restriction c57bl/6 mice are altered by caloric nk cell maturation

of April 8, 2018.This information is current as

RestrictionC57BL/6 Mice Are Altered by Caloric NK Cell Maturation and Function in

Elizabeth M. GardnerJonathan F. Clinthorne, Eleni Beli, David M. Duriancik and

http://www.jimmunol.org/content/190/2/712doi: 10.4049/jimmunol.1201837December 2012;

2013; 190:712-722; Prepublished online 14J Immunol 

Referenceshttp://www.jimmunol.org/content/190/2/712.full#ref-list-1

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The Journal of Immunology

NK Cell Maturation and Function in C57BL/6 Mice AreAltered by Caloric Restriction

Jonathan F. Clinthorne, Eleni Beli, David M. Duriancik, and Elizabeth M. Gardner

NK cells are a heterogenous population of innate lymphocytes with diverse functional attributes critical for early protection from

viral infections. We have previously reported a decrease in influenza-induced NK cell cytotoxicity in 6-mo-old C57BL/6 calorically

restricted (CR) mice. In the current study, we extend our findings on the influence of CR on NK cell phenotype and function in the

absence of infection. We demonstrate that reduced mature NK cell subsets result in increased frequencies of CD127+ NK cells in CR

mice, skewing the function of the total NK cell pool. NK cells from CR mice produced TNF-a and GM-CSF at a higher level,

whereas IFN-g production was impaired following IL-2 plus IL-12 or anti-NK1.1 stimulation. NK cells from CR mice were highly

responsive to stimulation with YAC-1 cells such that CD272CD11b+ NK cells from CR mice produced granzyme B and degranu-

lated at a higher frequency than CD272CD11b+ NK cells from ad libitum fed mice. CR has been shown to be a potent dietary

intervention, yet the mechanisms by which the CR increases life span have yet to be fully understood. To our knowledge, these

findings are the first in-depth analysis of the effects of caloric intake on NK cell phenotype and function and provide important

implications regarding potential ways in which CR alters NK cell function prior to infection or cancer. The Journal of Immu-

nology, 2013, 190: 712–722.

Caloric restriction (CR) is a dietary intervention that hasbeen shown to extend the life span of laboratory animals(1). Whereas excess energy intake has been associated

with increased incidence of disease, CR has been found to decreasethe severity of autoimmune disease, and decrease the incidence ofcardiac, kidney, or nervous system dysfunction (1–4). Other ben-efits of CR include decreased triglycerides and blood pressure,lower central adiposity, improved insulin sensitivity, and delayedage-related immunosenescence (5, 6). It has been established that,in laboratory conditions, CR reduces the incidence of spontaneoustumors and cancers in aged rodents, and slows the age-relateddecline in T cell proliferation, cytokine production, and CTL ac-tivity that is often observed during aging (7, 8). Lifelong CR ofmice preserves thymopoiesis in the face of aging, and has beenshown to enhance influenza-specific Abs and splenic lymphocyteproliferation after vaccination of mice with influenza (8, 9). Thesebeneficial changes to the adaptive immune system have been wellcharacterized; however, it has also been found that CR influencesinnate immune function (10, 11). Several decades ago, Weindruchet al. (12) reported that CR resulted in decreased splenic NK cellcytotoxicity compared with aged matched controls, although thiscould be ameliorated by polyinosinic:polycytidylic acid. Morerecently, we have shown CR results in increased susceptibility toprimary influenza infection and decreased influenza-induced NK

cell cytotoxicity in young and aged mice (13, 14). This was ac-companied by the observation that NK cell numbers and frequencyare decreased in the spleen of young CR mice (14). Overall, thesefindings have raised concerns about the effects of CR on innateimmunity, and may predispose CR individuals to suffer more se-vere primary infections (10, 15). However, at this time, few studieshave focused on understanding the effects of CR on innate immunecell development and function.NK cells are responsible for recognizing virally infected cells, as

well as transformed cells, including neoplasms and tumor cells(16–18). Development of NK cells takes place mainly in the bonemarrow (BM), and signals from stromal cells and cytokines resultin the microenvironment required for NK cell generation (19, 20).NK cell commitment takes place through upregulation of theshared IL-2/IL-15R b-chain (CD122), followed by acquisition ofthe NK cell marker NK1.1 in B6 mice (19, 21). Interactions withstromal cells within the BM regulate gene expression leading toprogrammed expression of surface molecules, including integrins,cytokine receptors, and a family of NK cell receptors (22–25). NKcell maturation is classified by using both surface phenotype andfunctional capacity. Phenotypic maturation takes place in stepwisefashion; expression of the integrin CD49b (DX5) is used to defineearly mature NK cells (26). Following acquisition of DX5, NKcells in the BM upregulate CD11b and CD43, which correlatesstrongly with the capability of a NK cell to produce large amountsof IFN-g (19). Once mature, NK cells seed various lymphoid andnonlymphoid peripheral tissues, with the majority of NK cellsexpressing high levels of DX5, CD11b, and CD43 (19, 27).After emigrating from theBMvia the blood and seeding peripheral

tissues, DX5+ NK cells continue to adapt to their environment; thedownregulation of CD27 and TRAIL and upregulation of killer celllectin-like receptor G1 (KLRG1) are associated with peripheral NKcell maturation (28, 29). The application of the marker CD27 hasallowed the DX5+ NK cell pool to be further divided into subsets inmice in which there is a linear progression from CD27+CD11b2

early mature NK cells to CD27+CD11b+ (double-positive [DP]) NKcells, followed by development into CD272CD11b+ NK cells. Thesephenotypic changes further reflect changes to NK cell function, as

Department of Food Science and Human Nutrition, Michigan State University, EastLansing, MI 48824

Received for publication July 3, 2012. Accepted for publication November 13, 2012.

This work was supported by National Institutes of Health Grant R01AG034949-01A1(to E.M.G.).

Address correspondence and reprint requests to Dr. Elizabeth M. Gardner, Depart-ment of Food Science and Human Nutrition, Michigan State University, 234C GMTrout Building, East Lansing, MI 48824. E-mail address: egardner@msu.edu

Abbreviations used in this article: AL, ad libitum; BM, bone marrow; CR, caloricrestriction; DN, double-negative; DP, double-positive; Eomes, eomesodermin;KLRG1, killer cell lectin-like receptor G1; LN, lymph node; MRI, magnetic reso-nance imaging; mTOR, mammalian target of rapamycin; NIA, National Institute onAging; PEM, protein energy malnutrition.

