Differential Regulation of Human NK Cell-Mediated Cytotoxicity by the Itk

This information is current as Dianne Khurana, Laura N. Arneson, Renee A. Schoon, of September 28, 2021. Christopher J. Dick and Paul J. Leibson J Immunol 2007; 178:3575-3582; ; doi: 10.4049/jimmunol.178.6.3575 http://www.jimmunol.org/content/178/6/3575 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Regulation of Human NK Cell-Mediated Cytotoxicity by the Tyrosine Kinase Itk1

Dianne Khurana, Laura N. Arneson, Renee A. Schoon, Christopher J. Dick, and Paul J. Leibson2

NK cells are effector lymphocytes that can recognize and eliminate virally infected and transformed cells. NK cells express distinct activating receptors, including an ITAM-containing FcR complex that recognizes Ab-coated targets, and the DNAX-activating protein of 10 kDa-containing NKG2D receptor complex that recognizes stress-induced ligands. The regulatory role of specific tyrosine kinases in these pathways is incompletely understood. In this study, we show that, in activated human NK cells, the tyrosine kinase IL-2-inducible T cell kinase (Itk), differentially regulates distinct NK-activating receptors. Enhanced expression of Itk leads to increases in calcium mobilization, granule release, and cytotoxicity upon stimulation of the ITAM-containing FcR, suggesting that Itk positively regulates FcR-initiated cytotoxicity. In contrast, enhanced Itk expression decreases cytotoxicity and Downloaded from granule release downstream of the DNAX-activating protein of 10 kDa-containing NKG2D receptor, suggesting that Itk is involved in a pathway of negative regulation of NKG2D-initiated granule-mediated killing. Using a kinase mutant, we show that the catalytic activity of Itk is required for both the positive and negative regulation of these pathways. Complementary experiments where Itk expression was suppressed also showed differential regulation of the two pathways. These findings suggest that Itk plays a complex role in regulating the functions initiated by distinct NK cell-activating receptors. Moreover, understanding how these pathways may be differentially regulated has relevance in the setting of autoimmune diseases and antitumor immune responses http://www.jimmunol.org/ where NK cells play key regulatory roles. The Journal of Immunology, 2007, 178: 3575–3582.

