Human Cancer Biology

Coengagement of CD16 and CD94 Receptors Mediates Secretion of and Induces Apoptotic Death of Naive Natural Killer Cells AnahidJewett,1Nicholas A. Cacalano,2 Christian Head,3 and Antonia Teruel1

Abstract Down-modulation of CD16 (FcgRIII) receptors and loss of natural killer (NK) cell function have been observed in oral cancer patients. However, neither the mechanisms nor the significance of the decrease in CD16 receptors have been fully understood. The cytotoxic activity and survival of NK cells are negatively regulated by antibodies directed against CD16 surface receptor. The addition of anti-CD94 antibody in combination with either F(abV)2 fragment or intact anti-CD16 antibody to NK cells resulted in significant inhibition of NK cell cytotoxic function and induction of apoptosis in resting human peripheral blood NK cells. Addition of interleukin-2 to anti-CD16 and/or anti-CD94 antibody-treated NK cells significantly inhibited apoptosis and increased the function of NK cells. There was a significant increase in tumor necrosis factor-a (TNF-a) but not IFN- g secretion in NK cells treated either with anti-CD16 antibody alone or in combination with anti-CD94 antibodies. Consequently, the addition of anti-TNF-a antibody partially inhibited apo- ptosis of NK cells mediated by the combination of anti-CD94 and anti-CD16 antibodies. Increase in apoptotic death of NK cells also correlated with an increase in type 2 inflammatory and in the induction of chemokines. Thus, we conclude that binding of antibodies to CD16 and CD94 NK cell receptors induces death of the NK cells and signals for the release of chemokines.

Natural killer (NK) cells exert important effector functions in receptor expression have been documented previously in the primary host defense against neoplasias. Furthermore, NK patients with cancer (2). Similarly, down-modulation of cells through secretion of cytokines are involved in the CD16 and CD94 NK cell receptors, loss of NK cell function, regulation of T-cell-mediated immune responses. Clearly, in and increased NK cell death were observed after their several disease models, the function and the numbers of NK interaction with oral tumor cells.4 Thus, triggering of CD16 cells are significantly decreased (1, 2). We have reported and CD94 receptors by ligands expressed on tumor cells may be previously that functional inactivation and death of NK cells are responsible for the functional abnormalities and death of NK observed following the interaction of NK cells with tumor- cells. Because specific tumor ligands for CD16 receptors have target cells or by triggering of CD16 receptors on NK cells been suggested but not characterized at present, we do not have (3, 4). However, neither the cellular mechanisms nor the the ability to study the role of these receptors on NK cell physiologic significance of death in NK cells have been fully function and death using tumor ligands (5). However, previous explored. studies made use of antibodies to CD16 receptor to establish Lymphocytes isolated from patients with cancer, particularly their significance in NK cell function. Furthermore, to study the head and neck, ovarian, renal, cervical, colorectal, prostate, and function of CD94 NK cell receptors, either clonal populations , have functional abnormalities and signaling defects of NK cells or NK cell lines expressing activating or inhibitory (1). Profound down-modulation of ~ chain and CD16 surface forms of these receptors were used. However, these studies, although quite important, have limitations in their application to naive polyclonal population of NK cells. Therefore, we were Authors’ Affiliations: 1The Jane and Jerry Weintraub Center for Reconstructive interested to explore the combination of CD16 and CD94 Biotechnology and Division of Oral Biology and Medicine, Jonsson Compre- 2 3 receptor modulation, as seen after the interaction of NK cells hensive Cancer Center; Departments of Radiation Oncology and Head and Neck with tumor-target cells, on NK cell function and survival. Surgery, School of Dentistry and Medicine, University of California at Los Angeles, Los Angeles, California A net balance between activating and inhibitory signals, Received 10/27/05; revised 12/22/05; accepted 1/23/06. which NK cells receive from tumor cells, dictate whether Grant support: National Institute of Dental and Craniofacial Research/NIH grant cytotoxic NK or T cells will become functionally activated or RO1-DE12880. inhibited in the presence of tumor-target cells. The list for both 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 activating and inhibitory signals delivered by different tumor with 18 U.S.C. Section 1734 solely to indicate this fact. cell ligands to NK cells through specific NK cell receptors has Requests for reprints: Anahid Jewett, Division of Oral Biology and Medicine, been increasing steadily (6). CD16 receptors are shown to University of California at Los Angeles School of Dentistry, 10833 Le Conte signal for the activation of NK cells, whereas CD94 receptors are Avenue, Los Angeles, CA 90095-1668. Phone: 310-206-3970; Fax: 310-794- 7109; E-mail: [email protected]. F 2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-2306 4 In preparation.