Copyright� 2013 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201837

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DP NK cells exhibit a greater responsiveness to in vitro culture withdendritic cells, whereas CD272CD11b+ KLRG1+ NK cells are ter-minally differentiated NK cells that are tightly regulated with re-duced capacity to proliferate or elicit effector function during viralinfection, such as murine CMV (29, 30).Recent studies have begun to identify the molecular mechanisms

that regulate NK cell development, egress, and differentiation. Thesestudies have identified transcription factors required for NK cellcommitment and differentiation into mature NK cells. Among thesetranscription factors, ID2, TOX, and E4BP4 are thought to regulatethe earliest developmental stages, whereas other studies haveestablished a role for the transcription factors T-bet, Eomesodermin(Eomes), Blimp-1, IFN regulatory factor-2, and Gata-3 in thegeneration of terminally differentiated CD43+ KLRG1+ NK cells(31–34). Similar to their role in T cell function, several of thesetranscription factors regulate aspects of NK cell function. Forexample, T-bet is involved in the expression of granzyme B in NKcells, whereas NK cells lacking Eomes are capable of producingmore TNF-a than their Eomes+ counterparts (31).Based on the above studies, we sought to understand how CR

impacts NK cell development and function independent of aging orinfection in 6-mo-old mice. In this study, we show that NK cells arereduced in frequency in peripheral tissues and exhibit an alteredphenotype in the spleens of CR mice. We characterize these changesby analyzing the expression of a variety of cell surface markersassociated with the maturation process, such as CD94, CD127, DX5,CD11b, CD43, and KLRG1. Most studies of murine NK cells focuson splenocytes, which normally express low levels of CD127.However, we discovered that CR results in an increased fraction ofNK cells that express CD127 in the spleen and lymph nodes (LN),accompanied with an increase in the frequency of NK cells in thethymus, of which the majority are CD127+. These cells are thoughtto have low cytotoxic potential and produce a variety of cytokines,and are normally found at the highest frequency in the thymus andLN. Comparison of the distribution of NK cell subsets between ALand CR mice revealed CR results in significant reductions to theCD272CD11b+ NK cell pool, suggesting caloric intake plays a rolein regulating peripheral NK cell maturation or homeostasis. We usedvarious stimuli to test the functional competence of NK cells fromCR mice, and found that alterations to NK cell function in CR miceare specific to the stimulus used. Our results suggest that CR sig-nificantly alters NK cell subset distribution, resulting in a heteroge-neous pool of NK cells displaying unique functional characteristics.

Materials and MethodsMice and diets

Specific pathogen-free young adult (6-mo) ad libitum (AL) and young adult(6-mo) CR male C57BL/6 mice were purchased from the National Instituteon Aging (NIA) colony maintained by Charles River Laboratories (Wil-mington, MA). The animal use protocol for this study was approved byMichigan State University Institutional Animal Care and Use Committee.Upon arrival, mice were housed individually in microisolator cages in theAmerican Association for the Accreditation of Laboratory Animal Care–accredited containment facility at Michigan State University and wereacclimated at least 10–14 d prior to the initiation of each experiment. BothCR (NIH-31/NIA-fortified) and AL (NIH-31) diets were purchased fromthe NIA, the compositions of which have been reported in detail previously(14). The composition of the CR diet is sufficient in micronutrients andminerals, but results in restriction of total energy intake. The CR regimeninitiated by the NIA is designed to gradually achieve 40% restriction inmice by 4 mo of age, such that they are weight stable upon arrival at 6 moof age. All experiments were repeated at least twice using four to five miceper diet treatment per experiment, unless otherwise noted.

Body composition, food intake, and metabolic profile

Body composition (fat, lean, water) was determined by daily magneticresonance imaging (MRI) during the feeding protocol. Mice were indi-

vidually housed, allowing food intake to be recorded each day. All micewere weighed daily between 0800 and 0900, after which they were fed. Theprotocol to assess body composition using the EchoMRI-500 (Echo MedicalSystems) has been validated and described in detail previously (35). Briefly,after calibration using a rapeseed oil standard, individual mice are placed ina MRI holding tube. The advantages of this system are that it is rapid, allowsfor repeatedmeasurements, and does not require anesthesia, enablingmice torecover immediately after MRI. Serum concentrations of corticosterone,albumin, and leptin were quantified by commercially available ELISA kits,according to the manufacturer’s instructions (Assaypro; Life Diagnostics,R&D Systems, respectively). Serum concentrations of glucose, cholesterol,and triglycerides were determined using colorimetric assays, as per themanufacturer’s instructions (Cayman Chemical). Plates were read at 450 nmwavelength using a Synergy HT plate reader (Bio-Tek), and concentrationswere determined using a standard curve for each respective assay.

Lymphocyte isolation

Following euthanasia, blood was collected by cardiac puncture into hepa-rinized syringes. Following cardiac puncture, spleens, lungs, LN (inguinal,auxiliary, and brachial), and thymus were excised and weighed. Isolation ofmononuclear cells from spleens and lungs has been previously described (35).Briefly, single-cell suspensions were obtained from spleens using homoge-nization. BM cells were isolated from the femur and tibia by flushing witha 25 5/8 needle and syringe containing RPMI 1640. The resulting cell sus-pensions were lysed of RBCs using an ammonium chloride buffer. Lungswere excised, weighed, and minced using a Miltenyi GentleMACs system.Cells were then incubated for 30 min at 37˚C in RPMI 1640 containing 5%FBS, 1 mg/ml collaganase D (Roche, Indianapolis, IN), and 80 Kuntz UnitsDNase (Roche). Cell suspensions from digested lungs or blood were dilutedwith PBS and layered onto 1083-Histopaque (Sigma-Aldrich) for isolationof mononuclear cells by density gradient centrifugation. Isolation of cellsfrom LN and thymus was performed by pressing the LN and thymus through40-mm cell strainers (BD Falcon). All cell suspensions were washed in PBSand resuspended for counting using trypan blue viability dye.