atural killer cells are innate effector lymphocytes that ITAM that recruits Syk family kinases upon activation, whereas eliminate cells infected by viruses. NK cells also have the DNAX-activating protein (DAP10)3 signal transducing subunit N the ability to destroy cells that have undergone malig- for the NKG2D receptor contains a motif that binds to both PI3K nant transformation. The principal mechanism used by NK cells to and Grb2 and triggers cell-mediated killing independently of Syk kill target cells is the secretion of preformed granules containing kinase activation. Grb2 couples to Vav1, phospholipase C perforin and granzymes that together induce apoptosis of the target (PLC)␥2, and SLP-76, whereas PI3K activation leads to Erk phos- by guest on September 28, 2021 cell. Upon direct contact with a target, NK cells can also activate phorylation (2, 3). The downstream pathways leading to granule apoptosis by engaging death receptor pathways. In addition, in- release remain incompletely characterized. flammatory cytokines, including IFN-␥ and TNF-␣, are secreted To understand the regulatory role of specific tyrosine kinases in by activated NK cells promoting NK cell cytotoxicity and shaping these pathways, we studied the Tec family tyrosine kinase IL-2- the innate and adaptive immune responses. NK cell function is inducible T cell kinase (Itk). Tec kinases are nonreceptor tyrosine regulated by a variety of activating and inhibitory cell surface re- kinases that modulate signals downstream of many hemopoietic ceptors. Thus, NK cell activation results from the triggering of surface receptors, including AgRs, cytokine receptors, integrins, activating receptors together with reduced signaling through in- and G protein-coupled receptors (4). Tec kinases are important hibitory receptors. Distinct activating receptors on NK cells in- components of signaling pathways leading to activation of PLC␥ ␥ clude the low-affinity receptor for IgG, Fc RIIIA (or CD16), here- and MAPKs, regulation of the actin cytoskeleton, adhesion, mi- after referred to as FcR, which recognizes Ab-coated target cells gration and transcriptional activation (5), pathways potentially rel- during Ab-dependent cell-mediated cytotoxicity, and the NKG2D evant to NK cell conjugate formation, granule polarization, and receptor that recognizes stress-inducible ligands on cells. exocytosis. Therefore, we hypothesized that Itk, the major Tec The FcR and NKG2D receptor both transmit an activation sig- family kinase expressed in NK cells, would have an important nal, resulting in granule release and cytotoxicity by NK cells, al- regulatory role in NK cell-mediated killing. Itk is expressed in T though these receptor-initiated events are differentially regulated cells, mast cells, and NK cells. In addition to its kinase activity, Itk (1, 2). The signal transducing subunit for the FcR contains an possesses multiple noncatalytic domains, including a pleckstrin homology (PH), Tec homology, Src homology 2 (SH)2, and SH3 domain, which have the capacity to bind to specific protein motifs. Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN 55905 A number of potential ligands and mediators of Itk function have Received for publication August 23, 2006. Accepted for publication December 28, 2006. 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. 3 Abbreviations used in this paper: DAP, DNAX-activating protein; BLT, N-benzy- 1 loxycarbonyl lysine thiobenzyl ester; IP3, inositol 1,4,5-trisphosphate; Itk, IL-2-in- This research was supported by the Mayo Foundation and by National Institutes of ducible T cell kinase; LU, lytic unit; PH, pleckstrin homology; PKC, Health Grant CA47752. C; PLC, phospholipase C; Rlk, resting lymphocyte kinase; SH, Src homology; 2 Address correspondence and reprint requests to Dr. Paul J. Leibson, Department of siRNA, small interfering RNA. Immunology, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org 3576 REGULATION OF HUMAN NK CYTOTOXICITY BY Itk been identified from studying the interactions of its various do- Inositol 1,4,5-trisphosphate (IP3) measurements mains in T cells in vitro. The PH domain of Itk has been demon- NK cells were infected with control vaccinia (WR) or recombinant vac- strated to bind to G protein ␤␥ subunits, a number of different cinia expressing Itk and stimulated with cross-linking anti-FcR or anti- protein kinase C (PKC) isoforms and inositol phospholipids (6, 7). NKG2D mAb as described previously (21). The reactions were stopped Furthermore, the PH domain targets Itk to the cell membrane (8). with ice-cold 20% perchloric acid, followed by 20-min incubation on ice. Membrane localization may be needed to regulate Itk function in The samples were centrifuged at 14,000 rpm for 15 min at 4°C, and su- pernatants were decanted and neutralized to pH 7.5 with ice-cold 1.5 M response to Src family tyrosine kinases (8, 9). The proline-rich KOH. KClO4 was sedimented by further centrifugation at 14,000 rpm for region in the Tec homology domain can bind to Grb2 and the SH3 15 min, and the decanted supernatants were used for measuring the amount domain of Src family members, including Fyn, Lyn, and Hck (10). of IP3 extracted. IP3 was measured using a competitive tritiated thymidine 3 Several proteins, including CD28, Cbl, and Wiskott-Aldrich syn- [ H]IP3 binding assay (Amersham Biosciences) in accordance with the manufacturer’s instructions. The amount of IP3 in each sample was deter- drome protein among others, have been shown to interact with the mined from measurement of the radioactivity and interpolation from a Itk SH3 domain in vitro (11, 12). The SH2 domain of Itk has been standard curve. shown to bind to the SLP-76 adaptor (13). Thus, it is apparent that Itk is involved in multiple signaling Cytotoxicity assays pathways, although its potential involvement in granule-dependent The 51Cr release assays were done as described previously (14). In all cell-mediated cytotoxicity is unknown. We report here the use of experiments, spontaneous release did not exceed 10% of maximum release. ϩ genetic, biochemical, and functional approaches to study the reg- In redirected cytotoxicity assays (2), NK clones killed the FcR P815 ulatory role of Itk downstream of two NK cell-activating receptors target cells only in the presence of the designated triggering mAbs. Lytic units (LUs) per 106 effector cells were calculated on the basis of 20% that trigger cytotoxicity by recruiting different proximal kinases. lysis of target cells, as expressed by the formula: number of LUs/106 Downloaded from Our analyses suggest that this Tec kinase has different regulatory effectors ϭ 106/T.Xp, where T is the number of target cells, p is the roles downstream of the FcR and the NKG2D receptor in human reference lysis level (20%), and Xp is the E:T ratio required to lyse 20% NK cells. of the targets (22). Cell stimulation and immunoblot analysis Materials and Methods Vaccinia infection of NK cells, cell stimulation, protein immunoprecipita- http://www.jimmunol.org/ Reagents, cells, and Abs tion, and detection of tyrosine phosphorylation were conducted as de- Unless otherwise indicated, reagents were obtained from Sigma-Aldrich. scribed previously (21). The murine mastocytoma P815 was obtained from American Type Culture Collection. Human NK cells were cloned and passaged as described pre- Measurements of granule release ␥ viously (14). The 3G8 mAb to human Fc RIIIA and the anti-EE murine High-affinity binding 96-well plates (Costar) were incubated overnight at mAb were purified from ascites by affinity chromatography over protein 4°C with either 1 and 5 ␮g/ml mAb to FcR or NKG2D or with IgG1 at the A-agarose. Monoclonal anti-NKG2D (R&D Systems), monoclonal Itk (BD same concentrations in 0.05 M carbonate buffer (pH 9.6) (100 ␮l per well Pharmingen), monoclonal anti-CD4 (BD Biosciences), purified mouse my- in triplicate). After washing the plates, NK cell suspensions (4 ϫ 106 cells/ eloma protein IgG1 (Bionetics), and anti-phosphorylated tyrosine (4G10; ml) were added to each well (150 ␮l/well) in RPMI 1640 medium (In- Upstate Signaling Solutions) were also used. Rabbit polyclonal antiserum

