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Clinical Cancer Research Vol. 12, 1994-2003, April 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Coengagement of CD16 and CD94 Receptors Mediates Secretion of Chemokines and Induces Apoptotic Death of Naive Natural Killer Cells

Anahid Jewett1, Nicholas A. Cacalano2, Christian Head3 and Antonia Teruel1

Authors' Affiliations: 1 The Jane and Jerry Weintraub Center for Reconstructive Biotechnology and Division of Oral Biology and Medicine, Jonsson Comprehensive Cancer Center; Departments of 2 Radiation Oncology and 3 Head and Neck Surgery, School of Dentistry and Medicine, University of California at Los Angeles, Los Angeles, California

Requests for reprints: Anahid Jewett, Division of Oral Biology and Medicine, University of California at Los Angeles School of Dentistry, 10833 Le Conte Avenue, Los Angeles, CA 90095-1668. Phone: 310-206-3970; Fax: 310-794-7109; E-mail: ajewett{at}ucla.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Down-modulation of CD16 (Fc{gamma}RIII) 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(ab')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-{alpha} (TNF-{alpha}) but not IFN-{gamma} secretion in NK cells treated either with anti-CD16 antibody alone or in combination with anti-CD94 antibodies. Consequently, the addition of anti-TNF-{alpha} antibody partially inhibited apoptosis 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 cytokines 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 the primary host defense against neoplasias. Furthermore, NK cells through secretion of cytokines are involved in the regulation of T-cell-mediated immune responses. Clearly, in several disease models, the function and the numbers of NK cells are significantly decreased (1, 2). We have reported previously that functional inactivation and death of NK cells are observed following the interaction of NK cells with tumor-target cells or by triggering of CD16 receptors on NK cells (3, 4). However, neither the cellular mechanisms nor the physiologic significance of death in NK cells have been fully explored.

Lymphocytes isolated from patients with cancer, particularly head and neck, ovarian, renal, cervical, colorectal, prostate, and melanoma, have functional abnormalities and signaling defects (1). Profound down-modulation of {zeta} chain and CD16 surface receptor expression have been documented previously in patients with cancer (2). Similarly, down-modulation of CD16 and CD94 NK cell receptors, loss of NK cell function, and increased NK cell death were observed after their interaction with oral tumor cells.4 Thus, triggering of CD16 and CD94 receptors by ligands expressed on tumor cells may be responsible for the functional abnormalities and death of NK cells. Because specific tumor ligands for CD16 receptors have been suggested but not characterized at present, we do not have the ability to study the role of these receptors on NK cell function and death using tumor ligands (5). However, previous studies made use of antibodies to CD16 receptor to establish their significance in NK cell function. Furthermore, to study the function of CD94 NK cell receptors, either clonal populations of NK cells or NK cell lines expressing activating or inhibitory 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 interested to explore the combination of CD16 and CD94 receptor modulation, as seen after the interaction of NK cells with tumor-target cells, on NK cell function and survival.

A net balance between activating and inhibitory signals, which NK cells receive from tumor cells, dictate whether cytotoxic NK or T cells will become functionally activated or inhibited in the presence of tumor-target cells. The list for both activating and inhibitory signals delivered by different tumor cell ligands to NK cells through specific NK cell receptors has been increasing steadily (6). CD16 receptors are shown to signal for the activation of NK cells, whereas CD94 receptors are capable of mediating both activation and inhibition of NK cell function depending on their association with NKG2C or NKG2A, respectively (7–11). Studies from several groups showed the recruitment and binding of tyrosine phosphatase SHP-1 to human NK cell inhibitory receptors and subsequent delivery of negative signals to NK cells (12, 13). Alternatively, the CD94/NKG2C complex associates with DAP12, which carries a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). Triggering of CD94/NKG2C by CD94 monoclonal antibodies (mAb) was shown to activate the function of clonal population of NK cells leading to increased production of IFN-{gamma}, increased cytotoxicity, and proliferation (11), an effect that was not seen when anti-CD94 antibody was added to naive polyclonal NK cells (see Results).