Clin Cancer Res 2006;12(7) April 1, 20 06 1994 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. NK Cell Apoptosis and CD16 and CD94 Receptors capable of mediating both activation and inhibition of NK cell CA). The FITC-conjugated anti-CD54, anti-CD16, and phycoerythrin- function depending on their association with NKG2C or conjugated anti-CD69 antibodies were obtained from Coulter/Immu- NKG2A, respectively (7–11). Studies from several groups notech (Miami, FL). TAPA-1 antibodies were obtained from Dr. Levy showed the recruitment and binding of tyrosine phosphatase (Stanford University, Stanford, CA). FITC-conjugated anti-Fas anti- bodies were obtained from PharMingen (San Diego, CA). Polyclonal SHP-1 to human NK cell inhibitory receptors and subsequent anti-TNF-a antibody was prepared in rabbits in our laboratory. The delivery of negative signals to NK cells (12, 13). Alternatively, anti-TNF-a mAbs (B154.9.1 and B154.7.1) were prepared in our the CD94/NKG2C complex associates with DAP12, which laboratory from hybridomas kindly supplied by Dr. G. Trinchieri carries a cytoplasmic immunoreceptor tyrosine-based activation (Wistar Institute, Philadelphia, PA). Anti–class II (L227 monomorphic motif (ITAM). Triggering of CD94/NKG2C by CD94 monoclo- ATCC HB.96), anti-LFA-1 (TS1.22.1.1.13.HB202), and anti-LFA-3 nal antibodies (mAb) was shown to activate the function of (TS2.9.1.4.3.HB205) antibodies were prepared from hybridomas clonal population of NK cells leading to increased production obtained from the American Type Culture Collection (Manassas, of IFN-g, increased cytotoxicity, and proliferation (11), an VA). Purified antibodies to CD16, CD56, CD18, and LFA-1 were V effect that was not seen when anti-CD94 antibody was added to purchased from Coulter/Immunotech. Purified F(ab )2 fragment of naive polyclonal NK cells (see Results). anti-CD16 antibody was purchased from Ancell (Bayport, MN). Control ascites IgG1 was derived from a control hybridoma (American There are conflicting results regarding CD16- and CD94- Type Culture Collection), and purified IgG1 was purchased from mediated NK cell death. Previous work has shown induction Coulter/Immunotech. of cell death in interleukin (IL)-2–activated NK cells by anti- Purification of NK cells. Peripheral blood mononuclear cells from CD16 antibodies (14, 15). However, we have shown healthy donors were isolated as described previously (3, 4). Purified NK elsewhere (4) and here that IL-2 rescues anti-CD16 anti- cells were negatively selected using a NK isolation (Miltenyi Biotech) body-treated NK cells from undergoing apoptotic cell death, and consisted of 85% to 95% of CD16+ cells. Approximately 10% to 15% an effect of IL-2 that is clearly described in T and NK cell of NK cell population was shown previously to have no or low CD16 + apoptosis and is in line with the major function of this expression on the surface of NK cells (20). The percentage of CD3 T cells + in many cancers (16–19). Thus, in this article, we and CD19 B cells determined by anti-CD3 and anti-CD19 antibodies in F F present several important novel findings regarding the death the purified NK samples was 2.3 3.2 and 3 4, respectively. These levels of antibody staining in purified NK samples were also observed of NK cells, an area of investigation that has received relatively after staining with isotype control antibodies, indicating the levels of little attention, although its significance has clearly been nonspecific binding of antibodies to purified NK cells. shown in many cancer patients, including those of the head 51Cr release assay, ELISA, surface staining of NK cells, and DNA and neck (1, 2). staining for apoptosis were all described previously (3, 4, 21). We examined the cytotoxic function, the surface phenotype, Multiplex cytokine and arrays. The fluorokine and the profiles of secreted cytokines and chemokines in the MAP cytokine and chemokine multiplex array kits were purchased from NK cells that were triggered to undergo apoptosis by anti- R&D Systems (Minneapolis, MN), and the procedures were conducted CD16 and/or anti-CD94 antibodies. We show that, in as suggested by the manufacturer. comparison with each of the anti-CD16 and anti-CD94 mAb treatment alone, the coaddition of anti-CD16 and anti-CD94 Results antibodies to naive NK cells results in the additive or synergistic induction of tumor necrosis factor-a (TNF-a) Increased induction of NK cell death by the addition of anti- secretion, inhibition of NK cell cytotoxicity, and induction of CD16and anti-CD94 antibodies in naive NK cells. The addition NK cell apoptotic death. Addition of IL-2 inhibited the of anti-CD16 antibody to naive NK cells induced significant cell apoptotic death induced by anti-CD16 and/or anti-CD94 death of NK cells as reported previously (4, 22). Cell death antibodies and increased the function of NK cells. We also induced by anti-CD16 antibody in NK cells was primarily report that the endogenous secretion of TNF-a is partly apoptotic (4, 22). Cell death induced in the presence of anti- responsible for the induction of NK cell death by anti-CD16 CD94 antibody alone at the same antibody concentration is and anti-CD94 antibodies. More importantly, the induction of significantly lower than that obtained in the presence of anti- cell death by CD16 and CD94 antibodies correlated with the CD16 antibody treatment (Table 1; Fig. 1). Likewise, the death secretion of chemokines, indicating the possibility of a role for induced in the presence of anti-CD94 antibody treatment is these receptors in the recruitment of fresh immune effectors to apoptotic (Fig. 1). The addition of a combination of anti-CD94 inflammatory infiltrates to replace those that are signaled to and anti-CD16 antibodies in comparison with each antibody die by the CD16 and CD94 receptors. alone to naive NK cells significantly increased the levels of cell death in NK cells (Table 1; Fig. 1). In contrast, cell death was Materials and Methods not observed when NK cells were treated with IL-2 (Fig. 1). Furthermore, the addition of IL-2 to either anti-CD16 antibody Cell lines and reagents. K562 target cells were cultured in RPMI treatment alone or the combination of anti-CD16 and anti- 1640, and SCC4, Cal33, and UMSCC tumor lines and UCLA-1 and CD94 antibody-treated NK cells significantly decreased the UCLA-2 primary oral tumors were cultured in either DMEM or RPMI levels of apoptotic cell death in NK cells (Fig. 1). The increased 1640 supplemented with 1% sodium pyruvate, 1% nonessential amino induction of cell death by a combination of anti-CD94 and acids, 1% glutamine, 1% penicillin-streptomycin (Life Technologies, anti-CD16 antibodies in NK cells was specific. Unlike CD94 Grand Island, NY), and 10% FCS (Irvine Scientific, Santa Ana, CA). UCLA-1 and UCLA-2 primary oral tumors were derived at University of antibody, the addition of anti-LFA-1, anti-LFA-3, and anti- California at Los Angeles from patients with tongue cancer (Dr. TAPA-1 antibodies in combination with anti-CD16 antibody- Christian Head). Recombinant IL-2 was obtained from Chiron Corp. treated NK cells had no enhancing effect on cell death (data not (Emeryville, CA). IFN-g was obtained from Peprotech (Piscataway, NJ). shown). Moreover, the addition of F(abV)2 fragment of CD16 The NK purification kit was obtained from Miltenyi Biotech (Auburn, antibody and intact mAb induced cell death of NK cells in the

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À indicated that CD94+ but not CD94 NK subset was the V Ta b l e 1. F(ab )2 fragment and intact anti-CD16 subpopulation that underwent augmented cell death (data not antibodies in combination with anti-CD94 antibody shown). These findings show that the coaddition of anti-CD94 induce death of NK cells and decrease the cytotoxic and anti-CD16 antibodies to NK cells results in a significant function of NK cells increase in the frequency of NK cell death. Furthermore, the CD94 receptor-positive NK cell subset was the subpopulation Treatment Control Anti-CD16 Anti-CD16 that underwent augmented cell death in the presence of anti-