vitrogen Life Technologies) containing 10% calf serum. After incubation at by guest on September 28, 2021 to Itk was obtained after immunization of rabbits with KLH-conjugated 37°C for 4 h, supernatants were evaluated for secretion using a standard Itk peptide 145–170 (QYDPTKNASKKPLPPTPEDNRRPLWE; Cocalico N-benzyloxycarbonyl lysine thiobenzyl ester (BLT) esterase assay (23). ␥ Biologicals). Rabbit polyclonal Abs to PLC 2, Vav1, and Zap70 have been For maximum secretion, 150 ␮l of cells was freeze-thawed three times in described previously (15–17). liquid nitrogen. Spontaneous secretion was from cells in wells with me- dium alone. Percent secretion was calculated as (sample Ϫ spontaneous)/ Recombinant vaccinia (maximum Ϫ spontaneous) ϫ 100%.

Full-length Itk containing an EE-epitope tag was subcloned into the pSP11 Itk suppression vector as described previously (18). A point mutation in the Itk kinase domain (K391R), which prevents ATP binding and thereby Itk activation, Itk-specific SMARTpool siRNA was obtained from Upstate Signaling So- was generated with the QuikChange Site-Directed Mutagenesis (Strat- lutions and transduced into NK cells using the Amaxa nucleofector system agene) in accordance with the manufacturer’s instructions. Recombinant as previously described (2). Nucleofected cells were returned to a fresh vaccinia viruses encoding the wild-type Itk or the kinase inactive form of flask of the original culture medium that had been reserved from the day Itk (K391R) were generated by insertion of the pSP11 constructs into the the cells were passed. Nonsilencing dsRNA (AF488; Qiagen) was used as WR strain of vaccinia virus via homologous recombination (18–20). The a negative control. Functional assays with whole-cell lysates were per- Flag-tagged CD4 chimeric receptors were generated by PCR and standard formed 48 h after nucleofection. Cell viability at this time was measured molecular biology techniques as described previously (1). with a Vi-cell viability analyzer (Beckman Coulter), and in all experiments, viability after nucleofection exceeded 75%. Protein suppression was quan- ϩ Ca2 mobilization assays tified by densitometry using the LabWorks analysis software (UVP).

2ϩ Changes in the levels of intracellular Ca were monitored by flow cy- Statistics tometry using cells loaded with the Ca2ϩ-sensitive fluorescent dye, indo- 1-AM (Calbiochem-Novabiochem). To express specific proteins, human In assays in which the percent 51Cr release is plotted, error bars represent NK cells were infected with the indicated nonrecombinant (WR) or re- SD of triplicate samples. Where LUs are plotted, error bars represent SD combinant vaccinia viruses at a multiplicity of infection of 20:1 for5hin obtained from nonlinear regression analysis to the exponential fit y ϭ A ϫ a humidified 37°C incubator. For the last hour of the infection, the cells (1 Ϫ eϪkx) as described previously (24). Where normalized LUs or secre- were loaded with 5 ␮M indo-1. In suppression experiments, NK cells tion are plotted, error bars represent the SD of the averages of multiple nucleofected with either negative control dsRNA (Qiagen) or Itk small experiments. Comparison between groups was performed with the paired interfering RNA (siRNA) (SMARTpool; Upstate Signaling Solutions) 48 h Student t test. A p value of Ͻ0.05 was considered statistically significant. earlier were incubated for 60 min at 37°C with 5 ␮M indo-1. The cells were washed and kept on ice until analyzed. For analysis, the indo-1-loaded NK Results cells were warmed for 10 min in a 37°C water bath and then stimulated with Abs to either the endogenous FcR or NKG2D receptor, together with Tyrosine phosphorylation of Itk after stimulation of the FcR and Ј goat anti-mouse IgG F(ab )2 (Cappel). The samples were analyzed imme- NKG2D receptor in NK cells diately by flow cytometry using a UV laser for excitation with violet (405 nm) and blue (530 nm) fluorescence emissions recorded. The data plots Upon FcR ligation, NK cells initiate Ab-dependent cell-mediated were generated using the FlowJo software program (Tree Star). Ca2ϩ data cytotoxicity, whereas a pathway of natural cytotoxicity is triggered are presented as mean values. after NKG2D ligation (25). Although each pathway is initiated by The Journal of Immunology 3577

FIGURE 1. Itk is tyrosine phosphorylated after both FcR and NKG2D stimulation. A, Cloned human NK cells were stimulated for the indicated times at 37°C with either isotype control, anti-FcR mAb, or anti-NKG2D

