images of the conjugates are shown for normal (N) and WAS NK cells. Location of NFAT2 (A1, a and f) and NF-κB (RelA) (B1, a and e) within the unconjugated IL-2-activated NK cell is also shown. Analysis of 30-min NK conjugates in A1 revealed three combinations for actin polarization (p, polarized; np, nonpolarized) and NFAT2 location (cy, cytoplasmic; n, nuclear). Representative images of each of these combinations in normal (A1, c–e) and WAS (A1, h–j) NK conjugates are shown along with the percentage of conjugates with each of these combinations in the pie diagrams (A3). Pie diagram representing the percentage of normal and WAS NK cell conjugates demonstrating nuclear or cytoplasmic location of NFAT2 (A2) or NF-κB (B2) at 10 and 30 min is also shown. Data in this figure are representative of 50–75 conjugates analyzed for each combination and time point.

FIGURE 2.

Temporal relationship between actin cytoskeleton and transcription factors in normal and WAS NK cells. NK cell conjugates analyzed at 10 min (A1, b and g; B1, b, c, f, and g) and 30 min (A1, c–e and h–j; B1, d and h) were triple labeled with the indicated reagents (in green, red, and blue), and the representative 2D images of the conjugates are shown for normal (N) and WAS NK cells. Location of NFAT2 (A1, a and f) and NF-κB (RelA) (B1, a and e) within the unconjugated IL-2-activated NK cell is also shown. Analysis of 30-min NK conjugates in A1 revealed three combinations for actin polarization (p, polarized; np, nonpolarized) and NFAT2 location (cy, cytoplasmic; n, nuclear). Representative images of each of these combinations in normal (A1, c–e) and WAS (A1, h–j) NK conjugates are shown along with the percentage of conjugates with each of these combinations in the pie diagrams (A3). Pie diagram representing the percentage of normal and WAS NK cell conjugates demonstrating nuclear or cytoplasmic location of NFAT2 (A2) or NF-κB (B2) at 10 and 30 min is also shown. Data in this figure are representative of 50–75 conjugates analyzed for each combination and time point.

Close modal
FIGURE 3.

Western blot analysis of nuclear and cytoplasmic extracts from normal and WAS NK cells. Representative Western blots of nuclear (nu) and cytoplasmic (cyto) extracts prepared from normal and WAS NK cells after stimulating with anti-NKp46-coated beads (a and c) or PMA/I (b) for the indicated time are shown. Equal volume (10 μl) of extracts obtained from 1 × 106 cells was loaded per lane. Blots were probed with the indicated Abs. β-actin loading control was used for each condition shown. Representative β-actin blots for indicated lysates obtained from normal and WAS NK cells are shown for both 10- and 30-min time points in the NKp46 stimulation condition. Appropriate ladder markers (data not shown) were simultaneously run with the samples to confirm the correct size of the bands. The data in the figure are representative of at least two independent experiments for each condition.

FIGURE 3.

Western blot analysis of nuclear and cytoplasmic extracts from normal and WAS NK cells. Representative Western blots of nuclear (nu) and cytoplasmic (cyto) extracts prepared from normal and WAS NK cells after stimulating with anti-NKp46-coated beads (a and c) or PMA/I (b) for the indicated time are shown. Equal volume (10 μl) of extracts obtained from 1 × 106 cells was loaded per lane. Blots were probed with the indicated Abs. β-actin loading control was used for each condition shown. Representative β-actin blots for indicated lysates obtained from normal and WAS NK cells are shown for both 10- and 30-min time points in the NKp46 stimulation condition. Appropriate ladder markers (data not shown) were simultaneously run with the samples to confirm the correct size of the bands. The data in the figure are representative of at least two independent experiments for each condition.

Close modal

Under nonphysiological conditions, unconjugated normal and WAS NK cells were stimulated with PMA/I for 10 and 30 min in the presence of 0.8 mM [Ca2+]ex. These experiments were done to evaluate the ability of membrane receptor-independent pathways to restore NFAT translocation. At 10 and 30 min, ∼90% of WAS and normal NK cells demonstrated complete nuclear (∼70%) or nuclear plus cytoplasmic (∼25%) localization of NFAT2 (Fig. 4,a showing 30-min conjugate, and data not shown for 10-min conjugate). Western blot analysis of the nuclear and cytoplasmic extracts obtained from these WAS NK cells demonstrated similar magnitude and kinetics of NFAT2 nuclear translocation compared with normal NK cells (Fig. 3 b, left panel).

