Functional Role of Phosphatidylinositol 3-kinase in Direct Tumor Lysis by Human Natural Killer Cells

Immunobiol. (2002) 205, pp. 74 – 94 © 2002 Urban & Fischer Verlag http://www.urbanfischer.de/journals/immunobiol

1

Immunology Program, H. Lee Moffitt Cancer Center, Department of Interdisciplinary Oncology, University of South Florida College of Medicine, Tampa, FL, USA, 2Second Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan, and 3Cellular Cytotoxicity Laboratory, Austin Research Institute, Heidelberg, Australia.

Functional Role of Phosphatidylinositol 3-kinase in Direct Tumor Lysis by Human Natural Killer Cells BIN ZHONG1, JIN HONG LIU1, DANIELLE L. GILVARY1, KUN JIANG1, MASATO KASUGA2, CONNIE A. RITCHEY1, JOSEPH A. TRAPANI3, AND SHENG WEI1 Received August 20, 2001 · Accepted in revised form December 11, 2001

Abstract Cytotoxicity is a key function of natural killer (NK) and T cells; yet the molecular mechanism is unclear. We have biological, biochemical and molecular evidence to demonstrate that phosphatidylinositol (PI) 3-kinase is critical for direct NK lysis of tumor cells, via control of intracellular granule movement. Tumor cell engagement rapidly activated PI 3-kinase in NK cells within 5 min, as demonstrated by p85 subunit tyrosine phosphorylation and its ability to generate phosphatidylinositol 3-phosphate, PI(3)P, from PI. Wortmannin and LY294002 effectively inhibited NK cells to lyse 51 Cr-labeled tumor cells at the same doses that blocked PI-phosphorylating function in tumor-activated NK cells. Immunostaining demonstrated that tumor engagement for only 5 min mobilized perforin and granzyme B from NK cells unidirectionally towards the target, and prior treatment of NK cells with either PI 3-kinase inhibitor effectively stopped this intracellular polarization. Lastly, ectopic expression of dominant-negative p85 or p110 mutant markedly suppressed NK lytic capacity. These results taken together demonstrate that PI 3-kinase may control NK lytic function via granule polarization towards the contacted target cell.

Introduction Natural killer (NK) and T cells constitute two separate but complementary mechanisms in the host to eliminate aberrant tumor cells or virus- and parasite-infected cells. Molecular signals that control these processes are well-defined in T cells but not in NK cells. NK cells release lytic granules containing perforin and granzyme B upon contact with their targets (1, 2). The signal transduction pathways that trigger the redistribution Abbreviations: LGL = large granular lymphocytes; MAPK = mitogen-activated protein kinase; NK = natural killer; PI 3-kinase = phosphatidyl inositol 3-kinase. 0171-2985/02/205/01-074 $ 15.00/0

Control of NK lytic function by PI 3-kinase · 75

of the lytic granules with their concentrated constituents to the area of cell-cell contact, where they are released to execute the lysis of target cells, are not well-defined. In T cells, it is clear that engagement of the T cell receptor with an antigen presented in the context of MHC molecules, together with binding of other co-receptors to their ligands, can ignite multiple signaling components (3). One substrate that becomes activated as a consequence of this signal cascade is PI 3-kinase (4, 5). PI 3-kinase is a heterodimer, consisting of a regulatory subunit, p85, and a catalytic subunit, p110, and, when activated, it acquires the ability to phosphorylate inositol phospholipids on the 3-position of the inositol ring (5). The PI 3-kinase metabolites, including phosphatidyl inositol (PI)3,4-biphosphate and PI3,4,5-triphosphate, stimulate calcium-independent isoforms of protein kinase C. It can also serve as a serine/threonine kinase to phosphorylate its substrate, Akt protein kinase (6–8). Existence of a SH3 domain and two SH2 domains in the p85 subunit allows for binding to both the proline-rich and phosphotyrosine regions, consisting of YXXM motifs, that are present on various growth factor receptors and protein kinases (9, 10). These associations link PI 3-kinase to a number of cellular processess, including the proliferative response to growth factors (11–13), inhibition of apoptosis (14), trafficking of intracellular vesicles (15), cell adhesion (16, 17), membrane ruffling (18) and chemotaxis (19, 20). The role of PI 3-kinase in NK cell-mediated target lysis is controversial (21). Although the pivotal role of PI3K has been indicated in NK activation, it has been reported that pharmacological inhibitors of PI 3-kinase lack any effect on spontaneous cytotoxicity mediated by some cloned human NK cells (22, 23). Since PI 3-kinase is the most diversified molecule and linked to many signal pathways, it is therefore critical to define the role of PI 3-kinase in tumor target induced lytic granule movement in NK cells. Both perforin and serine esterases such as granzyme B are constitutively synthesized and stored as active moieties within NK granules (1, 2). Upon conjugate formation with an appropriate target cell, the perforin- and granzyme-containing granules move to the site of cell-cell contact (“polarization”), and then exocytose their contents in a vectorial fashion towards the target cell. Perforin polymerization and insertion into the membrane punctures the target cell, allowing granzyme B to diffuse into the target cell or facilitating its entry via the endocytic (24–26). Granzyme B can then execute cell death via direct cleavage and activation of several caspases (27). Caspase-independent pathways are also triggered by granzyme B to contribute to cell death (28–30). There is strong evidence that perforin and granule contents can be released into the extracellular environment from NK cells upon ligation of their FcR, and this process is dependent on PI 3-kinase (31). A recent report indicated that MAPK may also regulate granule exocytosis in FcR crosslinked NK cells (32). However, the mechanism by which direct tumor contact induces intracellular granule polarization is unclear. Syk70 has been shown to be critical for NK lysis of sensitive target cells but no association has been made with the movement of granules (22). We thus set out to identify the signal pathways that control granule movement and recently identified that a ras-independent MAPK/ERK activation to be an important signal (33, 34). We now provide biological, biochemical and genetic evidence that PI 3-kinase also controls the direct lytic process and this control involves the intracellular mobilization of perforin and granzyme B in NK cells towards the relevant target cell.

