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Constitutive Expression and Role of the TNF Family Ligands in Apoptotic Killing of Tumor Cells by Human NK Cells¹

Yoshiro Kashii^2,*,, Roberto Giorda^*,, Ronald B. Herberman^*,,, Theresa L. Whiteside^*,, and Nikola L. Vujanovic^3,*,

Departments of ^* Pathology and Medicine, and University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Natural killer cells mediate spontaneously secretory/necrotic killing against rare leukemia cell lines and a nonsecretory/apoptotic killing against a large variety of tumor cell lines. The molecules involved in nonsecretory/apoptotic killing are largely undefined. In the present study, freshly isolated, nonactivated, human NK cells were shown to express TNF, lymphotoxin (LT)-, LT-ß, Fas ligand (L), CD27L, CD30L, OX40L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), but not CD40L or nerve growth factor. Complementary receptors were demonstrated to be expressed on the cell surface of solid tumor cell lines susceptible to apoptotic killing mediated by NK cells. Individually applied, antagonists of TNF, LT-₁ß₂, or FasL fully inhibited NK cell-mediated apoptotic killing of tumor cells. On the other hand, recombinant TNF, LT-₁ß₂, or FasL applied individually or as pairs were not cytotoxic. In contrast, a mixture of the three ligands mediated significant apoptosis in tumor cells. These findings demonstrate that human NK cells constitutively express several of the TNF family ligands and induce apoptosis in tumor cells by simultaneous engagement of at least three of these cytotoxic molecules.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent studies have defined a large family of pleiotropic biologically important cell membrane-bound and secreted molecules, named the TNF family of ligands. The major known members of this family are TNF, LT-,⁴ LT-ß, Fas (APO-1, CD95)L, CD27L, CD30L, CD40L, OX40L, 4-1BBL, NGF, TRAIL (APO-2L), APO-3L, and receptor activator of NF-B ligand (RANKL) (1, 2, 3, 4). Most of these molecules are type II transmembrane proteins, with an intracellular N terminus, a single transmembrane-spanning domain, and a homotrimeric extracellular C terminus, containing a homologous receptor-binding region of 150 amino acids on each monomer. The trimeric extracellular regions of the TNF family ligands are not only expressed as membrane-bound molecules, but are also secreted as soluble proteins after proteolytic cleavage. The exception is LT-, which only consists of the C terminus domain, with no intracytoplasmic or retained transmembrane domains, and thus is either directly secreted as a homotrimer or anchored to the membrane when incorporated into a heterotrimeric complex with LT-ß transmembrane protein (1, 2).

A large family of the complementary molecules, the TNF family of receptors, has been defined in parallel (1, 5). The major known family members are: TNFR1 (p60, CD120a), TNFR2 (p80, CD120b), Fas (APO-1, CD95), LT-ßR (TNFR-related protein), CD27, CD30, CD40, OX40, 4-1BB, the low affinity NGFR (5), DR3 (6), five receptors for TRAIL (DR4, DR5, TR2, DCR1, and DCR2) (7, 8), and receptor activator of NF-B (RANK) (4). All of these receptors are type I membrane-bound proteins and have characteristic extracellular cysteine-rich pseudo repeats that share significant intersubunit sequence homology. Although the cytoplasmic domains of these receptors are generally short and lack sequence homology, there is one region of homology, known as the death domain, which is shared by the apoptosis-transducing receptors TNFR1, Fas, DR3, DR4, and DR5 (1, 5, 6, 7, 8). The signaling pathways by which these receptors induce apoptosis are rather similar. Ligand binding to receptor induces a cascade of consecutive molecular interactions, including receptor trimerization, recruitment of an adaptor protein to the receptor death domain through homotypic interaction, binding of a proximal caspase to the adaptor protein via their dead effector domains, autocleavage and activation of the caspase, and induction of a serial activation of the apoptotic effector mechanisms, including distal caspases (9).

The TNF family ligands are predominantly expressed on activated immune cells and are distributed throughout the immune system (2). In addition, FasL is expressed in the immune privileged sites, such as eye, brain, testis, and tumors (10). In contrast, the corresponding receptors are more broadly distributed and have been found on a large variety of normal, virally infected, and cancer cells (5). Interactions between these ligands and corresponding receptors have been shown to play an important role in the development and regulation of the immune system and in inflammatory responses (1, 2, 5). A shared, unique feature of these ligand-receptor interactions is the ability to directly induce apoptotic death in cells (2, 5). Recent studies have shown that ligation of not only TNFR1, Fas, DR3, DR4, or DR5, which contain the cytoplasmic death domain, but also ligation of TNFR2, LT-ßR, CD30, or CD40, which do not have a similar motif, can efficiently signal apoptotic death in tumor cells, but not in most normal cells (2, 6, 7, 8, 11, 12, 13, 14, 15). Thus, certain members of the TNF family ligands and cells expressing these molecules might have a significant role in host immune defense against cancer.

CTLs and NK cells are major immune effector cells responsible for antitumor and antiviral host immune defense (16, 17). In this regard, NK cells have a unique ability to spontaneously perform such activities. In contrast, CTLs can do so only after Ag-specific activation (16, 17). Because of this ability, NK cells have been considered as the first line of immune defense against viral infections and cancer (16, 17). Until recently, antiviral and antitumor functions of NK cells have been attributed to their well-known secretory cytolytic activity. This killing mechanism includes receptor-ligand-mediated interactions between NK cells and target cells; Ca²⁺-dependent triggering of NK cell secretory activity, manifested by the release of cytoplasmic granules containing perforin and granzymes; formation of pores in the cell membrane of target cells by perforin insertion; and induction of necrosis in target cells (16, 18). However, recent studies have indicated that nonactivated NK cells express and utilize membrane-bound FasL to kill certain lymphoid cell targets expressing Fas (19, 20). Further studies have shown that human immature and mature NK cells differentially utilize TRAIL or FasL to kill susceptible target cells (21). In addition, it has been shown that activated NK cells express membrane-bound and/or secrete several of the TNF family ligands, including TNF, LT-₁ß₂, and FasL (22, 23, 24, 25), and are able to induce lysis of TNF- or FasL-susceptible targets in prolonged cytotoxicity assays (24, 26). Our recent studies, using several different selective assays for apoptosis, have demonstrated that freshly isolated, mature, nonactivated human peripheral blood NK cells have a much broader antitumor cytotoxic activity than that previously observed in the conventional 4-h ⁵¹Cr release cytotoxicity assays. We have found that NK cells rapidly (during 1 h) induced apoptosis in 25 different solid tumor cell lines tested (27). The effector molecules and receptors involved in this apoptotic killing mediated by nonactivated NK cells are completely unknown. We postulated that these effector molecules and receptors might belong to the TNF families of ligands and receptors.

