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C57BL/6 Mice Are Altered by Caloric
NK Cell Maturation and Function in
Elizabeth M. Gardner
Jonathan F. Clinthorne, Eleni Beli, David M. Duriancik and
2013; 190:712-722; Prepublished online 14
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The Journal of Immunology
NK Cell Maturation and Function in C57BL/6 Mice Are
Altered 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 ofinfection. 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.
nology, 2013, 190: 712–722.
with increased incidence of disease, CR has been found to decrease
the severity of autoimmune disease, and decrease the incidence of
cardiac, 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 delayed
age-related immunosenescence (5, 6). It has been established that,
in laboratory conditions, CR reduces the incidence of spontaneous
tumors and cancers in aged rodents, and slows the age-related
decline in T cell proliferation, cytokine production, and CTL ac-
tivity that is often observed during aging (7, 8). Lifelong CR of
mice preserves thymopoiesis in the face of aging, and has been
shown to enhance influenza-specific Abs and splenic lymphocyte
proliferation after vaccination of mice with influenza (8, 9). These
beneficial changes to the adaptive immune system have been well
characterized; however, it has also been found that CR influences
innate immune function (10, 11). Several decades ago, Weindruch
et al. (12) reported that CR resulted in decreased splenic NK cell
cytotoxicity compared with aged matched controls, although this
could be ameliorated by polyinosinic:polycytidylic acid. More
recently, we have shown CR results in increased susceptibility to
primary influenza infection and decreased influenza-induced NK
The Journal of Immu-
aloric restriction (CR) is a dietary intervention that has
been shown to extend the life span of laboratory animals
(1). Whereas excess energy intake has been associated
cell cytotoxicity in young and aged mice (13, 14). This was ac-
companied by the observation that NK cell numbers and frequency
are decreased in the spleen of young CR mice (14). Overall, these
findings have raised concerns about the effects of CR on innate
immunity, and may predispose CR individuals to suffer more se-
vere primary infections (10, 15). However, at this time, few studies
have focused on understanding the effects of CR on innate immune
cell 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 bone
marrow (BM), and signals from stromal cells and cytokines result
in the microenvironment required for NK cell generation (19, 20).
NK cell commitment takes place through upregulation of the
shared IL-2/IL-15R b-chain (CD122), followed by acquisition of
the NK cell marker NK1.1 in B6 mice (19, 21). Interactions with
stromal cells within the BM regulate gene expression leading to
programmed expression of surface molecules, including integrins,
cytokine receptors, and a family of NK cell receptors (22–25). NK
cell maturation is classified by using both surface phenotype and
functional capacity. Phenotypic maturation takes place in stepwise
fashion; expression of the integrin CD49b (DX5) is used to define
early mature NK cells (26). Following acquisition of DX5, NK
cells in the BM upregulate CD11b and CD43, which correlates
strongly with the capability of a NK cell to produce large amounts
of IFN-g (19). Once mature, NK cells seed various lymphoid and
nonlymphoid peripheral tissues, with the majority of NK cells
expressing high levels of DX5, CD11b, and CD43 (19, 27).
tissues, DX5+NK cells continue to adapt to their environment; the
downregulation of CD27 and TRAIL and upregulation of killer cell
lectin-like receptor G1 (KLRG1) are associated with peripheral NK
cell maturation (28, 29). The application of the marker CD27 has
allowed the DX5+NK cell pool to be further divided into subsets in
mice in which there is a linear progression from CD27+CD11b2
early mature NK cells to CD27+CD11b+(double-positive [DP]) NK
phenotypic changes further reflect changes to NK cell function, as
Department of Food Science and Human Nutrition, Michigan State University, East
Lansing, 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
Address correspondence and reprint requests to Dr. Elizabeth M. Gardner, Depart-
ment of Food Science and Human Nutrition, Michigan State University, 234C GM
Trout Building, East Lansing, MI 48824. E-mail address: firstname.lastname@example.org
Abbreviations used in this article: AL, ad libitum; BM, bone marrow; CR, caloric
restriction; 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 on
Aging; PEM, protein energy malnutrition.
by guest on October 17, 2015
DP NK cells exhibit a greater responsiveness to invitro culture with
dendritic 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 viral
infection, such as murine CMV (29, 30).