We then extended this analysis to assess the fate of NF-κB p65 (RelA) in WAS NK cells (Fig. 2,B1). At 10 min, the majority of normal NK conjugates demonstrated nuclear RelA (b and c), whereas majority of WAS NK conjugates (f and g) demonstrated cytoplasmic RelA. However at 30 min, similar to NFAT2, the majority of normal and WAS NK cell conjugates now demonstrated complete or partial nuclear location of RelA (d and h). Furthermore, Western blot analysis of the nuclear and cytoplasmic extracts of WAS NK cells conjugated with anti-NKp46 beads for 10 and 30 min confirmed our imaging findings (Fig. 3,a, right panel). Again similar to the NFAT2 activity, the magnitude and kinetics of RelA nuclear translocation were similar in normal and WAS NK cells stimulated with PMA/I (Fig. 3 b, right panel). Therefore, NFAT2 and RelA defects in WAS NK cells are rescued by the PMA/I-mediated signals.

In summary, deficiency of WASp results in delayed nuclear translocation of NFAT2 and NF-κB (RelA) in NK cells only during cell-cell interaction and NKp46 receptor-mediated signaling.

Having demonstrated the dual functions of WASp on NK cell cytoskeletal remodeling and activity of transcription factors, we asked whether WASp regulates these two functions sequentially or independently of each other.

NK cell conjugates triple labeled with F-actin, histones, and NFAT2 were analyzed at 10 and 30 min (Fig. 2, A1 and A3). At 10 min, the majority of normal NK conjugates concurrently demonstrated polarized F-actin and nuclear (partial or complete) NFAT2 (Fig. 2, A1b and A3, normal blue pie). In contrast, the majority of WAS NK conjugates concurrently demonstrated nonpolarized F-actin and cytoplasmic NFAT2 (Fig. 2, A1g and A3, WAS red pie). However, 8–10% of conjugates (normal or WAS) did demonstrate nuclear NFAT2 in the absence of polarized F-actin (Fig. 2,A3, green pie), and conversely, another 8–10% of conjugates demonstrated cytoplasmic NFAT2 even when F-actin was actively polarized (Fig. 2,A3, yellow pie). Therefore, F-actin polarization and NFAT activity are disconnected in ∼20% of the conjugates at 10 min. At 30 min, 25% of normal and 38% WAS conjugates demonstrated nuclear NFAT2 even when F-actin was nonpolarized (Fig. 2, A1, e and j, and A3, green pies). Therefore, while the number of WAS conjugates with polarized F-actin did not increase over time (from 27 to 29%), there was a significant increase in conjugates demonstrating nuclear NFAT2 (from 27 to 67%), indicating that NFAT2 activation is occurring independently of the changes in actin cytoskeleton.

To further test the requirement of intact F-actin in NFAT2 activation, we perturbed actin cytoskeleton with the depolymerizing agent Cyt-D. Cyt-D treatment of NK cells results in decreased number of conjugates formed with target cells (i.e., 5–10% for Cyt-D treated vs 20–30% for control). Cyt-D-treated normal NK cells conjugated with 721.221 cells were analyzed at 30 min (Fig. 4). Polarization of F-actin was severely reduced (13% conjugates) in Cyt-D-treated NK cells compared with untreated NK cells (55% conjugates); however, the majority of conjugates (∼80%) under either condition showed nuclear NFAT2 (Fig. 4,a). In contrast, Cyt-D-treated normal NK cells lost their ability to kill 721.221 cells (Fig. 4 d).

Extending this analysis, Cyt-D-treated normal NK cells were stimulated with PMA/I for 30 min and analyzed for NFAT2 translocation. The majority (85–95%) of both untreated and Cyt-D-treated NK cells demonstrated nuclear NFAT2 by imaging analysis (Fig. 4,a, top panel). Western blot analysis of the nuclear and cytoplasmic extracts derived from these NK cells demonstrated similar kinetics and magnitude of nuclear NFAT2 under either condition (Fig. 4,b). Furthermore, production and secretion of GM-CSF and IFN-γ were unimpaired in the normal NK cells incubated with Cyt-D and PMA/I for 16 h (Fig. 4,c). Similarly, nuclear translocation of RelA was unaffected in Cyt-D-treated NK cells stimulated with PMA/I (data not shown). Given the possibility of alteration in cell:cell couple formation by Cyt-D treatment, we analyzed the events in Cyt-D-treated NK cells conjugated with NKp46 beads by imaging. At both time points (i.e., 10 and 30 min), disruption of F-actin did not affect the ability of NFAT2 to translocate to the nucleus (Fig. 4 e).