We report in this study that human peripheral blood NK cells constitutively express a variety of the cell surface-bound TNF family ligands and are capable of simultaneously utilizing them to ligate corresponding receptors on tumor cells and to rapidly induce DNA fragmentation and apoptosis in these target cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

The following Abs were used in this study: anti-human TNF rabbit polyclonal Abs (PCA), anti-human LT- rabbit PCA (Genzyme, Cambridge, MA), anti-human TNF (IgG1) mouse mAb, anti-human LT- (IgG2b) mouse mAb (Endogen, Boston, MA), anti-human TNFRp60 (IgG1) mouse mAb, anti-human TNFRp80 (IgG2b) rat mAb (Genzyme), anti-human TNFRp60 (IgG1) mouse mAb, anti-human TNFRp80 (IgG2b) rat mAb (Immunex, Seattle, WA), B9.C9 anti-human LT-ß (IgG1) mouse mAb, BDA8 anti-human LT-ßR (IgG1) mouse mAb (Biogen, Cambridge, MA), anti-human LT-ßR goat PCA (University of California, Riverside, CA), NOC-1 anti-human FasL (IgG1) mouse mAb, NOC-2 anti-human FasL (IgG1) mouse mAb (PharMingen, San Diego, CA), 4H9 anti-human FasL (IgG1) hamster mAb (Osaka Bioscience Institute, Osaka, Japan), M3 anti-human Fas (IgG1) mouse mAb, M31 anti-human Fas (IgG1) mouse mAb (Immunex), anti-human CD27L (IgG1) mouse mAb, anti-human CD27 (IgG1) mouse mAb (LT Workshop V), anti-human CD30L (IgG1) mouse mAb (Immunex), anti-human CD30 (IgG1) mouse mAb (Becton Dickinson, San Jose, CA), anti-human CD40L (IgG1) mouse mAb (Immunex), anti-human CD40 (IgG1) mouse mAb (PharMingen), M180 anti-human TRAIL (IgG1) mouse mAb (Immunex), anti-human CD18 (LFA-1, ß₂ integrin) mouse mAb, anti-human CD29 (ß₁ integrin) (AMAC, Westbrook, ME), normal rabbit serum (Endogen), biotinylated anti-mouse IgG (H+L) goat PCA, biotinylated anti-rat IgG (H+L) goat PCA, biotinylated anti-hamster IgG (H+L) goat PCA, biotinylated anti-rabbit IgG (H+L) goat PCA, biotinylated anti-goat IgG (H+L) rabbit PCA, biotinylated anti-human IgG (H+L) goat PCA (Vector, Burlingame, CA), FITC-conjugated anti-human CD3 (IgG1) mouse mAb, PE-conjugated anti-human CD56 (IgG1) mouse mAb, FITC-conjugated anti-human CD16 (IgG1) mouse mAb, FITC-conjugated anti-human CD14 (IgG1) mouse mAb, FITC-conjugated anti-human CD19 (IgG1) mouse mAb, isotype control mAbs (Becton Dickinson), anti-human CD3 (IgG1) mouse mAb, anti-human CD5 (IgG1) mouse mAb, anti-human CD14 (IgG1) mouse mAb, anti-human CD19 (IgG1) mouse mAb, and anti-glycophorin A (IgG1) mouse mAb (Dako, Carpenteria, CA). PE-conjugated streptavidin was obtained from Sigma (St. Louis, MO). We utilized in this investigation the following fusion proteins: dimeric human TNFRp80:Fc fragment of human IgG1, dimeric human LT-ßR:Fc fragment of human IgG1, dimeric mouse Fas:Fc fragment of human IgG1, dimeric human IL-4R:Fc fragment of human IgG1 (Immunex), and dimeric human LT-ßR:Fc fragment of human IgG1 (Biogen).

The following cytokines and ligands were used: human rIL-2 (Chiron-Cetus, Emeryville, CA), human trimeric rFasL (Immunex), human trimeric rFasL, human trimeric rFlag-FasL (Alexis Biochemicals, San Diego, CA), human trimeric rTNF (Endogen), human trimeric rCD40L (Immunex), human rLT-₁ß₂ (Biogen), and human rIFN- (RusselUclaf, Romainvile, France).

Reagents from Immunex were gifts provided by Dr. Tony Troutt. Reagents from Biogen were gifts provided by Dr. Jeffrey Browing (Biogen, Cambridge, MA). The Abs from UC were provided by Dr. Carl F. Ware as a gift. The Abs from OBI were gifts provided by Dr. Shigekazu Nagata (Osaka Bioscience Institute).

Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-Fmk) inhibitor of caspase-3 was purchased from Enzyme Systems Products (Livermore, CA).