76 · B. ZHONG et al. Materials and Methods Cells

A human natural killer leukemia cell line, YT, (a gift from Dr. ECKHARD PODACK, University of Miami, Miami, FL, USA) has previously been used by us to investigate cytolytic function (33). Its ability to serve as an NK effector cell against a human B lymphoma cell line, Raji (American Type Culture Collection (ATCC), Rockville, MD, USA), was exploited in this study. Another NK-sensitive cell line, K562, used for measuring function in fresh NK cells, and two NK-resistant tumor cell lines, Jurkat and HL60, were all obtained from ATCC. All cells were cultured in RPMI 1640 containing 10% fetal calf serum with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 5 mM HEPES buffer (GIBCO Laboratories, Grand Island, NY, USA), which will be referred to as medium in the text. Isolation of large granular lymphocytes (LGL) from peripheral blood

Peripheral blood mononuclear cells were first obtained by Ficoll-Hypaque density centrifugation of leukocyte buffy coats, obtained from normal volunteers at the Southwest Florida Blood Bank. The mononuclear cells were washed with PBS twice and allowed to adhere to plastic for 1 h at 37°C in medium. The recovered nonadherent cells were further depleted of adherent cells by incubation on nylon wool columns for 30 min at 37 °C. The cells passing through the columns were then placed on a four-step discontinuous gradient with a range of Percoll from 40% to 47.5%, as previously described (35). The cells recovered from 42.5–45.0% Percoll were visually assessed for LGL morphology on Giemsa-stained cytocentrifuged slides to routinely contain 75%–90% LGL and were used to test for NK function. Chemical reagents and antibodies

The PI 3-kinase inhibitors, wortmannin and LY294002 (Calbiochem, La Jolla, CA, USA) were dissolved in dimethylsulfoxide (DMSO) and stored in aliquots at –20oC . The stock was diluted to the desired working concentration in RPMI 1640 immediately prior to use. Monoclonal anti-p85 and anti-phosphotyrosine (4G10), as well as rabbit anti-lyn, were purchased from Upstate Biotechnology Inc., (Lake Placid, NY, USA). Monoclonal anti-granzyme B (2C5), was generated as previously described (36). Monoclonal anti-perforin was purchased from Endogen/T cell Sciences, Woburn, MA, USA. Anti-phosphorylated Akt and anti-panAkt antibodies were purchased from Cell Signaling Technology Inc.( Beverly, MA, USA). Cytotoxicity assay

A 51Cr-release assay was performed as previously described, using Raji tumor cells as targets for YT effector cells and K562 tumor cells for fresh LGL (35, 37). Briefly, target tumor cells were labeled with 200 mCi of sodium [51Cr] chromate (Amersham, Arlington Heights, IL, USA) in 0.2 ml of medium at 37oC in a 5% CO2 atmosphere for 1 h. The cells were then washed three times and added to effector cells at 5 × 103 cells/well in 96-well round-bottom microplates, resulting in effector:target (E:T) ratios ranging from 50:1 to 3:1 in a final volume of 0.2 ml in each well. After 5 h incubation at 37oC, 100 ml of culture supernatants was harvested and counted in a gamma counter. The % specific 51Cr release was determined by the equation (experimental cpm – spontaneous cpm)/total cpm incorporated × 100. All determinations were done in triplicate, and the SEM of all assays was calculated and was typically around 5% of the mean or less. In assays using the PI 3-kinase inhibitors, YT cells or fresh LGL at 2.5 × 106 cells/ml, were incubated for 1 h at 37oC with serum-free RPMI 1640 or serial dilutions of wortmannin or LY294002 or an equal amount of DMSO used to dilute the highest concentration of inhibitor. The cells were then plated into triplicate wells of a 96 well microplate at various dilutions before 51Cr-labeled Raji tumor cells were added.

Control of NK lytic function by PI 3-kinase · 77 Fixation of target cells

Raji, Jurkat or HL60 tumor cells were washed with PBS once and incubated with 1% paraformaldehyde (methanol-free) in PBS, pH 7.4, on ice for 30 min. Then, the cells were washed four times with PBS to remove all paraformaldehyde. Immunoprecipitation and Western blotting