There are conflicting results regarding CD16- and CD94-mediated NK cell death. Previous work has shown induction of cell death in interleukin (IL)-2–activated NK cells by anti-CD16 antibodies (14, 15). However, we have shown elsewhere (4) and here that IL-2 rescues anti-CD16 antibody-treated NK cells from undergoing apoptotic cell death, an effect of IL-2 that is clearly described in T and NK cell apoptosis and is in line with the major function of this cytokine in many cancers (16–19). Thus, in this article, we present several important novel findings regarding the death of NK cells, an area of investigation that has received relatively little attention, although its significance has clearly been shown in many cancer patients, including those of the head and neck (1, 2).

We examined the cytotoxic function, the surface phenotype, and the profiles of secreted cytokines and chemokines in the NK cells that were triggered to undergo apoptosis by anti-CD16 and/or anti-CD94 antibodies. We show that, in comparison with each of the anti-CD16 and anti-CD94 mAb treatment alone, the coaddition of anti-CD16 and anti-CD94 antibodies to naive NK cells results in the additive or synergistic induction of tumor necrosis factor-{alpha} (TNF-{alpha}) secretion, inhibition of NK cell cytotoxicity, and induction of NK cell apoptotic death. Addition of IL-2 inhibited the apoptotic death induced by anti-CD16 and/or anti-CD94 antibodies and increased the function of NK cells. We also report that the endogenous secretion of TNF-{alpha} is partly responsible for the induction of NK cell death by anti-CD16 and anti-CD94 antibodies. More importantly, the induction of cell death by CD16 and CD94 antibodies correlated with the secretion of chemokines, indicating the possibility of a role for these receptors in the recruitment of fresh immune effectors to inflammatory infiltrates to replace those that are signaled to die by the CD16 and CD94 receptors.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents. K562 target cells were cultured in RPMI 1640, and SCC4, Cal33, and UMSCC tumor lines and UCLA-1 and UCLA-2 primary oral tumors were cultured in either DMEM or RPMI 1640 supplemented with 1% sodium pyruvate, 1% nonessential amino acids, 1% glutamine, 1% penicillin-streptomycin (Life Technologies, Grand Island, NY), and 10% FCS (Irvine Scientific, Santa Ana, CA). UCLA-1 and UCLA-2 primary oral tumors were derived at University of California at Los Angeles from patients with tongue cancer (Dr. Christian Head). Recombinant IL-2 was obtained from Chiron Corp. (Emeryville, CA). IFN-{gamma} was obtained from Peprotech (Piscataway, NJ). The NK purification kit was obtained from Miltenyi Biotech (Auburn, CA). The FITC-conjugated anti-CD54, anti-CD16, and phycoerythrin-conjugated anti-CD69 antibodies were obtained from Coulter/Immunotech (Miami, FL). TAPA-1 antibodies were obtained from Dr. Levy (Stanford University, Stanford, CA). FITC-conjugated anti-Fas antibodies were obtained from PharMingen (San Diego, CA). Polyclonal anti-TNF-{alpha} antibody was prepared in rabbits in our laboratory. The anti-TNF-{alpha} mAbs (B154.9.1 and B154.7.1) were prepared in our laboratory from hybridomas kindly supplied by Dr. G. Trinchieri (Wistar Institute, Philadelphia, PA). Anti–class II (L227 monomorphic ATCC HB.96), anti-LFA-1 (TS1.22.1.1.13.HB202), and anti-LFA-3 (TS2.9.1.4.3.HB205) antibodies were prepared from hybridomas obtained from the American Type Culture Collection (Manassas, VA). Purified antibodies to CD16, CD56, CD18, and LFA-1 were purchased from Coulter/Immunotech. Purified F(ab')2 fragment of anti-CD16 antibody was purchased from Ancell (Bayport, MN). Control ascites IgG1 was derived from a control hybridoma (American Type Culture Collection), and purified IgG1 was purchased from Coulter/Immunotech.

