of May 26, 2018.This information is current as

Productionγfor NK Cell IFN-Activation-Specific Metabolic Requirements

Tiphanie P. Vogel and Megan A. CooperMolly P. Keppel, Nermina Saucier, Annelise Y. Mah,

ol.1402099http://www.jimmunol.org/content/early/2015/01/16/jimmun

published online 16 January 2015J Immunol 

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The Journal of Immunology

Activation-Specific Metabolic Requirements for NK CellIFN-g Production

Molly P. Keppel,* Nermina Saucier,* Annelise Y. Mah,*,† Tiphanie P. Vogel,*,‡ and

Megan A. Cooper*,x

There has been increasing recognition of the importance of cellular metabolism and metabolic substrates for the function and

differentiation of immune cells. In this study, for the first time to our knowledge, we investigate the metabolic requirements

for production of IFN-g by freshly isolated NK cells. Primary murine NK cells mainly use mitochondrial oxidative phosphory-

lation at rest and with short-term activation. Remarkably, we discovered significant differences in the metabolic requirements of

murine NK cell IFN-g production depending upon the activation signal. Stimulation of NK cell IFN-g production was indepen-

dent of glycolysis or mitochondrial oxidative phosphorylation when cells were activated with IL-12 plus IL-18. By contrast,

stimulation via activating NK receptors required glucose-driven oxidative phosphorylation. Prolonged treatment with high-

dose, but not low-dose, IL-15 eliminated the metabolic requirement for receptor stimulation. In summary, this study demonstrates

that metabolism provides an essential second signal for induction of IFN-g production by activating NK cell receptors that can be

reversed with prolonged high-dose IL-15 treatment. The Journal of Immunology, 2015, 194: 000–000.

Natural killer cells are innate immune lymphocytes thatprovide a first line of defense against infection, partic-ularly viruses, and can recognize and kill tumor cells that

have downregulated self-MHC or express activating ligands (1–3).NK cell effector functions can be triggered by inflammatory cyto-kines, such as IL-12, IL-15, and IL-18, or by engaging germline-encoded activating NK receptors whose ligands are displayed byinfected and/or tumor cells (3–5). In response, NK cells produceinflammatory cytokines, principally IFN-g, and kill target cells.Although many triggers of NK cell activation and subsequent NKcell effector responses have been well characterized, the meta-bolic fuels required to drive NK cell functional responses arelargely unknown.Metabolism is the biochemical process used by cells to break

down fuels for energy production (i.e., ATP) or to generate critical

biomolecules. There are two primary and overlapping metabolicpathways for generating ATP from metabolic fuels, anaerobic gly-colysis and mitochondrial oxidative phosphorylation (OXPHOS)(6). Glucose fuels both pathways and is first metabolized in thecytoplasm via glycolysis to produce two molecules of pyruvate, fora net yield of two ATP molecules. Pyruvate can then be convertedto lactate (anaerobic metabolism) or, in the presence of oxygen,transported into the mitochondria to fuel OXPHOS for a net yield of30+ ATP molecules. Other metabolic substrates that can fuel mi-tochondrial OXPHOS include fatty acids (fatty acid oxidation) andglutamine (glutaminolysis). Metabolic pathways, including glycol-ysis, are attractive pharmacologic targets for disease therapy, par-ticularly cancers (7, 8). Furthermore, metabolic fuels are globallyaltered in many disease states such as metabolic syndrome and sepsis,and probably locally altered in certain microenvironments, includingsites of infection and tumors (9–11). Thus, it is important to considerthe effect of changes in metabolic fuels on the immune system.Metabolic pathways in immune cells, including dendritic cells,

neutrophils, and T cells, have been shown to be critical for cellularactivation and differentiation (11–13). For example, differentiation ofmemory T cells is critically dependent on a switch in energy meta-bolism from primarily OXPHOS to glycolysis, whereas quiescentmemory cells revert back to OXPHOS (11, 14). In CD4+ effectorT cells, a glycolytic switch is required for synthesis of IFN-g proteinvia the release of a posttranscriptional block in IFN-g processing (15).In this study, for the first time to our knowledge, we investigate

the basic metabolic requirements for resting NK cell IFN-g produc-tion via two different pathways, cytokine- and receptor-mediatedactivation. We hypothesized that NK cells would require meta-bolic fuels for activation and production of IFN-g. Our results dem-onstrate that, unlike T cells, NK cells do not require a glycolyticswitch for efficient IFN-g production. Rather, we observed activa-tion-specific metabolic requirements for NK cell IFN-g production.