Flow cytometry

Cells from various tissues were resuspended in FACS buffer (0.1% sodiumazide, 1% FBS, in Dulbecco’s PBS) at a concentration of 23 107 cells/ml.A total of 1–43 106 cells was incubated on ice for 10 min with anti-CD32/CD16 Ab (2.4G2) (BD Biosciences) to block FcgRII/III-mediated non-specific binding. Samples were then incubated with a mixture containingvarious combinations of the following fluorochrome-conjugated Abs(eBioscience, BD Biosciences, or BioLegend) at optimal concentrationsdetermined in our laboratory: NK1.1 (allophycocyanin or PE-Cy7), CD3(Alexafluor700 [500A2]), CD94/NKG2 (PE [HP-3D9]), CD27 (PE orPerCP-eFluor710 [LG.7F9]), CD127 (PE or PerCP-Cy5.5 [A7R.34]),CD51 (biotin [RMV-7]), CD49b (allophycocyanin or PE-Cy7 [DX5]),CD11b (PE-Cy7 or V500 [M1/70]), glucocorticoid-induced TNFR-relatedprotein (FITC [DTA-1]), B220 (allophycocyanin [RA3-6B2]), CD43(allophycocyanin-Cy7 [1B11]), Ly49C/I/F/H (FITC or PE [14B11]), Ly49-G2 (allophycocyanin [4D11]), Ly49D (FITC [4E5]), Ly49H (biotin [3D10]),and KLRG1 (allophycocyanin [2F1]). Biotinylated Abs were detected usingstreptavidin-conjugated PerCP-Cy5.5 or allophycocyanin-Cy7. Cells wereincubated in staining cocktails on ice in the dark for 30 min. To detecttranscription factor expression, cells were fixed and permeabilized usingeBioscience Foxp3 staining kit, according to the manufacturer’s instructions,and then incubated with Abs against T-bet (PE-Cy7 [4B10]) and Eomes(Alexafluor488 [Dan11mag]) (eBioscience). Viable lymphocytes were gatedbased on light-scattering properties, after which NK cells were characterizedas NK1.1+ CD32, unless otherwise noted. Samples were analyzed using anLSR II flow cytometer (BD Biosciences) or a FACS Canto II flow cytometer(BD Biosciences) with FlowJo software (Tree Star).

Cytokines, granzyme B production, and degranulation

NK cell capacity to produce IFN-g and degranulate was measured usingflow cytometry, according to previously published methods (36). Briefly,high-affinity 96-well plates (Thermo-Fisher) were coated with a mAbagainst NK1.1 (25 mg/ml [PK136]) or NKp46 (15 mg/ml [29A1.4]) for18 h at 4˚C. Plates were then washed with PBS three times, and freshlyprepared splenocytes (1–4 3 106) in complete media were added. Alter-natively, splenocytes in complete media were added to uncoated 96-wellplates, and IL-2 (1000 U) plus IL-12 (10 ng/ml) or YAC-1 cells (10:1 E:Tratio) were added. Anti-CD107a (FITC or PE-Cy7 [1D4B]), a marker ofdegranulation, was also added to NK1.1 and YAC-1–stimulated splenocytecultures (36, 37). Plates were incubated for 4–8 h, during which brefeldinA and monensin were added after the first 30 min. To elicit GM-CSF and

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TNF-a production, NK cells were incubated with IL-2 (1000 U) and IL-12(10 ng/ml) for 18 h, followed by PMA (50 ng/ml) and ionomycin (1 mg/ml), with brefeldin A being added during the last 4 h to block cytokinesecretion and raise intracellular cytokine stores, as reported by Vosshenrichet al. (34). Following incubation, cells were stained with lineage-specificAbs and then fixed and permeabilized using BD cytofix/cytoperm kits,according to manufacturers’ protocol. Intracellular cytokines and gran-zyme B were detected using mAbs against IFN-g (FITC or PE-Cy7[XMG1.2]), granzyme B (PE [GB11]), TNF-a (PE [MP6-XT22]), andGM-CSF (FITC [MP1-22E9]) (BD Biosciences).

Statistics

Statistics were performed using GraphPad Prism 4 software (La Jolla, CA).Values in text are means 6 SEM. Body composition, food intake, weight,serum metabolic profile, immune cell populations, and NK cell functionwere analyzed using Student t test to determine significant differencesbetween diet groups. Statistical significance was set at p , 0.05.

ResultsPhysiological parameters influenced by CR

CR is initiated at 14 wk of age (10% restriction), and at 15 wk of agethe restriction is increased to 25%. Finally, at 16 wk, mice are feda 40% restricted diet that is maintained throughout the life of theanimal. For this study, our CR protocol supplied mice with 3 g fooddaily in the form of a vitamin- and mineral-supplemented cookiesupplied by the NIA, whereas AL mice consumed ∼4.53 g fooddaily (Fig. 1A). Our data indicate feeding CR mice with a 3 gcookie resulted in a 34% restriction rather than 40%; however, westill observed physiological changes indicating our CR protocolinduced CR characteristics, which is supported by the notion thateven mild CR (10–25%) is effective in increasing life span (1).Restriction resulted in mice with reduced body weight (Fig. 1A)achieved by a reduction in both lean and fat mass, as well as re-duced body fat percentages (Fig. 1B). True CR exists independentlyfrom protein energy malnutrition (PEM), a deficiency resulting in

nutritional stress shown to negatively impact the immune systemand increase circulating glucocorticoids (38). To determine whetherthis CR protocol induced PEM, we assessed circulating levels ofcorticosterone, the major endogenous glucocorticoid (Fig. 1C), andserum albumin levels, a marker of protein status (Fig. 1C) (38).Consistent with the reports of others, CR resulted in increased cir-culating corticosterone; however, we found no difference in serumalbumin levels between CR and AL mice, indicating NIH-31/NIA–fortified diet contains sufficient protein (18%). These findings sup-port the notion that CR is a nutritional stress resulting in increasedcirculating glucocorticoids, but any immunological observations areindependent of PEM (39). Other physiological parameters influ-enced by CR included reduced levels of serum cholesterol, triglyc-erides, and leptin, but normal blood glucose (Fig. 1C, 1D).