Recent studies have begun to identify the molecular mechanisms
studies have identified transcription factors required for NK cell
commitment and differentiation into mature NK cells. Among these
transcription factors, ID2, TOX, and E4BP4 are thought to regulate
the earliest developmental stages, whereas other studies have
established aroleforthe transcriptionfactors T-bet,Eomesodermin
(Eomes), Blimp-1, IFN regulatory factor-2, and Gata-3 in the
generation of terminally differentiated CD43+KLRG1+NK cells
(31–34). Similar to their role in T cell function, several of these
transcription factors regulate aspects of NK cell function. For
example, T-bet is involved in the expression of granzyme B in NK
cells, whereas NK cells lacking Eomes are capable of producing
more 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 or
infection in 6-mo-old mice. In this study, we show that NK cells are
reduced in frequency in peripheral tissues and exhibit an altered
phenotype in the spleens of CR mice. We characterize these changes
by analyzing the expression of a variety of cell surface markers
associated with the maturation process, such as CD94, CD127, DX5,
CD11b, CD43, and KLRG1. Most studies of murine NK cells focus
on splenocytes, which normally express low levels of CD127.
However, we discovered that CR results in an increased fraction of
NK cells that express CD127 in the spleen and lymph nodes (LN),
accompanied with an increase in the frequency of NK cells in the
thymus, of which the majority are CD127+. These cells are thought
to have low cytotoxic potential and produce a variety of cytokines,
and are normally found at the highest frequency in the thymus and
LN. Comparison of the distribution of NK cell subsets between AL
and CR mice revealed CR results in significant reductions to the
CD272CD11b+NK cell pool, suggesting caloric intake plays a role
in regulating peripheral NK cell maturation or homeostasis. We used
various stimuli to test the functional competence of NK cells from
CR mice, and found that alterations to NK cell function in CR mice
are 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 Methods
Mice 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 Institute
on Aging (NIA) colony maintained by Charles River Laboratories (Wil-
mington, MA). The animal use protocol for this study was approved by
Michigan State University Institutional Animal Care and Use Committee.
Upon arrival, mice were housed individually in microisolator cages in the
American Association for the Accreditation of Laboratory Animal Care–
accredited containment facility at Michigan State University and were
acclimated at least 10–14 d prior to the initiation of each experiment. Both
CR (NIH-31/NIA-fortified) and AL (NIH-31) diets were purchased from
the NIA, the compositions of which have been reported in detail previously
(14). The composition of the CR diet is sufficient in micronutrients and
minerals, but results in restriction of total energy intake. The CR regimen
initiated by the NIA is designed to gradually achieve 40% restriction in
mice by 4 mo of age, such that they are weight stable upon arrival at 6 mo
of age. All experiments were repeated at least twice using four to five mice
per diet treatment per experiment, unless otherwise noted.
Body composition, food intake, and metabolic profile
Body composition (fat, lean, water) was determined by daily magnetic
resonance imaging (MRI) during the feeding protocol. Mice were indi-
vidually housed, allowing food intake to be recorded each day. All mice
were weighed daily between 0800 and 0900, after which they were fed. The
protocol to assess body composition using the EchoMRI-500 (Echo Medical
Systems) has been validated and described in detail previously (35). Briefly,
after calibration using a rapeseed oil standard, individual mice are placed in
a MRI holding tube. The advantages of this system are that it is rapid, allows
recover 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 the
manufacturer’s instructions (Cayman Chemical). Plates were read at 450 nm
wavelength using a Synergy HT plate reader (Bio-Tek), and concentrations
were determined using a standard curve for each respective assay.