Materials and MethodsMice and NK cell isolation

All micewere on the C57BL/6 background.Wild-type micewere purchasedfrom the National Cancer Institute, and Rag-22/2gc

2/2 mice were pur-chased from Taconic. Mice expressing the congenic CD45.1 receptor

*Division of Rheumatology, Department of Pediatrics, Washington University Schoolof Medicine, St. Louis, MO 63110; †Medical Scientist Training Program, WashingtonUniversity School of Medicine, St. Louis, MO 63110; ‡Division of Rheumatology,Department of Internal Medicine, Washington University School of Medicine,St. Louis, MO 63110; and xDepartment of Pathology and Immunology, WashingtonUniversity School of Medicine, St. Louis, MO 63110

Received for publication August 15, 2014. Accepted for publication December 13,2014.

This work was supported by National Institutes of Health/National Institute of Al-lergy and Infectious Diseases Grant 1K08-AI085030, Child Health Research Centerat Washington University School of Medicine Grant K12-HD076224, the Children’sDiscovery Institute, and the St. Louis Children’s Hospital (to M.A.C.). A.Y.M. issupported by National Institutes of Health Training Grant 5T32GM007200, and T.P.V. issupported by National Institutes of Health Training Grant 2T32AR007279. Researchreported in this publication was supported by the National Institute of Arthritis andMusculoskeletal and Skin Diseases, part of the National Institutes of Health, underAward P30AR048335.

The content is solely the responsibility of the authors and does not necessarilyrepresent the official views of the National Institutes of Health.

Address correspondence and reprint requests to Dr. Megan A. Cooper, Department ofPediatrics, Washington University School of Medicine, 660 South Euclid, Box 8208,St. Louis, MO 63110. E-mail address: Cooper_m@kids.wustl.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: 2DG, 2-deoxy-glucose; ECAR, extracellular acid-ification rate; OCR, oxygen consumption rate; OXPHOS, oxidative phosphorylation;poly(I:C), polyinosine-polycytidylic acid; ROS, reactive oxygen species.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402099

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(CD45.1+) and Rag-12/2 animals were obtained from The Jackson Lab-oratory and bred to obtain CD45.1+Rag2/2 mice. All animals were usedbetween 6 and 14 wk of age. Animals were housed in specific pathogen-free conditions, and studies were approved by the Washington UniversityAnimal Studies Committee. NK cells were enriched from spleens usinga negative-selection magnetic bead kit (Miltenyi Biotec). NK cell purityranged from 66 to 98%, with an average purity of 85.5 6 5.9% (SD), andcontaminating cells were generally CD3 negative.

Extracellular flux and ATP assays

Extracellular flux assays were performed using a XF96 Analyzer (SeahorseBioscience). Ninety-six–well flux plates were coated with 10 ml poly-L-lysine (Sigma-Aldrich), and NK cells (5 3 105 cells/well) were adhered toplates for 30 min prior to the assay. NK cells were assayed in triplicatein nonbuffered RPMI 1640 (Sigma-Aldrich) supplemented with 2 mML-glutamine, 1% FBS (Sigma-Aldrich), glucose to a final concentration of25 mM, and 1 mM sodium pyruvate. Oxygen consumption rate (OCR) andextracellular acidification rate (ECAR) readings were taken every 2.5–3min, and OCR and ECAR results shown represent the average over 60 minor average readings at each time point. Intracellular ATP was measured inlysed NK cells by a luminescence assay (ATPLite; Perkin Elmer), asrecommended by the manufacturer.

Culture media and metabolic inhibitors

Complete media consisted of RPMI 1640 (Mediatech) supplementedwith 10% FBS (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich;final concentration 4 mM), 0.05 mM 2-ME (Sigma), and antibiotics(penicillin/streptomycin). Glutamine-free media was prepared withglutamine-free RPMI 1640 (Sigma-Aldrich). Glucose-free media wasprepared with a combination of PBS and glucose-free RPMI 1640 (LifeTechnologies; 15% PBS, 85% RPMI 1640) and supplemented with 10%dialyzed FBS containing ,50 mg/ml glucose by enzymatic assay (glucose[HK] assay; Sigma-Aldrich), 0.05 mM 2-ME, 2 mM L-glutamine (finalconcentration 4 mM), and antibiotics. For culture of cells in glucose orglutamine-free media, cells were washed in an excess of PBS and thenbrought up in the appropriate media prior to culture. Inhibitors were ob-tained from Sigma-Aldrich, including oligomycin A, antimycin A, eto-moxir, and 2-deoxy-glucose (2DG), and added to cultures at the indicatedconcentrations. Galactose, galactose oxidase, and H202 were obtained fromSigma-Aldrich and added at the indicated concentrations.

