cytokine-induced apoptosis of human natural killer cells identifies

Cytokine-Induced Apoptosis of Human Natural Killer Cells Identifies aNovel Mechanism to Regulate the Innate Immune Response

By Mary E. Ross and Michael A. Caligiuri

Interferon-g (IFN-g) is critical for an effective innate immune tokine-induced NK cell production of IFN-g and TNF-a, alsodecreased cytokine-induced NK cell apoptosis. Costimula-response against infection. A combination of interleukins

(ILs) derived from activated T cells (IL-2) and monocytes tion of a CD3ÏCD56" NK leukemia cell line with IL-2 andIL-12 or IL-15 and IL-12 induced apoptosis in vitro, which(IL-12), or monocytes alone (IL-15 and IL-12), induces optimal

production of IFN-g from natural killer (NK) cells. The mecha- increased when combined with a chemotherapeutic agent.In summary, costimulation of human NK cells via thenism by which human NK cells downregulate their produc-

tion of IFN-g is unknown. Here we show that the same cyto- IL-2 receptor and the IL-12 receptor induces significant IFN-g production, followed by NK cell apoptosis and a decline inkines that induce human NK cell IFN-g production

subsequently induce apoptosis of the NK cells. Fas, bcl-2, IFN-g production. Hence, cytokines that activate this innateimmune response may also serve to limit it via apoptosis.or bax do not appear to be involved in this process. The

mechanism of cytokine-induced apoptosis of human NK This novel observation may have implications for the regula-tion of the innate immune response during infection, thecells appears to involve NK cell production of tumor necrosis

factor-a (TNF-a). Neutralization of TNF-a or inhibition of toxicity of combination cytokine therapy, and the treatmentof NK cell leukemia.TNF-a binding to the p80 TNF-a receptor partially inhibited

apoptosis. Transforming growth factor-b, which inhibits cy- q 1997 by The American Society of Hematology.

N stantial NK cell IFN-g production. The mechanism by whichhuman NK cells can limit this response is unknown and was

ATURAL KILLER (NK) cells are large granular lym-phocytes (LGL) that lack rearrangement of both T-

cell receptor and immunoglobulin genes and constitutively investigated in this report.express a number of cell surface cytokine receptors. Theseproperties are consistent with their role as innate immune MATERIALS AND METHODSeffector cells, which provide interferon-g (IFN-g) as an early Cell isolation, culture conditions, and cell lines. Humanresponse to infection. NK cell production of IFN-g is pivotal CD30CD56/ NK cells were isolated from fresh blood of normalfor effective monocyte elimination of obligate intracellular volunteers (American Red Cross, Buffalo, NY). Peripheral bloodpathogens such as Listeria monocytogenes, Toxoplasma gon- mononuclear cells (PBMC) were isolated using a discontinuous ficoll

gradient and adhered to plastic for 12 hours in RPCI 1640 containingdii, and Leishmania major. NK cell production of IFN-g10% human AB (HAB) serum (C-Six Diagnostic, Inc, Mequon, WI)also appears to be important in the pathogenesis of septicand antibiotics without cytokines. Nonadherent cells were recoveredshock in various animal models (reviewed in Bancroft et al1

and incubated in the presence of monoclonal antibodies (MoAbs)and Biron and Gazzinelli2).reactive against CD3, CD4, and HLA-DR, washed twice, and sub-It is clear that interleukin-12 (IL-12) is required, yet notjected to two consecutive immunomagnetic bead depletions withinsufficient for optimal production of IFN-g by resting human2 hours as described.10 Nondepleted PBMC were next stained with

NK cells.3-5 Several laboratories have shown that the combi- anti-CD56–phycoerythrin (PE) (Coulter Immunology, Hialeah, FL)nation of T-cell–derived IL-2 with monocyte-derived IL-12 and sorted to greater than 97% purity on a FACstar Plus (Bectonprovides a strong stimulus for IFN-g production by NK Dickinson, San Jose, CA). Unless otherwise indicated, NK cellscells.4-6 This is consistent with the fact that NK cells constitu- were plated at 5 1 104 cells/well in a 96-well plate and cultured for

