JNK MAP kinase activation is required for MTOC and granule polarization in NKG2D-mediated NK cell cytotoxicity

Changlin Li, Baoxue Ge*, Matthew Nicotra, Joel N. H. Stern, Hernan D. Kopcow, Xi Chen, and Jack L. Strominger†

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138

Contributed by Jack L. Strominger, December 28, 2007 (sent for review October 5, 2006) Interaction of the activating receptor NKG2D with its ligands is a cells are stimulated with soluble NKG2D ligands (17) or when major stimulatory pathway for cytotoxicity of natural killer (NK) pNK cells or the NK tumor cell line YTS are stimulated through cells. Here, the signaling pathway involved after NKG2D ligation is CD28 by CD80 or CD86 on targets (38). JNK as well as ERK has examined. Either incubation of the NKG2D-bearing human NKL been briefly reported to be required for NKG2D-mediated lysis tumor cell line with K562 target cells or cross-linking with NKG2D in cytotoxic T lymphocytes (39). However, the high concentra- mAb induced strong activation of the mitogen-activated tion of the JNK inhibitor SP600125 used (25 ␮M) is far above (MAP) kinases. Selective inhibition of JNK MAP kinase with four that which is selective for JNK (40), and the concentration used different means of inhibition greatly reduced NKG2D-mediated decreases cell viability and induces apoptosis (41). No other cytotoxicity toward target cells and furthermore, blocked the supporting data were presented. movement of the microtubule organizing center (MTOC), gran- In this report, two specific inhibitors of JNK at low concen- zyme B (a component of cytotoxic granules), and paxillin (a scaffold tration, a dominant-negative mutant and siRNA, were used to protein) to the immune synapse. NKG2D-induced activation of JNK determine whether inhibiting the activation of JNK in addition kinase was also blocked by inhibitors of Src protein tyrosine to ERK interferes with the signaling of NKG2D-mediated ␥ kinases and phospholipase PLC , upstream of JNK. Similarly, a cytotoxicity and elements of synapse formation that may be second MAP kinase pathway through ERK was previously shown to involved. be required for NK cell cytotoxicity. Thus, activation of two MAP kinase pathways is required for cytotoxic granule and MTOC Results polarization and for cytotoxicity of human NK cells when NKG2D Expression of NKG2D and Its Functional Activity in NK Cells. Exami- is ligated. nation of the expression of the NKG2D activating receptor in three NK tumor cell lines revealed that both NK92 and NKL ͉ ͉ ͉ ͉ D-JNK-1 ERK paxillin siRNA synapse express NKG2D, whereas no significant expression of NKG2D was detected on YTS cells [supporting information (SI) Fig. 6]. atural killer (NK) cells are a population of lymphocytes that More than 90% of CD56ϩ peripheral blood NK cells (pNK) cells Nlack surface Ig and CD3, express CD16 and CD56, and expressed NKG2D on their cell surface (SI Fig. 6A, Right). To mediate the killing of some virus-infected and tumor cells (1–3). examine whether NKG2D plays a functional role in the killing The function of NK cells is controlled by a coordinated signal activity of these NK cells, K562 was used as a target. This target generated from the ligation of inhibitory and activating recep- cell line expressed two NKG2D ligands, ULBP2 and MICA/B, tors. The ligation of activating receptors leads to activation of a on its surface, but neither ULBP1 nor ULBP3 (SI Fig. 6B). At cascade of intracellular downstream signaling molecules, ulti- an E:T ratio of 20:1, NKL, NK92, YTS, and pNK all showed mately resulting in polarization and exocytosis of granules to lyse 30–50% lysis of K562 cells. The addition of blocking NKG2D the target cells (4–7). mAb significantly attenuated cytotoxicity of NKL, NK92, and NKG2D is a C-type lectin-like receptor whose activation can pNK, but not of YTS (SI Fig. 6C). Thus, NKG2D is a functional trigger NK-cell mediated killing of virus-infected and tumor cells activating receptor. NKL and primary pNK cells were blocked (8, 9). Its ligands on targets cells are cell surface major histo- more efficiently and were, therefore, chosen for further study. compatibility complex (MHC) I-related chain A and B (MI- CA/B) and UL16-binding (ULBP) 1, 2, 3, and 4 in Effect of Two Specific Inhibitors on Activation of JNK MAPK by NKG2D humans (8, 10–12), and retinoic acid early transcript-1, minor Stimulation and Cytotoxicity. NKL cells were cross-linked with histocompatibility antigen H-60, and murine ULBP-like tran- anti-NKG2D mAb for different times. All three major MAPKs, script-1 in mice (13–16). Stimulation of NK cells with soluble JNK, ERK, and p38, were phosphorylated (Fig. 1A, Left). NKG2D ligands causes activation of phosphatidylinositol 3- Similarly, JNK was activated in NKL cells after incubation with kinase (PI3K) and Akt signaling pathways and also JAK1 and K562 target cells (Fig. 1A, Right). STAT5 (17). Furthermore, cross-linking of NK cells with To examine which of those MAPK signaling pathways is anti-NKG2D mAb activates Vav1, Rho GTPases, and PLC-␥ involved in the NKG2D-mediated cytolytic activity of NK cells, (18–25). Mitogen-activated protein kinases (MAPKs) are a family of