Abs and NK cell stimulation assays

Fluorochrome-conjugated Abs recognizing NK1.1 (PK136; BD Bio-sciences), NKp46 (29A1.4; BD Biosicences), CD3 (145-2C11; Bio-Legend), and IFN-g (XMG1.2; BioLegend) were used to identify NKcell production of IFN-g. Viability was assessed by staining cells withZombie Yellow Stain (BioLegend) or Fixable Yellow Dead Stain (LifeTechnologies). NK cells were stimulated with 10 ng/ml murine IL-12(PeproTech), 10 or 100 ng/ml murine IL-15 (PeproTech), 50 ng/mlmurine IL-18 (MBL), 10 ng/ml PMA (Sigma-Aldrich), and/or 500ng/ml calcimycin (Sigma-Aldrich). Where indicated, NK cells werecultured in low-dose IL-12 (1 ng/ml) and IL-18 (1 ng/ml). Ab stimulationwas performed by culturing enriched NK cells in plates coated with20 mg/ml purified anti-NK1.1 (PK136; BioXcell), anti-Ly49D (4E4;courtesy of W. M. Yokoyama and prepared by the Rheumatic DiseasesCore Center), or IgG control (BioXcell). Intracellular IFN-g productionwas assayed using BD Cytofix/Cytoperm (BD Biosciences), as recom-mended, with brefeldin A added to cells after 1 h of culture to inhibitsecretion of IFN-g. For in vivo priming of NK cells, wild-type mice wereinjected i.p. with 300 mg polyinosine-polycytidylic acid [poly(I:C)HMW; InvivoGen] or control PBS, and splenocytes were harvested 14–16 h later.

Quantitative RT-PCR

NK cells (2–3 3 105) were activated with 1 ng/ml IL-12 and IL-18 orplate-bound anti-NK1.1 in the presence or absence of oligomycin (100nM). Cells were harvested between 0.5 and 6 h. Control cells were culturedin media or with plate-bound isotype control IgG. RNA was isolated fromNK cells (TRIzol, Life Technologies or RNeasy Mini, Qiagen), and cDNAwas generated with random hexamers (Promega). Ifng primers/probe (16)were from IDT, and b-actin primer/probe set was from Applied Bio-systems. Copy numbers of transcript were quantitated by generation ofplasmid clones of Ifng and b-actin amplicons for use as standards andquantitated by real-time quantitative PCR (TaqMan, 7500 Fast Real-TimePCR instrument; Life Technologies).

NK cell proliferation assays

NK cells were labeled with 1 mM CFSE or VioletTrace (Invitrogen) andcultured in 96-well plates with the indicated concentrations of murineIL-15. For in vivo proliferation, splenocytes from CD45.1+ Rag-12/2 mice(2–5 3 105/mouse) were labeled with CFSE and adoptively transferredby tail vein injection into congenic CD45.2+ Rag-22/2gc

2/2 hosts and assayed3 d later.

Flow cytometric analysis and statistics

Flow cytometric analysis was performed on a Cytek-modified (CytekDevelopment) eight-color BD FACScan or BD FACSAria Fusion (BDBiosciences). Analysis was performed using FlowJo software (Tree Star).Statistical analysis was performed with GraphPad Prism 6 (GraphPadSoftware). Student’s paired t test was used to compare two matchedgroups, or ANOVA analysis was performed for more than two groups witha p value ,0.05 considered significant.

ResultsNK cells primarily use glucose-fueled OXPHOS at rest andwith activation

To determine the basic metabolic profile of NK cells, we used anextracellular flux assay to measure OCR (a measure of OXPHOS)and ECAR (a measure of lactate and anaerobic glycolysis) offreshly isolated murine splenic NK cells (Fig. 1A, 1B). Baselinemetabolic activity of resting splenic NK cells was relatively low,consistent with another recent report (17). At rest, NK cellspreferentially use OXPHOS, as shown by the OCR:ECAR ratio.Short-term activation (4–6 h) with cytokines or Abs recognizingthe activating receptors NK1.1 or Ly49D did not induce sub-stantial changes in energy pathway usage. NK cell intracellularATP was also stable following activation with IL-12 plus IL-18 oranti-NK1.1 (Fig. 1C), suggesting that these activation signals donot significantly increase or deplete ATP. Inhibition of OXPHOSwith the ATP synthase inhibitor oligomycin, or inhibition of glucosemetabolism by 2DG, a competitive inhibitor of glycolysis, signifi-cantly reduced ATP in activated NK cells (Fig. 1C). These resultssuggest that glucose is the primary OXPHOS fuel used duringNK cell activation, because blockade of glucose metabolism re-duced intracellular ATP to the same degree as global inhibition ofOXPHOS. Consistent with this hypothesis, inhibition of fatty acidswith etomoxir, a fatty acid oxidation inhibitor that blocks carnitinepalmitoyltransferase-1, had no effect on NK cell ATP (Fig. 1C).