Taken together, our results indicate that WASp independently regulates its dual functions, i.e., actin cytoskeletal remodeling and transcriptional activation.

Because the defect in transcription factors was abrogated upon prolonged conjugation in WAS NK cells, we asked whether WAS-NKIS normalizes over time. As a measure of normalized NKIS, we selected F-actin (pSMAC marker), WIP (iSMAC marker), and PKC-θ (cSMAC marker) events and analyzed the WAS-NKIS at 30 min. Because 30-min conjugates will also include some newly formed conjugates (identified by hand-mirror or semilunar morphology), only well-formed (i.e., rounded) NK conjugates were included in this analysis. As shown in Fig. 5, all three markers were polarized to the contact and were appropriately segregated in the WAS-NKIS (compare Figs. 1 a and 5). Our data suggest a requirement of WASp in the timely formation of a mature cytolytic NKIS.

FIGURE 5.

Configuration of WAS cytolytic NKIS at 30 min. Representative 2D and 3D (i.e., NKIS) images of WAS NK cell conjugate triple labeled with the indicated reagents (in green, blue, and red) analyzed at 30 min are shown. The location of NK and .221 cells is indicated in the first image. Either green, red, or blue color was removed to better visualize individual events. The data in the figure are representative of at least 30 conjugates analyzed.

FIGURE 5.

Configuration of WAS cytolytic NKIS at 30 min. Representative 2D and 3D (i.e., NKIS) images of WAS NK cell conjugate triple labeled with the indicated reagents (in green, blue, and red) analyzed at 30 min are shown. The location of NK and .221 cells is indicated in the first image. Either green, red, or blue color was removed to better visualize individual events. The data in the figure are representative of at least 30 conjugates analyzed.

Close modal

Because the NFAT activity depends on calcium influx and activation of CN (18, 34), we evaluated the effect of WASp deficiency on the pathways upstream of calcium mobilization, i.e., activation of PLC-γ1 and its regulator ZAP70 (35) as well as the effect on CN.

Both polarization and enrichment of p-PLC-γ1 (Tyr1254) and CN were significantly reduced in WAS-NKIS (Fig. 1, a and c). We then asked whether the overall activation of these molecules was also reduced, in parallel with decreased polarization, by quantitating the total amount of p-PLC-γ1 and CN in conjugated NK cells (see Materials and Methods). Although the amount of p-PLC-γ1 in conjugated normal NK cell increased by 2.7 (±0.1, n = 31) above the baseline, the amount in conjugated WAS NK cell increased by 2.1 (±0.1, n = 35) (p < 0.01). Similarly, the amount of CN in conjugated normal NK cell increased by 2.5 (±0.03, n = 31) compared to an increase by 1.9 (±0.04, n = 34) in conjugated WAS NK cell (p = 0.03).

Extending these experiments, normal and WAS NK cells were conjugated with anti-NKp46-coated beads for 10 and 30 min, and cytoplasmic extracts were analyzed for p-ZAP70 (Tyr319), p-PLC-γ1 (Tyr783, required for inositol 1,4,5-trisphosphate (InsP3) formation), and p-PLC-γ1 (Tyr1254, required for maximum InsP3 generation) (36) by Western blot. At 10 min, the intensity of bands of active ZAP70 and PLC-γ1 was reproducibly decreased in WAS NK cells compared with normal. At 30 min, strong signals of these active proteins were now detected in WAS NK cells, consistent with the overall delay in the maturation of WAS-NKIS (Fig. 3 c). Interestingly, however, weaker signals of these proteins were detected in normal NK cells at this late time point.

In summary, our data demonstrate modest impairment in the overall activation of PLC-γ1 and CN, but profound decrease in the degree of focal accumulation to the WAS-NKIS.