mAb mAb F(abV)2 CD16 mAbs. Inhibition of NK cell cytotoxic function by anti-CD16and anti- %Celldeath CD94 antibodies. The cytotoxic activity of purified naive NK Isotype control mAb 5 20 37 cells treated with anti-CD16 and/or anti-CD94 antibodies was Anti-CD94 mAb 8 37 40 assessed. Treatment of NK cells with anti-CD16 antibody signi- LU40/107 cells ficantly inhibited the NK cell cytotoxic function (Table 2A). The Isotype control mAb 22 F 1. 4 13. 5 F 0.7 1 F0.07 addition of anti-CD94 antibody to NK cells in the absence of Anti-CD94 mAb 17.5 F 0.7 3.7 F 0.07 0.0 F 0.0 anti-CD16 antibody had some inhibitory effect on cytotoxic function. However, the addition of anti-CD94 and anti-CD16  6 NOTE: NK cells at a concentration of 1 10 /mL were treated with isotype antibodies together significantly potentiated inhibition of control antibody (6 Ag/mL), anti-CD16 antibody (5 Ag/mL), anti-CD94 anti- NK cell cytotoxic function against K562 tumor-target cells body (5 Ag/mL), and anti-CD16 F(abV)2 fragment (5 Ag/mL) alone or in combination. The percentage of NK cell death and the levels of cytotoxicity (Table 2A). Treatment of NK cells with IL-2 augmented the NK mediated by NK cells were determined after12hours of incubation using stain- cytotoxicity, and the addition of anti-CD94 antibody in com- 51 7 ing with propidium iodide and Cr release assay, respectively. LU40/10 cells bination with IL-2 inhibited IL-2-mediated increase in cytotoxic is calculated by using the inverse of the number of effector cells needed to lyse 40% of tumor-target cells  10 0. activity. The addition of anti-CD94 antibody in combination with anti-CD16 antibody and IL-2 potentiated the inhibition of cytotoxic activity of NK cells against tumor-target cells when presence and absence of anti-CD94 antibody treatment treated NK cells were incubated for 12 to 18 hours before they excluding Fc receptor-mediated effects for the observed results were added to tumor-target cells (Table 2A). (Table 1). A significant inhibition of NK cell cytotoxicity could be Experiments were next done to determine whether the observed immediately after the addition of a combination of CD94+ NK cell subset was the subpopulation that underwent anti-CD94 and anti-CD16 antibodies (Fig. 2), whereas over- augmented apoptosis following treatment with anti-CD16 night pretreatment with each of the antibodies was needed to antibody. After staining with anti-CD94 antibody, CD94+ observe inhibition of cytotoxicity mediated by either intact anti- À and CD94 NK cell subpopulations were sorted (data not CD16 or anti-CD94 antibodies. However, inhibition of NK cell shown) and treated with anti-CD16 antibody. The results function could be observed immediately after the addition of

Fig. 1. IL-2 treatment decreases anti- CD16- and/or anti-CD94 antibody-induced apoptosis in NK cells. NK cells (1 Â10 6/mL) were treated with isotype control antibody (6 Ag/mL), anti-CD16 antibody (5 Ag/mL), IL-2 (500 units/mL), and anti-CD94 antibody (5 Ag/mL) alone or in combination. After 12 to 18 hours of incubation, the cells were washed, permeabilized, and stained with propidium iodide. To p r i g h t , percentages of NK cells in sub-G0-G1stage of cell cycle. Representative of one of four experiments. Student’s t test paired P for control versus anti-CD16 antibody and anti-CD16 antibody versus anti-CD16 antibody + anti-CD94 antibody is <0.05.

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Ta b l e 2 .

A. Inhibition of NKcell cytotoxic function against oral tumors by anti-CD16 and anti-CD94 antibodies LU40/107 cells Control IL-2 Anti-CD16 antibody Anti-CD16 + IL-2 SCC4 Isotype control mAb 16 F 1.1 5 8 F 66F 427F 1.1 Anti-CD94 antibody 12 F 1. 3 2 6 F 0.8 8 F 1. 4 10 F 0.9 Cal33 Isotype control mAb 2 F 0.2 17 F 20.0F 1. 5 4 F 1. 2 Anti-CD94 mAb 0.0 F 0.5 3 F 0.6 0.8 F 0.9 2 F 0.5 UCLA-1 Isotype control mAb 0.0 F 0.0 20 F 0.3 0.0 F 0.2 7 F 0.0 Anti-CD94 mAb 0.0 F 1. 2 3 F 0.8 0.0 F 1. 4 0 .0 F 0.9 UCLA-2 Isotype control mAb 6.5 F 1. 8 9 9 . 5 F 16 0.0 F 1. 5 12 F 1. 2 Anti-CD94 mAb 5 F 1 22.5 F 3.5 0.0 F 0.9 0.0 F 0.5 UMSCC Isotype control mAb 0.0 F 1. 4 47 F 70.0F 0.1 10 F 1. 6 Anti-CD94 mAb 0.0 F 0.0 9.6 F 0.0 0.0 F 0.0 1 F0.5 K562 Isotype control mAb 87 F 1. 5 2 75 . 5 F 49 36 F 780F 1. 4 Anti-CD94 mAb 83 F 2.8 187 F 39 16.5 F 0.7 28 F 5.6

B. Combination of anti-CD16 and anti-CD94 antibodies block NKcell ^ mediated inhibition ofVEGF secretion by UCLA-2 oral tumor cells Tr e a t m e n t FUCLA-2 VEGF (MFI) ÀNK+NK Control +IL-2 CD16 mAb CD16 mAb + IL-2 +UCLA-2 784 F 18 ö ö ö ö Isotype control mAb ÀUCLA-2 ö 17 F 248F 1. 5 2 0 F 136F 0.0 +UCLA-2 ö 392 F 10 292.5 F 83,724F 200 441 F10 Anti-CD94 mAb ÀUCLA-2 ö 20 F 243.5F 121.5F 0.0 37 F 1 +UCLA-2 ö 1,369 F 20 311 F84,764F 15 0 579 F 10

NOTE: A. Purified NK cells (1 Â10 6/mL) were treated with isotype control antibody (6 Ag/mL), anti-CD16 antibody (5 Ag/mL), IL-2 (500 units/mL), and anti-CD94 antibody (5 Ag/mL) alone or in combination, and the cells were incubated overnight at 37jC. An equal volume of NK cells from each sample was removed after12hours of incubation and added to 51Cr-labeled oral tumor-target cells or K562 cells and the NK cell cytotoxicity was determined in a 4-hour 51Cr release assay. B. Isotype control antibody (6 Ag/mL), anti-CD16 antibody (5 Ag/mL), anti-CD94 antibody (5 Ag/mL), and IL-2 (500 units/mL) alone or in combination were added to either NK cells alone or in the cocultures of NK cells (1 Â10 6/mL) with UCLA-2 primary oral tumor cells at an E:Tratio of 1:1, and after 16 hours of coincubation, the levels ofVEGFsecretion were determined in the supernatants using a sensitive and specific ELISA. Abbreviation: MFI, meam fluorescence intensity.