OXPHOS is required for receptor-stimulated NK cell IFN-g

Because NK cells primarily use OXPHOS, we hypothesized thatinhibition of this pathway might alter NK cell function. We testedthe ability of fresh splenic NK cells to produce IFN-g whenstimulated with cytokines or activating receptors in the presenceof increasing concentrations of oligomycin. NK cell survival wasintact during all assays, but was significantly reduced if NK cellswere cultured in the presence of oligomycin for .12 h (data notshown). Remarkably, treatment with 10 nM oligomycin almostcompletely abolished receptor-stimulated NK cell IFN-g produc-tion, but had minimal effect on IL-12 plus IL-18–stimulated IFN-gproduction (Fig. 2A). Inhibition of receptor-stimulated IFN-g byoligomycin was dose dependent (Fig. 2B), whereas there was onlyminimal decrease in IFN-g production by IL-12 plus IL-18–stimulated NK cells when cultured with up to 1 mM oligomycin(Fig. 2B and data not shown). The mean fluorescence intensity ofIFN-g in cytokine-producing NK cells was similar between un-treated and treated cells, suggesting that metabolic inhibition didnot alter strength of activation with IL-12 plus IL-18 (Fig. 2C). Inaddition, there was no decrease in NK1.1 expression with oligo-mycin that might account for the inability to stimulate NK cellsvia this receptor (data not shown).

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Preservation of IFN-g response with OXPHOS inhibition wasunique to IL-12 plus IL-18 stimulation, because activation withanother cytokine combination (IL-12 + IL-15) or with PMA andcalcimycin, a stimulus that results in a percentage and strength ofIFN-g response similar to IL-12 plus IL-18, was impaired. How-ever, the most profound defects in NK cell activation were inresponse to activating receptors, NK1.1 and Ly49D (Fig. 2D,normalized to maximum signal for each stimulus). Similar resultswere obtained with antimycin, an inhibitor of the electron transportchain and OXPHOS (data not shown), confirming that OXPHOS iscritical for receptor-mediated activation of NK cells.

OXPHOS results in the production of reactive oxygen species(ROS), which have been shown to be signaling molecules, in-cluding for Ag-specific expansion of T cells and IL-2 production(18). Whereas oligomycin inhibits OXPHOS, it has also beenshown to increase ROS (19). We therefore tested whether additionof ROS affected receptor-mediated NK cell activation. There wasno change in the percentage of IFN-g–positive NK cells afterNK1.1 stimulation when the culture media was supplemented withROS (Supplemental Fig. 1) with or without oligomycin. Thesedata suggest that ROS do not inhibit or enhance receptor-mediatedNK cell activation.

FIGURE 1. Metabolism of resting and activated NK cells. Extracellular flux assays were used to measure resting and activated NK cell OCR, a measure

of mitochondrial OXPHOS, and ECAR, a measure of glycolysis. (A) Cytokine activation (4 h) or (B) receptor stimulation (6 h) did not significantly change

OCR, ECAR, or the OCR:ECAR ratio. Results represent the mean 6 SEM of triplicate wells from three independent experiments. (C) Intracellular ATP

(pM/cell) after 6-h culture of NK cells with cytokines or plate-bound anti-NK1.1 in complete media (–) or with the metabolic inhibitors oligomycin (oligo,

1 mM), etomoxir (300 mM), and 2DG (50 mM). Statistics represent the comparison between stimulation alone versus stimulation with the indicated in-

hibitor for IL-12 plus IL-18 or anti-NK1.1–activated NK cells (one-way paired ANOVA). Results represent the mean 6 SEM of triplicate wells from four

independent experiments. *p # 0.05, **p # 0.01.

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Glucose-dependent NK cell activation

Our findings suggest that metabolism functions as a second signal forreceptor activation. We considered the possibility that the differencesobserved in IL-12 plus IL-18 versus receptor stimulation withmetabolic inhibitors were due to the high number of IFN-g–positivecells with this cytokine combination (.90% IFN-g+) as comparedwith receptor stimulation (∼30–40% IFN-g+). NK cells were

therefore cultured in low concentrations (1 ng/ml) of IL-12 and

IL-18. This resulted in a similar percentage of IFN-g–producing

NK cells as anti-NK1.1 stimulation, but, similar to data with

higher dose cytokines, there were no defects in IFN-g production

with oligomycin (Fig. 3A).Because we observed minimal effect of OXPHOS inhibition,

even with low-dose IL-12 plus IL-18 activation, we hypothesized

FIGURE 2. Inhibition of mitochondrial OXPHOS

significantly impairs receptor-stimulated NK cell IFN-g

production. (A–C) NK cells were stimulated for 6 h

with anti-NK1.1 or IL-12 plus IL-18 with or without

the OXPHOS inhibitor oligomycin. (A) Representative

flow plot of IFN-g production by NK cells in response

to anti-NK1.1 (top) or IL-12 plus IL-18 (bottom)

stimulated without (left) or with (right) 10 nM oligo-

mycin. (B) Percentage of NK cells producing IFN-g

measured by intracellular flow cytometry in response to

IL-12 plus IL-18 (left) or anti-NK1.1 (right) in the

presence of increasing doses of oligomycin. (C) Geo-

metric mean fluorescence intensity (MFI) of IFN-g

measured in IFN-g+ NK cells in the presence of oli-

gomycin compared with no inhibitor (–). (D) Percent-

age of maximal IFN-g–positive NK cells following 4-

to 6-h culture with 100 nM oligomycin normalized to

percentage of IFN-g+ NK cells without inhibitor for

each stimulus (cytokines [IL-12 + IL-15 or IL-12 + IL-

18], receptors [anti-NK1.1 or anti-Ly49D], or PMA +

calcimycin). Results represent the mean 6 SEM or

individual data points in (C), from three to four inde-

pendent experiments. *p # 0.05, **p # 0.01. na, not

available (too few events); ns, not significant.