4 days at 377C. Medium consisted of RPMI 1640 (Sigma, St Louis,tively express receptors for IL-2 (IL-2R)7 and possiblyMO) containing 10% HAB, antibiotics, recombinant human (rhu)IL-12 (IL-12R).8 IL-15 activates resting NK cells via compo-cytokines, and MoAbs where indicated. The NK-92 LGL leukemianents of the IL-2R. Both IL-15 and IL-12 are produced bycell line11 was generously provided by Dr H.-G. Klingemann (Uni-lipopolysaccharide (LPS)-activated monocytes, and in com-versity of British Columbia, Vancouver, Canada), and was main-bination, they also provide a potent costimulus for NK celltained in RPCI 1640 medium supplemented with 20% fetal bovineIFN-g production.6,9 Thus, in both T-cell–dependent and T-serum (FBS), antibiotics (GIBCO, Grand Island, NY), and either

cell–independent activation of NK cells, dual signaling via IL-2 or IL-15 at a concentration of 10 ng/mL.components of the IL-2R and the IL-12R can provide sub- Cytokines, MoAbs, and measurement of cytokines. All assays

described in this report contained rhu cytokines. IL-15 was providedby Immunex Corp (Seattle, WA) and was used at a concentrationof 15 ng/mL. IL-12 (specific activity of 4.5 1 106 U/mg) was pro-From the Division of Medicine, Departments of Hematologic On-

cology and Bone Marrow Transplantation, Molecular Medicine, and vided by Genetics Institute (Andover, MA) and was used at a concen-tration of 3 ng/mL. IL-2 (specific activity 1.53 1 107 U/mg) wasMolecular Immunology, Roswell Park Cancer Institute, Buffalo, NY.

Submitted June 3, 1996; accepted September 17, 1996. provided by Hoffmann LaRoche (Nutley, NJ) and used at a concen-tration of 15 ng/mL. c-kit ligand was provided by Amgen CorpSupported by National Institutes of Health Grant No. CA-68458,

Bethesda, MD and the Coleman Leukemia Research Fund, St Paul, (Thousand Oaks, CA) and used at 100 ng/mL. Tumor necrosis factor-a (TNF-a; specific activity 2 1 103 U/mg) was obtained from theMN.

Address reprint requests to Michael A. Caligiuri, MD, Roswell Asahi Chemical Corporation (Tokyo, Japan) and IFN-g was obtainedfrom Genentech (San Francisco, CA). Purified porcine transformingPark Cancer Institute, Buffalo, NY 14263.

The publication costs of this article were defrayed in part by page growth factor; (TGF)-b2 was used at a concentration of 2 ng/mL(R & D Systems, Minneapolis, MN). Doxorubicin (Adria Labs, Indi-charge payment. This article must therefore be hereby marked

‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to anapolis, IN) was used at a concentration of 600 ng/mL (1 mmol/L). For the experiments with the NK-92 cells, IL-12 and IL-2 wereindicate this fact.

q 1997 by The American Society of Hematology. purchased from R & D Systems. The TNF-a receptor-Fc fusionprotein (TNFR-Fc) and the IL-4 receptor-Fc fusion protein (IL-4R-0006-4971/97/8903-0011$3.00/0

910 Blood, Vol 89, No 3 (February 1), 1997: pp 910-918

AID Blood 0030 / 5h2c$$$581 01-10-97 18:41:48 bldal WBS: Blood

For personal use only.on February 17, 2018. by guest www.bloodjournal.orgFrom

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CYTOKINE-INDUCED APOPTOSIS OF NK CELLS 911

Fc) were provided by Immunex.12 A blocking MoAb to the p80 chain Surface staining of TNF-aR. Flow cytometric analysis of restingNK cell TNF-aR expression was evaluated using methods describedof the TNF-a receptor (TNF-aR) (M1, rat) and a blocking MoAb

to the p60 chain of the TNF-aR (M50, mouse) were both provided previously.10 Briefly, cells were incubated with anti-p80 TNF-aRMoAb, anti-p60 TNF-aR MoAb, or species-specific isotype controlby Immunex.13 For measurement of IFN-g and TNF-a production

in NK cell cultures, supernatants were removed from duplicate wells MoAb, washed, and incubated with goat antimouse fluorescein iso-thiocyanate (FITC) (1:100) or goat antirat FITC (1:200). Cells wereonly once without disturbing the cell pellet and frozen at 0707C

until assayed by commercial enzyme-linked immunosorbent assay washed and counterstained with NKH1-RD1, washed, fixed in 1%formalin and analyzed by FACScan (Becton Dickinson).(ELISA) (IFN-g: GIBCO-BRL, Gaithersburg, MD; TNF-a: R &

D) following the manufacturers’ recommendation. The total IFN-gconcentration in culture supernatants was determined by ELISA.