serine/threonine protein kinases consisting of three subgroups: Author contributions: C.L. and B.G. contributed equally to this work; C.L., B.G., and J.L.S. extracellular signal-regulated protein kinase (ERK), c-Jun N- designed research; C.L., B.G., M.N., and X.C. performed research; J.N.H.S. and H.D.K. terminal kinase (JNK), and p38 (26–32). They have been shown contributed new reagents/analytic tools; C.L., B.G., and X.C. analyzed data; and C.L., B.G., to play important roles in a variety of cellular functions, including and J.L.S. wrote the paper. cell proliferation, apoptosis, oncogenesis, differentiation, in- The authors declare no conflict of interest. flammation, cell migration, and cytoskeleton remodeling (33– *Present address: Institute of Health Science, CAS, Shanghai 200031, China. 36). In NK cells, ERK is in the pathway PI3K–Rac1–PAK– †To whom correspondence should be addressed. E-mail: [email protected]. MEK–ERK that regulates granule polarization mediated by This article contains supporting information online at www.pnas.org/cgi/content/full/

reorientation of the microtubule organizing center (MTOC) to 0712310105/DC1. IMMUNOLOGY the synapse (6, 37, 38). Furthermore, ERK is activated when NK © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712310105 PNAS ͉ February 26, 2008 ͉ vol. 105 ͉ no. 8 ͉ 3017–3022 Downloaded by guest on October 2, 2021 Anti-NKG2D + +++ NKL + K562 A A B 35 Time (min) 0 5153060 Time 0251030(min) 30 P*-JNK P*-JNK GFP- JNKdn 25 JNK 20 P*-p38 JNK JNK 15 p38 10 P*-ERK