CR results in altered NK cells in distribution in peripheraltissues

We have previously shown that the percentage of NK1.1+ lym-phocytes in the spleens, but not the lungs, is reduced in CR mice(14). However, it is possible that in this prior analysis NKTcells were included. Therefore, we assessed NK cell percentages(NK1.1+ CD32) in the spleen, blood, BM, LN, and lungs of 6-mo-old AL and CR mice and found that CR results in a significantreduction in the frequency of cells that are NK cells in the spleen,lungs, and blood (Fig. 2A). In contrast, NK cells were found to bepresent at normal frequencies in LN and at an increased frequencyin the BM (Fig. 2A). Because of differences in body weight andspleen mass, we normalized the absolute number of NK cells invarious tissues to the weight of each tissue harvested (Fig. 2C–E).As NK cell frequency was reduced in the lungs and spleen, weexpected to find a reduced number of NK cells after normalizingcell numbers to the weight of the respective tissues. Indeed, therewere fewer NK cells in the lungs and spleens of CR than AL mice

FIGURE 1. Food intake and physiological

parameters altered by CR. (A) Food intake and

body weight were recorded daily for 7 d, and

averages for AL and CR mice are shown. (B)

Body composition was assessed by MRI on the

day animals were sacrificed, and body fat per-

centage was calculated as the portion of fat

mass relative to total mass. (C) Circulating

levels of corticosterone, albumin, and leptin

were determined in serum from AL and CR

mice by ELISA on the day of sacrifice. (D)

Serum glucose, triglycerides, and cholesterol

from AL and CR mice were measured on day

of sacrifice by colorimetric assays. Data are

means 6 SEM. *Indicates significance, p ,0.05 (n = 8–10 mice/group).

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on a per mg of tissue basis (Fig. 2D, 2E). There was no detectabledifference in NK cell numbers in the BM of CR mice comparedwith AL mice (Fig. 2C). Thus, the reduced frequency of NK cellsin CR is not due to an increase in another cell population, butrather reflects a direct change to NK cells resulting from CR asboth frequency and numbers of NK cells are reduced.

CR alters expression of NK cell maturation markers

NK cells are a heterogeneous population of cells, undergoinga developmental process within the BM before seeding peripheraltissues (19, 29). Based on our observation that CR results inchanges to NK cell frequency in the spleen, we investigatedwhether this was due to a reduction in total NK cells or reflectedthat a specific stage of maturation was reduced in CR. We foundCR results in decreased percentages of splenic NK cells expressingthe maturation markers CD11b, CD43, and KLRG1, but not DX5(Fig. 3A). Similarly, fewer NK cells expressed Ly49C/I/F/H, in-dicating that NK cells in CR mice exhibit an immature phenotype(Fig. 3B). We also found NK cells from CR mice displayed in-creased expression of av integrin (CD51), CD127, and CD94 (Fig.3A), markers normally not expressed at high levels on mature NKcells (19). Further phenotypic marker examination showed that NKcells from CR mice exhibited higher expression of several acti-vation markers such as CD69, B220, and glucocorticoid-inducedTNFR-related protein (Fig. 3B). Overall, CR results in NK cellswith an activated and immature phenotype, leading us to hypoth-esize that a lack of mature NK cells is the cause for reduced NKcell frequency and number in peripheral tissues of CR mice.

Activating and inhibitory receptor expression is altered by CR

Our data indicate that the frequency of mature NK cells is altered byCR; therefore, we investigated whether expression of receptors

involved in NK cell function was also influenced by CR. We foundno difference in the median fluorescence intensity of the activatingreceptors NKp46 and NKG2D, but did observe a decreased fre-quency of NK cells stained with a mAb that recognizes Ly49C/I/F/H (40). Furthermore, we observed a decrease in the frequencyof NK cells expressing Ly49D, Ly49H, and Ly49G2 (data notshown). Because Ly49s are acquired during the maturation pro-cess, we investigated whether CR influenced the Ly49 receptorrepertoire on mature (CD11b+) and immature (CD11b2) NK cellsfrom CR and AL mice. CD11b+ NK cells from CR mice expressedslightly, but significantly reduced levels of Ly49H, Ly49G2, andLy49C/I/F/H than CD11b+ NK cells from AL mice (Fig. 3C).Similarly, CD11b2 NK cells from CR mice expressed signifi-cantly lower levels of Ly49H, Ly49G2, and Ly49C/I/F/H, al-though neither group showed significantly different expression ofLy49D (Fig. 3C).

CD127+ NK cells are increased in frequency, but not innumber, in the BM, spleen, and LN of CR mice

NK cells expressing the IL-7Ra (CD127) are normally found ata high frequency in the thymus and LN; however, the origin ofCD127+ NK cells remains to be fully resolved (34, 41). Athymic(foxn12/2) mice demonstrate a significant reduction in the fre-quency of CD127+ NK cells in the spleen and LN, supporting thenotion that the thymus is a major source of CD127+ NK cells inperipheral tissues (34). However, it has also been postulated thethymic NK cell developmental pathway is an extension of a path-way normally occurring in the BM (21). Analysis of CD127 ex-pression on NK cells from the spleen, LN, and BM of CR micerevealed a significantly greater proportion of NK cells in CR miceexpressed CD127 in all three tissues (Fig. 4A). Because we ob-served a significant increase in CD127 expression on NK cells fromCR mice (Fig. 3A), but changes in the frequency of total NK cells(Fig. 2A), we compared the absolute number of CD127+ NKcells between AL and CR mice (Fig. 4B). Whereas CD127+ NKcells represented a larger percentage of the total cell pool (data notshown), when we compared the absolute number of CD127+ NKcells present in AL and CR mice, we found no difference inCD127+ cell numbers in the spleen, LN, or BM (Fig. 4B), sug-gesting that CD127+ NK cell numbers are maintained in CR mice,whereas other NK cell subsets are reduced. The surface phenotypeof CD127+ NK cells in CR mice also differed slightly fromCD127+ NK cells from AL mice: in the spleen, DX5 and CD11bwere both expressed at higher levels on CD127+ NK cells from CRmice than CD127+ NK cells from AL mice (Fig. 4C), whereasthese cells had low expression of Ly49s compared with CD1272

NK cells from either diet group. Similar to the spleen, DX5 ex-pression was found to be higher on BM CD127+ NK cells from CRmice compared with CD127+ BM NK cells from AL mice (Fig.4C). Because the thymus is known to be important for the gen-eration of CD127+ NK cells, we assessed CD127+ NK cell fre-quencies in the thymi of CR and AL mice by first gating NK1.1+

CD32 cells (Fig. 4D) and comparing the frequency of NK cellsexpressing CD127, ∼80% of NK cells in both AL and CR (Fig.4D). Compared with AL mice, NK cells were increased in fre-quency (Fig. 4E) in the thymi of CR mice, but not in number aftercorrecting for differences in thymic size (Fig. 4E), whereas totalcells within the thymus were significantly reduced with CR (Fig.4F). Taken together, the fact that CD127+ NK cells are present incomparable numbers between AL and CR mice, combined withsimilar numbers of CD127+ NK cells per mg thymus, suggeststhymic output of NK cells is normal in CR mice and the increasedfrequency represents changes in frequencies of other NK cellpopulations in CR.