5 F(abV)2 fragment of CD16 antibody. Functional inhibition was completely (Fig. 2). However, if NK cells were treated with also observed in IFN-a-treated NK cells but not in IL-2-treated the combination of IL-2, anti-CD16, and anti-CD94 antibodies NK cells when a combination of anti-CD16 and anti-CD94 for 12 to 18 hours before they were cocultured with K562 or antibodies was added to NK cells immediately before their oral tumor cells (SCC4, Cal33, UCLA-1, UCLA-2, and UMSCC), cocultures with K562 tumor cells (Fig. 2). The addition of anti- significant inhibition of NK cell cytotoxic function could still CD56 or MHC class II antibody to anti-CD16 antibody-treated be observed even in the presence of IL-2 (Table 2A). Thus, it NK cells did not have any effect on NK cell cytotoxicity (data seems that IL-2 treatment delays the kinetics of NK cell not shown). Thus, in cultures in which NK cells were treated inactivation by anti-CD16 and anti-CD94 antibody treatment. with the combination of IL-2, anti-CD94, and anti-CD16 The inhibitory function of CD16 and CD94 antibodies in the antibodies immediately before they were added to K562 tumor absence of IL-2 treatment and its prevention in the presence of cells, IL-2 was able to prevent anti-CD16 and anti-CD94 IL-2 treatment in NK cells was also determined using a novel antibody-mediated inhibition of NK cell function nearly assay system. Both NK cell sensitive and resistant oral tumors secreted significant amounts of vascular endothelial growth 5 In preparation. factor (VEGF) in the supernatants.4 In contrast, no induction of

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VEGF secretion could be seen by naive or IL-2-activated NK obtained by each of anti-CD16 and anti-CD94 antibody alone cells (Table 2B). The levels of tumor-derived VEGF secretion in (Table 2B). IL-2 treatment was capable of preventing anti-CD16 the cocultures of NK cells with sensitive oral tumors (Table 2B) and/or anti-CD94 antibody-mediated increase in VEGF secre- correlated with the magnitude of cytotoxicity induced by the tion (Table 2B). Therefore, decrease in VEGF secretion in oral NK cells (Table 2A). Thus, naive and IL-2-treated NK cells were tumors is likely due to lysis of tumor cells by the NK cells able to decrease the levels of VEGF secretion in the cocultures because treatment of NK cells with anti-CD16 and anti-CD94 of NK cells with sensitive oral tumors (Table 2B). As expected, antibodies individually or in combination, which induced IL-2-treated NK cells decreased VEGF secretion by the oral death of NK cells, increased tumor-derived VEGF secretion. tumors to a greater extent than untreated NK cells (Table 2B). Increase in the secretion of TNF-a and IL-6but not that of Thus, modulation of tumor-derived VEGF secretion in the IFN-g by anti-CD94 and anti-CD16antibody-treated NK cells. cocultures of NK cells with oral tumors could be used as a We have reported previously that treatment of NK cells with measure of NK cell functional activity against oral tumors. anti-CD16 antibodies resulted in the induction of TNF-a secre- Therefore, to assess the effect of NK cell death on VEGF sec- tion and that TNF-a was responsible in part for the induction retion by the sensitive oral tumors in the cocultures of NK cells of apoptosis in naive NK cells (4, 22). Because the addition with tumor cells, isotype control antibody, anti-CD16 anti- of anti-CD94 and anti-CD16 antibodies augmented cell death, body, anti-CD94 antibody, IL-2, or their combination (Table 2B) we hypothesized that these antibodies may also augment secre- were added to the cocultures of NK cells with UCLA-2 primary tion of TNF-a. Treatment of NK cells with anti-CD16 antibody oral tumor cells, and after 16 hours of coincubation, the levels resulted in the augmented secretion of TNF-a but not IFN-g of VEGF secretion were determined in the supernatants by (Table 3A). The coaddition of anti-CD94 and anti-CD16 anti- ELISA. Under the conditions where no significant cell death bodies to NK cells significantly potentiated the secretion of could be observed in NK cells, both naive and IL-2-treated NK TNF-a (Table 3A). The addition of anti-CD94 antibody alone to cells inhibited tumor-derived VEGF secretion (Table 2B). untreated control NK cells had also some potentiating effect on However, VEGF secretion was significantly increased after the TNF-a secretion. The induction of TNF-a secretion by a combi- treatment of NK cells with anti-CD16 antibody and, to a lesser nation of anti-CD94 and anti-CD16 antibodies was specific for extent, anti-CD94 antibody (Table 2B). In addition, treatment these antibodies and was not observed when other antibodies, of NK cells with the combination of anti-CD16 and anti-CD94 such as anti-MHC class II, anti-CD56, and LFA-3 antibodies, antibodies resulted in an even greater induction of VEGF were used (data not shown). Therefore, these findings sug- secretion by the tumor cells when compared with those gested that TNF-a may have a role in death of NK cells induced by a combination of anti-CD94 and anti-CD16 antibodies. We thus examined whether secretion of TNF-a is responsible for the induction of NK cell death induced by the anti-receptor antibodies. Treatment of NK cells with anti-CD16 and anti- CD94 antibodies in the presence of anti-TNF-a antibody for 18 hours inhibited NK cell death partially (Fig. 3). These findings show that anti-CD16 and/or anti-CD94 antibody-mediated induction of NK cell death is due in part to the induction of TNF-a secretion by the NK cells. Because only partial inhibition of NK cell death could be obtained in the presence of anti-TNF-a antibody, it is likely that other factors in addition to TNF-a are involved in induction of death and inactivation of NK cell function. We have recently shown that IL-6 is an important negative regulator of NK cell function (23). Therefore, we were interested to assess whether treatment of NK cells with anti-CD16 and anti-CD94 receptor antibodies was able to induce IL-6 secretion (23). In addition to IL-6 secretion, the levels of IL-4, IL-5, IL-1h, IL-10, IL-13, and IFN-g secretion were determined after the treatment of NK cells with anti-receptor antibodies in the presence and absence of IL-2 treatment using multiplex cytokine bead array system to determine the possibility of a shift from type 1 to type 2 cytokine profiles in NK cells triggered to undergo cell death. Naive NK cells treated with anti-CD16 and/or anti-CD94 antibodies secreted significant levels of IL-6, IL-1h, IL-10, and IL-13 in addition to TNF-a (Table 3B). However, no secretion of IFN-g Fig. 2. Rapid inhibition of NK cell cytotoxicity by the treatment of NK cells with a could be observed by NK cells with the exception of those treated combination of anti-CD16 and anti-CD94 antibodies. Purified NK cells (1 Â10 6/mL) with IL-2 in the presence and absence of receptor antibodies. were treated with isotype control antibody (6 Ag/mL), IL-2 (500 units/mL), IFN-a (500 units/mL), anti-CD16 (5 Ag/mL), and anti-CD94 (5 Ag/mL) alone or in Secretion of both IL-4 and IL-5 by the treated NK samples was combination, and immediately thereafter, the treated NK cells were used for below the detection level (data not shown). Thus, these results 51 cytotoxicity with Cr-labeled K562 cells.The P for isotype control antibody or indicated that naive NK cells treated with antibodies to CD16 anti-CD16 antibody treatment versus the combination of anti-CD16 + anti-CD94 antibody treatment is <0.05 for untreated or IFN-a-treated NK cells and 0.48 for and CD94 NK receptors showed more of an inhibitory or type 2 IL-2-treated NK cells. Bars, SD. cytokine profile than an activating or type 1 cytokine profile.