Ј Downloaded from mAb, followed by a secondary goat anti-mouse IgG F(ab )2. Itk immuno- precipitates were resolved by SDS-PAGE, transferred to a membrane, and probed with either anti-phosphotyrosine mAb (upper panel, pTyr) or anti- Itk mAb (lower panel, Itk). B, NK cells were infected with vaccinia virus encoding the EE-Itk construct. Using anti-EE mAb, expressed EE-Itk was immunoprecipitated from NK cells after anti-FcR or anti-NKG2D cross- linking for the indicated times at 37°C. The immunoprecipitates were re- solved, transferred, and blotted with either anti-phosphotyrosine mAb (up- http://www.jimmunol.org/ per panel, pTyr) or anti-EE mAb (lower panel, EE). The data shown are representative of three independent experiments. receptor phosphorylation by a Src family tyrosine kinase, the sub- sequent signals leading to cytotoxicity for each receptor are initi- ated by different proximal kinases (1). To determine whether Itk is coupled to either pathway, we first evaluated whether endog- enously expressed Itk is tyrosine phosphorylated during activation by guest on September 28, 2021 of each receptor. For these and all subsequent studies, we used homogeneous, cloned populations of nontransformed human NK cells. NK cells were stimulated with cross-linking Abs to either the FcR or the NKG2D receptor to selectively trigger each pathway. FIGURE 2. Itk enhances the tyrosine phosphorylation of PLC␥2, IP3 Selective FcR or NKG2D cross-linking on NK cells induced the release, and intracellular calcium downstream of both the FcR and NKG2D tyrosine phosphorylation of endogenous Itk (Fig. 1A) or recom- receptor. A, NK cells were infected with control vaccinia (WR) or with binant vaccinia encoded Itk (Fig. 1B) with similar kinetics. Al- recombinant vaccinia encoding either wild-type Itk (EE.Itk) or the cata- though Itk was found to be activated downstream of both path- lytically inactive Itk mutant (EE.K391R). Cells were then stimulated for ways, the tyrosine phosphorylation observed after NKG2D the indicated times with either anti-FcR or anti-NKG2D, and then PLC␥2 stimulation was weaker relative to the signal observed after FcR immunoprecipitates were resolved and blotted. Densitometry (bar graph) stimulation. was expressed as the ratio of the density of the tyrosine-phosphorylated band to the density of the corresponding band in the PLC␥2 blot. Numbers Contribution of Itk to PLC␥2 activation, IP3 release, and refer to individual lanes. The data shown are representative of five inde- calcium signaling after stimulation of either the FcR pendent experiments. B, NK cells infected with control vaccinia or recom- or NKG2D binant vaccinia encoding Itk were stimulated with cross-linking anti-FcR or anti-NKG2D mAb for the indicated times. IP3 release in stimulated A key downstream effect of Tec kinases in hemopoietic cells is to samples was determined. The data shown are representative of two inde- fully activate PLC␥ contributing to a sustained release of calcium pendent experiments. C, NK cells infected with the indicated nonrecom- in the cell (26, 27). PLC␥-dependent calcium mobilization in NK binant (WR) or recombinant vaccinia viruses were labeled with indo-1 cells is required for granule exocytosis and killing. The specific and then stimulated with either anti-FcR mAb or anti-NKG2D mAb, Ј contribution of Itk to FcR or NKG2D-mediated phosphorylation of followed by goat anti-mouse IgG F(ab )2. Calcium release was mea- PLC␥2, the predominantly expressed isoform in NK cells, has not sured by flow cytometry. Expression of Itk was determined by immu- noblotting (inset): lane 1, control WR vaccinia; lane 2, recombinant been described previously. When we expressed recombinant Itk vaccinia encoding Itk; and lane 3, recombinant vaccinia encoding cat- and stimulated either the FcR or NKG2D, tyrosine phosphoryla- alytically inactive Itk (K391R). The data are representative of two in- ␥ tion of PLC 2 was enhanced downstream of both pathways com- dependent experiments. pared with either cells infected with the control WR strain vaccinia virus or with cells expressing the kinase inactive (K391R) mutant (Fig. 2A). The enhanced activation of PLC␥2 was principally seen at 5 min after receptor stimulation for each pathway, a finding infected with control WR vaccinia vs cells expressing the recom- consistent with the ability of Tec kinases to sustain calcium sig- binant kinase mutant (K391R) was comparable in five independent naling. The level of tyrosine phosphorylation of PLC␥2 in cells experiments (data not shown). 3578 REGULATION OF HUMAN NK CYTOTOXICITY BY Itk Downloaded from