PCR primers were designed by us to be 18–24 nucleotides long and to have a 100% homology with the particular regions of the genes coding characteristic extracellular regions of the molecules, according to gene sequences. The gene sequences were obtained from Dr. Shigekazu Nagata (FasL, OBI), Dr. Tony Troutt (TNF, LT-, LT-ß, CD27L, CD30L, and CD40L; Immunex), and the GenBank (TRAIL, OX40L, 4-1BBL, and NGF) using the OLIGO Primer Analysis Software, Version 5.0 (NBA, Software and Research Services for Tomorrow’s Discoveries, National Biosciences, Plymouth, MN). PCR oligomers were produced at the University of Pittsburgh Cancer Institute Oligonucleotide Synthesis Facility. All utilized PCR primers and their product lengths are: ß-actin sense, 5'-GGGTCAGAAGGATTCCTATG-3', and antisense, 5'-GGTCTCAAACATGATCTGGG-3' (237 bp); TNF sense, 5'-ACAAGCCTGTAGCCCATGTT-3', and antisense, 5'-AAAGATGACCTGCCCAGACT-3' (427 bp); LT- sense, 5'-GCTCACCTCATTGGAGACCC-3', and antisense, 5'-GGTGAGCTGGAACGCAGCCCC-3' (332 bp); LT-ß sense, 5'-GCCCACCTCATAGGCGCTCC-3', and antisense, 5'-GAGCTGCACCAGGCCGCCGAA-3' (371 bp); FasL sense, 5'-GGATTGGGCCTGGGGATGTTTCA-3', and antisense, 5'-TTGTGGCTCAGGGGCAGGTTGTTG-3' (344 bp); CD27L sense, 5'-GAGCTGCAGCTGAATCACAC-3', and antisense, 5'-GGCCAGGGGCGTCAGGCGCTG-3' (312 bp); CD30L sense, 5'-GGGCCTACCTCCAAGTGGCAAA-3', and antisense, 5'-GCTTCCCTGCCTGCTATCTGAAAGA-3' (576 bp); CD40L sense, 5'-ATTGCG GCACATGTCATAAGT-3', and antisense, 5'-CAATTCAAATACTCCTCCCAA-3' (329 bp); OX40L sense, 5'-GGCCTCTGTAATTCAGGGACTGGG-3', and antisense, 5'-GGAGGCTCCTTCACTTGCAATGAA-3' (520 bp); 4-1BBL sense, 5'-GCTCAGGCTCCGTTTCACTTGC-3', and antisense, 5'-ACCAGCCTCGGCAATGTCGA-3' (581 bp); NGF sense, 5'-GTCAAAGGCAGGATCAGGTTCCC-3', and antisense, 5'-CAGGGCTCGCTTCAGACTTCCA-3' (584 bp); and TRAIL sense, 5'-TGCACTTGAGGAATGGTGAACTGG-3', and antisense, 5'-TGTCCCTTAAGGAAACCTGGAGGC-3' (717 bp).

NK and T cells

NK cells were purified from normal human PBMC as 98% pure populations of CD5^-CD3^- TCR-ß^- CD14^-CD19^-CD56⁺CD16⁺ lymphocytes, using a modification of the previously described negative immunoselection technique with Ab-coated magnetic beads (28). Briefly, PBMC were separated on Ficoll-Hypaque gradients and incubated with anti-CD5, anti-CD3, anti-CD14, anti-CD19, and anti-Glycophorin A (Dako) mAbs. The Ab-pretreated PBMC were washed, and then consecutively incubated four times with magnetic beads coated with goat anti-mouse Ig, twice with beads obtained from Advanced Magnetics (Boston, MA) and twice with Dynabeads (Dynal, Lake Success, NY). After each of these incubations, mixtures of PBMC and magnetic beads were exposed to a magnet (Bio-Mag separator; Advanced Magnetics, Boston, MA), to eliminate mAb-coated PBMC. Then, freshly isolated, nonactivated NK cells were tested for purity by two-color flow cytometry. Aliquots of NK cells were kept overnight at 4°C in RPMI 1640 medium supplemented with 2% FBS (both from Life Technologies, Long Island, NY). These NK cells were used for both the flow cytometry studies of expression of the TNF family ligand proteins and the assessment of antitumor cytotoxicity. To avoid confounding results in RT-PCR due to the possible presence of non-NK cells, additional purification of the immunomagnetic bead-purified populations of NK cells was performed by sorting of CD3^-CD56⁺ lymphocytes in the FACStar^Plus cell sorter (Becton Dickinson), following their staining with FITC-conjugated anti-CD3 and PE-conjugated anti-CD56 mAbs. Sorted NK cells were 100% pure CD3^-CD56⁺ cells.

IL-2-activated NK (A-NK) cells were obtained from purified NK cells by IL-2-induced adherence, selection, activation, and 14-day expansion, as previously described (29). A-NK cells were shown to be homogenous population of CD3^-CD56⁺ cells. Activated T cells were obtained by 5-day in vitro Con A-induced activation and proliferation of peripheral blood T cells enriched on nylon wool columns (29). These Con A-activated T cells were 98% CD3⁺CD56^- cells.

Tumor cell lines

All tumor cell lines were of human origin. They included K562 myeloid leukemia; BT-20, MCF-7, SK-BR-3, and HTB-126 breast carcinomas; OVCAR-3 ovarian carcinoma (American Type Culture Collection, Manassas, VA); LS-174 colon carcinoma (NeoTx Corp.); HR gastric carcinoma; and PCI-1 and PCI-50 squamous cell carcinoma of the head and neck (SCCHN; UPCI, Pittsburgh, PA). The cell lines were grown under standard cell culture conditions, as previously described (27). The leukemic cell lines were grown as single cell suspensions and used in experiments when they were in the log phase of growth. The solid tissue-derived tumor cell lines were grown as adherent monolayers and utilized in experiments when they were 70% confluent. Cell suspensions of tumor cells grown as monolayers were prepared by mild trypsinization, using 0.05%/0.53 mM trypsin/EDTA in HBSS (Life Technologies) and 2-min incubation at 37°C, followed by one wash in ice-cold RPMI 1640 supplemented with 10% FBS (RPMI-10) and a trypsin inhibitor (Cell Systems, Kirkland, WA) and two washes in RPMI-10 alone.