Lysis (E:T=20:1) % 5 ERK 0 GFP GFP JNKdn p38dn B NKL+K562 C D 35 35 40 D-JNK-1 (

0 5 10 20 10 0 5 30 Cp38JNK1 30 RNAi 25 25 30 JNK1 JNK2 20

20 µ 15

20 M) p38 15 10 Lysis % 10 10 Lysis (E:T=20:1) % 5 0 5 C p38

Lysis (E:T=20:1) % JNK1 RNAi RNAi 0 0 C SB SP PD 0.0 5.0 10.0 15.0 20.0 E: T ratio Fig. 2. Inhibition of JNK activity by a JNK dominant-negative mutant or depletion of JNK by RNAi reduces NK killing activity. (A) NKL cells were pNK+K562 70 NKL+P815 transiently transfected with the GFP-JNKAF (JNKdn) mutant by using the 60 60 Amaxa nucleofection system. Expression of GFP-JNKdn was confirmed by FACS 50 50 analysis (Left) and Western blot analysis with anti-JNK antibody (Right). The lower band is endogenous JNK, and the upper band is the fusion protein 40 40 GFP-JNKdn. (B) NKL cells expressing JNKdn together with NKL cells expressing 30 30 GFP or GFP-p38AF (p38dn) as control were tested for lysis of 51Cr-labeled K562 20 20 cells (E:T ϭ 20:1). (C) NKL cells were transfected with siRNAs specific for JNK-1, 10 p38, or negative control siRNA. Depletion of JNK-1 expression was confirmed Lysis (E:T=20:1) % Lysis (E:T=20:1) % 10 0 by Western blotting with anti-JNK antibody (Upper) or with anti-p38 as a 0 Anti- IgG C C SB SP PD Anti- control (Lower). (D) NKL cells treated with JNK-1, p38, or negative control NKG2D NKG2D +SP siRNAs were tested for lysis of 51Cr-labeled K562 cells (E:T ϭ 20:1, Right). Data are representative of three or more experiments. C Anti-NKG2D C SP (10µM) C D-JNK-1 (20µM) Time (min) 0 5 10 30 0 5 10 30 0 5 10 30 0 5 10 30 P*-JNK P*-JNK inhibition of ERK by the upstream MAPK kinase MEK1/2 inhibitor PD98059 (PD) also partially decreased cytolytic activ- JNK ERK ity, but the p38 inhibitor SB203580 (SB) had no significant effect in either NKL or pNK cells (Fig. 1B, Upper and Lower Left). A SP (µM) 0 0 20 10 3 1 redirected cytolytic assay was also performed by using NKL cells D (min) Anti-NKG2D 0 5 5 5 5 5 pretreated with anti-NKG2D as effector cells and P815 cells P*-JNK expressing Fc receptor as target cells. SP600125 blocked the NKG2D-redirected cytotoxicity of NKL cells against P815 cells ERK (Fig. 1B, Lower Right). ␮ ␮ Fig. 1. Activation of JNK is required for NKG2D-mediated cytotoxicity. (A) Treatment of NKL cells with 10 M SP600125 or 20 M NKL cells were cross-linked with anti-NKG2D mAb for the indicated times at D-JNK-1 also efficiently blocked the phosphorylation of JNK 37°C. (Left) Activation of MAPKs was detected with anti-phospho-JNK, p38, or (Fig. 1C). Substantial inhibition of JNK phosphorylation was ERK, and total proteins were measured by blotting with anti-JNK, p38, and observed even at a very low concentration (3 ␮M) (Fig. 1D). ERK. (Right) NKL cells were cultured with K562 cells at a 1:1 ratio for 0–30 min. Activation of JNK was detected by anti-phospho-JNK. Anti-JNK was used as a Effect of a Dominant-Negative JNK Mutant and JNK siRNA on NKG2D- loading control. (B) Percentage of lysis of target cells by NK cells. NKL (Upper) or pNK (Lower Left) cells, pretreated for 30 min at 37°C with either DMSO, 20 Mediated Killing Activity. To examine further whether JNK activity ␮M p38 MAPK inhibitor SB, 10 ␮M MEK1/2 inhibitor PD, 10 ␮M JNK inhibitor is specifically required for killing of NK cells, GFP-JNKdn SP600125 (SP), or potent peptide inhibitor D-JNK-1 were tested for lysis of (S183A,Y185F), a dominant-negative (dn) mutant of JNK (43), 51Cr-labeled K562 cells. (Lower Right) Murine mastocytoma P815 cells labeled was transfected into NKL cells. The killing of NKL cells was with 51Cr were coated with anti-NKG2D and then incubated with NKL cells greatly reduced by the expression of the GFP-JNKdn mutant but pretreated with JNK inhibitor SP at E:T ratio of 20:1. (C) NKL cells pretreated not by similarly transfected GFP-p38dn (Fig. 2A and B). ␮ ␮ with 10 MSPor20 M D-JNK-1 were cross-linked with anti-NKG2D mAb for Additionally, NKL cells were transfected with siRNAs specific the indicated times. Phosphorylation of JNK was then detected by anti- phospho-JNK antibody. Total JNK or ERK was measured as a loading control. for JNK-1. Transfection of JNK-1 siRNA markedly reduced the (D) NKL cells pretreated with JNK inhibitor SP at different concentrations were expression of JNK-1 protein (Fig. 2C). NKL cells transfected cross-linked with anti-NKG2D mAb for 5 min. Phosphorylation of JNK was then with siRNA targeting JNK-1 showed markedly reduced killing of detected by anti-phospho-JNK antibody. Total ERK was measured as a loading K562 cells, whereas depletion of p38 had no significant effect control. These bands were run in the same gel as C, Right, and the controls for (Fig. 2D). the two experiments are the same. Data shown in A–D are representative of three or more individual experiments. Effects of Specific Inhibition of JNK Activation on Conjugation and Synapse Formation. Cytolytic activity of NK cells is mediated specific inhibitors of MAPKs were used. Specific inhibition of through effector cell–target cell conjugation, synapse formation JNK activation by 10 ␮M SP600125 or by the potent peptide including actin ring formation, polarization of the MTOC and inhibitor D-JNK-1 (42) significantly reduced cytotoxicity of cytolytic granules, and finally exocytosis of cytolytic proteins NKL on K562 targets (Fig. 1B, Upper Left and Right). Specific including perforin and granzymes (6, 44). Inhibition of JNK