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 of
Briefly, single-cell suspensions were obtained from spleens using homoge-
nization. BM cells were isolated from the femur and tibia by flushing with
a 25 5/8 needle and syringe containing RPMI 1640. The resulting cell sus-
pensions were lysed of RBCs using an ammonium chloride buffer. Lungs
were 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 Units
DNase (Roche). Cell suspensions from digested lungs or blood were diluted
with PBS and layered onto 1083-Histopaque (Sigma-Aldrich) for isolation
of mononuclear cells by density gradient centrifugation. Isolation of cells
from LN and thymus was performed by pressing the LNand thymus through
40-mm cell strainers (BD Falcon). All cell suspensions were washed in PBS
and resuspended for counting using trypan blue viability dye.
Cells from various tissues were resuspended in FACS buffer (0.1% sodium
azide, 1% FBS, in Dulbecco’s PBS) at a concentration of 2 3 107cells/ml.
A total of 1–4 3 106cells 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 containing
various combinations of the following fluorochrome-conjugated Abs
(eBioscience, BD Biosciences, or BioLegend) at optimal concentrations
determined in our laboratory: NK1.1 (allophycocyanin or PE-Cy7), CD3
(Alexafluor700 [500A2]), CD94/NKG2 (PE [HP-3D9]), CD27 (PE or
PerCP-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-related
protein (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 using
streptavidin-conjugated PerCP-Cy5.5 or allophycocyanin-Cy7. Cells were
incubated in staining cocktails on ice in the dark for 30 min. To detect
transcription factor expression, cells were fixed and permeabilized using
eBioscience 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 gated
based on light-scattering properties, after which NK cells were characterized
as NK1.1+CD32, unless otherwise noted. Samples were analyzed using an
LSR 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 using
flow cytometry, according to previously published methods (36). Briefly,
high-affinity 96-well plates (Thermo-Fisher) were coated with a mAb
against NK1.1 (25 mg/ml [PK136]) or NKp46 (15 mg/ml [29A1.4]) for
18 h at 4˚C. Plates were then washed with PBS three times, and freshly
prepared splenocytes (1–4 3 106) in complete media were added. Alter-
natively, splenocytes in complete media were added to uncoated 96-well
plates, and IL-2 (1000 U) plus IL-12 (10 ng/ml) or YAC-1 cells (10:1 E:T
ratio) were added. Anti-CD107a (FITC or PE-Cy7 [1D4B]), a marker of
degranulation, was also added to NK1.1 and YAC-1–stimulated splenocyte
cultures (36, 37). Plates were incubated for 4–8 h, during which brefeldin
A and monensin were added after the first 30 min. To elicit GM-CSF and
The Journal of Immunology713
by guest on October 17, 2015
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 cytokine
secretion and raise intracellular cytokine stores, as reported by Vosshenrich
et al. (34). Following incubation, cells were stained with lineage-specific
Abs 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]), and
GM-CSF (FITC [MP1-22E9]) (BD Biosciences).
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 function
were analyzed using Student t test to determine significant differences
between diet groups. Statistical significance was set at p , 0.05.
Physiological parameters influenced by CR
CR is initiated at 14 wk of age (10% restriction), and at 15 wkof age
the restriction is increased to 25%. Finally, at 16 wk, mice are fed
a 40% restricted diet that is maintained throughout the life of the
animal. For this study, our CR protocol supplied mice with 3 g food
daily in the form of a vitamin- and mineral-supplemented cookie
supplied by the NIA, whereas AL mice consumed ∼4.53 g food
daily (Fig. 1A). Our data indicate feeding CR mice with a 3 g
cookie resulted in a 34% restriction rather than 40%; however, we
still observed physiological changes indicating our CR protocol
induced CR characteristics, which is supported by the notion that
even 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 independently
from protein energy malnutrition (PEM), a deficiency resulting in
nutritional stress shown to negatively impact the immune system
and increase circulating glucocorticoids (38). To determine whether
this CR protocol induced PEM, we assessed circulating levels of
corticosterone, the major endogenous glucocorticoid (Fig. 1C), and
serum 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 serum
albumin 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 increased
circulating glucocorticoids, but any immunological observations are
independent 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 peripheral
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 NKT
cells 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 significant
reduction in the frequency of cells that are NK cells in the spleen,
lungs, and blood (Fig. 2A). In contrast, NK cells were found to be
present at normal frequencies in LN and at an increased frequency
in the BM (Fig. 2A). Because of differences in body weight and
spleen mass, we normalized the absolute number of NK cells in
various tissues to the weight of each tissue harvested (Fig. 2C–E).