RESULTSDaily production of IFN-g was calculated as follows: total IFN-g in supernatants from day x supernatant 0 mean total IFN-g in Kinetics of cumulative and daily IFN-g production. NKsupernatants from day (x-1). cells (¢ 97% pure) were isolated from fresh blood and cul-

Assessment of viability and apoptosis. Cell number and viability tured for 4 days in the presence of two monocyte-derivedwere determined by vital dye exclusion using a standard hemocytom- cytokines, IL-15 and IL-12. Total NK cell IFN-g productioneter. Methods used to detect apoptosis in cultured NK cells (ie, was noted to plateau by day 4 (Fig 1A). IFN-g stabilitypropidium iodide [PI] nuclear staining and ethidium bromide gel testing and culture in the presence of an anti-IFN–g receptorelectrophoresis) have been described elsewhere.14 As these experi-

MoAb showed that this plateau in NK cell production ofments were all initiated with a sorted (ú97%) population ofIFN-g was not due to protein degradation or consumptionCD30CD56/ NK cells, the fluorescence-activated cell sortingby NK cells, respectively (not shown). The amount of(FACS) analyses for PI fluorescence after 3 days of culture wereIFN-g produced each day was next calculated by subtractingperformed without a gate. Morphologic analysis was performed us-

ing Wright-Giemsa staining on cytocentrifuge preparations. the IFN-g of identical cultures on the previous day from the

Fig 1. Cumulative and daily IFN-g production andsurvival of purified CD3ÏCD56" human NK cells. (A)Cumulative NK cell IFN-g production in the presenceof 15 ng/mL IL-15 alone (●) or IL-15 and 3 ng/mLIL-12 (j). (B) Cumulative NK cell IFN-g production inthe presence of 15 ng/mL IL-2 alone (●) or IL-2 andIL-12 (j). (C) NK cell daily IFN-g production in thepresence of 15 ng/mL IL-15 alone (●) or IL-15 and 3ng/mL IL-12 (j). (D) NK cell daily IFN-g productionin the presence of 15 ng/mL IL-2 alone (●) or IL-2and IL-12 (j). (E) Enumeration of viable NK cells asdetermined by vital dye exclusion assay in the pres-ence of IL-15 alone (●), or IL-15 and IL-12 (j). (F)Enumeration of viable NK cells in the presence ofIL-2 alone (●), or IL-2 and IL-12 (j). Results representthe mean cell count for duplicate wells, with õ15%standard deviation. Four independent experimentsshowed similar results. Results were generally morepronounced in the CD3ÏCD56bright " subset of NKcells.





ROSS AND CALIGIURI912

total amount of IFN-g in the well on the current day. NK specifically required the survival signal delivered via compo-nents of the IL-2R, CD56brightNK cells were incubated withcell daily IFN-g production peaked on day 3 and underwent

a dramatic decline to nearly zero by day 4 (Fig 1C). In c-kit ligand, another NK cell survival factor that mediatesits effect via the constitutively expressed tyrosine kinasecontrast, culture of NK cells in IL-15 alone yielded minimal

amounts of daily IFN-g production cumulatively or daily receptor c-kit.15 Culture of NK cells with c-kit ligand plusIL-12 did not induce cell death when compared with NK(Fig 1A and C). Identical results were obtained in both sets

of experiments when IL-2 was substituted for IL-15 (Fig 1B cells cultured with c-kit ligand alone. Similarly, culture ofNK cells in the presence of c-kit ligand and IL-12 did notand D). Culture of fresh, resting NK cells in the presence

of IL-12 alone also yielded low levels of IFN-g (õ1 ng/ induce substantial (ie, ng/mL) IFN-g production (notshown). Thus, IL-12 interrupts NK survival mediated viamL).6

Cell death accounts for the decline in daily IFN-g produc- components of the IL-2R, but not NK survival mediated viac-kit.tion. We sought to determine the reason why daily NK cell

IFN-g production decreased dramatically following 3 days The reproducible decrease in NK cell numbers seen fol-lowing costimulation via the IL-2R and the IL-12R sug-of culture in the presence of excess IL-15 and IL-12 or

IL-2 and IL-12. Daily cell counts were performed by vital gested that the mechanism of cell death might involve activa-tion-induced apoptosis. Staining of cellular DNA with PIdye exclusion and indicated a sharp decline in viable cell

number by day 4 in cultures containing IL-15 and IL-12. In provided a quantitative measurement of apoptosis by de-tecting the fluorescence of individual nuclei using flow cy-contrast, NK cells cultured in IL-15 alone, had no decrease

in viable cell number (Fig 1E). Concentrations of IL-15 as tometry.16 PI fluorescence of cells with the normal diploidDNA content falls within a characteristic peak of constantlow as 0.01 ng/mL will maintain NK cell survival for days.14a