3018 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712310105 Li et al. Downloaded by guest on October 2, 2021 A pNK+K562 A pNK+K562

pNK

K562

C SP

K562 B C 80 35 30 pNK 60 25 20 Actin JNK (upper) Merge 40 15

P*-JNK(lower) zation NKIS to 10 % paxillin% polari-

20 Lysis (E:T=20:1) % 5 0 0 B CSP GFP GFP- Pax(S178A) Medium D C M D-JNK-1 µ 10 Media D-JNK-1 70 p=0.02 p=0.04 SP 60 50 Actin GFP- Granzyme Merge 40 Pax(S178A) 30 20 E 10 Mock % of polarization to NKIS 0 GFP-RNAi 40 Pax RNAi MTOC granules 30 Fig. 3. Effect of specific inhibition of JNK on synapse formation between NK cells and K562 cells. (A) Fixed pNK-K562 conjugates were stained with rabbit 20 polyclonal anti-JNK that also reacts with phospho-JNK or anti-phospho-JNK, Lysis % followed by the staining with Alexa Fluor 568-conjugated goat anti-rabbit 10 IgG. (B) NKL cells pretreated with 10 ␮M D-JNK-1 for 30–45 min at 37°C were 0 incubated with K562 cells for 30 min, allowing formation of cell–cell conju- 0 246 81012 gates. After cell fixation and permeabilization, microtubules (MT) were stained with anti-␣-tubulin mAb, followed by Alexa Fluor 568-conjugated Fig. 4. JNK and paxillin. (A) pNK cells treated with 10 ␮M SP600125 (SP) or anti-mouse IgG (red). F-actin (green) was stained with phalloidin-Alexa Fluor without (C, control) JNK inhibitor were incubated with K562 cells and then 488, and granules (blue) were stained with Alexa Fluor 647-conjugated anti- stained with mouse anti-actin (green) and monoclonal anti-paxillin (red) in granzyme B mAb. Representative images are shown. Average percentages of both NK cells (upper cell) and target cell (lower cell). (B) Percentage of paxillin NKL conjugates with MTOC or granule polarization are shown with SD and P polarization to the synapse of pNK cells treated or not treated with JNK values after counting Ͼ50 conjugates. Data are representative of three or inhibitor. For each effector–target cell, 40 conjugates were examined. (C) NKL more independent experiments. See Materials and Methods and SI Fig. 8B for cells were transfected with pBABE-GFP-PaxS178A mutant and then tested for details of determination of polarization. the lysis of 51Cr-labeled K562 cells (E:T ϭ 20:1). (D) GFP-PaxS178A-transfected NKL cells were treated or not treated with JNK inhibitor and then incubated with K562 cells. The conjugates were stained with Alexa Fluor 488-conjugated activation by SP600125 had no effect on conjugation between phalloidin (green) and Alexa Fluor 647-conjugated anti-granzyme B (blue). (E) NK cells and target cells (SI Fig. 7). NKL cells were transfected with paxillin siRNA, GFP siRNA, or control siRNA Both JNK (Upper) and phospho-JNK (Lower) accumulated at (mock). (Inset) Depletion of JNK expression was confirmed by Western blot- the synapse of pNK-K562 conjugates (Fig. 3A). Importantly, ting with anti-paxillin antibody. NKL cells treated with these siRNAs were 51 inhibition of JNK activity by D-JNK-1, SP600125, or JNK-1- tested for lysis of Cr-labeled K562 cells. Data are representative of three or more experiments. specific siRNA blocked polarization of granzyme B, a compo- nent of cytolytic granules (blue circle), and the MTOC (␥- tubulin, red dot) to the synapse, although it had no effect on the and B). Cytotoxicity of NKL cells on K562 targets was decreased localization of actin to the synapse (Fig. 3B and SI Fig. 8). JNK after NKL cells were transfected with specific siRNA for human is localized to the centrosome (an alternative name for MTOC) paxillin (Fig. 4E). JNK phosphorylates Ser-178 on paxillin, and during the cell cycle (45). this phosphorylation regulates cell migration (44, 47). However, Paxillin, a scaffold protein, also moves to the MTOC of T cells, overexpression of PaxS178A (GFP-PaxS178A) had no effect on where it colocalizes with ␣- and ␥-tubulin (46–48). Paxillin killing by NKL cells of K562 cells (Fig. 4C). Moreover, GFP-

moved to the synapse in association with the MTOC of NK cells PaxS178A still moved to the synapse and was still excluded in the IMMUNOLOGY also, and this localization was inhibited by SP600125 (Fig. 4 A presence of SP600125 (Fig. 4D).