Purification of NK cells. Peripheral blood mononuclear cells from healthy donors were isolated as described previously (3, 4). Purified NK cells were negatively selected using a NK isolation kit (Miltenyi Biotech) and consisted of 85% to 95% of CD16+ cells. Approximately 10% to 15% of NK cell population was shown previously to have no or low CD16 expression on the surface of NK cells (20). The percentage of CD3+ T cells and CD19+ B cells determined by anti-CD3 and anti-CD19 antibodies in 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 after staining with isotype control antibodies, indicating the levels of nonspecific binding of antibodies to purified NK cells.

51Cr release assay, ELISA, surface staining of NK cells, and DNA staining for apoptosis were all described previously (3, 4, 21).

Multiplex cytokine and chemokine protein arrays. The fluorokine MAP cytokine and chemokine multiplex array kits were purchased from R&D Systems (Minneapolis, MN), and the procedures were conducted as suggested by the manufacturer.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Increased induction of NK cell death by the addition of anti-CD16 and anti-CD94 antibodies in naive NK cells. The addition of anti-CD16 antibody to naive NK cells induced significant cell death of NK cells as reported previously (4, 22). Cell death induced by anti-CD16 antibody in NK cells was primarily apoptotic (4, 22). Cell death induced in the presence of anti-CD94 antibody alone at the same antibody concentration is significantly lower than that obtained in the presence of anti-CD16 antibody treatment (Table 1 ; Fig. 1 ). Likewise, the death induced in the presence of anti-CD94 antibody treatment is apoptotic (Fig. 1). The addition of a combination of anti-CD94 and anti-CD16 antibodies in comparison with each antibody alone to naive NK cells significantly increased the levels of cell death in NK cells (Table 1; Fig. 1). In contrast, cell death was not observed when NK cells were treated with IL-2 (Fig. 1). Furthermore, the addition of IL-2 to either anti-CD16 antibody treatment alone or the combination of anti-CD16 and anti-CD94 antibody-treated NK cells significantly decreased the levels of apoptotic cell death in NK cells (Fig. 1). The increased induction of cell death by a combination of anti-CD94 and anti-CD16 antibodies in NK cells was specific. Unlike CD94 antibody, the addition of anti-LFA-1, anti-LFA-3, and anti-TAPA-1 antibodies in combination with anti-CD16 antibody-treated NK cells had no enhancing effect on cell death (data not shown). Moreover, the addition of F(ab')2 fragment of CD16 antibody and intact mAb induced cell death of NK cells in the presence and absence of anti-CD94 antibody treatment excluding Fc receptor-mediated effects for the observed results (Table 1).


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  		Table 1. F(ab')2 fragment and intact anti-CD16 antibodies in combination with anti-CD94 antibody induce death of NK cells and decrease the cytotoxic function of NK cells

 

Figure 1
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  		Fig. 1. IL-2 treatment decreases anti-CD16- and/or anti-CD94 antibody-induced apoptosis in NK cells. NK cells (1 x 106/mL) were treated with isotype control antibody (6 µg/mL), anti-CD16 antibody (5 µg/mL), IL-2 (500 units/mL), and anti-CD94 antibody (5 µg/mL) alone or in combination. After 12 to 18 hours of incubation, the cells were washed, permeabilized, and stained with propidium iodide. Top right, percentages of NK cells in sub-G0-G1 stage 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.

 
Experiments were next done to determine whether the CD94+ NK cell subset was the subpopulation that underwent augmented apoptosis following treatment with anti-CD16 antibody. After staining with anti-CD94 antibody, CD94+ and CD94– NK cell subpopulations were sorted (data not shown) and treated with anti-CD16 antibody. The results indicated that CD94+ but not CD94– NK subset was the subpopulation that underwent augmented cell death (data not shown). These findings show that the coaddition of anti-CD94 and anti-CD16 antibodies to NK cells results in a significant increase in the frequency of NK cell death. Furthermore, the CD94 receptor-positive NK cell subset was the subpopulation that underwent augmented cell death in the presence of anti-CD16 mAbs.