As NK cell frequency was reduced in the lungs and spleen, we
expected to find a reduced number of NK cells after normalizing
cell numbers to the weight of the respective tissues. Indeed, there
were fewer NK cells in the lungs and spleens of CR than AL mice
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).
Food intake and physiological
714CR ALTERS NK CELL PHENOTYPE AND FUNCTION
by guest on October 17, 2015
on a per mg of tissue basis (Fig. 2D, 2E). There was no detectable
difference in NK cell numbers in the BM of CR mice compared
with AL mice (Fig. 2C). Thus, the reduced frequency of NK cells
in CR is not due to an increase in another cell population, but
rather reflects a direct change to NK cells resulting from CR as
both frequency and numbers of NK cells are reduced.
CR alters expression of NK cell maturation markers
NK cells are a heterogeneous population of cells, undergoing
a developmental process within the BM before seeding peripheral
tissues (19, 29). Based on our observation that CR results in
changes to NK cell frequency in the spleen, we investigated
whether this was due to a reduction in total NK cells or reflected
that a specific stage of maturation was reduced in CR. We found
CR results in decreased percentages of splenic NK cells expressing
the 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 avintegrin (CD51), CD127, and CD94 (Fig.
3A), markers normally not expressed at high levels on mature NK
cells (19). Further phenotypic marker examination showed that NK
cells from CR mice exhibited higher expression of several acti-
vation markers such as CD69, B220, and glucocorticoid-induced
TNFR-related protein (Fig. 3B). Overall, CR results in NK cells
with an activated and immature phenotype, leading us to hypoth-
esize that a lack of mature NK cells is the cause for reduced NK
cell frequency and number in peripheral tissues of CR mice.
Activating and inhibitory receptor expression is altered by CR
CR; therefore, we investigated whether expression of receptors
involved in NK cell function was also influenced by CR. We found
no difference in the median fluorescence intensity of the activating
receptors 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 frequency
of NK cells expressing Ly49D, Ly49H, and Ly49G2 (data not
shown). Because Ly49s are acquired during the maturation pro-
cess, we investigated whether CR influenced the Ly49 receptor
repertoire on mature (CD11b+) and immature (CD11b2) NK cells
from CR and AL mice. CD11b+NK cells from CR mice expressed
slightly, but significantly reduced levels of Ly49H, Ly49G2, and
Ly49C/I/F/H than CD11b+NK cells from AL mice (Fig. 3C).
Similarly, CD11b2NK 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 of
Ly49D (Fig. 3C).
CD127+NK cells are increased in frequency, but not in
number, in the BM, spleen, and LN of CR mice
NK cells expressing the IL-7Ra (CD127) are normally found at
a high frequency in the thymus and LN; however, the origin of
CD127+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 the
notion that the thymus is a major source of CD127+NK cells in
peripheral tissues (34). However, it has also been postulated the
thymic 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 mice
revealed a significantly greater proportion of NK cells in CR mice
expressed CD127 in all three tissues (Fig. 4A). Because we ob-
served a significant increase in CD127 expression on NK cells from
CR mice (Fig. 3A), but changes in the frequency of total NK cells
(Fig. 2A), we compared the absolute number of CD127+NK
cells between AL and CR mice (Fig. 4B). Whereas CD127+NK
cells represented a larger percentage of the total cell pool (data not
shown), when we compared the absolute number of CD127+NK
cells present in AL and CR mice, we found no difference in
CD127+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 phenotype
of CD127+NK cells in CR mice also differed slightly from
CD127+NK cells from AL mice: in the spleen, DX5 and CD11b
were both expressed at higher levels on CD127+NK cells from CR
mice than CD127+NK cells from AL mice (Fig. 4C), whereas
these 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 CR
mice 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+
CD32cells (Fig. 4D) and comparing the frequency of NK cells
expressing 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 after
correcting for differences in thymic size (Fig. 4E), whereas total
cells within the thymus were significantly reduced with CR (Fig.