Thus, the combination of IL-15 with IL-12 appeared to inter- intensity (set here at 200). Apoptotic nuclei undergoing en-donucleolytic cleavage of DNA appear within the hypodip-fere with the IL-15–mediated survival pathway. Identical

results were obtained when IL-2 was substituted for IL-15 loid region to the left of the diploid peak in the DNA histo-gram. Incubation of fresh human NK cells with the(Fig 1F). To determine whether this decrease in cell number

Fig 2. Apoptosis of NK cells following costimula-tion with IL-15 and IL-12. (A) Purified NK cells cul-tured for 3 days in medium containing 15 ng/mLIL-15 alone have only 1% of nuclei within the hypo-diploid DNA fraction. (B) A parallel culture of NKcells from the same individual in the presence ofIL-15 and 3 ng/mL IL-12 shows 39% of nuclei in thehypodiploid DNA region that characterizes apopto-sis. (C) Agarose gel electrophoresis of total DNA fromCD3ÏCD56" NK cells that were cultured for 3 days inIL-15 alone (lane 1) or IL-15 and IL-12 (lane 2). Geno-mic DNA was isolated from an equal number of wellsinitially containing 1.4 Ì 106 sorted NK cells. MWindicates a molecular weight standard. The DNA lad-dering effect characteristic of apoptosis is seen inlane 2. (D) Cytospin preparation of CD3ÏCD56" NKcells cultured 3 days in IL-15 alone or (E) IL-15 andIL-12, with nuclear fragmentation and membraneblebbing characteristic of apoptosis. (Wright Giemsastain, original magnification Ì 330.) These resultsare representative of four independent experiments.Similar results were obtained when IL-2 was substi-tuted for IL-15 (data not shown).






combination of IL-15 and IL-12 or IL-2 and IL-12 consis- IL-2 and IL-12 is a potent inducer of TNF-a production,compared with stimulation by either cytokine alone.6 In fact,tently resulted in the induction of DNA fragmentation, as

determined by nuclear staining with PI (Fig 2A and B). daily NK cell TNF-a production parallels that of IFN-g,with a peak by day 3 and rapid decline on day 4 (dataElectrophoretic analysis of DNA from NK cells cultured in

IL-15 and IL-12 showed bands in multiples of 200 bp, a not shown). The costimulation experiments were, therefore,repeated in the presence of soluble TNFR-Fc, which neutral-‘‘DNA ladder’’ characteristic of apoptosis. No DNA ladder

was present in cells cultured in IL-15 alone (Fig 2C). Simi- izes TNF-a,12 in an attempt to block the autocrine contribu-tion of TNF-a production to the cytokine-induced apopto-larly, morphologic evaluation of NK cells cultured in IL-15

and IL-12 showed a significant percentage of cells exhibiting sis. As shown in Fig 4A and B, the addition of solubleTNFR-Fc resulted in a greater than 50% reduction of NKthe classical characteristics of apoptosis: nuclear fragmenta-

tion, chromatin condensation, and membrane blebbing (Fig cell programmed cell death following costimulation withIL-15 and IL-12, which correlated with a reduction in TNF-2E), whereas cultures in IL-15 alone contained few cells

undergoing apoptosis (Fig 2D). a protein detected in culture supernatants. Combination ofanti-Fas MoAb and soluble TNFR-Fc did not further inhibitThe percentages of NK cells undergoing apoptosis when

cultured in IL-2, IL-2 plus IL-12, IL-15, or IL-15 plus apoptosis (data not shown).We investigated which TNF-aR might be responsible forIL-12 are shown for 18 random normal donors in Table 1.

Initiation of the process of apoptosis as determined by PI the induction of NK cell apoptosis. Indirect staining offreshly isolated NK cells demonstrated the presence of thestaining began within 48 hours of culture and required at

least 18 hours of exposure to IL-12 in the presence of p80 TNF-aR (Fig 5A), with absent to low density expressionof p60 TNF-aR (Fig 5B). The p80 TNF-aR protein has beenIL-15 or IL-2. We performed a dose-response curve to de-

termine how much IL-12 was required to interrupt the shown to be markedly upregulated on the surface of NKcells following exposure to IL-2 or IL-12.19 Indeed, in theIL-15–mediated NK survival. Increases in NK cell apoptosis

were seen at IL-12 concentrations as low as 0.02 ng/mL and presence of an MoAb, which blocked TNF-a binding to thep80 TNF-aR, a marked reduction in NK cell apoptosis wasachieved a plateau at 0.2 ng/mL (Fig 3). Tenfold decreases

in the number of NK cells per well or NK cell density observed in the presence of IL-15 and IL-12. An MoAbwhich blocked TNF-a binding to the p60 TNF-aR had noresulted in only modest (10% to 15%) reductions in cytokine-

induced apoptosis (data not shown). effect on cytokine-induced apoptosis (Fig 4C through F).Further, neutralization of IFN-g had no effect (data notCytokine-induced apoptosis is mediated in part by an au-

tocrine TNF/TNFR pathway. We next tried to determine shown).Costimulation of NK cells with IL-15 and IL-12 leads tothe mechanism by which the combination of IL-15 and