YT cells were cultured in serum-free medium for 4 h at 37oC prior to use, in order to reduce the background phosphorylation. Then the rested YT cells (1 × 107/ml) were treated with either serum-free RPMI 1640, DMSO, wortmannin or LY294002 for 1 h at 37oC, washed, and then mixed with an equal number of paraformaldehyde-fixed Raji target cells. The cells were rapidly pelleted at 1000 rpm in a microcentrifuge at 4oC followed by incubation for 0–15 min at 37oC. Then, the cells were solubilized by incubation at 4oC for 30 min in lysis buffer containing 1% Nonidet P-40, 10 mM Tris, 140 mM NaCl, 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 10 mM iodoacetamide, 50 mM NaF, 1 mM ethylenediamine-tetraacetic (EDTA), 0.4 mM Na orthovanadate, 10 mg/ml leupeptin, 10 mg/pepstatin, and 10 mg/ml aprotinin. Cell lysates were centrifuged at 15,000 × g for 10 min to remove nuclei and cell debris. For immunoprecipitation, lysates were precleared of non-specific binding proteins via sequential 1 h incubation at 4oC with normal mouse IgG and Protein A Sepharose beads respectively. Lysates containing 1.0 mg protein/ml were then incubated with 5–10 mg of the indicated antibody for 2 h. Immune complexes were collected with Protein A-Sepharose beads and washed 3 times with washing buffer (0.1% NP-40, 10 mM Tris, 140 mM NaCl, 0.1 mM PMSF, 10 mM iodoacetamide, 50 mM Na fluoride, 1 mM EDTA, 0.4 mM Na orthovanadate). Samples were then boiled for 5 min in loading buffer and separated by 10% SDS polyacrylamide gel electrophoresis followed by Western blot analysis with the desired antibody. The proteins were detected by the enhanced chemiluminescence detection system (ECL, Amersham, Arlington Heights, IL, USA). PI 3-kinase assay

YT cells, pretreated with or without wortmannin or LY294002 for 1 h at 37oC, were mixed with 1% paraformaldehyde-fixed Raji tumor cells for 5 min at 37oC. Cell lysates were then prepared and incubated with 4ug of anti-phosphotyrosine, 4G10, with overnight rotation at 40C. Immunoprecipitates were collected by 2 h incubation with 50 ul of Protein A Sepharose beads at 4oC. After washing thrice with lysis buffer, the immunoprecipitates were mixed with sonicated phosphatidyl inositol, PI (Advanti Polar-Lipids, Inc., Alabaster, AL, USA) in 20 mM HEPES (pH 7.5) with 1 mM EDTA, on ice for 20 min. Finally, 10 uCi of [m-32P]ATP (3,000 Ci/mmol), 40 uM ATP and 10 mM MgCl2 were added to the mixture for 30 min at 300C in a shaking waterbath. PI (3)P was extracted with phenol/chloroform and separated on a TLC plate. The plate was analyzed by autoradiography. Vaccinia viral delivery of dominant negative PI 3-kinase

Recombinant vaccinia viruses encoding dominant negative p85 and p110 subunits of PI 3-kinase (38–40) were constructed using the vector pSP11 in recombination with the WR strain of vaccinia. Vaccinia expressing CD56, a large granular lymphocyte-specific surface marker, was used as a vector control. The procedure of vaccinia viral infection has been described previously (34). Briefly, YT cells were incubated with various vaccinia constructs for 2 h at 37oC in serum-free medium at a multiplicity of infection (MOI) of 4. Cells were then further incubated in serum-containing medium for 4 h at 37oC before testing for cytotoxicity against Raji tumor cells in a 5 h 51Cr-release assay. Infection efficiency was checked via infection of vaccinia constructs containing green fluorescent protein (GFP) that can be detected by flow cytometric analysis. Immunostaining

YT cells, pretreated with 500 nM wortmannin, 100 uM LY294002 or DMSO for 1 h at 37oC, were added to Raji cells at a 1:1 ratio in a total volume of 100 ul, spun rapidly at 1000 rpm for 1 min in a

78 · B. ZHONG et al. cold microcentrifuge, and then incubated for 0–5 min at 37oC. The cells were then centrifuged onto a microscope slide and fixed at –20oC with methanol/acetone (3:1) for 20 min (33). The slides were air-dried and rehydrated for 2 h in several changes of PBS. All procedures were at room temperature. Polyclonal rabbit anti-lyn kinase or monoclonal anti-human perforin or granzyme B, diluted 1:200 with 0.1% NP-40 in 1%BSA in PBS, was applied to the slide for 1 h. After several washes with PBS for 2 h, the slides were incubated for 25 min with goat anti-rabbit IgG TRITC-labeled antibody (SIGMA) diluted 1:80 or goat anti-mouse Ig FITC-labeled antibody (SIGMA), diluted 1:100 in 0.1% NP-40 in PBS containing 1% BSA. The slides were then washed several times with PBS and covered with coverslips in mounting media of antifade/DAPI. Immunofluorescence was observed with a Leitz Orthoplan 2 microscope and images were captured by a CCD camera with the Smart Capture Program (Vysis, Downers Grove, IL, USA). On each slide, 100 YT/Raji conjugates were evaluated for perforin or granzyme B mobilization. Several controls were performed, i.e., YT cells alone or Raji tumor cells alone stained only with FITC-labeled goat anti-mouse Ig or with TRITC-labeled goat anti-rabbit IgG, to check for nonspecific binding of the secondary antibodies. Nonspecific binding was not detected and the results were omitted from the figures for clarity.