FIGURE 2. Tissue weight and distribution of NK cells in CR mice. (A)

The percentage of NK1.1+ CD32 cells of total lymphocytes was deter-

mined in various tissues known to contain NK cells and was found to be

significantly reduced in the lungs, blood, and spleen (SPL) of CR mice. (B)

Wet tissue weights from AL and CR mice were taken immediately fol-

lowing sacrifice of AL and CR mice. NK cell numbers from BM (C),

spleen (D), and lungs (E) of CR and AL mice expressed as the number of

NK cells per femur or per mg tissue. The absolute number of NK cells was

calculated based on the frequency of NK cells of total cells analyzed by

flow cytometry and divided by the wet tissue weight. Experiments were

repeated twice. Data are means 6 SEM. *Indicates significance, p , 0.05

(n = 5 mice/group/experiment).

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CD127+ NK cell cytokine production, but not cytotoxicity, issignificantly altered by CR

NK cells can be classified into distinct functional subsets based onthe cell surface phenotype and degree of cytokine production andcytotoxicity exhibited. In humans, CD16brightCD56dim NK cells areenriched in LN and produce high levels of cytokines, but havelimited cytotoxicity, and it has been reported murine CD127+ NKcells have similar functional attributes (34). Because we observedan increased frequency of CD127+ NK cells in the spleen and LNof CR mice, we measured the capacity of LN and splenic NK cells

to produce TNF-a and GM-CSF. Following stimulation, signifi-

cantly more NK cells from CR mice stained positive for TNF-a

and GM-CSF (Fig. 5A, 5B), correlating with the increased fre-

quency of CD127+ NK cells (Fig. 4A). When gating on CD127+

NK cells from either the spleen or LN of CR mice, we found these

cells produced TNF-a and GM-CSF at a higher frequency than

CD127+ NK cells from AL mice (Fig. 5C), indicating a direct

change in the activity of these cells on a per cell basis. Next,

because we observed an increase in cytokine production by

CD127+ NK cells from CR mice, we investigated whether these

FIGURE 3. Characterization of surface phenotype of splenic NK cells in CR mice. Histograms are representative and contain either percentage of NK

cells within the positive gate for the indicated cell surface Ag or the median fluorescence intensity of the indicated marker (when no gate is shown). (A)

Expression of surface markers associated with NK cell maturation on splenic NK cells gated NK1.1+ CD32 from 6-mo-old AL and CR mice. (B) Ex-

pression of NK cell receptors and activation markers on splenic NK cells from CR and AL mice. (C) Ly49 repertoire on both CD11b+ (top) and CD11b2

(bottom) NK cells. Data are mean 6 SEM. Experiments were repeated twice. *Indicates significance, p , 0.05 (n = 5 mice/group/experiment).

FIGURE 4. A greater fraction of NK cells from CR mice expresses CD127. (A) CD127 expression on spleen (SPL), LN, and BM NK cells from AL and

CR mice. (B) The absolute number of CD127+ NK cells (NK1.1+ CD32) in the spleen, LN, and BM of AL and CR mice. (C) Surface phenotype of splenic

and BM CD127+ NK cells from gates indicated in (A). Filled gray histogram represents CD127+ NK cells from AL; solid line represents CD127+ NK cells

from CR; and dotted line represents splenic CD1272 NK cells from AL mice. (D) Gating strategy for identification of thymic NK cells that are identified as

NK1.1+ CD32 (top) and CD127+ (bottom). (E) Frequency of NK cells in the thymus represented both as frequency of thymocytes (top) and number of NK

cells per mg thymus collected (bottom). (F) Absolute counts of various cell populations identified in the thymus of AL and CR are shown. Flow plots are

representative and contain the percentage of NK cells positive for the indicated gates. Experiments were repeated twice. Data are mean 6 SEM. *Indicates

significance, p , 0.05 (n = 5 mice/group/experiment).

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cells exhibited higher cytotoxic potential than CD127+ NK cellsfrom AL mice when stimulated with YAC-1 cells. In general,CD127+ NK cells produced less granzyme B and degranulated ata lower frequency than CD1272 NK cells in both CR and AL mice(data not shown); however, we observed no difference in thefrequency of CD127+ NK cells staining positive for granzyme Bor CD107a between CR and AL mice (Fig. 5C).

NK cell subset distribution is sensitive to energy intake

In mice, upregulation of CD11b on NK cells is associated withfunctional maturity; however, Hayakawa and Smyth (28) haveproposed that CD11b+ NK cells can be divided into functionalsubsets based on expression of CD27. We found that terminallydifferentiated NK cells (CD272CD11b+) made up a significantlysmaller portion of NK cells in both the spleen and BM of CR mice(Fig. 6A), although terminally differentiated NK cells are found at

a relatively low frequency in the BM (Fig. 6A). Immature NKcells (CD11b2) and NK cells coexpressing CD27 and CD11b(DP) represented a larger portion of the total NK cell pool in CRmice compared with AL mice (Fig. 6A). To determine whetherimmature and DP NK cells were actually increased or whether thiswas due to a decrease in mature NK cells, we compared the fre-quency of NK cell subsets of total splenocytes and we found a2-fold reduction in the frequency of DP NK cells and a 4-foldreduction in CD272CD11b+ NK cells in the spleen of CR mice(Fig. 6B). Comparison of the frequency of CD272CD11b2

(double-negative [DN]) or CD27+CD11b2 NK cells among totalsplenocytes revealed no differences between CR and AL. Uponassessing the frequency of NK cell subsets in the BM relative tototal cells harvested, we found CD27+CD11b2 and DP NK cellswere increased in CR mice. This finding can be extrapolated to theobserved increased frequency of NK cells in the BM of CR mice(Fig. 2A), which is due to an increase in CD27+CD11b2 and DPNK cells. These data suggest that whereas modest changes to NKcells exist within the BM of CR mice, the majority of differencespresent in CR mice are found in peripheral NK cell tissues such asthe spleen.