Inhibition of NK cell cytotoxic function by anti-CD16 and anti-CD94 antibodies. The cytotoxic activity of purified naive NK cells treated with anti-CD16 and/or anti-CD94 antibodies was assessed. Treatment of NK cells with anti-CD16 antibody significantly inhibited the NK cell cytotoxic function (Table 2A ). The addition of anti-CD94 antibody to NK cells in the absence of anti-CD16 antibody had some inhibitory effect on cytotoxic function. However, the addition of anti-CD94 and anti-CD16 antibodies together significantly potentiated inhibition of NK cell cytotoxic function against K562 tumor-target cells (Table 2A). Treatment of NK cells with IL-2 augmented the NK cytotoxicity, and the addition of anti-CD94 antibody in combination with IL-2 inhibited IL-2-mediated increase in cytotoxic 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 treated NK cells were incubated for 12 to 18 hours before they were added to tumor-target cells (Table 2A).


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  		Table 2.

 
A significant inhibition of NK cell cytotoxicity could be observed immediately after the addition of a combination of anti-CD94 and anti-CD16 antibodies (Fig. 2 ), whereas overnight pretreatment with each of the antibodies was needed to observe inhibition of cytotoxicity mediated by either intact anti-CD16 or anti-CD94 antibodies. However, inhibition of NK cell function could be observed immediately after the addition of F(ab')2 fragment of CD16 antibody.5 Functional inhibition was also observed in IFN-{alpha}-treated NK cells but not in IL-2-treated NK cells when a combination of anti-CD16 and anti-CD94 antibodies was added to NK cells immediately before their cocultures with K562 tumor cells (Fig. 2). The addition of anti-CD56 or MHC class II antibody to anti-CD16 antibody-treated NK cells did not have any effect on NK cell cytotoxicity (data not shown). Thus, in cultures in which NK cells were treated with the combination of IL-2, anti-CD94, and anti-CD16 antibodies immediately before they were added to K562 tumor cells, IL-2 was able to prevent anti-CD16 and anti-CD94 antibody-mediated inhibition of NK cell function nearly completely (Fig. 2). However, if NK cells were treated with the combination of IL-2, anti-CD16, and anti-CD94 antibodies for 12 to 18 hours before they were cocultured with K562 or oral tumor cells (SCC4, Cal33, UCLA-1, UCLA-2, and UMSCC), significant inhibition of NK cell cytotoxic function could still be observed even in the presence of IL-2 (Table 2A). Thus, it seems that IL-2 treatment delays the kinetics of NK cell inactivation by anti-CD16 and anti-CD94 antibody treatment.


Figure 2
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  		Fig. 2. Rapid inhibition of NK cell cytotoxicity by the treatment of NK cells with a combination of anti-CD16 and anti-CD94 antibodies. Purified NK cells (1 x 106/mL) were treated with isotype control antibody (6 µg/mL), IL-2 (500 units/mL), IFN-{alpha} (500 units/mL), anti-CD16 (5 µg/mL), and anti-CD94 (5 µg/mL) alone or in combination, and immediately thereafter, the treated NK cells were used for cytotoxicity with 51Cr-labeled K562 cells. The P for isotype control antibody or anti-CD16 antibody treatment versus the combination of anti-CD16 + anti-CD94 antibody treatment is <0.05 for untreated or IFN-{alpha}-treated NK cells and 0.48 for IL-2-treated NK cells. Bars, SD.