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Ta b l e 3 .

A. Augmentation of NKcell death and TNF- A secretion but not IFN-; in anti-CD16 and/or anti-CD94 antibody-treated NKcells Experiment no. Treatment Isotype control mAb Anti-CD16 mAb %Cell TNF-A (pg/mL) IFN-; (pg/mL) % Cell TNF-A (pg/mL) IFN-; (pg/mL) death death 1 Isotype control mAb 3 337 F 29 1.6 F 0.5 14 713 F 16 9 F 3.3 Anti-CD94 mAb 6 380 F 14 5.2 F 0.0 44 1,589 F 53 (0.02)* 10 F 2 2 Isotype control mAb 9 27 F 4.2 0.0 F 1. 3 2 6 16 8 F 38 12 F 6.4 Anti-CD94 mAb 14 58 F 14 0.0 F 667401F14 (0.0 5)* 5 6 F 0.9

B. Increased secretion of IL-6, IL-1B,IL-10,andIL-13butnotIFN-; in the presence of anti-CD16 and/or anti-CD94 antibody treatment Treatment IL-6 IL-1B IL-10 IL-13 IFN-; Isotype control mAb 687 102 <50 <50 <50 Anti-CD16 mAb >3,000 617 94 258 <50 IL-2 2,314 182 92 88 5,404 Anti-CD16 + IL-2 >3,000 >3,000 259 593 5,727 Anti-CD94 mAb >3,000 1,192 181 431 <50 Anti-CD16 + anti-CD94 mAbs >3,000 >3,000 353 572 <50 Anti-CD94 mAb + IL-2 >3,000 >3,000 186 843 6,529 Anti-CD16 + anti-CD94 >3,000 >3,000 413 1,030 4,519 mAbs + IL-2

C. Increased secretion of chemokines by NKcells when treated with anti-CD16 and/or anti-CD94 antibody

Treatment ENA (pg/mL) MCP1 (pg/mL) MIP-1a (pg/mL) MIP-1b (pg/mL) Rantes (pg/mL)

Isotype control mAb 409 151 233 164 403 Anti-CD16 mAb 967 587 5,015 25,385 3,010 IL-2 536 274 534 806 751 Anti-CD16 + IL-2 894 572 20,969 35,839 3,851 Anti-CD94 mAb 841 321 4,485 13,551 1,391 Anti-CD16 + anti-CD94 mAbs 816 412 5,613 27,262 2,181 Anti-CD94 mAb + IL-2 675 419 12,070 37,076 3,906 Anti-CD16 + anti-CD94 1,057 492 34,628 43,882 4,738 mAbs + IL-2

NOTE: A. NK cells at a concentration of (1 Â10 6/mL) were treated with isotype control antibody (6 Ag/mL), IL-2 (500 units/mL), anti-CD16 antibody (5 Ag/mL), and anti-CD94 antibody (5 Ag/mL) alone or in combination.The frequency of NK cell death was determined after12 to 18 hours of incubation using staining with propidium iodide. The levels ofTNF-a and IFN-g secretion in the supernatants were determined after 12 to 18 hours of incubation using specific ELISAs. B. NK cells (1 Â10 6/mL) were treated with isotype control antibody (6 Ag/mL), anti-CD16 antibody (5 Ag/mL), IL-2 (500 units/mL), and anti-CD94 antibody (5 Ag/mL) alone or in combination, and the cells were incubated for12 to 18 hours at 37jC.The amounts of released cytokines were measured using a fluorescent base multiplex cytokine array kit. For the simplicity of the presented data, SDs are not included. Representative of one of three experiments. C. NK cells (1Â10 6/mL) were treated with isotype control antibody (6 Ag/mL), anti-CD16 antibody (5 Ag/mL), IL-2 (500 units/mL), and anti-CD94 antibody (5 Ag/mL) alone or in combination, and the cells were incubated12to18 hours at 37jC.The amounts of released chemokines were measuredusing a fluorescent base multiplex chemokine array kit. For simplicity, SDs are not included. Representative of one of three experiments. *Numbers in parentheses are Ps for the differences between anti-CD16 antibody and anti-CD16 antibody in combination with anti-CD94 antibody-treated NK cells.

Increased induction of chemokine secretion by anti-CD16and/ pared with either anti-CD16 or anti-CD94 antibody-treated NK or anti-CD94 antibody treatment. We next examined the cells. Anti-CD94 antibody treatment alone also was able to secretion of chemokines in the supernatants recovered from increase the levels of chemokine secretion, but the levels were NK cells treated with and without anti-CD16 and/or anti-CD94 less when compared with those obtained from the anti-CD16 antibodies in the presence and absence of IL-2 treatment. The antibody-treated NK cells (Table 3C). It is important to note results indicated that anti-CD16 antibody-treated NK cells that the levels of secreted chemokines closely correlated with secreted the highest levels of chemokines and this increase was the magnitude of cell death in naive NK cells. further augmented in the presence of IL-2 treatment. Moreover, Effect of anti-CD94 antibody on the expression of cell surface IL-2-treated NK cells in the absence of receptor antibodies activation antigens on NK cells when combined with IL-2, anti- secreted substantially lower levels of chemokines when com- CD16antibody, and IL-2 plus anti-CD16antibody. Addition of