RT-PCR for mRNA expression of the TNF family ligands

RNA was extracted from highly purified populations of NK cells, A-NK cells, and Con A-activated T cells (3 x 10⁶ of each). Extraction was performed by the acid-guanidinium phenol-chloroform method (TRI REAGENT; Molecular Research Center, Cincinnati, OH). RT-PCR was performed using RNA PCR kit (Perkin-Elmer, Norwalk, CT). Cellular RNA (100 ng) was reverse transcribed into cDNA in a reaction mixture containing 5 mM MgCl₂, 1 mM dNTP, 2.5 µM oligo(dT) primer, 1 U RNase inhibitor, and 2.5 U reverse transcriptase. After incubation at 42°C for 15 min, the reaction was terminated by heating at 95°C for 5 min. PCR was performed on the cDNA using the sense/antisense primers listed above. The PCR reaction buffer (25 µl), consisting of 2 mM MgCl₂, 0.5 µM of each primer, 1 µCi [-³²P]dCTP, and 1 U Ampli Taq DNA polymerase (5 µl of each reverse-transcriptase solution), was added to an amplification tube. The amplification was performed for ß-actin, TNF, LT-, FasL, CD30L, TRAIL, 4-1BBL, OX40L, and NGF using the following protocol. The reaction was started by a 10-min cycle at 94°C, followed by 35 cycles of 1-min denaturation at 94°C, 1-min annealation at 60°C, and 1-min DNA chain extension at 72°C; and finished by a 10-min cycle at 72°C. The RT-PCR conditions were slightly different for detection of LT-ß, CD27L, and CD40L mRNAs, performing annealation at 45°C. Twenty-microliter aliquots of the amplified product were separated on 5% polyacrylamide gels. After electrophoreses, the gels were dried and exposed to BioMax film (Kodak, Rochester, NY) for 1 h. Densitometry analysis was performed using Bio Analysis software (London, U.K.), following scanning of the PCR bands.

Flow cytometry of the TNF family ligands and their receptors

For membrane staining, freshly harvested NK cells or tumor cells were suspended in ice-cold PBS containing 0.1% sodium azide and 1% FBS (PBS-AF). To increase sensitivity of the assay and to detect these proteins on the cell surface and/or within the cell, NK cells or tumor cells were fixed with 1% (w/v) paraformaldehyde for 15 min on ice, washed with PBS, and permeabilized by placing the cells in PBS containing 0.1% saponin, 0.1% sodium azide, and 1% FBS. The cells (0.2 x 10⁶/0.1 ml) were then incubated on ice for 30 min with unlabeled primary mAbs, polyclonal Abs, or receptor fusion proteins (10 µg/ml). Thus, NK cells were incubated with Abs specific for TNF, LT-, LT-ß, FasL, CD27L, CD30L, CD40L, or TRAIL, or with LT-ßR:Fc fusion protein. On the other hand, tumor cells were incubated with Abs against human TNFR1, TNFR2, LT-ßR, Fas, CD27, CD30, and CD40. Negative controls were cells incubated without Abs, or incubated with isotype-matched nonreactive Igs. The cells were then washed twice with PBS-AF and incubated on ice for 30 min in the presence of appropriate biotinylated secondary Abs (1/500 dilution). The cells were again washed and incubated on ice for 30 min in the presence of PE-conjugated streptavidin (1/20 dilution). Finally, the cells were washed twice in PBS-AF, fixed with 1% (w/v) paraformaldehyde/PBS solution, and analyzed by flow cytometry, as previously described (29). In addition, to better evaluate and compare the levels of the TNF family ligands or their receptors expressed on the cell surface and/or within the cell, ratios between the mean fluorescence intensities (MFIs) obtained from cells stained with specific Abs vs isotype-matched control Abs were calculated and compared for untreated and permeabilized cells, respectively.

Cytotoxicity assays

⁵¹Cr release cytotoxicity assay was performed with target cells in suspensions, as previously described (27).

[3H]Thymidine release (JAM) assay was performed as previously described (27). Briefly, target cells, growing either as suspensions or monolayers, were labeled with 5 µCi/ml of methyl [³H]thymidine (New England Nuclear, Boston, MA; 147.9 GBq/mmol) for 24 h at 37°C. Suspensions of labeled target cells were then harvested, washed twice in RPMI-10, and seeded in 96-well U-bottom plates (Costar, Cambridge, MA) at a density of 5000 cells/well. Effector cells were washed once in the same medium and added to the target cells. Six replicates were prepared for each control and experimental condition. Plates were centrifuged at 65 x g for 3 min and incubated for 1 h at 37°C. After incubation, cells were disrupted by freezing (at -20°C) and thawing (at room temperature) three times. The disrupted cells were harvested onto fiber glass filters, using a semiautomatic cell harvester (Scatron, Sterling, VA). The filters were dried and immersed in liquid scintillation fluid, and their radioactivity was determined in a LKB Betaplate counter (Pharmacia, Gaithersburg, MD). The percentage of specific [³H]thymidine release (% cytotoxicity) was determined using the following formula: % cytotoxicity = 100 x (C-E)/C, in which E (experimental) is cpm of target cells in the presence of effector cells and C (control) is cpm of target cells alone.

MTT assay was performed as previously described (30).

Blocking of the interactions between the TNF family ligands and their receptors

NK cells were preincubated for 60 min at room temperature with neutralizing Abs against TNF, LT-, or with isotype-matched nonreactive Abs (final concentration 20 µg/ml), or with TNFRp80:Fc or LT-ßR:Fc fusion proteins (final concentration 4 µg/ml). On the other hand, target cells were preincubated with anti-TNFRp60, anti-TNFRp80, or anti-Fas mAbs (final concentration 20 µg/ml). The effectors and targets were then mixed in a 20:1 E:T ratio, and cytotoxicity assays were performed, as described above.

Killing mediated by the TNF family ligands

Cytotoxicity was performed using the same three assays described above for assessment of NK cell killing. However, instead of NK cells, various dilutions of recombinant TNF, LT-₁ß₂, and FasL were added to radiolabeled or unlabeled target cells either individually or in various combinations.

Statistical analysis

Statistical analyses of the results were performed using the Wilcoxon’s signed-rank pair and Mann-Whitney U tests. Differences were considered significant when the p value was <0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
To begin to investigate the underlying mechanisms and to identify the molecules involved in NK cell-mediated nonsecretory/apoptotic killing, we studied whether human NK cells express and utilize membrane-bound TNF family cytotoxic ligands to induce this apoptosis in tumor cells. We also examined the role of simultaneous engagement of diverse cell membrane-bound TNF family ligands in this apoptotic killing.