4F). Taken together, the fact that CD127+NK cells are present in
comparable numbers between AL and CR mice, combined with
similar numbers of CD127+NK cells per mg thymus, suggests
thymic output of NK cells is normal in CR mice and the increased
frequency represents changes in frequencies of other NK cell
populations in CR.
The percentage of NK1.1+CD32cells 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).
Tissue weight and distribution of NK cells in CR mice. (A)
The Journal of Immunology715
by guest on October 17, 2015
CD127+NK cell cytokine production, but not cytotoxicity, is
significantly altered by CR
NK cells can be classified into distinct functional subsets based on
the cell surface phenotype and degree of cytokine production and
cytotoxicity exhibited. In humans, CD16brightCD56dimNK cells are
enriched in LN and produce high levels of cytokines, but have
limited cytotoxicity, and it has been reported murine CD127+NK
cells have similar functional attributes (34). Because we observed
an increased frequency of CD127+NK cells in the spleen and LN
of 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
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+CD32from 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).
Characterization of surface phenotype of splenic NK cells in CR mice. Histograms are representative and contain either percentage of NK
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 CD1272NK 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).
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
716 CR ALTERS NK CELL PHENOTYPE AND FUNCTION
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cells exhibited higher cytotoxic potential than CD127+NK cells
from AL mice when stimulated with YAC-1 cells. In general,
CD127+NK cells produced less granzyme B and degranulated at
a lower frequency than CD1272NK cells in both CR and AL mice
(data not shown); however, we observed no difference in the
frequency of CD127+NK cells staining positive for granzyme B
or 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 with
functional maturity; however, Hayakawa and Smyth (28) have
proposed that CD11b+NK cells can be divided into functional
subsets based on expression of CD27. We found that terminally
differentiated NK cells (CD272CD11b+) made up a significantly
smaller 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 NK
cells (CD11b2) and NK cells coexpressing CD27 and CD11b
(DP) represented a larger portion of the total NK cell pool in CR
mice compared with AL mice (Fig. 6A). To determine whether
immature and DP NK cells were actually increased or whether this
was due to a decrease in mature NK cells, we compared the fre-
quency of NK cell subsets of total splenocytes and we found a
2-fold reduction in the frequency of DP NK cells and a 4-fold
reduction in CD272CD11b+NK cells in the spleen of CR mice
(Fig. 6B). Comparison of the frequency of CD272CD11b2
(double-negative [DN]) or CD27+CD11b2NK cells among total
splenocytes revealed no differences between CR and AL. Upon
assessing the frequency of NK cell subsets in the BM relative to
total cells harvested, we found CD27+CD11b2and DP NK cells
were increased in CR mice. This finding can be extrapolated to the
observed increased frequency of NK cells in the BM of CR mice
(Fig. 2A), which is due to an increase in CD27+CD11b2and DP
NK cells. These data suggest that whereas modest changes to NK
cells exist within the BM of CR mice, the majority of differences
present in CR mice are found in peripheral NK cell tissues such as
CR results in differential expression of T-bet and Eomes in NK
Although the precise molecular mechanisms that regulate NK cell
maturation remain to be elucidated, it has been suggested that
terminal maturation of NK cells is at least partially dependent on
the transcription factors Eomes and T-bet (31). To understand
whether changes in NK cell maturation were related to altered
transcription factor expression, we analyzed the expression pattern
of T-bet and Eomes within splenic NK cell subsets from CR and
AL mice (Fig. 6C). With respect to Eomes, we found fewer DN,
CD27+CD11b2, and DP NK cells from CR mice expressed Eomes
when compared with the corresponding NK cell subsets from AL
mice (Fig. 6C). There was no difference in the expression of T-bet
when comparing CD27+CD11b2or DP NK cells from CR or AL
mice. However, we detected DN and CD272CD11b+NK cells
from CR mice expressed significantly lower levels of T-bet than
CD272CD11b+NK cells from AL mice (Fig. 6C). Finally, after