FIGURE 4. Itk enhances cytotoxicity stimulated through ITAM-me- diated pathways and suppresses killing triggered through DAP10. A, The chimeric receptors are each tagged with a Flag epitope and contain the extracellular and transmembrane portions of the CD4 molecule. http://www.jimmunol.org/ Each receptor is distinguished by the intracellular portion correspond- ing to the cytoplasmic tails of the ␨, ␥, DAP12, or DAP10 adaptors. B,NK FIGURE 3. Itk differentially regulates cell-mediated cytotoxicity. A, cells were coinfected with recombinant vaccinia virus encoding the indi- NK cells were infected with either control WR vaccinia or recombinant cated chimeric receptor and either control vaccinia (WR) or vaccinia en- vaccinia encoding Itk. Infected NK cells were then incubated with 51Cr- coding Itk. Cells were incubated with 51Cr-labeled P815 cells in the pres- labeled P815 cells and either 0.14 ␮g/ml anti-FcR mAb or anti-NKG2D ence of anti-CD4 mAb to trigger cytotoxicity through the chimeric mAb. The E:T ratios used were 20:1, 10:1, 5:1, and 2.5:1. Supernatants receptors or anti-FcR or anti-NKG2D mAb to initiate killing through the were assayed for 51Cr release after4hat37°C. The data are expressed as endogenous receptors. After4hat37°C, the amount of 51Cr released into LUs Ϯ 1 SD. Data shown are representative of multiple independent cy- the supernatants was measured. Data shown are representative of three B totoxicity experiments. , NK cells infected with the indicated vaccinia by guest on September 28, 2021 independent experiments. viruses were incubated with P815 cells coated with anti-FcR or anti- NKG2D as in A. Itk expression was determined by immunoblotting (inset): lane 1, control WR vaccinia; lane 2, recombinant vaccinia encoding Itk; Regulation of NK cell-mediated cytotoxicity and granule and lane 3, recombinant vaccinia encoding kinase inactive Itk (K391R). C, Infected cells were incubated with 51Cr-labeled 721 target cells (natural release by Itk cytotoxicity assay). Data shown are representative of three independent To determine whether the enhancement of FcR- and NKG2D-in- experiments. duced calcium signals with Itk overexpression had a functional consequence, we assessed the influence of Itk on NK cell-mediated Activated PLC␥ hydrolyzes its substrate, phosphatidylinositol killing. We infected NK cells with either control vaccinia or re- 4,5-bisphosphate, generating the second messengers IP3 and diac- combinant vaccinia encoding Itk. To specifically initiate NK cell ylglycerol (28). When we measured IP3 release in NK cells ex- killing through either the FcR or NKG2D, infected NK cells were pressing recombinant Itk and stimulated through either the FcR or used as effectors in a redirected cytotoxicity assay against P815 NKG2D, an ϳ1.5-fold increase in IP3 was observed downstream tumor targets that bind either anti-FcR mAb or anti-NKG2D mAb. of either pathway compared with the amount of IP3 generated in We found that, consistent with increased calcium, cells expressing the stimulated control infected cells (Fig. 2B). Because IP3 acts to recombinant Itk enhanced FcR-initiated killing (Fig. 3A). Surpris- open intracellular calcium stores promoting an initial, transient rise ingly, the same cells expressing recombinant Itk decreased killing in intracellular calcium, we next compared calcium fluxes in NK triggered through NKG2D (Fig. 3A). We tested over 20 different cells. Consistent with the increased tyrosine phosphorylation of NK clones in independent experiments and consistently observed PLC␥2 and IP3 generation observed, we found that intracellular this differential regulation of cytotoxicity with Itk overexpression calcium was enhanced for both the FcR and NKG2D pathways (data not shown). To determine whether the catalytic activity of Itk when recombinant Itk was expressed (Fig. 2C). Notably, the cal- was required for the modulation of killing, the catalytically inac- cium waveform was different downstream of the NKG2D receptor tive form of Itk was expressed. As seen before, wild-type Itk en- as has been reported previously (21). The waveform had a rela- hanced killing of P815 cells triggered through the FcR and sup- tively delayed onset and lower amplitude; however, its amplitude pressed cytotoxicity initiated through NKG2D (Fig. 3B). Mutation was increased by recombinant Itk expression. Calcium fluxes were of the catalytic site fully abrogated that modulation, suggesting the same in NK cells infected with control vaccinia or in cells that activation of Itk is required for both the positive and negative expressing the recombinant kinase mutant, consistent with their regulation of cytotoxicity (Fig. 3B). Although Itk has the potential similar tyrosine phosphorylation of PLC␥2. Taken together, these to positively regulate both pathways through calcium, only FcR- results suggest that Itk enhances the proximal signaling pathways initiated killing is enhanced, whereas NKG2D-initiated cytotoxic- leading to calcium release downstream of both the FcR and ity is actively suppressed. Interestingly, NK cell killing of the 721 NKG2D receptor. tumor, which does not express the NKG2D ligands MICA/B (data The Journal of Immunology 3579

FIGURE 5. Granule secretion triggered through the FcR and NKG2D receptor is differentially regulated by Itk. Cloned human NK cells infected with the indicated vaccinia viruses were stimulated for4hat37°C with either 1 or 5 ␮g/ml plate-bound mAb (anti-FcR and anti-NKG2D). Granule release was measured by a BLT esterase assay.