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that NK cells might require aerobic glycolysis to respond to thisstimulus. However, culture of NK cells in glucose-free mediaresulted in minimal decreases in IFN-g–producing NK cells withlow-dose IL-12 plus IL-18, but significant defects with anti-NK1.1(Fig. 3A) or anti-Ly49D (Supplemental Fig. 2). In contrast toOXPHOS inhibition, glycolytic inhibition more selectively inhib-ited receptor-stimulated IFN-g because there were only modestdecreases in PMA/calcimycin or IL-12 plus IL-15 activation

(Supplemental Fig. 2). We tested the ability of NK cells to useother OXPHOS fuels by extracellular flux assay. Indeed, inhi-bition of glucose metabolism with 2DG resulted in decreasedglycolysis (measured by ECAR) with compensatory increasednonglucose-fueled OXPHOS (measured by OCR) (SupplementalFig. 2). However, inhibition of alternative OXPHOS fuels, fattyacids and glutamine, had minimal effect on receptor activation(Supplemental Fig. 3). These findings suggest that glucose is theprimary OXPHOS fuel required for receptor activation.

IL-12 plus IL-18 stimulation is metabolism independent

Having confirmed that IL-12 plus IL-18 stimulation is independentof glucosemetabolism or OXPHOS at low- or high-dose stimulation,we next evaluated whether cytokines, but not receptors, inducemetabolic plasticity and allow NK cells to efficiently use eitherglycolysis or mitochondrial OXPHOS. To test this, NK cells werecultured with inhibitors of both glycolysis and OXPHOS. BecauseATP is required for cell survival, NK cells did not live when culturedin the presence of both oligomycin and 2DG (data not shown).Therefore, NK cells were activated with low concentrations of IL-12plus IL-18 in the presence of two different doses of the glycolyticinhibitor 2DG (10 or 50 mM) plus etomoxir in glutamine-free mediato simultaneously block glycolysis and the other two major mito-chondrial OXPHOS fuels (Fig. 3B). Consistent with the hypothesisthat NK cells can produce IFN-g relatively independent of metab-olism, NK cell IFN-g production with IL-12 plus IL-18 was com-pletely preserved when both glycolysis and each of the other majorOXPHOS fuels, fatty acids or glutamine, were blocked (Fig. 3B).By contrast, inhibition of glycolysis and the other major fuels ofOXPHOS led to a significant impairment in anti-NK1.1–mediatedNK cell activation (Fig. 3C), which was not significantly differentfrom oligomycin or glucose-free media alone (Fig. 3A, 3C).We next tested whether IL-12 plus IL-18 induced a glycolytic

switch upon treatment with oligomycin that was absent in receptor-stimulated NK cells. Oxygen consumption (OCR) decreased andglycolysis (ECAR) increased with oligomycin treatment, as ex-pected (Fig. 4A–D). However, there was no difference in OCR orECAR readings from oligomycin-treated NK cells with or withoutstimulation (IL-12 plus IL-18 or NK1.1). Similarly, the OCR:ECAR ratios of oligomycin-treated cells stimulated with IL-12plus IL-18 or anti-NK1.1 were equivalent (Fig. 4E). Together,these data suggest that IL-12 plus IL-18 does not induce signifi-cant metabolic plasticity that would account for the observedmetabolism-independent activation.

IL-12 plus IL-18 activation of NK cell IFN-g production istranscriptionally regulated

To determine whether OXPHOS inhibition affected receptor-mediated transcription of Ifng, we measured levels of transcriptin IL-12 plus IL-18 or anti-NK1.1–stimulated NK cells with orwithout oligomycin at different time points by quantitative RT-PCR (Fig. 5A, 5B). There was delayed transcription of Ifng inlow-dose IL-12 plus IL-18–stimulated NK cells with oligomycin,which normalized by 4 h (Fig. 5A). However, whereas the per-centage and mean fluorescence intensity of IFN-g–producing NKcells were similar between low-dose IL-12 plus IL-18 and anti-NK1.1–activated NK cells at 6 h (Fig. 5C and data not shown),receptor stimulation resulted in very little upregulation of Ifngtranscript (Fig. 5B). The low levels of Ifng transcription weredelayed with oligomycin in NK1.1-activated NK cells, and, sim-ilar to cytokine stimulation, normalized by 4 h. These data suggestthat production of IFN-g protein is transcriptionally regulated withcytokine activation, but posttranscriptionally regulated with re-ceptor stimulation. Consistent with this hypothesis, IL-12 plus IL-