Expression of the TNF family ligand gene transcripts in NK cells

First, using RT-PCR, we examined NK cell lysates for the presence of mRNAs for the various TNF family ligands. In these experiments, RNA was isolated from highly purified populations of NK cells, IL-2-activated NK (A-NK) cells, and Con A-activated T cells. The experiments showed (Fig. 1) that NK cells contained mRNA for several known TNF family ligands, but not for CD40L and NGF. Thus, expression of mRNA was detected using primers specific for TNF, LT-, FasL, CD30L, 4-1BBL, TRAIL, LT-ß, CD27L, and OX40L. In comparison, activated NK cells and Con A-activated T cells showed slightly different patterns of expression of mRNA of the TNF family ligands. Hence, while NK cells did not contain detectable CD40L or NGF mRNA, activated NK cells contained the CD40L mRNA, and activated T cells contained both the CD40L and NGF mRNA. Both activated NK cells and activated T cells expressed mRNA of all other TNF family ligands found to be expressed in nonactivated NK cells. These results showed that NK cells constitutively express messages for several TNF family ligands, and that in these cells IL-2 stimulation induces de novo expression of the CD40L mRNA.

View larger version (36K): [in this window] [in a new window]   FIGURE 1. Expression of mRNA for the TNF family ligands in NK cells. Total RNA was extracted from samples of highly purified populations of freshly isolated human peripheral blood nonactivated NK cells, A-NK cells, and Con A-activated T cells (3 x 106). RT-PCR was performed with 100 ng of the cellular RNA, using specific oligoprimers listed in Materials and Methods and 35 amplification cycles in the presence of [-32P]dCTP. The amplified cDNA products were separated using polyacrylamide gel electrophoreses and analyzed by autoradiography.

Next, we assessed by flow cytometry whether the constitutive transcription of the TNF family ligand genes in NK cells was accompanied by their translation and protein expression. Expression of epitopes specific for several TNF family ligands, including TNF, LT-, LT-ß, FasL, CD27L, CD30L, and TRAIL, but not CD40L, was consistently detected (Fig. 2) on intact (i.e., presumably on the cell surface) and/or permeabilized (i.e., presumably on the cell surface and/or within the cell) NK cells. TNF, LT-, FasL, and CD27L showed relatively high levels of expression on intact and similar levels of expression on permeabilized NK cells (i.e., presumably high and selective expression on the cell surface); LT-ß and CD30L were detected at low levels on intact NK cells, but at high levels in permeabilized NK cells (i.e., presumably low expression on the cell surface, but high expression within the cell); and TRAIL was not detected on intact NK cells, but was found at significant levels on permeabilized NK cells (i.e., no apparent expression on the cell surface, but significant expression within the cell) (Fig. 2). Expression of TNF, LT-, and FasL was confirmed by using additional Abs (data not shown). The finding of coexpression of LT- and LT-ß on the cell surface of NK cells indicated that the membrane-bound LT-ß heterotrimer might be present on these effector cells. These data are in accordance with those for mRNA expression (Fig. 1) and provide clear evidence that human NK cells constitutively express many different TNF family ligands, several of which might be able to induce cell death in susceptible cell targets.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Expression of the TNF family ligand proteins by NK cells. Purified human peripheral blood nonactivated NK cells, either untreated (intact) or paraformaldehyde fixed and saponin treated (permeabilized), were consecutively coincubated with: 1) primary Abs specific for the TNF family ligands (anti-TNF PCA, anti-LT- PCA, anti-LT-ß mAb, anti-FasL NOC-2 mAb, anti-CD27L mAb, anti-CD30L mAb, anti-CD40L mAb, and anti-TRAIL mAb or isotype-matched control Ig); 2) biotinylated secondary Abs; and 3) PE-conjugated streptavidin, to label their cell surface or cytoplasmic proteins, respectively. The labeled cells were analyzed in a FACScan flow cytometer using the Reproman program. The data are from a representative experiment of three performed, showing overlays of single-color histograms of isotype control (open histograms) and specific Ab (filled histograms) fluorescence. For TNF, LT-, and FasL, similar results to those presented were obtained with anti-human TNF mAb (Endogen), anti-human LT- mAb (Endogen), and two anti-human FasL mAbs (NOC-1, Pharmingen; 4H9, OBI), respectively (data not shown). In addition, LT-ßR:Fc fusion protein showed significant binding to the cell surface of NK cells, presumably by interacting with LT-1ß2 and/or LIGHT (data not shown).

If the nonsecretory apoptotic killing by NK cells is mediated via membrane-bound forms of the TNF family ligands, solid tumor cell targets, which are susceptible to this mechanism of killing, should express the corresponding cell membrane receptors. Therefore, we examined the expression of the TNF family receptors on human tumor cell lines, which have been previously shown to be susceptible to nonsecretory apoptotic killing mediated by NK cells (27). Using immunostaining with specific Abs and flow-cytometric analysis, nine of these human tumor cell lines, including four breast carcinomas, one ovarian carcinoma, one colon carcinoma, one gastric carcinoma, and two SCCHN, were tested and found to express several TNF family receptors on the cell surface (data for the OVCAR-3 ovarian cancer and the BT-20 breast cancer cell lines are shown in Fig. 3). All the tested tumor cell lines expressed significant, but variable, levels of TNFRp60 (low), LT-ßR (intermediate to high), Fas (intermediate to high), CD30 (intermediate), and CD40 (intermediate to high). In addition, low levels of TNFRp80 and CD27 were found to be expressed on some of the tested tumor cell lines (TNFRp80, on OVCAR-3, LS-174, and HR; CD27, on OVCAR-3 and SK-BR-3; data not shown). The MFI for TNFRp60, TNFRp80, and Fas, compared with that of isotype-matched controls, were found to be 3- to 10-fold higher in permeabilized than on intact tumor cells, suggesting that these molecules are present at higher levels within the cell than on the cell surface. In addition, to confirm these findings obtained with the amplified, three-step immunofluorescence technique (i.e., consecutive incubations of cells with unconjugated primary Ab, biotinylated secondary Abs, and PE-conjugated streptavidin), we performed one- and two-step immunofluorescence assays with directly labeled Abs. The one- and two-step assays showed lower background than the three-step assays. However, the former assays also demonstrated 5- to 10-fold lower sensitivity than the latter assays, and were unable to detect TNFR1 or TNFR2 on intact cells, showing low but significant levels of these proteins only after permeabilization of the cell (data not shown). Similarly, the other TNF family receptors on tumor cells or the TNF family ligands on NK cells, which demonstrated a range of expression from low to high levels in the three-step assays, showed in one- and two-step assays absence or low levels of expression, respectively. These findings demonstrate that tumor cells express several of the TNF family receptors and that most of these receptors are expressed on the cell surface at relatively low levels. The data indicate that a high turnover of at least some of the TNF family receptors might occur in tumor cells. It also appears that the TNF family ligands on NK cells have similar properties. Therefore, NK cells and tumor cells constitutively express on the cell surface several cytotoxic ligands and their receptors, respectively, which interaction can potentially induce apoptotic cell death in tumor cells.