finding CD272CD11b+NK cells expressed altered levels of T-bet,
we compared the frequency of CD272CD11b+NK cells from AL
and CR mice expressing terminal maturation markers KLRG1 and
CD43 (Fig. 6D), as the upregulation of these markers is thought to
be at least partially dependent on T-bet expression. Consistent
with reduced T-bet expression, both CD43 and KLRG1 were de-
creased on CD272CD11b+NK cells from CR mice, suggesting
terminal 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 immature
and have altered distribution of functional subsets, and therefore
beganaseries ofexperimentstodeterminewhether thesecells were
also functionally immature. We found that BM NK cells from CR
and AL mice were equally capable of IFN-g production, although
this tended to be lower in CR mice (Fig. 7A), possibly due to the
decrease in CD272CD11b+NK cells in the CR BM (Fig. 6A). In
contrast, stimulation of splenic NK cells with IL-2 plus IL-12 or
anti-NK1.1 resulted in significantly fewer NK cells producing
IFN-g from CR mice (Fig. 7A, 7B). To determine whether the
observed changes in NK cell IFN-g production were due to
a functional impairment or simply because of altered distribution
of NK cell subsets, we compared IFN-g production by CD27+
CD11b2, DP, and CD272CD11b+NK cells from CR and AL mice
following stimulation known to elicit IFN-g production by mature
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 mean 6 SEM. *Indicates
significance, p , 0.05 (n = 5 mice/group/experiment).
Functional characterization of CD127+NK cells from CR
The Journal of Immunology717
by guest on October 17, 2015
NK cells (IL-2 + IL-12) (Fig. 7A) (42). We observed no difference
in IFN-g production between NK cell subsets from AL and CR
mice, suggesting the observed changes in IFN-g production are
due to alterations in NK cell subset distribution. NK cells from CR
mice stimulated with plate-bound anti-NK1.1 or anti-NKp46
degranulated 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 cells
from CR mice following activation receptor ligation by Abs (Fig.
7B, 7C). Interestingly, we found stimulation with YAC-1 cells
resulted in increased surface CD107a and IFN-g by NK cells from
CR mice (Fig. 7D) as well as enhanced CD69 expression (data not
shown) compared with NK cells from AL mice. Although the use
of anti-NK1.1 or anti-NKp46 to activate NK cells resulted in
a decreased frequency of NK cells from CR mice producing
granzyme 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 less
functional following stimulation with cytokines or plate-bound
Abs, but retain high levels of responsiveness when faced with
CR results in functional changes to CD272CD11b+NK cells
Whereas IFN-g production by NK cell subsets was comparable
following cytokine stimulation, we observed NK cells from CR
mice responded robustly to YAC-1 cells in vitro (Fig. 7D) and
asked whether any of our previous observations, such as decreased
expression 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 with
YAC-1 cells and measured the functionality of individual NK cell
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).
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
718 CR ALTERS NK CELL PHENOTYPE AND FUNCTION
by guest on October 17, 2015
subsets (Fig. 7E). The function of CD27+CD11b2and DP NK
cells was comparable between AL and CR mice; however, CD272
CD11b+NK cells from CR mice produced granzyme B and
degranulated at a higher frequency than CD272CD11b+NK cells
from AL controls (Fig. 7E). IFN-g production was not different
between any of the NK cell subsets analyzed following YAC-1
stimulation (data not shown), suggesting the increase in IFN-g+
NK cells from CR mice was related to changes in NK cell subset
It 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 food
components have all been suggested to influence NK cell cyto-
toxicity or NK cell development (43–51). However, detailed
analysis of how dietary manipulation influences NK cell function
and 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 have
been understudied. Prompted by our observation that NK cells are
reduced in frequency and numbers in most peripheral tissues of
CR mice, we further investigated functional and developmental
changes 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 data
indicate CR mainly influences the homeostasis of mature NK cell
subsets in mice, as DN and CD27+CD11b2NK cells represented
a comparable fraction of total splenocytes, but CD11b+NK cells
were significantly reduced.