Using ELISA, IL-2, IFN-γ, TNF-∝, and GM-CSF were quantitated in IL-2-activated WAS and normal NK cells. Normal and WAS NK cells not stimulated with PMA/I produce minimally detectable amount of cytokines (data not shown). PMA/I-stimulated WAS NK cells produce similar amount of IFN-γ as normal NK cells; however, GM-CSF production was reduced when determined by ELISA (Fig. 6,a) and intracellular FACS analysis (Fig. 6,b). Although ∼80% of WAS and normal NK cells were IFN-γ+ (data not shown), only ∼15% of WAS NK cells were GM-CSF+ compared with 78% normal NK cells, although the majority of WAS NK cells expressed the activation marker, CD69 (Fig. 6 b). The amount of IL-2 and TNF-α produced was similar, although negligible, in both normal and WAS NK cells. In summary, our data suggest a link between WASp deficiency and differential effect on GM-CSF, but not IFN-γ production in the IL-2-activated NK cells.

FIGURE 6.

Cytokine secretion profile of normal and WAS NK cells. a, Dot plot representing quantitative analysis of secreted cytokines, i.e., GM-CSF, IFN-γ (left panel), and IL-2, TNF-α (right panel) for PMA/I-activated normal (N) and WAS NK cells are shown. Data from seven normal donors and one WAS patient (WAS-1, analyzed from two independent samples) are shown. b, FACS profile of same NK cells from a normal donor (top panel) and WAS patient (bottom panel) dual labeled either with GM-CSF (y-axis) and CD69 (x-axis) (right panels) or with isotype control Abs (left panels) along with the percentage of cells in each quadrant is shown.

FIGURE 6.

Cytokine secretion profile of normal and WAS NK cells. a, Dot plot representing quantitative analysis of secreted cytokines, i.e., GM-CSF, IFN-γ (left panel), and IL-2, TNF-α (right panel) for PMA/I-activated normal (N) and WAS NK cells are shown. Data from seven normal donors and one WAS patient (WAS-1, analyzed from two independent samples) are shown. b, FACS profile of same NK cells from a normal donor (top panel) and WAS patient (bottom panel) dual labeled either with GM-CSF (y-axis) and CD69 (x-axis) (right panels) or with isotype control Abs (left panels) along with the percentage of cells in each quadrant is shown.

Close modal

The cytolytic ability of WAS NK cells is unaffected, as evidenced by efficient killing of a HLA-class I-negative target (i.e., 721.221) by FACS-purified IL-2-activated NK clones and polyclonal NK cells generated from three WAS patients (Fig. 7).

FIGURE 7.

Cytotoxicity profile of WAS NK cells. Cytotoxicity of IL-2-activated normal and WAS NK clones and polyclonal NK cells against 721.221 target cells as measured in a 4-h 51Cr release assay is shown for three WAS patients. The number (n) of NK clones tested for each WAS patient is shown in parentheses, and the percentage of cytotoxicity is shown as an average value ± SEM. NK clones were not tested in WAS-3 patient and are indicated in the figure as “nd.”

FIGURE 7.

Cytotoxicity profile of WAS NK cells. Cytotoxicity of IL-2-activated normal and WAS NK clones and polyclonal NK cells against 721.221 target cells as measured in a 4-h 51Cr release assay is shown for three WAS patients. The number (n) of NK clones tested for each WAS patient is shown in parentheses, and the percentage of cytotoxicity is shown as an average value ± SEM. NK clones were not tested in WAS-3 patient and are indicated in the figure as “nd.”

Close modal

Our study describes a novel role for WASp in regulating nuclear translocation of NFAT2 and NF-κB p65 (RelA) during cell-cell interaction and upon cross-linking the natural cytotoxicity receptor NKp46. These effects of WASp are independent of WASp-regulated F-actin polymerization. Furthermore, total absence of WASp results in disorganized F-actin and mislocation of the signaling molecules in WAS-NKIS. In particular, CN and components involved in calcium influx, i.e., PLC-γ1 and ZAP70, display delayed activation and recruitment to the WAS-NKIS. Our study therefore offers an explanation for the well-established phenomenon of decreased calcium influx in WAS lymphocytes (7, 14).