Li et al. PNAS ͉ February 26, 2008 ͉ vol. 105 ͉ no. 8 ͉ 3019 Downloaded by guest on October 2, 2021 ␥ A 30 5D), suggesting PLC- is upstream of JNK in the pathway of 25 NKG2D-mediated cytotoxicity. 20 Discussion 15 NK cells express diverse families of activating receptors that can 10 trigger lysis of the target cells upon recognition of specific 5 Lysis (E:T=20:1) % ligands. NKG2D is an activating receptor that plays a significant 0 DMSO Wortmannin role in NK cell-mediated tumor cell recognition and cytolysis. Stimulation of NKG2D results in binding of DAP10 and recruit- B Wortmannin ++++ ment of PI3K through its PI3K-binding motif YNIM (20) and PMA + then triggers a Syk-independent pathway involving PI3K, PLC-␥ Anti-NKG2D ++++ ++++ Grb2, Vav1, and Rho family GTPase (18, 25). The present Time (min) 0251010 02510 studies have extended this signaling pathway to include the P*-JNK MAPK JNK in the general picture. Selective inhibition of JNK JNK activation by a JNK inhibitor significantly attenuated NKG2D- mediated cytotoxicity of NK cells. Inhibition of ERK by a selective inhibitor of its MAPK kinase also blocked cytotoxicity, C although to a less extent at the concentration used. Inhibition of 50 p38 had no significant effect on NKG2D-mediated cytolysis of 40 NK cells. Because use of high concentrations of MAPK inhib- itors may decrease their specificity (40), low concentrations of 30 inhibitors were used in the present experiments to avoid the nonspecific inhibition of other kinases. Furthermore, a domi- 20 nant-negative JNK-1 mutant or depletion of JNK expression by siRNA significantly impaired NKG2D-mediated cytotoxicity, Lysis (E:T=20:1) % 10 but importantly, neither treatment of p38 had any effect. Thus, 0 C PP2 Pice Bis U73122 JNK is a key downstream player in the NKG2D signaling pathway. In addition, inhibition of JNK also reduced the cyto- C PP2 U73122 lytic activity of YTS cells, which do not express NKG2D but D Anti-NKG2D Time (min) 0 5 10 30 0 5 10 30 0 5 10 30 depend on CD28 for killing activity (38). Thus, the role of JNK P*-JNK in the regulation of cytolytic activity is general in NK cells. However, the reports from Chini et al. (49) and Trotta et al. (50) ERK provided evidence for p38 involvement in development of NK Fig. 5. Upstream regulators for the NKG2D-induced JNK activation. (A) NKL cytotoxicity. In particular, Trotta et al. (50) excluded a major role cells were preincubated with 200 nM wortmannin and then incubated with for JNK-1 in this process. Such a discrepancy could be caused by 51Cr-labeled K562 cells at E:T ϭ 10:1 for4hat37°C. Percentage of lysis was the use of different analysis methods or cell lines. The data measured as 51Cr release. (B) NKL cells were pretreated with 200 nM wort- shown in Figs. 1 and 2 suggest that p38 may not be involved but mannin and then stimulated by cross-linking with anti-NKG2D monoclonal should not be regarded as definitive proof of lack of involvement antibody at 37°C for 0–30 min. NKL were also treated with phorbol 12- of this kinase in NK cytotoxicity without further study. myristate 13-acetate (PMA) and NaCl as positive controls. Activation of JNK Phosphorylation of paxillin, a focal adhesion-associated pro- was detected by anti-phospho-JNK (Upper), and the total protein was mea- sured by anti-JNK (Lower). (C) NKL cells, treated with DMSO, 20 ␮M Src PTK tein, at Ser-178 by JNK has been shown to be essential for cell inhibitor PP2, 100 ␮M Syk inhibitor piceatannol (Pice), 2 ␮M PKC inhibitor migration (43). Paxillin has also been shown to localize to the bisindolylmaleimide (Bis), or 10 ␮M PLC inhibitor U73122 were incubated with MTOC of T cells, where it colocalizes with ␣- and ␥-tubulin (47). 51Cr-labeled K562 cells (ratio 20:1) to measure percentage of lysis. (D) NKL cells In NK cells, both JNK and activated phospho-JNK localized to pretreated with DMSO, PP2, and U73122 were cross-linked with anti-NKG2D the synapse of NK cells together with the MTOC after specific mAb for the indicated times. Activation of JNK was detected with anti- recognition of target cells. The MTOC, granzyme B (cytolytic phospho-JNK antibody, and total JNK protein was examined by using anti-JNK granules), and paxillin all moved to the synapse, and inhibition antibody. Results are representative of three experiments. of JNK activation blocked these movements. Moreover, siRNA knockdown of paxillin greatly impaired cytotoxicity of NK cells. However, expression of a S178A mutant of paxillin (PaxS178A) Multiple Signaling Pathways Are Required for NKG2D-Mediated Cy- did not inhibit the cytotoxicity. Thus, the role of paxillin in totoxicity of NK Cells. Binding of the p85 regulatory subunit of synapse formation of NK cells is not mediated through the PI3K to the tyrosine-phosphorylated YNIM motif of DAP10 phosphorylation of the Ser-178 residue. JNK might phosphor- couples NKG2D-initiated signaling downstream to PI3K (18, 20, ylate other serine, threonine, or tyrosine sites of paxillin to 25). Although inhibition of PI3K in the ERK pathway by regulate the movement of paxillin directly, or mediate its effect wortmannin greatly reduced the killing of K562 by NKL cells through another downstream target protein to affect paxillin (Fig. 5A), it had no effect on NKG2D-induced JNK activation localization indirectly. Detailed analysis of the six Ser/Thr and (Fig. 5B). four Tyr phosphorylation sites in paxillin (46) is required in the ␥ Because Src, Syk, PLC- , and PKC have been implicated in future. some modes of lymphocyte activation, their involvement in PI3K has been demonstrated to be a pivotal player in the NKG2D-induced JNK activation was examined. Pretreament of regulation of NK cytotoxicity by controlling the activation of NKL cells with the Src inhibitor PP2 or the PLC-␥ inhibitor ERK (18, 37, 38). However, NKG2D-induced JNK activation U73122 [but neither with the inactive analog U73343 (data not was independent of PI3K, but was partly dependent on Src shown), the Syk inhibitor piceatannol, nor the PKC inhibitor kinase and PLC. Caraux et al. (51) have recently shown that bisindolylmaleimide] markedly reduced their killing activity PLC-␥2 is pivotal for the cytotoxicity of NK cells, but MTOC against K562 cells (Fig. 5C). Moreover, pretreatment of NKL polarization was shown to be normal in PLC-␥2-deficient NK cells with PP2 or U73122 followed by stimulation of the cells with cells. JNK activation might depend on other PLC isoforms. NKG2D mAb attenuated NKG2D-induced JNK activation (Fig. Taken together, two signaling pathways, Src–PI3K–ERK and