www.aacrjournals.org 1999 Clin Cancer Res 2006;12(7) April 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Human Cancer Biology anti-CD16 antibody to NK cells did not increase the expression Discussion of CD69 activation antigen on NK cells. Addition of either CD94 antibody or a combination of anti-CD16 and anti-CD94 anti- It is well established that patients with head and neck cancers bodies was also unable to increase expression of CD69 on NK exhibit elevated numbers of apoptotic immune effectors in cells (data not shown). However, significant augmentation of their peripheral blood (24, 25). Furthermore, lymphocytes and CD69 activation antigen could only be observed in IL-2-treated dendritic cells obtained from the peripheral blood of these NK cells. Addition of either anti-CD16 or anti-CD94 antibody in patients exhibit significantly lower cytotoxicity and a decrease combination with IL-2 decreased CD69 expression on NK cells in surface expression of CD16 and ~ chains (24, 25). In addi- when compared with that obtained in the presence of IL-2 tion, when NK cells were cocultured with resistant oral tumors, treatment alone (data not shown). In addition, the expression of a large proportion of the NK cells underwent apoptotic cell CD54 adhesion molecule was significantly increased in the death and exhibited profound down-modulation of CD16 and presence of IL-2 treatment of NK cells, and the addition of either CD94 NK cell receptors.4 However, it is still unclear whether anti-CD16 or anti-CD94 antibodies or both was unable to triggering of these receptors individually or in combination by increase CD54 expression on NK cells (Fig. 4). IL-2-mediated specific tumor ligands is responsible for inducing cell death of augmentation of CD54 expression was significantly decreased in NK cells. Despite these important observations, surprisingly the presence of either anti-CD16 antibody alone or the very few studies have been conducted to delineate either the combination of anti-CD16 and anti-CD94 antibodies (Fig. 4). physiologic significance or the mechanisms responsible for the We also examined the induction of and induction of cell death in NK cells since the initial character- expression on NK cells. The coaddition of anti-CD16 and anti- ization of CD16 receptor-mediated NK cell death (4, 14, 15). CD94 antibodies augmented surface Fas receptor (CD95) and Therefore, studies were done to delineate the overall contribu- Fas ligand expression (data not shown). However, the addition tion of CD16 and CD94 receptors in NK cell death by using of anti-Fas receptor and anti–Fas ligand antibodies individually specific receptor antibodies. Thus, in this report, we present or in combination did not inhibit anti-CD94 plus anti-CD16 evidence that coaddition of antibodies specific for CD16 and antibody-mediated NK cell death (data not shown). CD94 NK cell receptors mediates functional inactivation and induces death of naive NK cells. Furthermore, treatment of NK cells with a combination of anti-CD94 and anti-CD16 anti- bodies results in an increase in TNF-a synthesis and secretion. The role of TNF-a in the regulation of NK cell apoptosis was evident, as a correlation between the levels of secreted TNF-a and the frequency of cell death in NK cells could be observed. Therefore, the addition of anti-TNF-a antibody could partially inhibit NK cell death. Collectively, these findings indicate a role for CD94 and CD16 receptors in TNF-a-induced death of naive NK cells and suggest the potential involvement of these receptors in ligand-mediated inactivation and death of NK cells by tumor cells in oral cancer patients. Treatment of NK cells with a combination of anti-CD16 and anti-CD94 antibody mediated inactivation of NK cell cytotox- icity with accelerated kinetics when compared with each of the antibodies alone. Inhibition of cytotoxicity measured by 51Cr release assay could be observed immediately, within minutes after the addition of a combination of anti-CD94 and anti- CD16 antibodies to NK cells, whereas overnight treatment of NK cells with each of the intact anti-CD16 or anti-CD94 antibodies was needed before their addition to 51Cr-labeled K562 tumor cells to observe significant inactivation of NK cell cytotoxic function (Fig. 2). However, although the inhibition of NK cell cytotoxic function occurred rapidly within minutes after the addition of a combination of anti-CD16 and anti-CD94 antibodies, NK cell apoptosis, on the other hand, was mea- surable only after 12 hours of incubation. In contrast, the addition of F(abV)2 fragment of CD16 antibody in the presence or the absence of anti-CD94 antibody inhibited the function of naive NK cells completely and induced significantly higher Fig. 3. Inhibition of anti-CD16 and anti-CD94 antibody-induced cell death in NK levels of cell death in NK cells, which was measurable right after cells by anti-TNF-a antibody. NK cells were treated with isotype control antibody a short-term incubation with the antibody when compared (1:100 dilution), anti-CD16 antibody (5 Ag/mL), IL-2 (500 units/mL), anti-CD94 with intact anti-CD16 antibody.6 Therefore, these experiments antibody (5 Ag/mL), and anti-TNF-a antibody (1:100 dilution) alone or in combination.The levels of NK cell death were determined using staining with indicated that the ability to detect cell death immediately after propidium iodide after an overnight incubation period.The paired Psforthe difference between anti-TNF-a-treated and untreated NK samples treated with anti-CD16 or anti-CD16 antibody in combination with anti-CD94 antibody are <0.05. Bars, SD. 6 Submitted for publication.

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Fig. 4. CD54 expression on NK cells treated with anti-CD16 and/or anti-CD94 antibodies. NK cells (1 Â10 6/mL) were treated with isotype control antibody (6 Ag/mL), anti-CD16 antibody (5 Ag/mL), IL-2 (500 units/mL), and anti-CD94 antibody (5 Ag/mL) alone or in combination, and the cells were incubated for 12 to 18 hours at 37jC. The levels of CD54 expression were determined using EPICS Elite flow cytometer. To p r i g h t , percentages of antibody-stained cells.The cursor was set based on isotype control antibodies.