FIGURE 3. Receptor stimulation is also dependent on glycolysis,

whereas IL-12 plus IL-18 activation is metabolism independent. (A) NK

cells were stimulated for 6 h with low-dose IL-12 plus IL-18 (1 ng/ml

each) or anti-NK1.1 in complete media (black), in the presence of 100 nM

oligomycin (white), or in glucose-free media (gray). (B and C) NK cells

were stimulated for 6 h with (B) low-dose IL-12 plus IL-18 (1 ng/ml each)

or (C) anti-NK1.1 in complete media (–) or in glutamine-free (Glut-free)

media with 10 or 50 mM 2DG plus an inhibitor of fatty acid oxidation

(etomoxir). Results represent the mean 6 SEM of four independent

experiments. *p # 0.05, **p # 0.01. ns, not significant.

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18–stimulated NK cells had delayed production of IFN-g proteinwith oligomycin, which normalized by 6 h. By contrast, there was

very little production of IFN-g protein with oligomycin treatment

of anti-NK1.1–stimulated NK cells, even at 6 h, when transcript

levels of Ifng were the same. Thus, although oligomycin had a

similar effect on Ifng transcription for both stimuli (i.e., delayed

upregulation), there are significant differences in the transcrip-

tional and posttranscriptional control of IFN-g protein production

between the two stimuli.

Short-term cytokine priming cannot compensate for theabsence of an OXPHOS signal for receptor activation

To determine whether IL-12 or IL-18 could prime cells to respondto receptors independent of metabolism, fresh NK cells werepretreated with different combinations of cytokines, followedby activation with anti-NK1.1 with or without oligomycin(Fig. 6A). All cells were cultured with IL-15 (10 ng/ml low doseor 100 ng/ml high dose) to maintain survival. Although the base-

line IFN-g production with NK1.1 stimulation was higher with

FIGURE 4. IL-12 plus IL-18 stimulation does not induce a significant metabolic switch in NK cells. Extracellular flux analysis was used to measure OCR

and ECAR of NK cells following 6-h culture with (A and C) IL-12 plus IL-18 or (B and D) anti-NK1.1, with or without oligomycin (100 nM). There was no

significant difference in OCR or ECAR of oligomycin-treated NK cells in the presence or absence of cytokine or receptor stimulation. Statistics over error

bars represent comparison with untreated cells (first bar); statistics above the line represent comparison of oligomycin treatment to oligomycin plus NK cell

activation (one-way ANOVA). (E) OCR:ECAR ratio of receptor- or cytokine-stimulated NK cells. Results represent the mean 6 SEM of three independent

experiments. *p # 0.05, **p # 0.01. ns, not significant.

FIGURE 5. Differences in Ifng transcription with

oligomycin treatment. NK cells were stimulated with

low-dose IL-12 plus IL-18 (1 ng/ml each) or anti-

NK1.1 in the presence or absence of oligomycin

(oligo). (A and B) Absolute copy number of Ifng and

b-actin (Bact) was quantitated by real-time PCR be-

tween 0.5 and 6 h. Results represent the ratio of Ifng to

Bact 3 100 and the mean 6 SEM of duplicate wells

from three to four independent experiments. Statistics

represent differences between activated NK cells with-

out or with oligomycin. (C) Intracellular IFN-g pro-

tein in the presence of brefeldin A was measured by

flow cytometry at the same time points. Results rep-

resent the mean 6 SEM of five independent experi-

ments. Statistics represent differences between activated

NK cells without or with oligomycin for each stim-

ulus (IL-12 + IL-18 or anti-NK1.1). *p # 0.05, **p #

0.01, ***p # 0.001, ****p # 0.0001 (two-way

ANOVA analysis).

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cytokine priming, there was still a defect in activation in thepresence of oligomycin (Fig. 6A). Similar results were obtainedwhen cytokines were added at the start of receptor stimulation (datanot shown). Thus, these results suggest that receptor-mediated ac-tivation of NK cells requires a metabolically derived second signalthat is not required for, or stimulated by, IL-12 or IL-18 activation.

NK cell metabolic requirements for receptor stimulation arereversed with prolonged IL-15 priming

IL-15 is critical for NK cell differentiation and survival and alsoprimes NK cells for cytotoxicity and in vivo effector functions (4,20–22). Long-term treatment with high-dose IL-15 or IL-2 dif-ferentiates lymphokine-activated killer cells that have enhancedantitumor responses (23). We hypothesized that one mechanismby which IL-15 might enhance and prime NK cell function is byaltering the metabolic requirements for NK cell activation. Indeed,following 72-h culture with 100 ng/ml IL-15, NK cells had nodefect in IFN-g production with OXPHOS inhibition in responseto cytokine, receptor, or PMA and calcimycin activation (Fig. 6B).