IL-12 or IL-2 and IL-12 could trigger programmed cell death the endogenous production of IFN-g and TNF-a, followedby induction of NK cell apoptosis. Blocking TNF-a de-in human NK cells. In data not shown, incubation of NK

cells in IL-2 and IL-12 did not alter the expression of bcl-2 creases the cytokine-induced apoptosis. However, culture ofNK cells in the presence of IL-15 and IFN-g or IL-15 andor bax protein. Cytokine-induced apoptosis was not inhibited

by blocking Fas, a member of the TNF-aR family known TNF-a did not lead to induction of apoptosis (Fig 5C). Thus,while TNF-a appears to participate in cytokine-inducedto induce apoptosis following stimulation by its natural li-

gand.17 The TNF-a–mediated program of apoptosis can be apoptosis, simultaneous signaling via components of theIL-2R and the IL-12R appear to be critical for induction ofdelayed for days following receptor activation,18 raising the

possibility that this death pathway might be involved. In- programmed cell death in NK cells. Other studies from ourlaboratory have demonstrated that TGF-b2 suppressesdeed, costimulation of NK cells by IL-15 and IL-12 orTNF-a, IFN-g, and chemokine production by NK cells cul-tured in IL-15 and IL-12 or IL-2 and IL-12.20,21 NK cells

Table 1. Percent Apoptosis of Human NK Cells cultured for 3 days in IL-2, IL-12, and TGF-b2 showed areduction in the percentage of cells undergoing apoptosisDonor IL-2 / Donor IL-15 /

No. IL-2 IL-12* No. IL-15 IL-12* by 50% or greater as quantitated by PI. A representativeexperiment is shown in Fig 6A through C. However, addition1 5 36 10 1 39of exogenous TNF-a to this culture did not reverse the partial2 8 63 11 10 50

3 19 40 12 10 63 inhibition of apoptosis (data not shown). This suggests that4 20 74 13 10 70 the inhibitory effect of TGF-b on cytokine-induced NK cell5 23 58 14 18 62 apoptosis is not mediated by an inhibition of TNF-a secre-6 16 69 15 8 67 tion.7 18 53 16 19 52

Cytokine-induced apoptosis of NK cell leukemia. The8 21 48 17 20 49

ability to induce apoptosis in normal human NK cells follow-9 28 63 18 23 64ing costimulation with IL-15 and IL-12 or IL-2 and IL-12

Purified NK cells were cultured for 3 days with IL-2 (15 ng/mL) or suggested that this might have potential application in theIL-2 plus IL-12 (3 ng/mL); IL-15 (15 ng/mL) or IL-15 plus IL-12. The

treatment of NK cell malignancies with a CD30CD56/ phe-percentage of cells undergoing apoptosis was determined by nuclear

notype. The NK-92 cell line was developed from a patientstaining with PI using flow cytometry. Results from 20 individual do-with rapidly progressive non-Hodgkin’s lymphoma whonors are shown. The standard deviation of the mean for experimentsfailed standard chemotherapy and died during the leukemicdone in triplicate (donors 6 to 9 and 15 to 18) was õ8%.

* P Å .004 as calculated by the Exact Wilcoxon Signed Rank Test. phase of his disease.11 NK-92 cells are CD30CD56/,






seen at the single cell level, implying that neither cell-to-cell contact nor cell-mediated cytotoxicity were required.Clones of cells surviving the first exposure to IL-2 and IL-12 showed susceptibility upon reexposure (data not shown).Further, synergy in cell kill was seen in the presence of IL-2, IL-12, and the antineoplastic agent doxorubicin (Fig 7C).Similar results were obtained when IL-15 was substitutedfor IL-2 (data not shown).