CR results in differential expression of T-bet and Eomes in NKcells

Although the precise molecular mechanisms that regulate NK cellmaturation remain to be elucidated, it has been suggested thatterminal maturation of NK cells is at least partially dependent onthe transcription factors Eomes and T-bet (31). To understandwhether changes in NK cell maturation were related to alteredtranscription factor expression, we analyzed the expression patternof T-bet and Eomes within splenic NK cell subsets from CR andAL mice (Fig. 6C). With respect to Eomes, we found fewer DN,CD27+CD11b2, and DP NK cells from CR mice expressed Eomeswhen compared with the corresponding NK cell subsets from ALmice (Fig. 6C). There was no difference in the expression of T-betwhen comparing CD27+CD11b2 or DP NK cells from CR or ALmice. However, we detected DN and CD272CD11b+ NK cellsfrom CR mice expressed significantly lower levels of T-bet thanCD272CD11b+ NK cells from AL mice (Fig. 6C). Finally, afterfinding CD272CD11b+ NK cells expressed altered levels of T-bet,we compared the frequency of CD272CD11b+ NK cells from ALand CR mice expressing terminal maturation markers KLRG1 andCD43 (Fig. 6D), as the upregulation of these markers is thought tobe at least partially dependent on T-bet expression. Consistentwith reduced T-bet expression, both CD43 and KLRG1 were de-creased on CD272CD11b+ NK cells from CR mice, suggestingterminal differentiation of NK cells in CR mice is incomplete.

NK cells from CR mice have altered functional responses

We found NK cells from CR mice to be phenotypically immatureand have altered distribution of functional subsets, and thereforebegan a series of experiments to determinewhether these cells werealso functionally immature. We found that BM NK cells from CRand AL mice were equally capable of IFN-g production, althoughthis tended to be lower in CR mice (Fig. 7A), possibly due to thedecrease in CD272CD11b+ NK cells in the CR BM (Fig. 6A). Incontrast, stimulation of splenic NK cells with IL-2 plus IL-12 oranti-NK1.1 resulted in significantly fewer NK cells producingIFN-g from CR mice (Fig. 7A, 7B). To determine whether theobserved changes in NK cell IFN-g production were due toa functional impairment or simply because of altered distributionof NK cell subsets, we compared IFN-g production by CD27+

CD11b2, DP, and CD272CD11b+ NK cells from CR and AL micefollowing stimulation known to elicit IFN-g production by mature

FIGURE 5. Functional characterization of CD127+ NK cells from CR

mice. (A) Analysis of cytokine production by NK cells (NK1.1+ CD32)

from AL and CR mice in nonstimulated (NS) controls (top), cells isolated

from spleen (middle), and LN (bottom) stimulated with IL-2 (1000 U/ml),

IL-12 (10 ng/ml), and PMA (50 ng/ml) plus ionomycin (1 mg/ml). (B)

Summary of the frequency of cytokine-producing NK cells in the spleen

(top) and LN (bottom) of AL and CR mice. (C) Splenic lymphocytes were

gated NK1.1+ CD32CD127+ and analyzed for cytokine production and

cytotoxicity. Histograms of TNF-a and GM-CSF production following IL-

2 plus IL-12 stimulation (top) and granzyme B and CD107a (bottom)

staining following stimulation with YAC-1 cells (10:1 E:T ratio) in

CD127+ NK cells from CR and AL mice. Filled gray histogram represents

AL; solid line represents CR; and dotted line represents cells with no

stimulation (NS) from CR mice. Flow plots and histograms are represen-

tative and contain the percentage of NK cells positive for the indicated

gates. Experiments were repeated twice. Data are mean6 SEM. *Indicates

significance, p , 0.05 (n = 5 mice/group/experiment).

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NK cells (IL-2 + IL-12) (Fig. 7A) (42). We observed no differencein IFN-g production between NK cell subsets from AL and CRmice, suggesting the observed changes in IFN-g production aredue to alterations in NK cell subset distribution. NK cells from CRmice stimulated with plate-bound anti-NK1.1 or anti-NKp46degranulated at a lower frequency than NK cells from AL mice,as defined by reduced surface CD107a (Fig. 7B, 7C). Similarly,granzyme B production was significantly diminished in NK cellsfrom CR mice following activation receptor ligation by Abs (Fig.7B, 7C). Interestingly, we found stimulation with YAC-1 cellsresulted in increased surface CD107a and IFN-g by NK cells fromCR mice (Fig. 7D) as well as enhanced CD69 expression (data notshown) compared with NK cells from AL mice. Although the useof anti-NK1.1 or anti-NKp46 to activate NK cells resulted ina decreased frequency of NK cells from CR mice producinggranzyme B, we found no difference in granzyme B production

between CR and AL NK cells following YAC-1 stimulation (Fig.7D). Taken together, our data suggest CR influences the distri-bution of NK cell subsets and results in NK cells that are lessfunctional following stimulation with cytokines or plate-boundAbs, but retain high levels of responsiveness when faced withtarget cells.

CR results in functional changes to CD272CD11b+ NK cells

Whereas IFN-g production by NK cell subsets was comparablefollowing cytokine stimulation, we observed NK cells from CRmice responded robustly to YAC-1 cells in vitro (Fig. 7D) andasked whether any of our previous observations, such as decreasedexpression of markers of terminal maturation on CD272CD11b+

NK cells from CR mice (Fig. 6D), were related to increased re-sponsiveness. We stimulated NK cells from AL and CR mice withYAC-1 cells and measured the functionality of individual NK cell

FIGURE 6. Altered distribution of NK cell subsets in the BM and spleen of CR mice. (A) Distribution of NK cell (NK1.1+ CD32) subsets based on

expression of CD27 and CD11b in the BM (top) and spleens (bottom) of AL and CR mice. (B) Summary of the frequency of NK cells in each subset both as

a percentage of NK cells (top) and as a percentage of total cells recovered (bottom). NK cell subsets were defined as CD272CD11b2 (DN), CD27+CD11b2,

CD27+CD11b+ (DP), and CD272CD11b+. (C) Gating strategy for transcription factor analysis (left) and summary of transcription factor expression in

splenic NK cells from AL and CR mice (right). (D) Expression of KLRG1 and CD43 on splenic CD272CD11b+ NK cells from AL and CR mice. Flow

plots are representative and contain the percentage of NK cells positive for the indicated gates. Experiments were repeated twice. Data are mean 6 SEM.

*Indicates significance, p , 0.05 (n = 4–5 mice/group/experiment).

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subsets (Fig. 7E). The function of CD27+CD11b2 and DP NKcells was comparable between AL and CR mice; however, CD272

CD11b+ NK cells from CR mice produced granzyme B anddegranulated at a higher frequency than CD272CD11b+ NK cellsfrom AL controls (Fig. 7E). IFN-g production was not differentbetween any of the NK cell subsets analyzed following YAC-1stimulation (data not shown), suggesting the increase in IFN-g+

NK cells from CR mice was related to changes in NK cell subsetdistribution.