Results Suppression of tumoricidal function in NK cells by inhibition of PI 3-kinase

In order to examine the involvement of PI 3-kinase in NK lysis, we first tested the effect of two structurally unrelated but specific inhibitors of PI 3-kinase, wortmannin and LY294002 (41, 42), on lysis of Raji tumor cells by the NK cell line, YT (37). A representative experiment of 4 that were performed is shown in Figure 1. YT cells were pretreated with a range of concentrations of wortmannin from 10nM to 500 nM or LY294002 from 25 uM to 200 uM for 1 h at 37o C prior to incubation with 51Crlabeled Raji tumor cells for 5 h at 37oC to test for lysis. A dose-dependent inhibition by wortmannin was observed at all the effector/target ratios tested. Inhibition was detected, starting at 10nM, with 50% inhibition at 50–100 nM and almost complete inhibition at 500 nM (Fig. 1A). In the case of LY294002, inhibition began at 25 uM, reaching 50% inhibition at 50–100 nM, and was complete at 200 uM (Fig. 1B). Assessment of viability by trypan blue exclusion indicated that none of the concentrations of wortmannin or LY294002 were toxic and all cells were recovered at 100% viability. DMSO, tested at a concentration equivalent to that used to dilute the highest concentration of either wortmannin or LY294002, had little toxic effect. The results therefore indicate that inhibition of PI 3-kinase function interfered with NK effector function. To confirm that PI 3-kinase is involved in NK lysis, we also tested the effect of wortmannin and LY294002 on freshly-isolated NK cells, enriched as large granular lymphocytes by Percoll density gradients from peripheral blood mononuclear cells of a normal donor (35). These peripheral blood NK cells were treated with 10 nM to 500 nM of wortmannin or 10 uM to 100 uM of LY294002 for 1 h at 37oC prior to testing for lysis of 51Cr-labeled K562 tumor cells. Figure 1C and D show one of four experiments performed, that both PI 3-kinase inhibitors blocked fresh NK cell lysis of tumor cells, with similar doses as those effective against YT effector cells. Because PI 3-kinase has been implicated in several growth factor-mediated cell proliferative responses (5), we next examined if the continuous growth of YT cells main-

Control of NK lytic function by PI 3-kinase · 79

Figure 1. Inactivation of NK lysis of tumor cells by wortmannin and LY294002. YT cells were untreated or treated 1 h at 37oC with 10–500 nM of wortmannin (A), or 25–200 uM of LY294002 (B), or an amount of DMSO used to dilute the highest concentration of each PI 3-kinase inhibitor. The cells were then tested in triplicate wells for lysis of 51Cr-labeled Raji tumor cells at the effector:target ratios indicated. (C) and (D), Highly-enriched human large granular lymphocytes, freshly isolated by Percoll gradient centrifugation of nonadherent peripheral blood mononuclear cells, were preincubated with wortmannin (C) or LY294002 (D) at the indicated concentrations for l h at 37oC, prior to testing for lysis against 51Cr-labeled K562 tumor cells. The SEM for each mean % cytotoxicity of Raji tumor cells was less than 5% of the mean and was not included.

80 · B. ZHONG et al. tained in fetal calf serum-containing medium could be inhibited by wortmannin or LY294002. YT cells, pretreated with the same range of concentrations of the two PI 3kinase inhibitors as shown in Figure 1, were cultured in medium for 24 h, with 3H-thymidine added for another 6 h. Remarkably, none of the concentrations used for both reagents had any adverse effect on YT cell proliferation (data not shown). Even 500 nM of wortmannin and 200 uM of LY294002, which completely blocked NK lytic function, did not cause cell cycle arrest. These results suggest that PI 3-kinase may function selectively to control effector function and not proliferative function in YT cells. Induction of tyrosine phosphorylation in PI 3-kinase within NK cells by interaction with target cells

The effective suppression of NK function by two independent PI 3-kinase inhibitors suggests that this kinase must become activated and functional in NK cells upon target ligation. As the result of activation, the p85 subunit is known to become tyrosine-phosphorylated (5). Therefore, the next step was to attempt to detect tyrosine phosphorylation in the p85 component of PI 3-kinase before and after YT cell interaction with Raji tumor target cells. YT cells were incubated with paraformaldehyde-fixed Raji tumor cells at 37oC for 0–15 min and the mixture was then lysed and immunoprecipitated with anti-phosphotyrosine, 4G10. The immunoprecipitate was then resolved in a 10% polyacrylamide gel and blotted with 4G10 to detect tyrosine-phosphorylated proteins. Figure 2A shows that a phosphorylated protein band at 85 kDA was readily detected within minutes of YT cell interaction with Raji cells, and was maximally detected at 5 min (lane 3, top panel). Immunoprecipitation with isotype matched control IgG did not produce any band with anti-phosphotyrosine Western blotting (lane 5, top panel). Immunoprecipitates of YT cells or Raji cells alone also produced no tyrosine-phosphorylated band (data not shown). Presence of equivalent amounts of p85 in the whole cell lysates from each treated group was checked prior to immunoprecipitation (bottom panel). Thus, YT cells, upon direct ligation with target cells, rapidly phosphorylated a protein band that comigrated at the same molecular weight as p85/PI 3-kinase.

˜ Figure 2. Detection of tyrosine-phosphorylated p85 protein in Raji-activated YT effector cells. (A). YT cells were cultured with paraformaldehyde-fixed Raji cells at a 1:1 ratio, for 0–15 min at 37oC. The cells were then lysed and immunoprecipitated with monoclonal anti-phosphotyrosine, 4G10 or isotype-matched IgG. (top panel). The immunoprecipitates were then probed with 4G10 by Western blotting. Presence of equivalent amounts of p85 was checked in whole cell lysates prior to immunoprecipitation (bottom panel). (B). YT cells, mixed with fixed Raji tumor cells for 0–15 min at 37 oC, were lysed. The lysates were immunoprecipitated with anti-phosphotyrosine, 4G10, or control isotype matched IgG, and then probed with anti-p85 (top panel). Presence of equivalent amounts of p85 was checked in whole cell lysates prior to immunoprecipitation (bottom panel). (C). Akt activation triggered by Raji ligation. YT cells, mixed with equal numbers of fixed Raji tumor cells for 0–15 min at 37 oC, were lysed. The lysates were analyzed by Western blotting with antibodies to active Akt (top panel ) and reprobed with antibodies to pan-Akt to check for equal loading (bottom panel). Raji mixed with equal numbers of fixed YT cells (lane 6) and Raji or YT alone (lanes 7 and 8) were also included as controls.