not shown), was also enhanced by recombinant Itk (Fig. 3C). How- ever, the receptors and ligands that trigger natural cytotoxicity in this assay are not known. Downloaded from To determine whether the difference in killing seen with en- hanced Itk expression was a result of the signaling motifs in the FIGURE 6. Suppression of Itk in human NK cells with siRNA. A,We cytoplasmic tails of each receptor, recombinant vaccinia encoding introduced control dsRNA or serial dilutions of Itk-specific siRNA into NK Flag-tagged CD4 chimeric receptors were generated. The cytoplas- clones with the Amaxa nucleofector system. For titrated Itk siRNA sam- ples, control dsRNA was supplemented to have 300 pmol of total siRNA mic tails of chimeric receptors either had ITAMs (␥, ␨, and per sample. Equal protein from whole-cell lysates was resolved by SDS- DAP12) or the PI3K/Grb2 motif YINM (DAP10) (Fig. 4A). We PAGE 48 h after nucleofection, and Itk expression was determined by http://www.jimmunol.org/ have previously shown that these chimeric receptors, when ex- immunoblot and densitometry (below blot). The results of three indepen- pressed in human NK cells and cross-linked with a CD4 Ab, ini- dent experiments are shown. B, The membrane with greatest Itk suppres- tiate the same signaling pathways as the endogenous ITAM- or sion (experiment 1; 83% suppression) was reblotted with rabbit polyclonal non-ITAM-coupled receptors (1). The different signaling pathways antisera to PLC␥2, Vav, and Zap70. downstream of the chimeric receptors are each activated with the same Ab, thereby excluding possible differences in Ab stimulation of the endogenous receptors. In NK cells expressing the chimeric Itk suppression in NK cells using siRNA receptors, coexpression of recombinant Itk enhanced cytotoxicity Genetic down-regulation of specific proteins has been useful in triggered through each of the ITAM-containing receptors. In con- discerning the function of many regulatory proteins. However, the by guest on September 28, 2021 trast, cytotoxicity initiated through either the DAP10 chimeric re- in vivo suppression of individual Tec family kinases has often ceptor or the endogenous NKG2D receptor was suppressed in cells been complicated by redundant functions between the family expressing recombinant Itk (Fig. 4B). These results suggest that Itk members (29). Despite this potential limitation, we used siRNA differentially regulates ITAM- vs DAP10-mediated cytotoxicity. delivered with the Amaxa nucleofector system to suppress Itk in To determine whether the modulation of killing was reflected by our cells. As a control, nonsilencing dsRNA was introduced into changes in granule release, the amount of granzyme A being re- NK cells in parallel. To demonstrate the specificity of the Itk- leased was quantified in a BLT esterase assay. In this assay, gran- targeting siRNA, we examined the protein levels 48 h after nucleo- ule secretion is triggered with Abs to each receptor coated on a fection. Expression of Itk was specifically reduced and was titrat- plate and granzyme A release quantitated by spectrophotometry. In able (Fig. 6A) whereas the expression of other key signaling this assay, granule release triggered by either the FcR or NKG2D molecules was not (Fig. 6B). Semiquantitative RT-PCR also de- have comparable sensitivity to calcium as determined by measur- tected suppression of Itk-specific mRNA in the siRNA-treated ing granule release after combining specific receptor stimulations cells, whereas no changes in Rlk-specific or Tec-specific mRNA with a range of concentrations of the calcium ionophore ionomycin were noted (data not shown). When we suppressed Itk in NK cells (data not shown). The same differential regulation of granule se- and compared calcium fluxes, we observed a small but consistent cretion occurred with expression of recombinant Itk as with cyto- decrease in calcium mobilization downstream of both FcR and toxicity, i.e., enhancement of granule release downstream of the NKG2D receptor (Fig. 7). The partial reduction in calcium is con- FcR and suppression of NKG2D-initiated granule secretion (Fig. sistent with Itk acting as an amplifier of calcium signaling and the 5). Similarly, expression of the kinase inactive mutant (K391R) potential for redundancy with other nonsuppressed Tec family ki- fully abrogated the modulation of granule secretion. Three inde- nases in the activation of PLC␥2. To determine the activation of pendent experiments with additional NK clones indicated that Itk other downstream pathways that may be regulated by Itk, we stim- overexpression significantly enhanced FcR-initiated secretion ulated Itk-suppressed cells through each receptor pathway and ( p Ͻ 0.05) and significantly inhibited NKG2D-initiated secretion used phospho-specific Abs to compare Erk activation. Although ( p Ͻ 0.05). Expression of the K391R did not significantly change Erk was inducibly tyrosine phosphorylated downstream of both secretion levels induced by either receptor as compared with the receptor pathways, we did not detect any differences in Erk phos- WR control. Expression of either wild-type Itk or the K391R mu- phorylation in Itk-suppressed NK cells, despite suppression of Itk tant Itk consistently, but modestly (Ͻ20%), increased spontaneous by greater than 80% (data not shown). This may indicate potential granule release (data not shown), suggesting a potential modula- functional redundancy in the regulation of Erk by other Tec family tory role for Itk independent of its catalytic activity. Taken to- kinases expressed in NK cells or the potential for receptor-initiated gether, these results suggest that Itk differentially regulates granule activation of Erk to proceed in a Tec family kinase-independent release and killing downstream of the FcR and NKG2D receptor. manner. 3580 REGULATION OF HUMAN NK CYTOTOXICITY BY Itk