View larger version (42K): [in this window] [in a new window]   FIGURE 3. Expression of the TNF family receptors on tumor cells. Suspensions of tumor cells were prepared, and similarly stained and analyzed by flow cytometry, as described in Fig. 2, for testing of expression of the TNF family ligand proteins on NK cells. Primary Abs were specific for the TNF family receptors. As similar results were obtained with all nine tested tumor cell lines, data are presented only for OVCAR-3 (ovarian carcinoma, A) and BT-20 (breast carcinoma, B) cell lines. The data for TNFRp60 and TNFRp80 were obtained with Immunex mAbs, for LT-ßR with BDA8 mAb from Biogen, and for Fas with M3 mAb from Immunex. Similar results were obtained with anti-TNFRp60 and anti-TNFRp80 mAbs from Genzyme, anti LT-ßR PCA from UC, and with M31 mAb from Immunex, respectively (data not shown). The data are from a representative experiment of three performed. Note, MFIs for TNFR1 were found to be 4- to 7-fold higher (OVCAR-3 and BT-20), for TNFR2 10-fold higher (OVCAR-3), and for Fas 3-fold higher (BT-20) on permeabilized than on intact tumor cells.

Nonsecretory/apoptotic killing mechanism of NK cells has been defined using 1-h [³H]thymidine release assay, transmission electron microscopy, in situ and flow cytometry TUNEL assays (27), and Annexin V-based flow cytometry assay (unpublished data). In addition, the apoptotic killing was confirmed by its blocking with Z-VAD-Fmk caspase-3 inhibitor and by demonstration that the apoptosis was followed by cell death in 18-h ⁵¹Cr release or MTT assays as well as in clonogenic assay (unpublished data). The obtained data in all of these various assays were comparable. To determine whether the nonsecretory/apoptotic killing is mediated by the interactions of the TNF family ligands expressed on NK cells with the corresponding receptors on tumor cell targets, we assessed the ability of neutralizing Abs and R:Fc fusion proteins, specific for the TNF family ligands or their receptors, to block this killing in 1-h [³H]thymidine release assay. These experiments were performed using four different human solid tumor-derived cell lines as targets: OVCAR-3 ovarian carcinoma, BT-20 breast carcinoma, LS-174 colon carcinoma, and PCI-1 SCCHN. The tumor cell lines have previously been shown to be susceptible to NK cell-mediated nonsecretory/apoptotic killing, but completely resistant to NK cell-mediated secretory/necrotic killing (27). NK cells were preincubated with individual Abs or R:Fc fusion proteins specific for the TNF family of ligands, while target cells were preincubated with individual Abs specific for the TNF family receptors. The effect of these treatments was assessed by testing cytotoxic activity of NK cells against tumor cell targets in 1-h [³H]thymidine release assay (27). Representative data from one of six similar experiments obtained with OVCAR-3 and BT-20 cell lines were shown in Fig. 4. It was found that each individual neutralizing reagent, specific for TNF, LT-, LT-ß, TNFRp60, TNFRp80, or Fas, which presumably blocked interactions between TNF-TNFR, LT-ß-LT-ßR, and FasL-Fas, almost completely blocked induction of apoptosis mediated by NK cells in the tumor cell lines. The levels of blocking of the apoptotic killing obtained by the individual antagonists of the TNF family ligands and their receptors could not be augmented by simultaneous treatment with two or more of these antagonists (data not shown). In addition, anti-LFA-1 (CD18) mAb inhibited apoptotic killing of tumor cells by NK cells, indicating that adhesion between LFA-1 on NK cells and ICAMs on target cells might have a role in this type of NK cell-mediated killing. Similar results were obtained using LS-174 or PCI-1 cell targets and/or 18-h MTT assays (data not shown). Results obtained with control reagents and in our additional experiments demonstrated that the inhibition of apoptotic killing by the complete Abs or R:Fc fusion proteins was a consequence of the specific blockade of the receptor-ligand interactions, but not of mutual elimination of NK cells by Ab-dependent cellular cytotoxicity via Abs bound to their cell surface. The supporting data for this conclusion are: 1) neither nonreactive isotype control Ig nor specifically reactive Abs to CD29 (ß₁ integrin) (Fig. 4) and CD56 or IL-4R:Fc fusion protein, which bind to the corresponding structures expressed on the cell membrane of NK cells (data not shown), suppressed NK cell-mediated apoptotic killing of cancer cells; 2) the blockade of NK cell-mediated apoptotic killing of tumor cells was not only achieved by preincubation of NK cells with the reagents specific for the ligands, but also by preincubation of tumor cells with Abs specific for the receptors (Fig. 4); 3) binding of the ligand-specific reagents to NK cells did not suppress their ability to mediate secretory/necrotic killing against K562 cell targets (data not shown); and 4) direct demonstration that blocking reagents used in our studies were unable to induce Ab-dependent cellular cytotoxicity in NK cells (data not shown). The observed differences in inhibition levels of NK cell-mediated apoptotic killing produced with different antagonists and/or in different tumor cell targets could be attributed to the variations of positions of the epitopes on the studied molecules in relation to their reactive domains and/or to the differences in cell surface exposure of the epitopes, respectively.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. NK cells simultaneously utilize several membrane-bound TNF family ligands to induce apoptosis in solid tissue-derived tumor cell targets. Freshly isolated human peripheral blood nonactivated NK cells were preincubated with isotype-matched control Ig, anti-CD18 mAb, anti-CD29 mAb, anti-TNF PCA, anti-LT- PCA, TNFRp80:Fc, or LT-ßR:Fc. Tumor cells were labeled with radioactive isotopes and preincubated with anti-TNFRp60, anti-TNFRp80, or anti-Fas (M3) mAbs. The effector and target cells were then mixed in a 20:1 E:T ratio, and cytotoxicity assays were performed. The data are percentages of specific killing. Mean SDs were less than 10% of the mean values. All differences between the experimental and control samples were statistically significant (p < 0.05–0.001). One-hour [3H]thymidine release assay was applied to assess apoptosis in tumor cells. Blocking of TNF and LT- was also performed with mAbs, and the results were similar to those obtained with PCA (data not shown). In addition, blocking of FasL was performed using Fas:Fc fusion protein, and the obtained results were similar as that with anti-Fas mAb (data not shown).