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82 · B. ZHONG et al. To confirm that the detected phosphoprotein at 85 kDa was the subunit of PI 3-Kinase, we next probed the anti-phosphotyrosine immunoprecipitates from YT/Raji conjugates after 0–15 min incubation at 37oC, with anti-p85. We detected maximal p85 phosphorylation in YT cells at 5 min of incubation with fixed Raji target cells ( Fig. 2B lane 3). Control immunoprecipitation with IgG pulled down no band (lane 6). Whole cell lysates (WCL) from YT effector cells alone were also run in the same gel (lane7) to provide a direct marker for p85. Equal amounts of PI 3-kinase were present in the samples prior to immunoprecipitation ( Fig. 2B bottom panel). In order to provide direct evidence that PI 3-kinase indeed becomes specifically activated after target ligation, we investigated whether PI 3-kinase was able to directly phosphorylate Akt. Akt is a known protein kinase which lies directly downstream of PI3K and is used as an indicator for PI3K activation. Phosphorylated Akt can be detected by using a commercially available anti-phospho-Akt antibody. As shown in Figure 2C, we found that target engagement induced Akt phosphorylation in YT cells within 2 min and peaked at 5–10 min indicating that ligation of YT cells with Raji target cells is responsible for PI 3-kinase activation. To exclude the possibility that the PI 3-kinase activity comes from the Raji cells rather than YT cells, we also fixed YT cells with paraformaldehyde and mixed them with unfixed Raji cells. After 5 min incubation, we were unable to detect any level of Akt phosphorylation (Fig. 2C lane 6). Control samples of either Raji or YT alone had no detectable levels of Akt phosphorylation (Fig. 2C lanes 7 & 8). Equal amounts of Akt were present after reprobing the membrane with anti-pan Akt ( Fig. 2C bottom panel). We then examined whether PI 3-kinase activation was specific. To answer this question, we tested YT cells against NK resistant tumor cells, HL60 and Jurkat. Neither targets can be lysed by YT cells in a 51Cr release assay (data not shown). YT cells, preincubated with either HL60 or Jurkat cells for 0 – 5 min at 37oC, were lysed and immunoprecipitated with anti-phosphotyrosine, 4G10, followed by Western blotting with antip85. Figure 3A shows that neither HL60 nor Jurkat could induce p85 tyrosine phosphorylation, while Raji could effectively do so in the same experiment (top panel). Whole cell lysates (WCL) from YT cells alone provided a direct marker for p85. Proteins were equally present in the samples, as depicted by anti-p85 Western blotting prior to immunoprecipitation (bottom panel). YT lysates from a similar experiment were then examined for their Akt phosphorylation. As shown in Figure 3B, only Raji target cells but neither HL60 nor Jurkat, could induce Akt phosphorylation in YT cells after 5 min ligation with target cells. Thus, it appears that only NK-sensitive target cells can trigger PI 3-kinase phosphorylation.

Evaluation of kinase function in PI 3-kinase isolated from YT cells

Tyrosine phosphorylation of PI 3-kinase is one measure of its activation, but it is essential to demonstrate that the activated PI 3-kinase does express function. For this purpose, an in vitro kinase assay using phosphatidyl inositol (PI) as the substrate was employed and the production of PI 3-phosphate, PI (3)P, was measured (43). YT cells, with or without 5 min incubation with Raji tumor cells, were lysed and the antiphosphotyrosine immunoprecipitates were tested for their ability to phosphorylate PI. Figure 4 A shows that PI (3)P was absent in YT cells that has been mixed with Raji

Control of NK lytic function by PI 3-kinase · 83

Figure 3. (A). YT cells were preincubated with 2 NK-resistant target cells, HL60 and Jurkat, for 0–5 min at 37oC prior to immunoprecipitation with anti-phosphotyrosine, 4G10, followed by Western blotting with anti-p85 (top panel). Incubation with Raji tumor cells was included as a positive control. Presence of equivalent amounts of p85 was checked in whole cell lysates prior to immunoprecipitation (bottom panel). (B). YT cells, mixed with equal numbers of HL60 or Jurkat for 0–5 min at 37oC, were lysed. The cell lysates were analyzed by Western blotting with antibodies to active Akt (top panel ) and reprobed with antibodies to pan-Akt to check for equal loading (bottom panel).

cells without preincubation (lane 1) while 5 min preincubation at 37oC of YT/Raji cells in DMSO was sufficient to trigger PI (3)P production (lane 2). Pretreatment of YT cells for 1 h at 37oC with 10 nM to 500 nM of wortmannin, prior to 5 min incubation with Raji cells (lanes 3–5) resulted in a dose-dependent inhibition of PI (3)P