the addition of antibodies to NK cells related to the type and antibodies is time dependent. When IL-2 is added in the potency of antibodies used in inducing death of NK cells combination with anti-CD16 and/or anti-CD94 antibodies at and not because of desensitization of NK cell function due to time 0 and immediately used to assess NK cell function, NK hyperactivation shown recently (26). Indeed, at no point after cell cytotoxicity was almost completely restored. However, if the addition of anti-receptor antibodies we could observe an NK cells treated with IL-2 and the receptor antibodies are increase in either IFN-g secretion or expression of CD69 and incubated for 12 to 18 hours before they are used in CD54 activation antigens, which were shown to be the cytotoxicity assays, significant inhibition of NK cell function characteristics of NK cell inactivation by receptor desensitiza- can be observed even in the presence of IL-2 treatment in NK tion (26). cells. Thus, these experiments indicated that IL-2 treatment We have shown previously that cell death induced by anti- delayed the kinetics of inactivation in NK cells when treated CD16 antibody is primarily apoptotic and is significantly with the antibodies to CD16 and CD94 surface receptors. inhibited when NK cells were treated with IL-2 (4). Similarly, Indeed, the role of IL-2 in prevention of apoptotic cell death is IL-2 treatment significantly inhibited anti-CD94 antibody- clearly shown in T cells (16, 18, 19). However, in NK cells, it mediated NK cell apoptotic death. Similar findings were also is not well established. IL-2 is known to increase the levels of obtained when NK cell death was determined after the phosphatidylinositol 3-kinase–dependent Akt and elevation addition of IL-2 to anti-CD16 and anti-CD94 antibody-treated in survival of IL-2-treated NK cells (17, 27). Another potential NK cells. Indeed, the addition of IL-2 but not IFN-a to the mechanism for prevention of NK cell death by IL-2 is combination of anti-CD16 and anti-CD94 antibody-treated activation of nuclear factor-nB transcription factor, which is NK cells prevented the loss of NK cell cytotoxicity completely known to increase antiapoptotic in a variety of effector in short-term assays, and it was capable of partially decreasing cell types (28–30). Indeed, IL-2-treated NK cells have decreased the loss of cytotoxicity in longer-term treatments of NK cells amounts of InB in their cell lysates (22). Moreover, we have with the antibodies. Further evidence for the inhibitory shown previously that expression of superrepressor of InB, function of IL-2 on cell death and prevention of functional which inhibits nuclear factor-nB in NK cells, increases cell death inactivation of NK cells was obtained when tumor-derived of NK and T cells (22, 31). VEGF secretion was used as a readout system for the surviving As indicated earlier, we were unable to block apoptosis tumor cells in the cocultures of primary oral tumor cells with induced in NK cells treated with the combination of anti-CD16 IL-2, anti-CD16, and anti-CD94 antibody-treated NK cells. and anti-CD94 antibodies by the addition of anti-Fas receptor Because the addition of anti-CD16 and/or anti-CD94 anti- or anti–Fas ligand antibodies. Moreover, the addition of anti- bodies significantly inhibited NK cell–mediated lysis of tumor TNF-a antibody could only block partially the death of NK cells. cells, the levels of VEGF secretion remained higher in Therefore, other mechanisms in addition to TNF-a-mediated cocultures of these samples with UCLA-2 tumor cells. Indeed, induction of cell death could be responsible for NK cell death addition of NK cells treated with the combination of anti- induced by receptor antibodies. In this regard, it seems less likely CD16 and anti-CD94 antibodies to UCLA-2 tumor cells that either perforin, granzyme B, or TNF-a-related apoptosis- enhanced the levels of VEGF secretion beyond that obtained inducing ligand (TRAIL) might have important roles because the when UCLA-2 tumor cells were cultured in the absence of NK levels of expression were either not changed or decreased in cells (Table 2B). Overall, these data indicate that IL-2 the presence of anti-CD16 antibody treatment, whereas IL-2 treatment prevents NK cells from undergoing rapid inactiva- treatment up-regulated all of the above-mentioned .7 tion by anti-CD16 and anti-CD94 antibodies and restores their function. However, restoration of function by IL-2 when NK cells are treated with anti-CD16 and/or anti-CD94 7 Submitted for publication.

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In agreement with our findings, previous studies also showed receptors. Interestingly, we observed loss of cytotoxic function a profound and significant inhibition of cytotoxic effector rather than an increase in function when antibodies to CD94 function in the presence of anti-CD94 antibody (32–34). The receptors were added to naive NK cells. Furthermore, IL-2- death induced by anti-CD94 antibody treatment could be due mediated up-regulation of CD54 adhesion molecules was to a fratricide mechanism; however, the addition of anti-LFA-1 significantly suppressed when treated with the combination or anti-CD54 antibody, which blocks cell-cell interaction, did of anti-CD16 and anti-CD94 antibodies. Additionally, no not affect the levels of cell death mediated by anti-CD16 significant induction of IFN-g secretion or increase in CD69 and anti-CD94 antibody-treated NK cells, suggesting an alter- activation antigen could be observed when naive NK cells were native mechanism for CD94-mediated NK cell death (data treated with anti-CD16 and/or anti-CD94 antibodies. However, not shown). Anti-LFA-1 antibody, however, did block phorbol significant induction of IL-6, IL-1h, IL-13, IL-10, and TNF-a 12-myristate 13-acetate/ionomycin–mediated induction of were observed in the presence of anti-CD16 and/or anti-CD94 NK cell death by inhibiting cell-cell interaction and aggrega- antibody treatment of naive NK cells, suggesting a type 2 tion (22). cytokine profile for anti-CD16 and anti-CD94 antibody-treated Addition of soluble HLA class I to NK cells induced cell death NK cells. on engagement of CD94/NKG2C killer-activating receptors Finally, the addition of antibodies to CD16 and/or CD94 (11). The cell death was observed after the addition of soluble cell surface receptors in the presence and absence of IL-2 HLA class I or on cross-linking of CD94 receptors by specific induced significant secretion of chemokines, indicating a role antibodies on clonal NK cell populations (11). In clonal for these NK cell receptors in the recruitment of fresh populations of NK cells, induction of activation-induced cell immune effector cells. Furthermore, there was a correlation death by activating CD94/NKG2C receptors on NK cells was between chemokine secretion and induction of cell death. proposed to be the mechanism for NK cell death because Indeed, this could be one of the important physiologic increased secretion of IFN-g and augmented cytotoxicity could functions of these receptors because additional immune be observed on cross-linking of CD94 receptors (11). However, effectors are required to replace the ones that are lost to cell in our studies using naive polyclonal populations of NK cells, death. Thus, one important function of these receptors may the results may represent the overall net effect between be to ensure recruitment of fresh immune effectors to the site activating and inhibitory signaling triggered by CD94 NK cell of inflammation to compensate for the loss of the dying cells receptors, because the antibodies to CD94 receptors do not by signaling for increased secretion of chemokines in NK discriminate between activating or inhibitory forms of these cells.