3020 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712310105 Li et al. Downloaded by guest on October 2, 2021 Src–PLC–JNK, acting in an additive rather than synergistic has a blank value of Ϸ25% because of random distribution within this sector manner, contribute to NKG2D-mediated cytotoxicity. JNK and and is therefore semiquantitative. This blank has not been subtracted from ERK are both phosphorylated during granule and MTOC the data shown. At least three independent experiments were performed to polarization using a variety of different NK cells and target cells, give a minimum of 50 conjugates counted for each cell–drug combination. A but only after activation of some NK surface receptors, thus two-tailed t test was used to compare the differences between groups. A P value Ͻ 0.05 was considered statistically significant. providing a definition of a NK-activating receptor and general- izing the results described here (52). Cell Activation and Immunoblot Analysis. In experiments involving NKG2D ␮ Materials and Methods cross-linking, NK cells were incubated for 30 min in ice with 10 g/ml anti- NKG2D before adding goat anti-mouse F(abЈ)2 at 37°C for the indicated period Antibodies and Reagents. NKG2D mAb was purchased from R&D Systems. of times. For experiments in which NK cells were activated by target cells, NK Anti-CD56 mAb, phycoerythrin-conjugated goat anti-mouse IgG1, and anti- cells were incubated with paraformaldehyde-fixed target cells at 37°C for the paxillin mAb were from BD PharMingen. Anti-ERK, JNK, and anti-phospho- indicated period of times. Activation of MAPK was detected by immunoblot- ERK, phospho-JNK, and phospho-p38 were from Cell Signaling. Rabbit anti- ting with anti-phospho-MAPK rabbit antibody, and total MAPK protein was p38 was a gift from Jiahuai Han (Scripps, La Jolla, CA). Anti-␥-tubulin mAb was blotted with anti-MAPK rabbit antibody. from Sigma. The JNK inhibitor SP600125 was from BioSource. The potent JNK peptide inhibitor D-JNK-1 was kindly provided by Xigen S.A. The MEK1/2 Cytotoxicity Assays. The 51Cr-release assays were done as described in ref. 53. inhibitor PD (upstream of ERK), p38 inhibitor SB, the PI3K inhibitor wortman- In redirected cytotoxicity assays, effector cells and target cells were mixed in nin, the Src inhibitor PP2, the Syk inhibitor piceatannol, the PKC inhibitor the presence of soluble anti-NKG2D or anti-IgG for 30 min. For experiments bisindolylmaleimide, and the PLC inhibitor U73122 were from Calbiochem. Plasmid pBABE-GFP-Paxillin(S178A) was constructed by PCR from pEGFP- with inhibitors, effector cells were incubated with inhibitors for 30 min at Paxillin(S178A), kindly provided by K. Jacobson (University of North Carolina, 37°C, 5% CO2 before the cytotoxic assay. Assays were typically performed Chapel Hill, NC) (43). three times, unless otherwise indicated.