Our data indicate that the primary role of WASp in facilitating nuclear translocation of NFAT2 and RelA is due to its effects on reorganization of the activating NKIS. Inappropriate positioning of the proteins within the WAS-NKIS, by preventing interaction with their binding partners, impairs the strength of receptor-proximal signals. Therefore, in the 10-min WAS-NKIS, enrichment of p-ZAP70 was reduced, resulting in impaired activation of PLC-γ1 (35). Decreased activity of PLC-γ1 by impairing calcium entry via InsP3-mediated receptor-operated channels and directly via store-operated channels could impact all aspects of calcium signaling (37) and account for impaired nuclear translocation of both NFAT and RelA in WAS NK cells (38). PLC-γ1-deficient Jurkat cells also fail to induce NF-κB activation (39). Although WASp deficiency impairs both NFAT2 and RelA activity, Itk deficiency impairs NFAT2 without affecting NF-κB (40), and PKC-θ and Vav1 deficiency impairs NF-κB without affecting NFAT (41, 42, 43). PMA/I, which directly increases [Ca2+]i levels and activates NFAT via Ras activation (18), at least partially rescues WAS NK cells from NFAT2 and RelA defects. PMA/I-induced abrogation of T cell defects was also observed in Itk−/−, WAS−/−, WIP−/−, and PKC-θ−/− murine T cells (7, 25, 40, 41). In contrast, cross-linking of NKp46 on WAS NK cells does not induce nuclear translocation of NFAT2 and NF-κB at the time when translocation of these factors normally occurs (i.e., within 10 min). Furthermore, decreased activity of PLC-γ1 could contribute to the formation of a disorganized WAS-NKIS, given its role in maintaining stable and mature Th IS (44).

The overall activation of CN, important for the activity of NFAT (17), was less affected. However, the accumulation of CN in the WAS-NKIS was significantly reduced compared with normal, suggesting that increase in focal concentration within the NKIS, rather than the global changes in CN concentration, must be important for regulating the signaling events through CN-mediated dephosphorylation. This concept is supported by the observation that synaptic accumulation of CN in the neurons controls receptor activation and desensitization (45).

We also find that WASp regulates the activity of transcription factors independently of the state of actin cytoskeleton. These observations are in agreement with studies of domain-deleted WASp mutants (19). In our studies of the normal NK cells, perturbation of the F-actin cytoskeleton by Cyt-D did not affect nuclear translocation of NFAT2 in the NK:B conjugates or NK:bead conjugates (Fig. 4), suggesting that calcium signals must remain maintained even in a depolymerized NK cell. Indeed, studies in excitable cells have shown no impact on the receptor-operated channel- and store-operated channel-mediated calcium responses upon extensive Cyt-D-induced actin cytoskeletal breakdown (46). Analysis of WAS NK conjugates at 30 min clearly demonstrates a lack of association between NFAT activity and actin polarization. Our data suggest that actin polymerization and the integrity of actin cytoskeleton are not essential for transmission of signals to the nucleus, at least at the time points studied. Similarly, accelerated apoptosis observed in WAS lymphocytes occurs independently of the defect in actin polymerization (31). It is therefore likely that activated WASp can simultaneously deliver multiple signals through its different domains.

In our studies, we noted differential effect of WASp deficiency on production of GM-CSF, but not IFN-γ, although transcription of both GM-CSF and IFN-γ genes is regulated by NFAT (18). A similar differential effect on cytokine production was observed in Itk−/− T cells (i.e., IL-4 defect, but normal IFN-γ) (40). Interestingly, studies in murine WAS−/− T cells have shown impaired transcription of IL-2 and IL-4 genes, but normal transcription of IFN-γ, yet the secretion of IFN-γ was impaired (47). However, our observation of reduced production of GM-CSF in PMA/I-stimulated WAS NK cells was unexpected in light of near normal activation of NFAT2 and RelA under this condition. It is likely that activation of GM-CSF promoter may require signals in addition to NFAT and RelA. Indeed, studies in PMA/I-stimulated T cells have shown a requirement for multiple simultaneous signals to activate the GM-CSF promoter (48).

The most significant cytoskeletal defect observed in WAS NK cells was the inability of F-actin to reorganize into the microdomains of NKIS, but not in the formation of F-actin polymers. Although the majority of WAS NK cells fail to polarize F-actin, in ∼35% of WAS NK cells, F-actin is actively polarized. Furthermore, F-actin enrichment in the WAS-NKIS is only modestly reduced (Fig. 1 c). Minimal effects on F-actin polymerization, polarization, and microvilli formation were also observed in WAS T cells (14, 31, 49, 50). Collectively, these data suggest that other molecules known to have Arp2/3-dependent actin-nucleating activity (51, 52) may partially compensate and enable at least some actin polymerization to occur locally in the absence of WASp. Alternatively, Arp2/3-independent actin polymerization by human zyxin via binding to Ena/VASP homology 1 domain-containing proteins (e.g., Homer-3) could also nucleate actin filament assembly (53). WAS NK cells demonstrate normal polarization and enrichment of Homer-3 to the NKIS, suggesting the latter’s role in initiating actin polymerization locally at the NKIS. These data fit well with a recent observation in T cells, in which polarization of Homer-3 precedes actin polarization (54). However, zyxin-mediated F-actin polymers are long and unbranched in contrast to WASp-Arp2/3-mediated actin filaments, which are highly branched (53). This F-actin morphology could further compound the actin segregation defect in the WAS-NKIS, resulting in disordered SMAC structures.