 
The inhibitory function of CD16 and CD94 antibodies in the absence of IL-2 treatment and its prevention in the presence of IL-2 treatment in NK cells was also determined using a novel assay system. Both NK cell sensitive and resistant oral tumors secreted significant amounts of vascular endothelial growth factor (VEGF) in the supernatants.4 In contrast, no induction of VEGF secretion could be seen by naive or IL-2-activated NK cells (Table 2B). The levels of tumor-derived VEGF secretion in the cocultures of NK cells with sensitive oral tumors (Table 2B) correlated with the magnitude of cytotoxicity induced by the NK cells (Table 2A). Thus, naive and IL-2-treated NK cells were able to decrease the levels of VEGF secretion in the cocultures of NK cells with sensitive oral tumors (Table 2B). As expected, IL-2-treated NK cells decreased VEGF secretion by the oral tumors to a greater extent than untreated NK cells (Table 2B). Thus, modulation of tumor-derived VEGF secretion in the cocultures of NK cells with oral tumors could be used as a measure of NK cell functional activity against oral tumors. Therefore, to assess the effect of NK cell death on VEGF secretion by the sensitive oral tumors in the cocultures of NK cells with tumor cells, isotype control antibody, anti-CD16 antibody, anti-CD94 antibody, IL-2, or their combination (Table 2B) were added to the cocultures of NK cells with UCLA-2 primary oral tumor cells, and after 16 hours of coincubation, the levels of VEGF secretion were determined in the supernatants by ELISA. Under the conditions where no significant cell death could be observed in NK cells, both naive and IL-2-treated NK cells inhibited tumor-derived VEGF secretion (Table 2B). However, VEGF secretion was significantly increased after the treatment of NK cells with anti-CD16 antibody and, to a lesser extent, anti-CD94 antibody (Table 2B). In addition, treatment of NK cells with the combination of anti-CD16 and anti-CD94 antibodies resulted in an even greater induction of VEGF secretion by the tumor cells when compared with those obtained by each of anti-CD16 and anti-CD94 antibody alone (Table 2B). IL-2 treatment was capable of preventing anti-CD16 and/or anti-CD94 antibody-mediated increase in VEGF secretion (Table 2B). Therefore, decrease in VEGF secretion in oral tumors is likely due to lysis of tumor cells by the NK cells because treatment of NK cells with anti-CD16 and anti-CD94 antibodies individually or in combination, which induced death of NK cells, increased tumor-derived VEGF secretion.

Increase in the secretion of TNF-{alpha} and IL-6 but not that of IFN-{gamma} by anti-CD94 and anti-CD16 antibody-treated NK cells. We have reported previously that treatment of NK cells with anti-CD16 antibodies resulted in the induction of TNF-{alpha} secretion and that TNF-{alpha} was responsible in part for the induction of apoptosis in naive NK cells (4, 22). Because the addition of anti-CD94 and anti-CD16 antibodies augmented cell death, we hypothesized that these antibodies may also augment secretion of TNF-{alpha}. Treatment of NK cells with anti-CD16 antibody resulted in the augmented secretion of TNF-{alpha} but not IFN-{gamma} (Table 3A ). The coaddition of anti-CD94 and anti-CD16 antibodies to NK cells significantly potentiated the secretion of TNF-{alpha} (Table 3A). The addition of anti-CD94 antibody alone to untreated control NK cells had also some potentiating effect on TNF-{alpha} secretion. The induction of TNF-{alpha} secretion by a combination of anti-CD94 and anti-CD16 antibodies was specific for these antibodies and was not observed when other antibodies, such as anti-MHC class II, anti-CD56, and LFA-3 antibodies, were used (data not shown). Therefore, these findings suggested that TNF-{alpha} 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-{alpha} 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-{alpha} 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-{alpha} secretion by the NK cells.


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  		Table 3.

 

Figure 3
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  		Fig. 3. Inhibition of anti-CD16 and anti-CD94 antibody-induced cell death in NK cells by anti-TNF-{alpha} antibody. NK cells were treated with isotype control antibody (1:100 dilution), anti-CD16 antibody (5 µg/mL), IL-2 (500 units/mL), anti-CD94 antibody (5 µg/mL), and anti-TNF-{alpha} antibody (1:100 dilution) alone or in combination. The levels of NK cell death were determined using staining with propidium iodide after an overnight incubation period. The paired Ps for the difference between anti-TNF-{alpha}-treated and untreated NK samples treated with anti-CD16 or anti-CD16 antibody in combination with anti-CD94 antibody are <0.05. Bars, SD.