A small difference in receptor stimulation without glucose per-sisted, and activation continued to be independent of fatty acidoxidation or glutamine (Fig. 6C). High-dose, 100 ng/ml, IL-15was required to induce metabolism-independent activation, aslower-dose (10 ng/ml) IL-15 had no effect on the metabolic re-quirement for receptor activation (Fig. 6D). Short-term stimula-tion was not sufficient to induce this effect, because there was nochange in the metabolic requirements after overnight treatmentwith 100 ng/ml IL-15 (Fig. 6A), and at least 48 h of stimulationwas required (data not shown).One major difference between stimulation with 10 versus 100

ng/ml IL-15 is that cells proliferate only with the higher dose (datanot shown). Thus, it is possible that the changes in metabolicrequirements were due to proliferation and not IL-15 treatment.However, when comparing IFN-g production by the least-dividedversus most-divided NK cells, as marked by CFSE dilution, bothpopulations produced normal amounts of IFN-g with oligomycin,and, in fact, we consistently observed higher IFN-g production inundivided cells (Fig. 6E). Furthermore, adoptive transfer of CFSE-

FIGURE 6. Exposure to high-dose IL-15, but not proliferation, alters the metabolic requirements for receptor-stimulated IFN-g production. (A) NK cells

were incubated for 14–16 h with IL-15 (10 or 100 ng/ml), IL-12, and/or IL-18; washed; and then stimulated for 6 h with anti-NK1.1 or control IgG in the

presence or absence of oligomycin (Oligo). Results represent the mean 6 SEM from four independent experiments; statistics represent comparison of anti-

NK1.1 versus anti-NK1.1 plus oligomycin. (B and C) NK cells were cultured for 72 h with 100 ng/ml IL-15, washed, and activated (6 h) with cytokines,

PMA plus calcimycin (PMA + Ca), or receptors (anti-NK1.1, anti-Ly49D, or control IgG) in the presence of (B) oligomycin (100 nM) or (C) glucose-free

(GF) media, glutamine-free media, etomoxir, or glutamine-free media. Results represent the mean 6 SEM from three independent experiments. (D) NK

cells were cultured for 72 h with 10 or 100 ng/ml IL-15, followed by anti-NK1.1 stimulation in the presence or absence of oligomycin. (E) Production of

IFN-g by highly divided and undivided NK cells, as measured by CFSE dilution following IL-15 activation (100 ng/ml). Results for (D) and (E) are

representative of three independent experiments. (F) OCR and ECAR of NK cells treated with IL-15 (100 ng/ml) for 72 h compared with freshly isolated

NK cells rested in media for 4 h. Results represent the mean 6 SEM of three to six replicate wells from two independent experiments. *p # 0.05, **p #

0.01, ****p # 0.0001.

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labeled splenocytes into alymphoid Rag22/2gc2/2 hosts and in-

duction of homeostatic proliferation had no effect on the meta-bolic requirement for IFN-g production (Supplemental Fig. 4). Wealso tested whether the TLR3 analog poly(I:C), which upregulatesdendritic cell IL-15/IL-15ra (4), could prime NK cell metabolicindependence. However, defects persisted in receptor stimulationof metabolically inhibited NK cells after in vivo activation over-night with poly(I:C) (Supplemental Fig. 4D), although to a lesserdegree than naive NK cells.Finally, we evaluated whether treatment with high-dose IL-15

affected NK cell metabolism. Indeed, NK cells significantly up-regulated OXPHOS and glycolysis as measured by OCR andECAR (Fig. 6F) after culture in high-dose IL-15, consistent withanother recent study (17). Although both metabolic pathways in-creased, there was a decreased dependence on OXPHOS in IL-15–treated NK cells, as measured by a decreased ratio of OCR:ECAR.

DiscussionMetabolism is critical for a wide array of cellular functions andhas been increasingly recognized to be important for immune cellfunction (12). To our knowledge, this represents the first study toinvestigate the metabolic fuels used by fresh NK cells for pro-duction of IFN-g. Remarkably, we demonstrate that a metabolism-driven second signal is required for receptor-mediated, but notIL-12 plus IL-18–stimulated, activation of NK cell IFN-g.NK cells have the ability to rapidly produce large amounts of

IFN-g protein within hours of activation. Surprisingly, NK cellswere relatively metabolically inactive at baseline and had nosignificant increase in their OXPHOS or glycolysis after short-term activation, as measured by an extracellular flux assay. How-ever, despite no increased metabolic activity with stimulation,inhibition of glucose/glycolysis or global OXPHOS inhibition re-sulted in near-complete abrogation of receptor-stimulated IFN-gproduction. The receptors tested in this study, NK1.1 and Ly49D,both partner with ITAM-bearing adapters (24), suggesting thatOXPHOS is a requisite second signal for ITAM-mediated IFN-gproduction. Inhibition of other OXPHOS fuels, including gluta-mine or fatty acid oxidation, had no effect on receptor-stimulatedIFN-g production, and glucose appears to be the primary fuelrequired to drive OXPHOS.The activation-specific metabolic requirements for NK cell