Cells and Tranfectants. pNK cells were isolated from peripheral blood from Antibodies. Anti-MICA/B mAb, anti-ULBP-1, 2, 3 mAb, and phycoerythrin anonymous healthy donors by using Ficoll either directly or after enrichment (PE)-conjugated anti-NKG2D antibody were purchased from R&D Systems. for NK cells with Rosettesep according to the manufacturer’s instructions (StemCell Technologies). Enriched NK cell populations were Ͼ85% CD56ϩ/ Flow Cytometry. For NKG2D, cells were stained with PE-conjugated anti-NKG2D CD3Ϫ with Ͻ0.5% CD3ϩ cells. NKL and NK92 tumor cells were grown in RPMI antibody. To detect surface expression of NKG2D ligands in K562 cells, the cells medium containing 100 units/ml IL-2 (PetroTech, Inc.), 10% FBS, 1% L- were incubated with unconjugated mAbs for 20 min and washed with PBS glutamine, 1% sodium pyruvate, and streptomycin/penicillin. K562 erythro- containing 2% FBS. PE-conjugated goat anti-mouse IgG was then used for further leukemia cells were used as target cells. Transfection of NKL cells was per- staining. Stained cells were analyzed with FACSCalibur (Becton Dickinson). formed by using the nucleofection technology (Amaxa). FACS-Based Conjugation Assay. Cell conjugate formation was determined by siRNA and siRNA Transfection. siRNAs for human p38 (sc-29433), JNK-1 (sc- flow cytometry as described (54, 55). NK cells at 5 ϫ 106 cells per ml were 29380), paxillin (sc-29439), and control (sc-37007) were purchased from Santa labeled with PKH67 (green; Sigma) for 2–5 min at 25°C; they were mixed with Cruz Biotechnology. Transfection of siRNA into NKL cells was performed K562-RFP cells transfected with RFP (red). After incubation at 37°C for 10–15 according to the manufacturer’s instructions. min, the cells were resuspended and then fixed by the addition of 0.5 ml of ice-cold PBS/0.5% paraformaldehyde. The relative proportion of conjugation Laser Scanning Confocal Microscopy and Analysis of Polarization and Conjuga- (red/green) events was promptly determined by two-color flow cytometric tion. The imaging work was done as described in ref. 44. To evaluate granule analysis. polarization, cell–cell conjugates showing F-actin polymerization were exam- ined by visually dividing the cell into four radial sectors (SI Fig. 8); the granules ACKNOWLEDGMENTS. We thank B. Rybalov for technical assistance with and MTOC were deemed polarized when they were oriented toward the FACS analysis and other laboratory members for help at all stages. This work conjugated cell within the sector in contact with the target cell. This method was supported by National Institutes of Health Research Grant AI50207.

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