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
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
(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+CD32cells 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
Function of NK cells from CR mice is altered after interrogation with various stimuli. (A) IFN-g production by BM and splenic NK cells
The Journal of Immunology719
by guest on October 17, 2015
phenotype, yet retain cytotoxicity against YAC-1 cells has been
reported previously (32, 52, 53), suggesting that cytotoxicity is
acquired at an early stage of NK cell development (52). Further-
more, the hyperresponsiveness of CR NK cells to YAC-1 cells is at
least partially related to the increased frequency of DP NK cells in
CR mice, as these cells are known to respond more robustly to
YAC-1 cells, have a lower activation threshold, and exhibit cyto-
toxicity against YAC-1 cells through both NKG2D-dependent and
independent mechanisms (28). We also observed enhanced re-
sponsiveness to YAC-1 cells in the CD272CD11b+NK cell subset
from CR mice compared with AL mice. This phenomenon appears
limited to NK cell activation mediated through cell–cell inter-
actions, as we did not observe differences between the function of
CD272CD11b+NK cells from AL and CR mice after stimulation
with cytokines or Abs against major activating receptors. Inves-
tigation of potential causes for this observation revealed lower
KLRG1 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 to
YAC-1 cells is due to both an increased frequency of DP NK cells
as well as increased cell–cell responsiveness of CD272CD11b+
Little is known about the molecular events required for acqui-
sition of KLRG1 and downregulation of CD27 on CD11b+NK
cells in the periphery; however, this process is thought to be
mediated through coordinated expression of several transcription
factors (55, 56). NK cell development and homeostasis rely on
numerous transcription factors, such as Id2, IFN regulatory factor-
2, Eomes, T-Bet, Gata-3, Blimp1, and E4BP4 (31, 55). It has been
suggested that terminal maturation of NK cells takes place in the
spleen in a T-bet–dependent manner, with upregulation of KLRG1
and 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 NK
cells (31, 56). In this study, we investigated whether T-bet or
Eomes deficiencies in NK cells from CR mice could explain the
observed reduction in terminally mature NK cells. We found T-bet
to be expressed at lower levels in DN and, importantly, CD272
CD11b+NK cells. Based on this finding, we further analyzed the
surface phenotype of CD272CD11b+NK cells in CR mice and
found that significantly fewer CD272CD11b+NK cells expressed
CD43 and KLRG1. Our data suggest dietary regimes, such as CR,
can result in altered expression of transcription factors important
for 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 factors
such as Eomes and T-bet in T cells. Inhibition of mTOR by
treatment of CD8+T cells with a CR mimetic, rapamycin, results
in the inhibition of IL-12–induced T-bet expression, suggesting
a direct relationship between energy status and development
of lymphocytes into effector subtypes (57). However, although
becoming increasingly established in T cells, the relationship
between metabolism and NK cell maturation and function is
somewhat less well understood. Leptin, a cytokine involved in the
upstream activation of PI3K and AKT, was shown in this study to
be reduced in CR mice (58, 59). Importantly, NK cells express the
leptin receptor (CD295), and leptin has been shown to be im-
portant in maintaining NK cell numbers, making our observations
pertaining to decreased leptin in CR mice a potential candidate
through which some of the immunomodulatory effects of CR are
mediated (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 changes
to NK cells, some of which are similar to the results reported in
this study in our model of CR, such as high expression of CD127
and 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 it
has been shown IL-15 signaling regulates NK cell development in
the BM and NK cell homeostasis and terminal maturation in the
periphery (65). Because we observed a significant reduction in NK
cells in CR mice, one hypothesis is that CR may result in reduced
levels of IL-15 trans-presentation. This is supported by the ob-
servation that CR results in apoptosis of senescent memory T cells
in aged mice, which are thought to be dependent on IL-7 and IL-
15 (65–67). However, we observed no difference in IL-15Ra
levels on splenic monocytes from CR mice (E. Gardner, unpub-
lished observations) and did not observe a significant reduction in
NK cells in the BM, resulting in inconclusive findings about the
implications of CR on IL-15 production or trans-presentation.