DiscussionIt has been put forth that NK cells are sensitive to dietary ma-nipulation; excessive and restricted energy intake, alcohol con-sumption, vitamins, and minerals as well as bioactive foodcomponents have all been suggested to influence NK cell cyto-toxicity or NK cell development (43–51). However, detailedanalysis of how dietary manipulation influences NK cell functionand homeostasis is limited. Although CR has been shown repeat-edly to have beneficial effects on T cell senescence, the implica-tions of CR on innate immunity and NK cell homeostasis havebeen understudied. Prompted by our observation that NK cells arereduced in frequency and numbers in most peripheral tissues ofCR mice, we further investigated functional and developmentalchanges to the NK cell pool in adult CR mice. In addition to re-duced frequency of total NK cells, our results suggest that gener-ation of NK cells in the BM is relatively unimpaired in CR,whereas the generation and/or maintenance of NK cells in pe-ripheral tissues such as the spleen appear most affected. Further-more, using CD27 and CD11b to classify NK cell subsets, our dataindicate CR mainly influences the homeostasis of mature NK cellsubsets in mice, as DN and CD27+CD11b2 NK cells representeda comparable fraction of total splenocytes, but CD11b+ NK cellswere significantly reduced.

In contrast to reduced mature NK cells, we observed an increased

frequency of CD127+ NK cells not only in LN of CR mice, but

also in the spleen and BM of CR mice, suggesting that CD127+

NK cells compose a larger portion of the NK cell pool in CR mice.

Because we observed comparable numbers of CD127+ NK cells

between AL and CR mice, we hypothesize that CD127+ NK cell

output is not impaired in CR, whereas classical mature NK cell

development is impaired, resulting in a greater frequency of NK

cells being CD127+. CD127+ NK cells are normally recognized as

having poor cytolytic potential, but high proinflammatory cyto-

kine production (34); thus, we were not surprised to find NK cells

from the LN and spleens of CR mice produced TNF-a and GM-

CSF at a higher frequency than NK cells from AL mice. However,

when we compared the capacity of CD127+ NK cells from AL

mice and CR mice to produce TNF-a and GM-CSF, we consis-

tently observed higher production of these cytokines by CD127+

NK cells from CR mice. The unique functional characteristics of

CD127+ NK cells have been attributed to their limited Ly49 re-

ceptor repertoire (34); however, we did not detect any differences

in the frequency of splenic CD127+ NK cells expressing Ly49C/I/

F/H. In contrast, we show DX5 and CD11b are expressed at higher

levels on CD127+ NK cells from CR mice, tempting us to spec-

ulate that perhaps increased cytokine production is related to a

more mature phenotype of these cells. Alternatively, BM CD127+

NK cells from CR mice expressed higher levels of Ly49 recep-

tors, possibly acquiring increased functional competence early in

development.In this study, we show NK cells from CR mice are impaired in

their ability to respond to stimulation through cytokine and acti-

vation receptors; however, we also demonstrate that NK cells from

CR mice respond more robustly to YAC-1 cells than NK cells from

AL mice. The observation that NK cells may harbor an immature

FIGURE 7. Function of NK cells from CR mice is altered after interrogation with various stimuli. (A) IFN-g production by BM and splenic NK cells

(NK1.1+ CD32) stimulated with IL-2 (1000 U/ml) plus IL-12 (50 ng/ml) (top) and the frequency of splenic NK cell subsets producing IFN-g from AL and

CR mice (bottom). (B–D) Splenic NK cells from AL and CR mice were stimulated with (B) anti-NK1.1 (25 mg/ml), (C) anti-NKp46 (15 mg/ml), and (D)

YAC-1 cells (10:1 E:T ratio), and DX5+ CD32 cells were analyzed for production of IFN-g, granzyme B, and surface CD107a. (E) Histograms representing

granzyme B and CD107a staining in NK cell subsets following stimulation of splenic NK cells with YAC-1 cells. NK cells from AL and CR mice were

gated DX5+ CD32, and the indicated NK cell subset was analyzed for granzyme B or CD107a expression. Filled gray histogram represents AL; solid line

represents CR; and dotted line represents cells from CR mice that received no stimulation (NS). Flow plots and histograms are representative and contain

the percentage of NK cells positive for the indicated gates. Experiments were repeated twice. Data are mean 6 SEM. *Indicates significance, p , 0.05 (n = 5

mice/group/experiment).

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phenotype, yet retain cytotoxicity against YAC-1 cells has beenreported previously (32, 52, 53), suggesting that cytotoxicity isacquired at an early stage of NK cell development (52). Further-more, the hyperresponsiveness of CR NK cells to YAC-1 cells is atleast partially related to the increased frequency of DP NK cells inCR mice, as these cells are known to respond more robustly toYAC-1 cells, have a lower activation threshold, and exhibit cyto-toxicity against YAC-1 cells through both NKG2D-dependent andindependent mechanisms (28). We also observed enhanced re-sponsiveness to YAC-1 cells in the CD272CD11b+ NK cell subsetfrom CR mice compared with AL mice. This phenomenon appearslimited to NK cell activation mediated through cell–cell inter-actions, as we did not observe differences between the function ofCD272CD11b+ NK cells from AL and CR mice after stimulationwith cytokines or Abs against major activating receptors. Inves-tigation of potential causes for this observation revealed lowerKLRG1 expression on CD272CD11b+ NK cells from CR mice,which is often associated with hyporesponsive NK cells (30, 54).Thus, it is likely the increased responsiveness of CR NK cells toYAC-1 cells is due to both an increased frequency of DP NK cellsas well as increased cell–cell responsiveness of CD272CD11b+

NK cells.Little is known about the molecular events required for acqui-

sition of KLRG1 and downregulation of CD27 on CD11b+ NKcells in the periphery; however, this process is thought to bemediated through coordinated expression of several transcriptionfactors (55, 56). NK cell development and homeostasis rely onnumerous transcription factors, such as Id2, IFN regulatory factor-2, Eomes, T-Bet, Gata-3, Blimp1, and E4BP4 (31, 55). It has beensuggested that terminal maturation of NK cells takes place in thespleen in a T-bet–dependent manner, with upregulation of KLRG1and CD43 being severely impaired in T-bet–deficient (tbx212/2)mice, whereas Eomes appears to play an opposite role by pro-moting downregulation of markers associated with immature NKcells (31, 56). In this study, we investigated whether T-bet orEomes deficiencies in NK cells from CR mice could explain theobserved reduction in terminally mature NK cells. We found T-betto be expressed at lower levels in DN and, importantly, CD272