DISCUSSION

Animal studies demonstrating that excessive productionof IFN-g by NK cells is responsible for death followingrepeated exposure to endotoxin provide the most compellingin vivo data to support the importance of timely negativefeedback during more limited infections.2 Monocytes acti-vated by infectious organisms provide TNF-a, IL-1b,IL-15, and IL-12 for optimal IFN-g production by NK

Fig 3. The effect of IL-12 concentration on induction of apoptosiscells.1-5,9 NK cells, in turn, provide monocytes with IFN-gof NK cells. Purified CD3ÏCD56" human NK cells were cultured for 3and TNF-a for an effective innate immune response againstdays in the presence of 15 ng/mL IL-15 and varying concentrations

of IL-12. The percent of NK cells undergoing apoptosis was deter- a number of obligate intracellular pathogens.1,2,4,5,22 Here wemined by PI-stained nuclei using flow cytometry. Results are repre- provide the first in vitro evidence that two of the same mono-sentative of three experiments.

cyte-derived cytokines, IL-15 and IL-12, that induce earlyIFN-g and TNF-a production by NK cells, can also limitthis response. The mechanism of downregulation involves anIL-2– or IL-15–dependent LGL leukemia cells that display

cytotoxic activity against standard NK targets11 (and M. Cali- apoptotic pathway that uses endogenous TNF-a in a negativeautocrine feedback loop. NK cells can undergo apoptosisgiuri, unpublished observation). The incubation of NK-92

cells in the presence of IL-2 and IL-12 for 24 hours resulted following activation with IL-2 and engagement of the lowaffinity FcgIIIR23,24 or following cell-mediated lysis of ain induction of apoptosis, compared with cells incubated in

IL-2 alone (Fig 7A and B). In clonal assays, this effect was specific leukemia cell line.25 However, the current report

Fig 4. Autocrine production of TNF-a contributesto cytokine-induced apoptosis of human NK cells viathe p80 TNF-aR. In each condition described below,purified CD3ÏCD56bright" NK cells were isolated fromfresh blood10 and cultured at 1 Ì 105 cells/well for 3days in medium containing 15 ng/mL IL-15 and 3 ng/mL IL-12, after which nuclei were stained with PI toobtain a quantitative measurement of apoptosis. (A)Culture in the presence of a nonreactive receptor fu-sion protein (IL-4R-Fc, 500 mg/mL) shows 54% of nu-clei in the hypodiploid peak, characteristic ofapoptosis, while (B) culture in the presence of aTNFR-Fc fusion protein (500 mg/mL) partially neutral-izes TNF-a and shows only 21% of nuclei in the hypo-diploid peak. (C) Cultures containing a nonreactiveisotype control MoAb (50mg/mL) show 55% of nucleiin the hypodiploid fraction of DNA while (D) incuba-tion with a blocking MoAb to the p80 TNF-aR (M1,50 mg/mL) shows 32% of nuclei in the hypodiploidDNA fraction. (E) Cultures containing a nonreactiveisotype control MoAb (50 mg/mL) show 64% of NKcell nuclei in the hypodiploid fraction of DNA and (F)incubation with a blocking MoAb to the p60 TNF-aR(M50, 50 mg/mL) shows no measurable effect com-pared with control. Results were obtained with NKcells from a single donor and are representative ofthree experiments using different donors.






provides evidence that monokines alone may have a role inlimiting the innate immune response via apoptosis of NKcells, in the absence of antigen-specific humoral immunityor NK cytotoxic activity against a tumor cell. The data alsooffer an explanation for the observations that resting or

Fig 5. Surface density expression of the (A) TNF-aR p80 and (B)TNF-aR p60 on fresh human NK cells by flow cytometry. Shadedarea in each histogram represents isotype control antibody binding.Experiments were conducted with fresh NK cells from a single donorand are representative of two separate experiments. (C) Requirementfor components of the IL-2R and IL-12R activation for the inductionof NK cell apoptosis. Purified NK cells were cultured for 3 days in thepresence of indicated cytokines at the following concentrations:IL-15 (15 ng/mL), TNF-a (5,000 U/mL), IFN-g (10 ug/mL), and IL-12 (3ng/mL). Results are expressed as the percent of cells undergoingapoptosis as determined by nuclear PI staining and flow cytometry.Standard deviation of the mean from duplicate wells was less than Fig 6. Reduction in cytokine-induced apoptosis by TGF-b. Purified2%. Results are representative of three individual experiments. CD3ÏCD56" human NK cells were cultured for 3 days in medium

containing (A) 15 ng/mL IL-2, (B) IL-2 and 3 ng/mL IL-12, or (C) IL-2,IL-12 and 2 ng/mL TGF-b2 . Nuclei were stained with PI and thenquantitated for apoptosis by flow cytometry. Results are representa-tive of three independent experiments.