 
Because only partial inhibition of NK cell death could be obtained in the presence of anti-TNF-{alpha} antibody, it is likely that other factors in addition to TNF-{alpha} 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-1ß, IL-10, IL-13, and IFN-{gamma} 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-1ß, IL-10, and IL-13 in addition to TNF-{alpha} (Table 3B). However, no secretion of IFN-{gamma} could be observed by NK cells with the exception of those treated with IL-2 in the presence and absence of receptor antibodies. Secretion of both IL-4 and IL-5 by the treated NK samples was below the detection level (data not shown). Thus, these results indicated that naive NK cells treated with antibodies to CD16 and CD94 NK receptors showed more of an inhibitory or type 2 cytokine profile than an activating or type 1 cytokine profile.

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

Effect of anti-CD94 antibody on the expression of cell surface activation antigens on NK cells when combined with IL-2, anti-CD16 antibody, and IL-2 plus anti-CD16 antibody. Addition of anti-CD16 antibody to NK cells did not increase the expression of CD69 activation antigen on NK cells. Addition of either CD94 antibody or a combination of anti-CD16 and anti-CD94 antibodies was also unable to increase expression of CD69 on NK cells (data not shown). However, significant augmentation of CD69 activation antigen could only be observed in IL-2-treated NK cells. Addition of either anti-CD16 or anti-CD94 antibody in combination with IL-2 decreased CD69 expression on NK cells when compared with that obtained in the presence of IL-2 treatment alone (data not shown). In addition, the expression of CD54 adhesion molecule was significantly increased in the presence of IL-2 treatment of NK cells, and the addition of either anti-CD16 or anti-CD94 antibodies or both was unable to increase CD54 expression on NK cells (Fig. 4 ). IL-2-mediated augmentation of CD54 expression was significantly decreased in the presence of either anti-CD16 antibody alone or the combination of anti-CD16 and anti-CD94 antibodies (Fig. 4).


Figure 4
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  		Fig. 4. CD54 expression on NK cells treated with anti-CD16 and/or anti-CD94 antibodies. NK cells (1 x 106/mL) were treated with isotype control antibody (6 µg/mL), anti-CD16 antibody (5 µg/mL), IL-2 (500 units/mL), and anti-CD94 antibody (5 µg/mL) alone or in combination, and the cells were incubated for 12 to 18 hours at 37°C. The levels of CD54 expression were determined using EPICS Elite flow cytometer. Top right, percentages of antibody-stained cells. The cursor was set based on isotype control antibodies.

 
We also examined the induction of Fas receptor and Fas ligand expression on NK cells. The coaddition of anti-CD16 and anti-CD94 antibodies augmented surface Fas receptor (CD95) and Fas ligand expression (data not shown). However, the addition of anti-Fas receptor and anti–Fas ligand antibodies individually or in combination did not inhibit anti-CD94 plus anti-CD16 antibody-mediated NK cell death (data not shown).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
It is well established that patients with head and neck cancers exhibit elevated numbers of apoptotic immune effectors in their peripheral blood (24, 25). Furthermore, lymphocytes and dendritic cells obtained from the peripheral blood of these patients exhibit significantly lower cytotoxicity and a decrease in surface expression of CD16 and {zeta} chains (24, 25). In addition, when NK cells were cocultured with resistant oral tumors, a large proportion of the NK cells underwent apoptotic cell death and exhibited profound down-modulation of CD16 and CD94 NK cell receptors.4 However, it is still unclear whether triggering of these receptors individually or in combination by specific tumor ligands is responsible for inducing cell death of NK cells. Despite these important observations, surprisingly very few studies have been conducted to delineate either the physiologic significance or the mechanisms responsible for the induction of cell death in NK cells since the initial characterization of CD16 receptor-mediated NK cell death (4, 14, 15). Therefore, studies were done to delineate the overall contribution of CD16 and CD94 receptors in NK cell death by using specific receptor antibodies. Thus, in this report, we present evidence that coaddition of antibodies specific for CD16 and 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 antibodies results in an increase in TNF-{alpha} synthesis and secretion. The role of TNF-{alpha} in the regulation of NK cell apoptosis was evident, as a correlation between the levels of secreted TNF-{alpha} and the frequency of cell death in NK cells could be observed. Therefore, the addition of anti-TNF-{alpha} antibody could partially inhibit NK cell death. Collectively, these findings indicate a role for CD94 and CD16 receptors in TNF-{alpha}-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 cytotoxicity 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 measurable only after 12 hours of incubation. In contrast, the addition of F(ab')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 levels of cell death in NK cells, which was measurable right after a short-term incubation with the antibody when compared with intact anti-CD16 antibody.6 Therefore, these experiments indicated that the ability to detect cell death immediately after the addition of antibodies to NK cells related to the type and the potency of antibodies used in inducing death of NK cells and not because of desensitization of NK cell function due to hyperactivation shown recently (26). Indeed, at no point after the addition of anti-receptor antibodies we could observe an increase in either IFN-{gamma} secretion or expression of CD69 and CD54 activation antigens, which were shown to be the characteristics of NK cell inactivation by receptor desensitization (26).