relatively constant despite suppression of Itk by 84%, which was the highest knockdown achieved. These results are consistent with Itk acting as a positive regulator of FcR-mediated cytotoxicity and suggest that Itk has a complex activity downstream of NKG2D potentially involving both positive and negative regulation. We have found that both receptor pathways display a similar depen- dence on calcium to trigger granule release and killing (data not shown). Therefore, the involvement of Itk in a pathway of negative regulation downstream of NKG2D is likely, since killing initiated FIGURE 7. Calcium signaling is decreased in Itk-suppressed NK cells. by NKG2D in Itk suppressed cells was maintained despite a de- NK clones expressing control dsRNA (Neg) or Itk-specific siRNA (sItk) crease in intracellular calcium. were stimulated 48 h after nucleofection with anti-FcR or anti-NKG2D, and calcium release was measured. Expression of Itk was determined by immunoblotting (inset) as follows: lane 1, negative control nonsilencing dsRNA; and lane 2, Itk-specific siRNA. Percent Itk suppression was 66%. Discussion The results shown are representative of two independent experiments. We have found that, in human NK cells, Itk plays a differential role in regulating distinct pathways of cell-mediated cytotoxicity. We show in multiple experimental settings that Itk plays a positive We then used Itk-suppressed NK cells as effectors in a redi-

regulatory role on ITAM-initiated killing, whereas Itk is involved Downloaded from rected cytotoxicity assay, initiating killing through either the FcR in a pathway of negative regulation downstream of the NKG2D or NKG2D receptor. A partial decrease in killing through the FcR receptor that initiates signaling through the non-ITAM transmem- was observed in Itk suppressed cells, whereas killing triggered by brane adaptor DAP10. We also show that the regulation of cyto- the same cells through the NKG2D pathway was maintained (Fig. toxicity by Itk involves a differential modulation of granule release 8A). These trends were confirmed in five independent experiments, and requires the catalytic activity of Itk. Our findings have poten- which show that the suppression of killing through the FcR in

tially important clinical implications as elaborated below. http://www.jimmunol.org/ ITK-suppressed cells was significant ( p Ͻ 0.005), whereas killing The role of Itk in peripheral cytotoxic lymphocytes has been through the NKG2D pathway by the same cells was not signifi- ϩ cantly different (Fig. 8B). In these experiments, increased suppres- investigated from studies of CD8 T cell function in Itk-deficient mice (30, 31). It has recently been shown that Tec kinases regulate sion of Itk correlated with a greater decrease in FcR-initiated kill- ϩ ing, whereas killing initiated by the NKG2D receptor remained the development of CD8 CTLs in this animal model (32, 33). Studies of CD8ϩ T cell function in Itk-deficient mice have re- ported a disparity between the in vitro responses of these cells and the in vivo responses of these mice to viral infections (30, 31). The TCR stimulation of CD8ϩ T cells lacking Itk or Itk and Rlk re- sulted in impaired phosphorylation and activation of PLC␥1, Erk, by guest on September 28, 2021 and p38 MAPK and loss of a sustained calcium response. These lead to defects in expansion and effector cytokine production. De- spite the substantial defects in CD8ϩ T cell responses observed in vitro, antiviral immune responses proceeded efficiently in the ab- sence of Itk and Rlk (31). The authors proposed that the T cell defects could be compensated for by the innate immune response to the infection. However, innate cytotoxic cells are also dependent on the activation of PLC␥ and intracellular calcium release for effector function. Our findings that Itk differentially regulates dis- tinct pathways of NK cell-mediated cytotoxicity may provide an explanation for this apparent disparity. It would be predicted from our work that killing of virally infected cells through the NKG2D pathway would be intact and possibly enhanced in Itk-deficient NK cells. It is also possible that Itk has a negative regulatory role in the function of CTLs, which, like NK cells, also express NKG2D. One can also speculate from our data that the antitumor FIGURE 8. The reduction of Itk suppresses FcR-initiated cytotoxicity, immune responses of NK cells toward tumors bearing ligands for whereas cytotoxicity triggered through NKG2D is maintained. A, FcR- and NKG2D may also be enhanced by Itk deficiency. This possibility NKG2D-mediated cytotoxicity. NK clones expressing control dsRNA (Neg) or Itk-specific siRNA (siItk) were incubated with P815 cells coated remains to be tested. with anti-FcR or anti-NKG2D at various E:T ratios (horizontal axis). Inset, Similar to what we have found in NK cells, a differential role for Immunoblot of Itk protein expression. Percent decrease in Itk expression Itk downstream of distinct T cell pathways has been reported pre- was 54%. B, NK clones expressing control dsRNA or Itk-specific siRNA viously. In T cells, Itk has been implicated in signaling pathways were evaluated in five individual experiments where Itk suppression was from both the ITAM-containing TCR and the CD28 costimulatory Ͼ 50%. LUs were normalized to those of the control dsRNA sample. Error receptor that contains a PI3K-binding motif (34, 35). The role of bars represent the SD of the averages of the experiments. Comparison Itk may differ downstream in these T cell pathways. Early char- between groups was performed with the paired Student t test using a two- tailed distribution. Levels of cytotoxicity were significantly less (p ϭ acterization of T cell function in Itk-deficient mice showed that 0.0046) for killing through the FcR in Itk-suppressed cells, whereas CD3-mediated proliferative responses were reduced (36), whereas ϩ NKG2D-initiated killing was not significantly different when Itk was sup- proliferation of Itk-deficient CD4 T cells in response to anti- pressed (p ϭ 0.9768). CD28 stimulation was enhanced, supporting a differential role for The Journal of Immunology 3581