Fig 7. Cytokine-induced apoptosis of a humanLGL leukemia cell line. NK-92 cells were plated ata concentration of 5 Ì 105/mL in culture mediumcontaining either (A) 15 ng/mL IL-2 alone or (B) IL-2and 3 ng/mL IL-12 for 24 hours, after which cellsunderwent cytocentrifuge preparation and Wright-Giemsa staining. Original magnification Ì 330. (C)NK-92 cells were first cultured at a concentration of1 Ì 105 cells/mL for 2 hours in medium containing:15 ng/mL IL-2; IL-2 and 600 ng/mL doxorubicin; IL-2and 3 ng/mL IL-12; IL-2, IL-12, and doxorubicin. Cellswere next washed three times with RPMI 1640 andplated in a 96-well plate at 10 cells/well in the pres-ence of IL-2 alone or IL-2 and IL-12, as indicated. After2 days, each well was washed three times with RPMI1640, and cultures were then continued in mediumcontaining IL-2 for 6 more days. Wells were then vi-sually scored for the presence of a viable colony.Similar results were obtained when IL-15 was substi-tuted for IL-2 in these experiments (data not shown).

IL-2–activated NK cells have decreased proliferation in the sion.31 While the p60 TNF-aR has been more commonlynoted to initiate the apoptotic signal, similar signaling haspresence of IL-12 and IL-2, compared with IL-2 alone.26,27

Downregulation of NK cell IFN-g production follows pro- been seen with the p80 TNF-aR. Interestingly, apoptosismediated via the p80 TNF-aR appears to be delayed for uplonged (18 hours) exposure to the same two cytokines that

induce IFN-g production and uses TNF-a and the p80 to 48 hours in other systems,18 as was seen in the currentstudy.TNF-aR to induce apoptosis. The requisite costimulation

(IL-15 and IL-12) of NK cells parallels that of the peripheral TGF-b can inhibit NK cell production of TNF-a andIFN-g and can limit the induction of cytokine-induced NKT-cell system where repeated antigen activation in combina-

tion with IL-2 induces apoptosis. Antigen-induced apoptosis cell apoptosis. This suggests that other factors may also serveto limit NK cell activation in vivo. There is good experimen-of T cells has been shown to be mediated via Fas,28 and

more recently, TNF-aR in vitro29 and in vivo.30 T cells and tal evidence that monocyte-derived IL-10 can act as a nega-tive regulator for NK cell IFN-g production in the mouse.4NK cells, therefore, appear to require costimulatory signals

to initiate the cascade leading to apoptosis. T cells require T- In humans, a similar mechanism involving IL-10 may beless likely, as IFN-g inhibits monocyte production ofcell receptor activation in combination with a single cytokine

receptor signal, while NK cells can accomplish this via two IL-10,32 and IL-10 can enhance NK cell production ofIFN-g.33 Nonetheless, the fact that negative regulators suchdistinct cytokine receptors in the absence of an antigen-

specific receptor signal. This is consistent with the NK cell’s as TGF-b can be identified suggests that cytokine-inducedapoptosis of NK cells may not be a primary mechanismrole as an innate immune effector cell responding to mono-

kines early and nonspecifically following microbial inva- of limiting IFN-g production following microbial infection.






listeriosis and interleukin 10 is a physiologic antagonist. Proc NatlRather, the pathway may become operative in vivo duringAcad Sci USA 90:3725, 1993more sustained infectious insults when other more reversible

5. Gazzinelli RT, Hieny S, Wynn TA, Wolf SF, Sher A: Interleu-mechanisms of negative feedback are inoperative or ineffec-kin 12 is required for the T-lymphocyte-independent induction oftive. One such example is that monocytes do not appear tointerferon g by an intracellular parasite and induces resistance in T-produce TGF-b following lipopolysaccharide (LPS) activa-cell-deficient hosts. Proc Natl Acad Sci USA 90:6115, 1993

tion.34 Lethal endotoxin-induced septic shock can occur as 6. Carson WE, Giri JG, Lindemann MJ, Linett ML, Ahdieha consequence of overwhelming bacterial infection and is M, Anderson D, Eisenmann J, Grabstein K, Caligiuri MA: In-thought to be mediated in part by massive endogenous pro- terleukin 15 is a novel cytokine that activates human naturalduction of IFN-g and TNF-a.2 This suggests that each of killer cells via components of the IL-2 receptor. J Exp Med

180:1395, 1994these postulated mechanisms of negative feedback can be7. Tsudo M, Goldman CK, Bongiovanni KF, Chan WC, Wintonovercome in vivo.