These data demonstrate that NK cells mediate nonsecretory/apoptotic killing of tumor cells via activity of their cell membrane-bound TNF family ligands. The data also indicate that simultaneous engagement of at least three different TNF family ligands (i.e., TNF, LT-ß, and FasL) expressed on NK cells with corresponding receptors expressed on tumor cells provides a signal(s) that is required to induce apoptosis in tumor cell targets.

Rapid induction of apoptosis in tumor cells by soluble ligands of the TNF family

To directly assess whether the simultaneous engagement of three different TNF family receptors on tumor cells by corresponding ligands is a necessary condition for induction of apoptosis in tumor cells, we examined the apoptosis-inducing ability of individual ligands at various concentrations and/or of two or three ligands in combinations. The tested combinations included various concentrations of one ligand and a single concentration of one or two other ligands. The ligand-mediated apoptotic killing was assessed against OVCAR-3, BT-20, LS-174, and PCI-1 human tumor cell lines, using 1-h [³H]thymidine release and 18-h MTT assays. The results obtained with these different targets and assays were similar. The representative data from 1 of 14 experiments are shown in Table I. The presented experiment was performed with OVCAR-3 tumor cell targets, using 1-h [³H]thymidine release assay. It showed that treatments of targets with either one (TNF, LT-₁ß₂, or FasL) or two (TNF + LT-₁ß₂, TNF + FasL, TNF + FLAG-FasL, or LT-₁ß₂ + FasL) ligands did not induce significant cytotoxic effects in a large range of ligand concentrations, including the ligand concentrations described by others to be significantly effective against susceptible targets (11, 31). In contrast, simultaneous treatments of tumor cells with three ligands (TNF + LT-₁ß₂ + FasL, or TNF + LT-₁ß₂ + FLAG-FasL) induced significant levels of apoptosis in tumor cells. This killing was inducible using a large range of concentrations of the ligands (i.e., from 0.01–10 ng or from 0.1–50 ng) and showed a different dose dependence for each of the three ligands. Thus, optimal killing of the three ligands was obtained using 0.1 ng of TNF, 1–10 ng of LT-₁ß₂, and 10–50 ng of FasL. As expected, FLAG-FasL gave a significantly greater killing than soluble FasL (Table I), possibly because of its better ability to polymerize Fas. Similar results to those obtained with the soluble ligands were also generated using immobilized Abs specific for TNFR1, LT-ßR, and Fas (data not shown). Thus, while the engagement of one or two receptors was without notable effects, the simultaneous engagement of all three receptors induced significant apoptosis in tumor cells. In addition, preincubation and/or coincubation of target cells with cytokines that are known to induce increases in cell surface expression and/or function of the TNF family receptors, such as IFN- (32) or CD40L (33), significantly increased cytotoxic activity of the three ligands (data not shown). However, it is unlikely that either IFN- or CD40L participated in nonsecretory/apoptotic killing mediated by nonactivated NK cells, measured in 1-h cytotoxicity assays, because only NK cells activated for 24 h or longer, but not nonactivated NK cells, express/secrete these cytokines (22 (Fig. 1). Comparative analysis of data demonstrated that the apoptotic killing of tumor cells induced by soluble TNF family ligands was remarkably similar to that mediated by NK cells. Thus, it could only be induced when all three ligand-receptor pairs were simultaneously engaged, and the absence or exclusion of the engagement of any one of the three ligand-receptor pairs effectively abolished killing activity of the remaining two. This finding indicates that each of the three ligand-receptor pairs is equally important in cytotoxic activity of the combination. In addition, the ligand-mediated killing, similar to NK cell-mediated nonsecretory/apoptotic killing, could be detected not only in 1-h [³H]thymidine release and 18-h MTT assays, but also in 1-h Annexin V-based flow cytometry and 18-h ⁵¹Cr release assays, and could be inhibited by Z-VAD-Fmk caspase-3 inhibitor (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We demonstrate for the first time that NK cells constitutively express most, and simultaneously utilize several, TNF family ligands to induce apoptosis in solid tumor cell targets.