CD11b+ NK cells. Based on this finding, we further analyzed thesurface phenotype of CD272CD11b+ NK cells in CR mice andfound that significantly fewer CD272CD11b+ NK cells expressedCD43 and KLRG1. Our data suggest dietary regimes, such as CR,can result in altered expression of transcription factors importantfor NK cell maturation such as Eomes and T-bet, possibly re-sulting in changes to NK cell phenotype and function.Recent studies have highlighted the central role of the energy-

sensitive serine/threonine kinase, mammalian target of rapamycin(mTOR), in regulating the expression of transcription factorssuch as Eomes and T-bet in T cells. Inhibition of mTOR bytreatment of CD8+ T cells with a CR mimetic, rapamycin, resultsin the inhibition of IL-12–induced T-bet expression, suggestinga direct relationship between energy status and developmentof lymphocytes into effector subtypes (57). However, althoughbecoming increasingly established in T cells, the relationshipbetween metabolism and NK cell maturation and function issomewhat less well understood. Leptin, a cytokine involved in theupstream activation of PI3K and AKT, was shown in this study tobe reduced in CR mice (58, 59). Importantly, NK cells express theleptin receptor (CD295), and leptin has been shown to be im-portant in maintaining NK cell numbers, making our observationspertaining to decreased leptin in CR mice a potential candidatethrough which some of the immunomodulatory effects of CR aremediated (60, 61). In NK cells, inhibition of PI3K signaling,a kinase with a central role in the integration of metabolic signals

upstream of mTOR, results in phenotypic and functional changesto NK cells, some of which are similar to the results reported inthis study in our model of CR, such as high expression of CD127and reduced terminal maturation (62, 63).In the BM and periphery, IL-15 has been firmly established to

play a critical role in the generation and maintenance of NK cells(64). Mice deficient in IL-15 have few detectable NK cells, and ithas been shown IL-15 signaling regulates NK cell development inthe BM and NK cell homeostasis and terminal maturation in theperiphery (65). Because we observed a significant reduction in NKcells in CR mice, one hypothesis is that CR may result in reducedlevels of IL-15 trans-presentation. This is supported by the ob-servation that CR results in apoptosis of senescent memory T cellsin aged mice, which are thought to be dependent on IL-7 and IL-15 (65–67). However, we observed no difference in IL-15Ralevels on splenic monocytes from CR mice (E. Gardner, unpub-lished observations) and did not observe a significant reduction inNK cells in the BM, resulting in inconclusive findings about theimplications of CR on IL-15 production or trans-presentation.Furthermore, CR has been shown to reduce chronic inflammationthat arises during aging or in models of chronic inflammationthrough reducing production of inflammatory cytokines such asC-reactive protein, IL-6, TNF-a, and leptin, as well as increasingcirculating glucocorticoids (68). Overall, these changes result ina significantly altered cytokine milieu in vivo, leading us to hy-pothesize that the changes to NK cells observed are due to amultitude of adaptations to the CR homeostatic environment,confounding the isolation of a single specific cause.It should be noted that the tissues we observed decrease in NK

cell frequency in CRmice generally house NK cells that have madesignificant maturational progress. For example, in AL mice, tissuessuch as the lungs and blood house mostly terminally differentiatedCD272CD11b+ NK cells (28), a subset of NK cells that we showto be significantly reduced in CR mice. This suggests the lack ofmature NK cells in CR mice contributes greatly to the decreasedfrequency of NK cells observed throughout the body of CR mice.The inability of NK cells to robustly populate the lungs and bloodof CR mice could also be related to decreased expression ofchemokine receptors, as CR has been previously shown to influ-ence chemokine receptor expression on T cells (69). Among thesechemokine receptors, S1P5 has been shown to regulate emigrationof NK cells from BM sinusoids in a T-bet–dependent manner,suggesting reduced T-bet expression may be the cause for in-creased BM NK cells in CR mice. However, we do not believe thisto be the case, as we only observed decreased T-bet expression inDN and CD272CD11b+ NK cells, rather than CD27+CD11b2 orDP NK cells, the likely candidates for BM egress. Furthermore,we do not believe impaired emigration from the BM of CR micecan completely explain the decreased frequency of NK cells inperipheral tissues of CR mice, as the increased frequency of NKcells in the BM did not result in differences between the totalnumber of NK cells recovered from the femurs of CR and ALmice.CR without malnutrition is a dietary intervention used in both

gerontological and oncological research, yet CR protocols arevaried, despite attempts at unifying and standardizing the dietaryintervention (38, 69). However, we employed the best documentedand studied CR protocols, established by the NIA, which havebeen repeatedly shown to increase the life span of laboratoryanimals when initiated early in life (1). Furthermore, our studies inyoung adult CR mice allow us to study the effect of CR on NKcells independent of aging. We and others have found adult micesubjected to CR early in life suffer increased susceptibility topathogens, thus raising the question of whether CR is only useful

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in a laboratory setting (10, 13, 15, 39, 70–72). Infection via thesepathogens results in substantial weight loss, which could be det-rimental to CR mice because of limited energy reserves (7).However, it is also plausible that CR initiated before adulthoodresults in immunological changes such as those presented in thiswork, that increase susceptibility to specific pathogens, limitingthe usefulness of this intervention in humans. However, futurestudies are required to determine whether this is specific forrespiratory viruses as we have shown, or whether other viralinfections such as HSV-1, mousepox, and murine CMV posea greater threat to CR mice as well (16, 73). Our study utilizingdietary manipulation in a mouse model led us to wonder whetherCR has a similar effect on NK cells in humans. Indeed, the NIAhas begun a series of human trials to determine the efficacy of CRin humans; this study, known as the Comprehensive Assessment ofthe Long-Term Effects of Reducing Intake of Energy (CALERIE),should allow for further study of the influence of CR on immunefunction in humans (74). Furthermore, because CR is designed asa dietary intervention to delay aging, it will be interesting to de-termine whether any age-related changes in NK cell phenotypethat occur in mice are ameliorated or exacerbated by CR inhumans and mice (36, 73).

AcknowledgmentsWe thank Dr. Jeannine Scott for insightful discussion regarding the manu-

script. We also thank Brooke Roman for technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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