Itk downstream of CD3 and CD28 (37). More recent work sug- possible that Tec family kinases have the capacity to both amplify gests that Itk is not essential for CD28 signaling in naive Itk- and attenuate signaling to fine tune signaling pathways. deficient CD4ϩ T cells (38), although the ability of Itk to nega- There are several implications of our work. First, this study tively influence CD28 may depend on the state of activation of the shows that human NK cells, a subpopulation of lymphocytes that cells. It remains to be determined whether Itk has a negative role play a role in tumor immunosurveillance, autoimmune and inflam- in pathways downstream of other transmembrane proteins and re- matory diseases, and transplantation biology, can be modulated by ceptors that contain a PI3K-binding motif in their cytoplasmic tails the Tec family kinase Itk. Specifically, the NKG2D pathway has such as TCR-interacting molecule and ICOS. been shown to have a deleterious effect in the development of In accordance with its involvement in T cell signaling down- certain autoimmune diseases. For example, in rheumatoid arthritis stream of the TCR and CD28, we show that in NK cells Itk be- (53) and celiac disease (3), abnormal expression of stress-induced comes tyrosine phosphorylated downstream of both the FcR and NKG2D ligands in inflamed tissues together with the induction of NKG2D receptor that use similar proximal kinases to initiate sig- NKG2D receptors on lymphocytes by local inflammatory cyto- naling. Notably, we found that tyrosine phosphorylation of Itk was kines allows these cells to become cytotoxic toward the stressed more prominent in the FcR pathway, which couples to Syk family tissues. Our findings suggest that Itk has a negative regulatory role kinases. Interestingly, earlier studies in Jurkat T cells found that Itk in NKG2D-initiated killing. In this context, elucidation of the pre- was dependent on the Syk family kinase Zap70 and the transmem- cise mechanism of inhibition of NKG2D-mediated cytotoxicity brane adaptor linker for activation of T cells for activation (39). would be expected to have significant therapeutic implications for This may suggest that the activation of Itk is different downstream these disorders. Second, our finding that expression of a catalyti- of the NKG2D receptor, which, unlike the FcR, does not couple to cally inactive form of Itk completely abrogated the modulation of Downloaded from either Syk family kinases or the adaptor linker for activation of T cytoxicity in both the FcR and NKG2D pathways suggests that the cells but instead initiates signaling through PI3K and Grb2 (1, 2). kinase domain of Itk may be a therapeutic target for regulating Consistent with the differences in proximal kinase activation, we these pathways. Third, our finding that Itk has a complex role again show that the calcium waveform differs downstream of the downstream of NKG2D involving both positive and negative reg- endogenous FcR and NKG2D receptor. FcR engagement produces ulation supports a potential role for this Tec kinase to serve as a

a rapid increase in intracellular calcium, whereas NKG2D activa- modulator of the activity of the NKG2D pathway in both resting http://www.jimmunol.org/ tion leads to a relatively delayed low amplitude signal. Intracellu- and activated cells. Taken together, our analyses suggest that, in lar calcium controls a wide variety of cellular functions in addition human NK cells, Itk plays a differential role in regulating the func- to granule-mediated killing, including cell adhesion, motility, gene tions initiated by distinct activating receptors. The modulation of expression, and proliferation. Many of these functions also involve NK cell function by targeting Itk has relevance not only for im- Tec kinases (40–42). Variations in calcium waveforms may have proving tumor immunosurveillance and viral immunity but also for different functional consequences as has been demonstrated in B reducing the severity of autoimmune diseases and graft rejection. lymphocytes (43). In these cells, the amplitude and duration of calcium signals has been reported to control differential activation Acknowledgments of the proinflammatory transcriptional regulators NF-␬B, JNK, We thank Drs. Daniel D. Billadeau and Dragan Jevremovic for the gen- by guest on September 28, 2021 and NFAT (44, 45). We show that Itk enhances the proximal sig- eration of recombinant vaccinia viruses used in this study. naling pathways leading to calcium release for both the FcR and NKG2D pathways, consistent with the function of the Tec kinase Disclosures Btk in amplifying proximal AgR signals in B cells (46). The dif- The authors have no financial conflict of interest. ferential regulation of FcR- and NKG2D-initiated pathways by Itk therefore suggests that Itk has additional functions downstream of References intracellular calcium or independently of calcium which differ for 1. Billadeau, D. D., J. L. Upshaw, R. A. Schoon, C. J. Dick, and P. J. Leibson. 2003. 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