References 1. Kuss I, Saito T, Johnson JT, Whiteside TL. Clinical for a negative regulation exerted by members of CD16-, CD16+ and IL-2-, IL-12-, IL-15- or IFN-a- significance of decreased ~ chain expression in periph- the inhibitory receptor superfamily. Blood 2002;99: activated natural killer cells differentially respond to eral blood lymphocytes of patients withhead and neck 17 0 6 ^ 14 . sphingosylphosphorylcholine, lysophosphatidylcho- cancer. Clin Cancer Res 1999;5:329^ 34. 11. SpaggiariGM,ContiniP,DonderoA,etal.Soluble line and platelet-activating factor. Eur J Immunol 2. Lai P, Rabinowich H, Crowley-Nowick PA, Bell HLA class I induces NK cell apoptosis upon the en- 2005;35:2699^708. MC, Mantovani G, Whiteside TL. Alterations in gagement of killer-activating HLA class I receptors 21. Mori S, Jewett A, Murakami-Mori K, et al. The par- expression and function of signal-transducing pro- through FasL-Fas interaction. Blood 2002;100: ticipation of the Fas-mediated cytotoxic pathway by teins in tumor-associated T and natural killer cells in 4098^107. natural killer cells is tumor-cell-dependent. Cancer patients with ovarian carcinoma. Clin Cancer Res 12. Olcese L, Lang P,Vely F, et al. Human and mouse Immunol Immunother 1997;44:282 ^ 90. 1996;2:161 ^ 73. killer-cell inhibitory receptors recruit PTP1Cand PTP1D 22. Jewett A. Activation of c-Jun N-terminal kinase in 3. Jewett A, Bonavida B. Target-induced inactivation protein tyrosine phosphatases. J Immunol 1996;156: the absence of NFnB function prior to induction of and cell death by apoptosis in a subset of human NK 4531^4. NK cell death triggered by a combination of anti-class cells. J Immunol 1996;156:907 ^ 15. 13. Campbell KS, Dessing M, Lopez-Botet M, et al. I and anti-CD16 antibodies. Hum Immunol 2001;62: 4. Jewett A, Cavalcanti M, Bonavida B. Pivotal role of Tyrosine phosphorylation of a human killer inhibitory 320^ 31. endogenousTNF-a in the induction of functional inac- receptor recruits protein tyrosine phosphatase 1C. 23. Jewett A, Cacalano NA,Teruel A, et al. Inhibition of tivation and apoptosis in NK cells. J Immunol 1997; JExpMed1996;184:93^100. nuclear factor nB(NFnB) activity in oral tumor cells 15 9 :4 815 ^ 22. 14. Ortaldo JR, Mason AT, O’Shea JJ. Receptor- prevents depletion of NK cells and increases their func- 5. Mandelboim O, Malik P, Davis DM, Jo CH, Boyson induced death in human natural killer cells: involve- tional activation. Cancer Immunol Immunother 2005; JE, Strominger JL. Human CD16 as a lysis receptor ment of CD16. JExp Med 1995;181:339 ^ 44. 18 :1 ^ 12. mediating direct cytotoxicity. Proc 15. Ida H, Robertson MJ, Voss S, et al. CD94 ligation 24. Hoffmann TK, Dworacki G, Tsukihiro T, et al. Natl Acad Sci U S A1999;96:5640 ^ 4. induces apoptosis in a subset of IL-2-stimulated NK Spontaneous apoptosis of circulating T lympho- 6. Lanier LL. Turning on natural killer cells. J Exp Med cells. J Immunol 1997;159:2154 ^ 60. cytes in patients with head and neck cancer and 2000;191:1259 ^ 62. 16. Fung MM, Chu YL, Fink JL, et al. IL-2- and STAT5- its clinical importance. Clin Cancer Res 2002;8: 7. Lazetic S, Chang C, Houchins JP, Lanier LL, Phillips regulated cytokine gene expression in cells express- 2553 ^ 62. JH. Human natural killer cell receptors involved in ing the Tax protein of HTLV-1. Oncogene 2005;24: 25. Hoffmann TK, Muller-Berghaus J, Ferris RL, et al. MHC class I recognition are disulfide-linked hetero- 4624 ^ 33. Alterations in the frequency of dendritic cell subsets in dimers of CD94 and NKG2 subunits. J Immunol 1996; 17. Jiang K, Zhong B, Ritchey C, et al. Regulation of Akt- the peripheral circulation of patients with squamous 157:4741 ^ 5. dependent cell survival by Syk and Rac. Blood 2003; cell carcinomas of the head and neck. Clin Cancer 8. Carretero M, Cantoni C, BellonT, et al.The CD94 and 101:236 ^ 4 4. Res 2002;8:1787^ 93. NKG2-A C-type lectins covalently assemble to form a 18. Guarini A, Riera L, Cignetti A, et al. Transfer of 26. Coudert JD, Zimmer J, Tomasello E, et al. Altered natural killer cell inhibitory receptor for HLA class I the interleukin-2 gene into human cancer cells NKG2D function in NK cells induced by chronic expo- molecules. Eur J Immunol 1997;27:563 ^ 7. induces specific antitumor recognition and restores sure to NKG2D ligand-expressing tumor cells. Blood 9. Brooks AG, Posch PE, Scorzelli CJ, Borrego F, the expression of CD3/T-cell receptor associated 2005;106:1711^ 7. Coligan JE. NKG2A complexed with CD94 defines a signal transduction molecules. Blood 1997;89: 27. Ahmed NN, Grimes HL, Bellacosa A, et al. Trans- novel inhibitory natural killer cell receptor. J Exp Med 212^ 8. duction of interleukin-2 antiapoptotic and proliferative 1997;185:795^800. 19. Rosenberg SA, Lotze MT. Cancer immunotherapy signals via Akt protein kinase. Proc Natl Acad Sci 10. Spaggiari GM, Contini P, Carosio R, et al. Soluble using interleukin-2 and interleukin-2-activated lym- U S A1997;94:3627 ^ 32. HLA class I molecules induce natural killer cell apo- phocytes. Annu Rev Immunol 1986;4:681 ^ 709. 28. Kawakami H,Tomita M, MatsudaT, et al.Transcriptional ptosis through the engagement of CD8: evidence 20. Jin Y, Damaj BB, Maghazachi AA. Human resting activation of survivin through the NF-nB pathway by

Clin Cancer Res 2006;12(7) April 1, 20 06 2002 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. NK Cell Apoptosis and CD16 and CD94 Receptors

humanT-cell type I tax. Int J Cancer and to increased cell apoptosis in response to mito- cell cytotoxicity by NKG2A: association of NKG2A 2005;115:967 ^ 74. genic stimuli, but not to abnormal thymopoiesis. with SHP-1and SHP-2 protein-tyrosine phosphatases. 29. Rivera-Walsh I, Cvijic ME, Xiao G, et al. The J Immunol 1999;162:6442 ^ 50. EurJ Immunol 1998;28:264 ^ 76. NF-nB signaling pathway is not required for Fas 31. Jewett A, Hume WR, Le H, et al. Induction of apo- 33. Zingoni A, Palmieri G, Morrone S, et al. CD69-trig- ligand gene induction but mediates protection from ptotic cell death in peripheral blood mononuclear gered ERK activation and functions are negatively reg- activation-induced cell death. J Biol Chem 2000; and polymorphonuclear cells by an oral bacterium, ulated by CD94/NKG2-A inhibitory receptor. Eur J 275:25222^30. Fusobacterium nucleatum. Infect Immun 2000;68: Immunol 2000;30:644^51. 30. Ferreira V, Sidenius N,Tarantino N, et al. In vivo inhi- 1893 ^ 8. 34. JinY, Fuller L, Esquenazi V, et al. Bone marrow cells bition of NF-n B inT-lineage cells leads to a dramatic 32. Le Drean E,Vely F, Olcese L, et al. Inhibition of anti- inhibit the generation of autologous EBV-specific CTL. decrease in cell proliferation and cytokine production gen-inducedTcell response and antibody-induced NK Hum Immunol 2000;61:538 ^ 47.

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Anahid Jewett, Nicholas A. Cacalano, Christian Head, et al.

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