IFN-g production shown in this work are quite distinct fromT cells, which require glycolysis, but not mitochondrial OXPHOS,for production of IFN-g in response to receptors, PMA and ion-omycin, or IL-12 plus IL-18 (15, 25). In this study, we discovereda major difference in the transcriptional upregulation of Ifng be-tween cytokine- and receptor-stimulated NK cells. It is well de-scribed that NK cells constitutively express Ifng transcript, butnot protein (26). Thus, it is likely that one mechanism by whichmetabolic inhibition impairs receptor-stimulated, but not IL-12plus IL-18–stimulated, IFN-g protein production is by inhibitionof translation of pre-existing transcript. In CD4 T cells, glycolysiswas shown to be important for the posttranscriptional processingof IFN-g due to consumption of the glycolytic enzyme GAPDH,which was otherwise bound to the 39 untranslated region of IFN-gtranscript (15). However, in contrast to our findings in this work,inhibition of OXPHOS with oligomycin had no effect on receptor-stimulated T cell production of IFN-g (15). Thus, consumption ofGAPDH is unlikely to explain our observed dependence on bothglycolysis and OXPHOS for NK cell receptor-stimulated IFN-gproduction. The results in this work suggest distinct cell-specificregulation of IFN-g protein production by innate and adaptiveeffector cells. However, one caveat to this conclusion is that NK

cells were studied directly ex vivo in this work, whereas priorwork with T cells was performed after in vitro culture/activation;thus, it is possible that activation alters T cell metabolic require-ments for IFN-g production.Prolonged culture of NK cells with high-dose IL-15 led to in-

creased NK cell metabolism and particularly glycolysis, as mea-sured by a decrease in the OCR:ECAR ratio. Cells cultured for.48 hin high-dose IL-15 no longer required OXPHOS for receptor-stimulated IFN-g production, an effect that was dose dependentand did not require proliferation. Interestingly, IL-15–stimulatedNK cells still demonstrated some dependence on glucose for IFN-gproduction. Perhaps this is a reflection of their overall increaseddependence on glycolysis for cellular metabolism, or, alternatively, anindication that they have switched to a regulatory mechanism moresimilar to T cell control of IFN-g posttranscriptional processing.Alterations of the metabolic requirements of NK cell activation withIL-15 are clinically relevant, because there are ongoing clinical trialsof IL-15 treatment of NK cells prior to adoptive immunotherapy, aswell as administration of IL-15 for cancer therapies targeting NKcells (27). Our data would suggest that, in addition to priming andexpansion of NK cells, IL-15 may also impart NK cells with en-hanced functionality in metabolically deprived locations such as tu-mor microenvironments.IL-15 is known to prime NK cell effector functions, including

cytokine production and cytotoxicity (4, 20). Recently, the Walzerlaboratory demonstrated that high-dose IL-15 activates mTOR,which stimulates NK cell glucose uptake, proliferation, and cyto-lytic responses (17). Similarly, Nandagopal et al. (28) reported thatIL-15–induced mTOR is important for NK cell IFN-g productionwhen costimulated with this cytokine. mTOR is an important met-abolic regulator of T cell function and upregulates glucose uptakeand glycolysis in activated T cells (29). Another recent study alsodemonstrated a dependence on glycolysis, but not OXPHOS, forIL-12 plus IL-2–stimulated IFN-g production by NK cells culturedin IL-15 for 7 d, and implicated IL-15–induced mTOR for glyco-lytic programming of NK cells (30). Thus, IL-15–induced mTORmay represent a potential mechanism whereby NK cells culturedwith high-dose IL-15 upregulate glycolysis. However, it remains un-clear how prolonged high-dose IL-15 signaling and increasedglycolysis alter the OXPHOS requirement for receptor-mediatedsignaling, as shown in this work.In summary, these findings demonstrate that NK cell IFN-g re-

sponses are dictated by the metabolic environment in an activation-specific manner. This suggests that NK cell function in vivo willbe affected by the availability of metabolic fuels and that drugstargeting metabolism, such as glycolytic inhibitors, will impactNK cell activation. The finding that prolonged high-dose IL-15alleviates the metabolic requirement for receptor-mediated NKcell stimulation suggests a potential mechanism to reverse themetabolic dependence of receptor activation. Additional investi-gation into the glucose-driven OXPHOS signal that is required forreceptor signaling, and how this signal interacts with known re-ceptor signaling pathways, will be important (31). Finally, it willbe interesting to explore the metabolic requirements for other NKcell functions, including cytotoxicity, licensing, and generation ofmemory NK cells (32–34).

AcknowledgmentsWe thank T. A. Fehniger and W. M. Yokoyama for helpful discussion and

reagents.

DisclosuresThe authors have no financial conflicts of interest.

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