IL-7 Is Essential for Homeostatic Control of
T Cell Metabolism In Vivo
Sarah R. Jacobs, Ryan D. Michalek and Jeffrey C. Rathmell
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References
J Immunol 2010; 184:3461-3469; Prepublished online 1
March 2010;
doi: 10.4049/jimmunol.0902593
http://www.jimmunol.org/content/184/7/3461
The Journal of Immunology
IL-7 Is Essential for Homeostatic Control of T Cell
Metabolism In Vivo
Sarah R. Jacobs, Ryan D. Michalek, and Jeffrey C. Rathmell
C
ontrol of T cell homeostasis is critical to maintain proper
immunity and to avoid autoimmunity or immunodeficiency. The social control model for cell and tissue homeostasis posits that cell extrinsic signals are required for cell
survival during development and to maintain cellular homeostasis
of mature tissues (1). In the absence of these signals or growth
factors, cells undergo a spontaneous programmed cell death via
the intrinsic apoptotic pathway. T cells are highly dependent on
cell extrinsic signals for survival and function both during development and when mature in the periphery. One mechanism by
which extrinsic signals may allow cells to evade apoptosis is
through the maintenance of cellular metabolism (2, 3). Acquisition of energy by individual cells in the form of sugars, lipids, or
amino acids can be regulated by growth factors (4–6) and is
critical to perform housekeeping functions required for survival
and production of essential molecules (7). If cell metabolism decreases, the ability of cells to grow and proliferate when stimulated may be diminished, and apoptosis may ensue (2). Although
it is clear that cell extrinsic signals are required for evasion of
apoptosis, it remains undetermined if the same signals are responsible to sustain basal cell metabolism in vivo and how these
pathways may influence T cell physiology and homeostasis.
Department of Pharmacology and Cancer Biology and Department of Immunology,
Sarah W. Stedman Center for Nutrition and Metabolism, Duke University Medical
Center, Durham, NC 27710
Received for publication August 7, 2009. Accepted for publication January 27, 2010.
This work was supported by National Institute of Allergy and Infectious Diseases
R01AI063345. R.D.M. was supported by The Irvington Institute Fellowship Program
of the Cancer Research Institute. J.C.R. is a Bernard Osher Fellow of the American
Asthma Foundation.
Address correspondence and reprint requests to Dr. Jeffrey C. Rathmell, Department
of Pharmacology and Cancer Biology, Duke University Medical Center, P.O. Box
3813, Durham, NC 27710. E-mail address: [email protected]
Abbreviations used in this paper: ER, estrogen receptor; MFI, mean fluorescence intensity; ND, not determined; PI, propidium iodide; WT, wild-type.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902593
Among the many signals received by T cells in vivo that may
provide survival and growth signals, the cytokine IL-7 has been
established as necessary for T cell development, homeostatic
proliferation, and survival (8). The absence of IL-7 or any of its
proximal signaling components leads to an SCID (9). IL-7 is
produced by stromal cells and detected by a two-part receptor on
lymphocytes consisting of the common g-chain that is shared by
multiple cytokines and a more specific receptor, IL-7Ra (IL-7R).
IL-7R signals through the Jak/STAT and PI3K/Akt signaling
pathways, both of which are known to have effects on cell survival, growth, and metabolism (10, 11). The specific role and
mechanism by which IL-7R may influence each of these processes
in vivo, however, has not been fully determined.
IL-7 may promote cell survival and growth through several
mechanisms. One important pathway involves regulation of Bcl-2
family members. Specifically, IL-7 signaling results in increased
expression of the antiapoptotic protein Bcl-2 (12), and overexpression
of Bcl-2 can partially rescue T cell development in IL-7R–deficient
animals (13, 14). The antiapoptotic Bcl-2 family member Mcl-1 has
also been connected to IL-7–induced cell survival, and IL-7–dependent cell survival was eliminated in the absence of Mcl-1 (15).
However, no single modification in apoptotic regulatory genes has
completely restored survival or repaired functional defects associated
with the loss of IL-7. This and evidence that IL-7 can inhibit cell
death even in Bcl-2–deficient cells (16) suggests that IL-7 may
also control cell function and survival through other pathways.