We have shown previously that cell death induced by anti-CD16 antibody is primarily apoptotic and is significantly inhibited when NK cells were treated with IL-2 (4). Similarly, IL-2 treatment significantly inhibited anti-CD94 antibody-mediated NK cell apoptotic death. Similar findings were also obtained when NK cell death was determined after the addition of IL-2 to anti-CD16 and anti-CD94 antibody-treated NK cells. Indeed, the addition of IL-2 but not IFN-{alpha} to the combination of anti-CD16 and anti-CD94 antibody-treated NK cells prevented the loss of NK cell cytotoxicity completely in short-term assays, and it was capable of partially decreasing the loss of cytotoxicity in longer-term treatments of NK cells with the antibodies. Further evidence for the inhibitory function of IL-2 on cell death and prevention of functional inactivation of NK cells was obtained when tumor-derived VEGF secretion was used as a readout system for the surviving tumor cells in the cocultures of primary oral tumor cells with IL-2, anti-CD16, and anti-CD94 antibody-treated NK cells. Because the addition of anti-CD16 and/or anti-CD94 antibodies significantly inhibited NK cell–mediated lysis of tumor cells, the levels of VEGF secretion remained higher in cocultures of these samples with UCLA-2 tumor cells. Indeed, addition of NK cells treated with the combination of anti-CD16 and anti-CD94 antibodies to UCLA-2 tumor cells enhanced the levels of VEGF secretion beyond that obtained when UCLA-2 tumor cells were cultured in the absence of NK cells (Table 2B). Overall, these data indicate that IL-2 treatment prevents NK cells from undergoing rapid inactivation 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 antibodies is time dependent. When IL-2 is added in combination with anti-CD16 and/or anti-CD94 antibodies at time 0 and immediately used to assess NK cell function, NK cell cytotoxicity was almost completely restored. However, if NK cells treated with IL-2 and the receptor antibodies are incubated for 12 to 18 hours before they are used in cytotoxicity assays, significant inhibition of NK cell function can be observed even in the presence of IL-2 treatment in NK cells. Thus, these experiments indicated that IL-2 treatment delayed the kinetics of inactivation in NK cells when treated with the antibodies to CD16 and CD94 surface receptors. Indeed, the role of IL-2 in prevention of apoptotic cell death is clearly shown in T cells (16, 18, 19). However, in NK cells, it is not well established. IL-2 is known to increase the levels of phosphatidylinositol 3-kinase–dependent Akt and elevation in survival of IL-2-treated NK cells (17, 27). Another potential mechanism for prevention of NK cell death by IL-2 is activation of nuclear factor-{kappa}B transcription factor, which is known to increase antiapoptotic proteins in a variety of effector cell types (28–30). Indeed, IL-2-treated NK cells have decreased amounts of I{kappa}B in their cell lysates (22). Moreover, we have shown previously that expression of superrepressor of I{kappa}B, which inhibits nuclear factor-{kappa}B in NK cells, increases cell death of NK and T cells (22, 31).