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Control of NK lytic function by PI 3-kinase · 85

production. Therefore, PI 3-kinase in YT cells becomes not only tyrosine-phosphorylated but also functionally active upon target ligation. This activation can be blocked with wortmannin. Similarly, LY294002 could block PI 3-kinase function in YT cells activated by 5 min incubation with Raji cells (Fig. 4B). The concentrations of wortmannin and LY294002 that inhibited PI 3-kinase function matched those required for blocking of NK lytic function against 51Cr labeled Raji tumor cells (Fig1). Again, the same concentrations of wortmannin and LY294002 shown above also inhibited Akt phosphorylation (Fig. 4C). Altogether, these results confirm the finding that PI 3-kinase becomes phosphorylated and enzymatically active upon engagement with Raji target cells. Infection of YT cells with dominant-negative PI 3-Kinase

The ability of wortmannin and LY294002 to suppress NK lysis of tumor cells and the observation that PI 3-kinase function is activated by NK target ligation offer strong supportive data for the involvement of PI 3-kinase in the NK lytic process. To provide yet

Figure 4. Analysis of PI 3-kinase function in Raji-activated YT effector cells. YT cells, untreated or treated for 1 h at 37oC with 10–500 nM of wortmannin (A) or 25–100 uM of LY294002 (B) or the highest concentration of DMSO used, were mixed with Raji tumor cells at a 1:1 ratio for 0–5 min at 37oC. The cells were then lysed and immunoprecipitated with anti-phosphotyrosine, 4G10. The immuno-precipitates were incubated with [g-32P]ATP and phosphatidyl inositol (PI) as a substrate, and the generation of PI 3-phosphate (PI3P) was analyzed by thin layer chromatography. (C). YT cells, following similar treatment as shown in (A) and (B), were incubated with Raji tumor cells for 0–5 min. The whole cell lysates were generated and analyzed by Western blotting with antibodies to active Akt (top panel) and reprobed with antibodies to pan-Akt to check for equal loading (bottom panel).

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Figure 5. Inhibition of NK function by transient infection with dominant-negative p85 and p110 in YT effector cells. (A) Equal aliquots of YT cells were mock-infected (left panel) or infected with vaccinia constructs containing GFP-fluorescence protein (right panel) to monitor infection efficiency. Infection was carried out for 6 h and the infected cells were then monitored for GFP-positivity by flow cytometric analysis. (B) Equal aliquots of YT cells were mock-infected or infected with a dominant negative p85 (dnp85) or p110 (dnp110). The irrelative gene, CD56, as a control were also used to infect YT cells. After 6 h at 37oC, mock-infected, CD56-infected and dn-p85 or dn-p110 infected YT cells were assessed for viability and adjusted to the appropriate concentrations of viable cells, before testing for lysis of 51Cr-labeled Raji tumor cells at the indicated effector:target ratios. The SEM of each mean % cytotoxicity was less than 5% and was not shown.

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another evidence for this pathway, we undertook a molecular approach. We tested if infection of YT cells with dominant-negative PI 3-kinase could interrupt their lytic capacity. We used the well-characterized p85 deletion mutant that lacks the p110 binding site, and p110 mutant that lack enzymatic activity in its kinase domain, thus preventing regulation and activation of the PI 3-kinase pathway (38–40). YT cells were either mock-infected or infected with vaccinia virus encoding dominant negative p85 (dn-p85) and p110 (dn-p110). Infection was carried out for 6 h, which provided sufficient time for ample protein production from viral delivery without loss of cell viability. CD56 was also introduced into a separate YT pool as a non-specific infection control. The infection efficiency was monitored by infection of YT cells with vaccinia constructs containing GFP-fluorescence protein. Figure 5A shows that after 6 h incubation the infection efficiency rate in YT cells is more than 88% when compared with uninfected YT cells (left panel), which is evidenced by the cell population becoming GFP positive as monitored by flow cytometric analysis (right panel). Infected YT cells were adjusted to the appropriate concentrations prior to testing against 51Cr-labeled Raji tumor cells and were compared to mock-infected YT cells that were similarly processed but without any virus infection. Figure 5B shows that only the YT cells infected with the mutant p85 and p110 showed marked deficiency in lysis of Raji cells, while YT cells expressing the irrelevant gene, CD56, had the same ability as mock control YT cells to lyse the tumor cells. Thus, functional PI 3-kinase appears to be essential for optimal NK function against tumor cells. PI 3-kinase control of perforin mobilization within NK cells

One hallmark of the NK lytic process is the release of perforin and granzyme B, which are concentrated to the zone of contact between the NK cell and its target (25). Although the coordinated effort between perforin, to produce pores in the target membrane and granzyme B, to trigger DNA fragmentation, is now known, the molecular signals that control the polarization of these molecules are not well-defined. Exocytosis of granule contents into the extracellular supernatant has been reported to be triggered by FcR crosslinking in NK cells and this release apparently requires PI 3-kinase (31). However, it is not clear if PI 3-kinase is also involved in intracellular redistribution within the cytoplasm towards the target contact site in direct NK target lysis. To answer this question, we evaluated the pattern of perforin distribution in YT cells under several conditions, including with and without target ligation or with and without pretreatment with either wortmannin or LY294002. Figure 6 shows immunostaining of a representative YT/Raji conjugate before and after each type of treatment. A selective marker was first identified for Raji cells, i.e., lyn kinase, that is absent in YT cells, while perforin was found only in YT cells. Thus, Raji cells did not stain with FITC-anti-perforin (A) but reacted with TRITC-anti-lyn (B). In contrast, YT cells did not stain with TRITC-antilyn (C) but reacted with FITC-anti-perforin in punctated forms (D). All cells stained blue with the nuclear marker. The DMSO-treated YT cell, when conjugated to a TRITC-labeled Raji tumor cell, with 0 min incubation (E), showed even distribution of FITC-perforin, as in the YT cells alone (D). However, upon 5 min incubation at 37°C, the DMSO-treated YT cell conjugated to a Raji tumor cell had completely mobilized its intracellular perforin to the zone of contact (F). Further incubation up to 15 min did not change this pattern (data not shown). When the YT cell was pretreated for 1 h at

88 · B. ZHONG et al.