Furthermore, like CD2-associated protein-deficient T cells (12), WAS-deficient NK cells demonstrate delayed tyrosine phosphorylation (i.e., of ZAP70 and PLC-γ1) and impaired cSMAC formation. Studies in the T cells, using the in silico model, have defined a pivotal role of cSMAC in facilitating rapid maturation of Th IS (55). Our studies on WAS-NKIS also support such a role for cSMAC in NKIS maturation. These results are in agreement with the proposed role of WASp in murine Th IS formation (12). Recently, however, WASp was shown to be important in the formation of Th IS only under low, but not high peptide concentration (49). Interestingly, in normal NK cells, we observed decreased intensity of p-PLC-γ1 (Tyr1254 and Tyr783) and p-ZAP70 bands at 30 min, after cross-linking NKp46 receptor (Fig. 3 c). Although delayed accumulation of p-PLC-γ1 and p-ZAP70 in WAS NK cells is consistent with the overall delay in WAS-NKIS maturation, the decreased accumulation of these proteins in the late normal-NKIS could perhaps result from localized degradation by ubiquitin ligases up-regulated upon sustained calcium-CN activity within the normal NKIS (44). Western blot analysis of extracts from normal NK cells stimulated with anti-NKp46 beads showed a persistent increase in the intensity of CN band at 30 min (data not shown).

Finally, our results also show that the overall natural cytotoxicity remains intact in the IL-2-activated WAS NK cells, in agreement with a previous study (23). Collectively, these data suggest that the minimum requirements for granule exocytosis must be maintained even in the face of immature WAS-NKIS. Indeed, T cell killing does not require a mature IS (56).

In summary, we have identified a novel role of WASp in mediating nuclear translocation of NFAT2 and NF-κB during NK cell:target cell interaction and NKp46-mediated signaling. Although WASp is not absolutely essential for the nuclear translocation of NFAT2 and NF-κB, this activity is dramatically delayed in the absence of WASp. Dysregulation of NFAT activity in certain SCID patients, resulting from impaired calcium flux, interestingly identifies a mechanism shared by these two immune deficiency disorders (57).

The authors have no financial conflict of interest

We thank Dr. Lloyd Old (Ludwig Institute for Cancer Research) for access to digital fluorescence microscope. We thank Dr. Michael K. Rosen and members of his laboratory for providing the GFP-WASp-GTPase-binding domain reagent. We also thank Richard J. O’Reilly for providing patient samples, and Hina Maniar and Richard B. Sisson for technical assistance.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grants NIAID-K08-AI 51402 (to Y.M.V.), R01-AI 50193 (to B.D.), and NCI-CA 08748 (Core Grant to Memorial Sloan-Kettering Cancer Center); by a Junior Faculty Scholar Award from the American Society of Hematology (to Y.M.V.); and by the Immune Deficiency Foundation (to Y.M.V.).

3

Abbreviations used in this paper: WAS, Wiskott-Aldrich syndrome; 2D, two-dimensional; 3D, three-dimensional; Arp, actin-related protein complex; CN, calcineurin; cSMAC, central supramolecular activation cluster; Cyt-D, cytochalasin D; Ena/VASP, Enabled/vasodilator-stimulated phosphoprotein; Grb2, growth factor receptor-bound protein 2; IFI, integrated fluorescence intensity; InsP3, inositol 1,4,5-trisphosphate; IS, immunological synapse; iSMAC, intermediate SMAC; MTOC, microtubule-organizing center; NKIS, NK cell IS; p, phosphorylated; PKC, protein kinase C; PLC, phospholipase C; PMA/I, PMA/ionomycin; pSMAC, peripheral SMAC; RE, relative enrichment; SMAC, supramolecular activation cluster; WASp, WAS protein; WIP, WASp-interacting protein; ex, extracellular.

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