As indicated earlier, we were unable to block apoptosis induced in NK cells treated with the combination of anti-CD16 and anti-CD94 antibodies by the addition of anti-Fas receptor or anti–Fas ligand antibodies. Moreover, the addition of anti-TNF-{alpha} antibody could only block partially the death of NK cells. Therefore, other mechanisms in addition to TNF-{alpha}-mediated induction of cell death could be responsible for NK cell death induced by receptor antibodies. In this regard, it seems less likely that either perforin, granzyme B, or TNF-{alpha}-related apoptosis-inducing ligand (TRAIL) might have important roles because the levels of gene expression were either not changed or decreased in the presence of anti-CD16 antibody treatment, whereas IL-2 treatment up-regulated all of the above-mentioned genes.7

In agreement with our findings, previous studies also showed a profound and significant inhibition of cytotoxic effector function in the presence of anti-CD94 antibody (32–34). The death induced by anti-CD94 antibody treatment could be due to a fratricide mechanism; however, the addition of anti-LFA-1 or anti-CD54 antibody, which blocks cell-cell interaction, did not affect the levels of cell death mediated by anti-CD16 and anti-CD94 antibody-treated NK cells, suggesting an alternative mechanism for CD94-mediated NK cell death (data not shown). Anti-LFA-1 antibody, however, did block phorbol 12-myristate 13-acetate/ionomycin–mediated induction of NK cell death by inhibiting cell-cell interaction and aggregation (22).

Addition of soluble HLA class I to NK cells induced cell death on engagement of CD94/NKG2C killer-activating receptors (11). The cell death was observed after the addition of soluble HLA class I or on cross-linking of CD94 receptors by specific antibodies on clonal NK cell populations (11). In clonal populations of NK cells, induction of activation-induced cell death by activating CD94/NKG2C receptors on NK cells was proposed to be the mechanism for NK cell death because increased secretion of IFN-{gamma} and augmented cytotoxicity could be observed on cross-linking of CD94 receptors (11). However, in our studies using naive polyclonal populations of NK cells, the results may represent the overall net effect between activating and inhibitory signaling triggered by CD94 NK cell receptors, because the antibodies to CD94 receptors do not discriminate between activating or inhibitory forms of these receptors. Interestingly, we observed loss of cytotoxic function rather than an increase in function when antibodies to CD94 receptors were added to naive NK cells. Furthermore, IL-2-mediated up-regulation of CD54 adhesion molecules was significantly suppressed when treated with the combination of anti-CD16 and anti-CD94 antibodies. Additionally, no significant induction of IFN-{gamma} secretion or increase in CD69 activation antigen could be observed when naive NK cells were treated with anti-CD16 and/or anti-CD94 antibodies. However, significant induction of IL-6, IL-1ß, IL-13, IL-10, and TNF-{alpha} were observed in the presence of anti-CD16 and/or anti-CD94 antibody treatment of naive NK cells, suggesting a type 2 cytokine profile for anti-CD16 and anti-CD94 antibody-treated NK cells.

Finally, the addition of antibodies to CD16 and/or CD94 cell surface receptors in the presence and absence of IL-2 induced significant secretion of chemokines, indicating a role for these NK cell receptors in the recruitment of fresh immune effector cells. Furthermore, there was a correlation between chemokine secretion and induction of cell death. Indeed, this could be one of the important physiologic functions of these receptors because additional immune effectors are required to replace the ones that are lost to cell death. Thus, one important function of these receptors may be to ensure recruitment of fresh immune effectors to the site of inflammation to compensate for the loss of the dying cells by signaling for increased secretion of chemokines in NK cells.


   Footnotes
 
Grant support: National Institute of Dental and Craniofacial Research/NIH grant RO1-DE12880.

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

4 In preparation. Back

5 In preparation. Back

6 Submitted for publication. Back

7 Submitted for publication. Back

Received 10/27/05; revised 12/22/05; accepted 1/23/06.


   References
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 Abstract
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 References
 

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A. Jewett, C. Head, and N.A. Cacalano
Emerging Mechanisms of Immunosuppression in Oral Cancers
Journal of Dental Research, December 1, 2006; 85(12): 1061 - 1073.
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