E, Yagita M, Grimm EA, Waldmann TA: The p75 peptide is theThe data presented in this report would suggest that con-receptor for interleukin 2 expressed on large granular lymphocytessiderable caution should be exercised before simultaneouslyand is responsible for the interleukin 2 activation of these cells. Procdelivering combinations of IL-2 and IL-12 or IL-15 andNatl Acad Sci USA 84:5394, 1987IL-12 in vivo. Prolonged exposure to both agents could not

8. Naume B, Gately MK, Desai BB, Sundan A, Espervik T: Syn-only induce very high levels of proinflammatory cytokines ergistic effects of interleukin 4 and interleukin 12 on NK cell prolif-such as TNF-a, IFN-g, and macrophage inflammatory pro- eration. Cytokine 5:38, 1993tein-1a (MIP-1a),21 but might also induce apoptosis in a 9. Carson WE, Ross ME, Baiocchi RA, Marien M, Baioni N,majority of NK cells. This, in turn, could leave the host Grabstein K, Caligiuri MA: Endogenous production of interleukin

15 by activated human monocytes is critical for optimal productiontransiently more susceptible to obligate intracellular patho-of interferon-g by natural killer cells in vitro. J Clin Invest 96:2578,gens such as Toxoplasma gondii or Mycobacterium tubercu-1995losis. Further, as IL-15 may be constitutively produced by

10. Matos ME, Schnier GS, Beecher MS, Ashman LK, Williamsa number of tissues in vivo,35,36 some of these effects couldDE, Caligiuri MA: Expression of a functional c-kit receptor on apotentially be seen with the exogenous administration ofsubset of natural killer cells. J Exp Med 178:1079, 1993IL-12 alone.

11. Gong JH, Maki G, Klingemann HG: Characterization of aApoptosis appears to be a final common pathway for a human cell line (NK-92) with phenotypical and functional character-

number of cancer therapies, including traditional chemother- istics of activated natural killer cells. Leukemia 8:652, 1994apy, radiation therapy, and cell mediated cytotoxicity.37 The 12. Mohler KM, Torrance DS, Smith CA, Goodwin RG, Stremlerability to costimulate a malignant counterpart of NK cells KE, Fung VP, Madani H, Widmer MB: Soluble TNF receptors are

effective therapeutic agents in lethal endotoxemia and function si-with IL-2 and IL-12 or IL-15 and IL-12 and the convergencemultaneously as both TNF carriers and TNF antagonists. J Immunolof their signaling pathways to induce apoptosis may provide151:1548, 1993new opportunities to combine cytokine therapy with chemo-

13. Ware CF, Crowe PD, Vanarsdale TL, Andrews JL, Graysontherapy for the treatment of NK cell leukemia. Careful con-MH, Jerzy R, Smith CA, Goodwin RG: Tumor necrosis factor (TNF)sideration would have to be given to the dose and schedule ofreceptor expression in T lymphocytes. Differential regulation of thesuch a regimen, given the potential toxicities of this cytokinetype I TNF receptor during activation of resting and effector T cells.

combination discussed above. J Immunol 147:4229, 199114. Baiocchi RA, Ross ME, Tan JC, Chou CC, Sullivan L, Haldar

S, Monne M, Seiden MV, Narula SK, Sklar J, Croce CM, CaligiuriACKNOWLEDGMENTMA: Lymphomagenesis in the SCID-Human mouse involves abun-We thank Drs Elizabeth Repasky, William E. Carson, Paul Ander-dant production of human interleukin-10. Blood 85:1063, 1995son, and Douglas Williams for their insightful comments; Drs Stan

14a. Carson WE, Fehniger TA, Haldar S, Eckhert K, LindemannKrajewski and John Reed for assistance with immunoblots. We thankMJ, Lai CF, Croce CM, Baumann H, Caligiuri MA: A potentialDr Eric Bluman, Yuko Kono, and other members of the laboratoryrole for interleukin-15 in the regulation of human natural killer cellfor their technical assistance, and the Department of Flow Cytometrysurvival. J Clin Invest (in press).for cell sorting. We thank Dr H.E. Klingemann for the NK-92 cell

15. Carson WE, Haldar S, Baiocchi RA, Croce CM, Caligiuriline.MA: The c-kit ligand suppresses apoptosis of human natural killercells through the upregulation of bcl-2. Proc Natl Acad Sci USA

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1997 89: 910-918

Mary E. Ross and Michael A. Caligiuri Novel Mechanism to Regulate the Innate Immune ResponseCytokine-Induced Apoptosis of Human Natural Killer Cells Identifies a

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cytokine-induced apoptosis of human natural killer cells identifies

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