Figure 6. Inhibition of perforin polarization in NK cells by wortmannin. YT cells, pretreated with DMSO, 500 nM of wortmannin or 100 uM of LY294002 for 1 h at 37oC, were incubated with an equivalent number of Raji tumor cells for 0–5 min at 37oC. The cells were then cytospinned onto microscope slides and stained with anti-perforin-FITC and/or anti-lyn-TRITC. (A) Raji tumor cells stained with anti-perforin-FITC, (B) Raji cells stained with anti-lyn-TRITC, (C) YT cells stained with anti-lyn-TRITC, (D) YT cells stained with anti-perforin-FITC, (E) a DMSO-pretreated YT cell that had bound a Raji tumor cell at 0 min and stained with anti-perforin-FITC/anti-lyn-TRITC, (F) a DMSO-pretreated YT cell that had bound a Raji tumor cell for 5 min at 37oC, and stained with antiperforin-FITC/anti-lyn-TRITC, (G) a wortmannin-pretreated YT cell that had bound a Raji tumor cell for 5 min at 37oC, and stained with anti-perforin-FITC/anti-lyn-TRITC, (H) a LY-294002-pretreated YT cell that had bound Raji tumor cells for 5 min at 37oC and stained with anti-perforinFITC/anti-lyn-TRITC.

Control of NK lytic function by PI 3-kinase · 89

Figure 7. Inhibition of granyzme B polarization in NK cells by LY294002. YT cells, pretreated with DMSO, 500 nM of wortmannin or 100 uM of LY294002 for 1 h at 37oC, were incubated with an equivalent number of Raji tumor cells for 0–5 min at 37oC. The cells were then cytospinned onto microscope slides and stained with anti-granzyme B-FITC and/or anti-lyn-TRITC. (A) Raji tumor cells stained with anti-granzyme B-FITC, (B) Raji cells stained with anti-lyn-TRITC, (C) YT cells stained with anti-lyn-TRITC, (D) YT cells stained with anti-granzyme B-FITC, (E) a DMSO-pretreated YT cell that had bound a Raji tumor cell at 0 min and stained with anti-granzyme B-FITC/anti-lyn-TRITC, (F) a DMSO-pretreated YT cell that had bound a Raji tumor cell for 5 min at 37oC, and stained with antigranzyme B-FITC/anti-lyn-TRITC, (G) a wortmannin-pretreated YT cell that had bound a Raji tumor cell for 5 min at 37oC, and stained with anti-granzyme B-FITC/anti-lyn-TRITC, (H) a LY294002-pretreated YT cell that had bound Raji tumor cells for 5 min at 37oC and strained with anti-granzyme B-FITC/anti-lyn-TRITC.

90 · B. ZHONG et al. 37oC with 500 nM of wortmannin prior to target conjugation for 5 min, it no longer could mobilize perforin upon target contact and showed even perforin distribution in the cytoplasm (G). Similarly, the YT cell that was pretreated with LY294002 under the same conditions could not demonstrate perforin mobilization when it was conjugated to a Raji tumor cell (H). Enumeration of 100 conjugates per slide indicated that the percentage of YT/Raji conjugates that showed mobilized perforin at 0 min was 6.8%, and upon 5 min incubation, was 29.0%. Wortmannin and LY294002 treatment reduced the percentage of YT/Raji conjugates with mobilized perforin to 9.0% and 11.0% respectively. Similar results were obtained in two other experiments (data not shown). PI 3-kinase control of granzyme B mobilization within NK cells

With the observation that PI 3-kinase was required for perforin mobilization in YT cells, we next examined if granzyme B was mobilized in the same manner and has the same requirements. Figure 7 shows a representative YT/Raji conjugate before and after treatment with the two PI 3-Kinase inhibitors. Raji cells showed no staining with FITC-antigranzyme B (A) but with TRITC-anti-lyn (B). YT cells showed no staining with TRITC-anti-lyn (C) but with FITC-anti-granzyme B (D). The DMSO-treated YT cells, conjugated to a Raji cell, without preincubation, showed no mobilization of granzyme B (E), but upon 5 min incubation, had completely redirected granzyme B towards the contact zone (F). As with perforin, 15 min incubation did not change this pattern (data not shown). The YT cell, that had been pretreated with either 500 nM of wortmannin (G) or 100 uM of LY294002 (H) for 1 h at 37oC, could not perform this function and retained an even distribution of granzyme B throughout its cytoplasm, even when it was conjugated to a Raji cell. Microscopic assessment of 100 YT/Raji conjugates per slide indicated that the percentage of YT/Raji conjugates with mobilized granzyme B was 5.0% with 0 min inucubation and climbed to 20.0% with 5 min incubation at 37oC. Wortmannin and LY294002 treatment reduced the percentage of YT/Raji conjugates with mobilized granzyme B to 5% and 8% respectively. These results were reproducible in two other experiments (data not shown). Therefore, granzyme B, like perforin is under the control of PI 3-Kinase. Discussion Cytotoxicity is a defining event in lymphocytes, specifically organized for the destruction of unwanted cells in the host. NK and cytotoxic T cells utilize granule-mediated apoptosis, which requires the coordinated actions of perforin and granzyme B (44). It is widely accepted that the initial contact with a target cell causes a directional exocytosis of granule contents from the effector cell to the junction with the target cell. With regards to the target cell, data are emerging to reveal that perforin may provide a means to deliver granzyme B into its cytosol. Upon endocytosis of granzyme B, the accompanying perforin inserted into the vesicle membrane can disrupt membrane integrity and allow granzyme B to escape into the cytosol (25). Granzyme B may also enter via a direct endocytic pathway. Once target cell entry is achieved, granzyme B can initiate the death process (27). In vitro, caspases 3,6,7,8,9, and 10 can all serve as substrates for granzyme B, and caspase activation by granzyme B is documented in vivo, although the exact