Furthermore, CR has been shown to reduce chronic inflammation
that arises during aging or in models of chronic inflammation
through reducing production of inflammatory cytokines such as
C-reactive protein, IL-6, TNF-a, and leptin, as well as increasing
circulating glucocorticoids (68). Overall, these changes result in
a significantly altered cytokine milieu in vivo, leading us to hy-
pothesize that the changes to NK cells observed are due to a
multitude 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 CR micegenerally house NK cells that have made
significant maturational progress. For example, in AL mice, tissues
such as the lungs and blood house mostly terminally differentiated
CD272CD11b+NK cells (28), a subset of NK cells that we show
to be significantly reduced in CR mice. This suggests the lack of
mature NK cells in CR mice contributes greatly to the decreased
frequency of NK cells observed throughout the body of CR mice.
The inability of NK cells to robustly populate the lungs and blood
of CR mice could also be related to decreased expression of
chemokine receptors, as CR has been previously shown to influ-
ence chemokine receptor expression on T cells (69). Among these
chemokine receptors, S1P5 has been shown to regulate emigration
of 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 this
to be the case, as we only observed decreased T-bet expression in
DN and CD272CD11b+NK cells, rather than CD27+CD11b2or
DP NK cells, the likely candidates for BM egress. Furthermore,
we do not believe impaired emigration from the BM of CR mice
can completely explain the decreased frequency of NK cells in
peripheral tissues of CR mice, as the increased frequency of NK
cells in the BM did not result in differences between the total
number of NK cells recovered from the femurs of CR and AL
CR without malnutrition is a dietary intervention used in both
gerontological and oncological research, yet CR protocols are
varied, despite attempts at unifying and standardizing the dietary
intervention (38, 69). However, we employed the best documented
and studied CR protocols, established by the NIA, which have
been repeatedly shown to increase the life span of laboratory
animals when initiated early in life (1). Furthermore, our studies in
young adult CR mice allow us to study the effect of CR on NK
cells independent of aging. We and others have found adult mice
subjected to CR early in life suffer increased susceptibility to
pathogens, thus raising the question of whether CR is only useful
720 CR ALTERS NK CELL PHENOTYPE AND FUNCTION
by guest on October 17, 2015
in a laboratory setting (10, 13, 15, 39, 70–72). Infection via these
pathogens 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 adulthood
results in immunological changes such as those presented in this
work, that increase susceptibility to specific pathogens, limiting
the usefulness of this intervention in humans. However, future
studies are required to determine whether this is specific for
respiratory viruses as we have shown, or whether other viral
infections such as HSV-1, mousepox, and murine CMV pose
a greater threat to CR mice as well (16, 73). Our study utilizing
dietary manipulation in a mouse model led us to wonder whether
CR has a similar effect on NK cells in humans. Indeed, the NIA
has begun a series of human trials to determine the efficacy of CR
in humans; this study, known as the Comprehensive Assessment of
the Long-Term Effects of Reducing Intake of Energy (CALERIE),
should allow for further study of the influence of CR on immune
function in humans (74). Furthermore, because CR is designed as
a dietary intervention to delay aging, it will be interesting to de-
termine whether any age-related changes in NK cell phenotype
that occur in mice are ameliorated or exacerbated by CR in
humans and mice (36, 73).
We thank Dr. Jeannine Scott for insightful discussion regarding the manu-
script. We also thank Brooke Roman for technical assistance.
The authors have no financial conflicts of interest.
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