Activated NK cells and CTLs are known to express several TNF family ligands, including TNF, LT-, LT-ß, and FasL (2, 22, 23, 24), and to kill susceptible cell targets not only via perforin and granzymes, but also via TNF or FasL (15, 24, 26, 34, 35, 36). On the other hand, there is limited evidence regarding expression and utilization of the TNF family ligands by nonactivated NK cells. Thus, only a differential expression of functional TRAIL on immature NK cells (21) and FasL on freshly isolated mature NK cells (19, 20) has been described. We demonstrate in this study that human nonactivated NK cells constitutively express, both at the message and protein levels, most of the known TNF family ligands, TNF, LT-, LT-ß, FasL, CD27L, CD30L, OX40L, 4-1BBL, and TRAIL. We also show that IL-2-activated NK cells express all of the constitutively expressed TNF family ligands and de novo express CD40L.

The TNF family ligands have been implicated in a variety of important biological processes, including growth regulation, development of the immune system, immune regulation, inflammatory responses, and antitumor function (1, 2, 5). Therefore, expression of these pleiotropic molecules by NK cells suggests that these cells might have a variety of related important biological roles. In this study, we demonstrate that at least some of the constitutively expressed TNF family ligands on NK cells are involved in NK cell-mediated apoptotic killing of tumor cell targets. Individually applied antagonists of either TNF, LT-ß, or FasL were able to substantially block apoptotic killing by NK cells, and such inhibition could not be augmented by any combination of reagents tested. On the other hand, neither recombinant TNF, LT-₁ß₂, or FasL nor immobilized Abs to TNFR1, LT-ßR, or Fas were significantly cytotoxic when applied individually or in combination of two different ligands or Abs. Only when at least three different ligands or Abs were simultaneously applied did we observe a significant induction of rapid apoptosis in tumor cells. These data demonstrate that disruption of the engagement of only one ligand-receptor pair is sufficient to prevent the induction of NK cell-mediated apoptosis in tumor cells. Therefore, this cytotoxic mechanism is susceptible to down-regulation. In addition, the engagement of only one or two ligand-receptor pairs is insufficient to induce apoptosis in tumor cells. Thus, this killing is not readily inducible. A possible explanation for these seemingly divergent findings might be that homotypic engagement of only one type of the TNF family receptors is insufficient to induce the apoptotic signal in most tumor cell targets, and that only simultaneous interaction of three or more different ligand-receptor pairs could generate sufficiently strong and/or appropriate quality signal(s) to rapidly induce the cascade of intracellular events necessary for induction of apoptosis in tumor cell targets (Fig. 5). Cell surface density and/or stability of the interacting molecules and/or signaling properties of the TNF family receptors on tumor cells might be important factors defining the efficiency of their engagement and function.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. The newly proposed model and the current model of the TNF family ligand-receptor interactions mediating apoptosis. The mechanism depicted in the new model is probably operative mostly when the TNF family ligands and/or their receptors are expressed at low levels. It indicates that simultaneous heterotypic aggregation and cross-linking of at least three different receptor types on tumor cells by corresponding ligands on effector cells lead to apoptosis in tumor cells. The mechanism depicted in the current model is most likely operative when the TNF family ligands and/or their receptors are expressed at high levels. It indicates that the homotypic aggregation and cross-linking of at least three TNF family receptors of one type on tumor cells by corresponding ligands on effector cells lead to apoptosis in tumor cells.

Our data indicate that CD40L is able to significantly increase cytotoxic activity of combined TNF, LT-ß, and FasL. Therefore, it will be of interest to test the role of the other ligands, shown to be expressed by NK cells, in spontaneous nonsecretory/apoptotic killing of tumor cells by NK cells.

We observed that some of the TNF family ligands or their receptors were expressed at higher levels within the cells than on the cell surface of NK cells or tumor cells, respectively. In addition, the levels of expression of the TNF family ligands on NK cells and the TNF family receptors on tumor cells may be increased by IL-2 activation and IFN- or CD40L, respectively (data not shown). These findings indicate that the cell surface expression of the ligands and/or receptors as well as apoptosis-inducing activity of NK cells against tumor cells might be rapidly increased by appropriate stimulation, similar to that recently shown for FasL in human monocytes or for Fas in tumor cells (2, 5, 37). Thus, a better understanding of the regulation of expression and/or function of these molecules on NK cells and/or tumor cells might lead to development of an improved NK cell-based immunotherapy. In addition, our demonstration that simultaneous application of three or more TNF family ligands in low concentrations efficiently induces apoptosis in tumor cells suggests that combined treatment with selected TNF family ligands might be exploited as a novel approach for cancer therapy.

In conclusion, our study demonstrates that human NK cells constitutively express 9 of the 11 tested TNF family ligands and simultaneously utilize at least three of them (i.e., TNF, LT-₁ß₂, and FasL) to ligate corresponding receptors and induce apoptosis in tumor cells. These and previously published (27) data redefine NK cells as potent constitutive immune effectors, which are able to utilize not only the perforin-mediated secretory/necrotic mechanism to kill rare leukemia cell targets, but also powerful TNF family ligand-mediated nonsecretory apoptotic mechanism to destroy most solid tumor cell targets.

	Acknowledgments

 
We thank Immunex Research and Development, Biogen, Dr. Shigekazu Nagata, and Dr. Carl F. Ware for providing Abs, Rc:Fc fusion proteins, and the rTNF family ligands. We also thank Dr. Shigekazu Nagata and Dr. Dan Johnson for critical reading of and comments on the manuscript.

	Footnotes

 
¹ This work was supported by National Institute of Health Grant 1-P60 DE13059-01 (N.L.V.), National Institute of Health Grant RO1-CA63513 (T.L.W.), and the Pathology Education and Research Foundation, Pittsburgh.

² Current address: Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan.

³ Address correspondence and reprint requests to Dr. Nikola L. Vujanovic, University of Pittsburgh Cancer Institute, Biomedical Science Tower W1045, 211 Lothrop Street, Pittsburgh, PA 15213. E-mail address:

⁴ Abbreviations used in this paper: LT, lymphotoxin; A-NK cells, IL-2-activated adherent NK cells; L, ligand; MFI, mean fluorescence intensity; NGF, nerve growth factor; PCA, polyclonal Ab; SCCHN, squamose cell carcinoma of the head and neck; TRAIL, TNF-related apoptosis-inducing ligand.

Received for publication October 28, 1998. Accepted for publication September 2, 1999.

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