Control of NK lytic function by PI 3-kinase · 91

sequence in which the caspases are cleaved and activated is not yet clearly defined (29, 44). Despite rapid development in the elucidation of the granule-initiated apoptotic cascade inside the target cell, few studies have concentrated on the mechanisms that govern granule redirection inside the effector cell itself, prior to release into the engaged target cell. In this study, we have identified the critical role of PI 3-kinase in the motility of perforin and granzyme B within the effector cell. PI 3-kinase activation occurs rapidly, peaking within 5 min, and requires a specific target cell to generate it. NK cells, upon binding to a relevant NK-sensitive target cell, Raji, immediately displayed PI 3-kinase activation as assessed by its tyrosine phosphorylation within the p85 subunit. Specificity of this reaction was proven by the inability of two NK-resistant target cells, Jurkat and HL60, to induce PI 3-kinase activation in the same NK cells. PI 3-kinase activation could be further substantiated by the functional ability of the PI 3-kinase isolated from Raji-activated NK cells to produce PI (3)P from phosphatidyl inositol in an in vitro kinase assay. The involvement of PI 3-kinase in lysis appeared to be universal in all NK cells, because LGL, freshly-isolated from 4 normal donors, were equally sensitive to wortmannin or LY294002 inhibition. Inhibition of NK lysis by wortmannin or LY294002, however, only provides associative evidence for PI 3-kinase and lytic function. Direct evidence was thus sought for, using a molecular approach. Transient infection experiments demonstrated that expression of dominant-negative p85 and p110 subunits of PI 3-kinase in NK cells clearly caused a drastic loss of lytic function, while expression of the irrelevant gene, CD56, had no such effect. In defining where the block by wortmannin or LY294002 might be, we hypothesized that granule mobilization towards the tumor cell could be the target. This was validated by immunostaining for perforin and granzyme B in NK:tumor conjugates. Within 5 min of conjugation of NK cells with a target cell, perforin, which was usually evenly distributed throughout the cytoplasm in non-conjugated NK cells, was found completely redistributed to the point of contact with the target cell. NK cells, pretreated with either wortmannin or LY294002, formed conjugates with target cells, but could not mobilize its cytoplasmic perforin. The same characteristic was repeated with granzyme B redistribution. Thus, our results taken together point to PI 3-kinase as a critical signal that controls intracellular cytoplasmic movement of granule components. Our finding of PI 3-kinase control of granule polarization adds yet another important function to this molecule. Use of wortmannin or LY294002 has recently demonstrated that deliberate inhibition of PI 3-kinase with these reagents in T cells could cause tolerance induction against B7-1 expressing allogeneic melanoma cells by interuption of CD28 triggering (45). Interestingly, this tolerance produced by PI 3-kinase could be reversed by IL-2 but not by IL-4 and IL-7, both of which share the common IL-2Rg with IL-2. Using intact B cells as antigen-presenting cells, wortmannin-treated T cells could not adequately respond to CD3 crosslinking and B cell stimulation, and this deficiency could be traced to suboptimal CD40L and ICAM-1 induction (46). The unavailability of these costimulatory molecules apparently caused the loss of ability by the T cells to activate B cells to proliferate or produce Ig. Thus, PI 3-kinase is intricately involved in the activation phase of T cells. Our study indicates that PI 3-kinase is also tightly associated with the effector phase. Binding of a relevant target cell induces PI 3-kinase activation in NK cells which sets in motion the granules components, perforin and granzyme B, to directionally head

92 · B. ZHONG et al. towards the receptor:ligand interaction site. Previous studies with NK cells have also shown that PI 3-kinase is involved in granule exocytosis, but this mechanism has mainly be resolved through the ADCC pathway whereby the target cells are ligated to effector cells via FcR (31). Moreover, these studies focused on generalized granule exocytosis into the supernatant and provide no information on intracellular movement. PI 3-kinase is known to control actin organization and affect the cytosketal structure (15, 19, 20, 39). How PI 3-kinase is affecting granule polarization towards the contact point is an important question that needs further investigation. We have recently shown that MAPK/ERK2 is also involved in control of NK lysis of tumor cells, and it appears to affect perforin and granzyme B movement (33, 34). MAPK has also been recently associated with FcR triggering of NK cells to affect extracellular granule release (32). Whether PI 3 Kinase and MAPK/ERK2 work within the same signal pathway or controls two separate processes that converge to result in perforin/granzyme B movement should be another important issue to resolve. Acknowledgements

This work was supported by National Cancer Institute Grant CA83146 and also supported by the Pathology Core, FACScan Core and Molecular Imaging Core facilities of the H. Lee Moffitt Cancer Center.

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