An additional function of IL-7 that is potentially essential for T cell
development and homeostasis may be regulation of basal T cell
metabolism. T cells cultured in the absence of normal environmental
signals have decreased glucose uptake and glycolysis. Culture of
T cells in the presence of rIL-7, however, can partially maintain
glucose uptake and surface levels of theglucose transporter Glut1 and
can wholly maintain T cell glycolytic flux (11, 17, 18). Glucose
metabolism is critical for T cell activation and likely also plays a role
in naive T cell homeostasis, survival, and ability to become effector
cells (19–21). No evidence, however, has yet emerged to show that
this regulation has a functional role in vivo with endogenous levels
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
It has become apparent that T cells require growth signals to maintain function and viability necessary to maintain proper immune
homeostasis. One means by which cell extrinsic signals may mediate these effects is by sustaining sufficient basal cell metabolism to
prevent cell atrophy. The role of metabolism and the specific growth factors essential to maintain metabolism of mature T cells
in vivo, however, are poorly defined. As IL-7 is a nonredundant cytokine required for T cell development and survival and can
regulate T cell metabolism in vitro, we hypothesized it may be essential to sustain metabolism of resting T cells in vivo. Thus,
we generated a model for conditional expression of IL-7R in mature T cells. After IL-7R deletion in a generally normal lymphoid
environment, T cells had reduced responses to IL-7, including abrogated signaling and maintenance of antiapoptotic Bcl-2 family
expression that corresponded to decreased survival in vitro. T cell survival in vivo was also reduced after loss of the IL-7R in a T cellintrinsic manner. Additionally, IL-7R deletion resulted in delayed growth and proliferation following stimulation. Importantly,
in vivo excision of IL-7R led to T cell atrophy that was characterized by delayed mitogenesis and reduced glycolytic flux. These
data are the first to identify an in vivo requirement for a specific cell extrinsic signal to sustain lymphocyte metabolism and suggest
that control of glycolysis by IL-7R may contribute to the well-described roles of IL-7 in T cell development, homeostatic proliferation, and survival. The Journal of Immunology, 2010, 184: 3461–3469.
3462
Materials and Methods
Mice
IL-7Rflox transgenic mice were produced by cloning a murine cDNA of IL7Ra that was flanked by two loxP sequences into the pLck.E2 vector and
microinjected into C57Bl6/J oocytes by the Duke Transgenic Mouse Facility (Durham, NC). Mice were screened by PCR with the following
primers: forward 59-AGCCGAGGCTCCCTCTGA-39 and reverse 59-GTAGCCATTGCAGCTAGGTG-39. Cre:estrogen receptor (ER) transgenic
animals expressed Cre-recombinase fused to ER under a ubiquitin promoter and were a generous gift of Eric Brown (University of Pennsylvania,
Philadelphia, PA). IL-7R2/2 mice were purchased (The Jackson Laboratory, Bar Harbor, ME) and IL-72/2 mice were a generous gift of Motonari
Kondo (Duke University, Durham, NC). Mice were backcrossed and
maintained on a C57Bl6/J background and housed at Duke University, and
the appropriate institutional boards approved all procedures. To activate
Cre:ER and excise the floxed transgene, mice were treated with 0.15 mg
tamoxifen (Sigma-Aldrich, St. Louis, MO) dissolved in corn oil by i.p.
injection for 2 consecutive d, and animals were sacrificed 3 d after the first
treatment unless otherwise indicated. Excision experiments were performed on same-sex littermates.
Adoptive transfers
Adoptive transfer experiments were performed by isolation of splenic T cells
from animals on a C57B16/J background. T cells injected into IL-72/2 hosts
were stained for CFSE prior to injection and examined 7 d posttransfer. For
in vivo cell survival assays, Thy1.2 T cells from C57B16/J or IL-7Rflox
animals were injected into Thy1.1 hosts, and animals were treated with
tamoxifen 12 h after the indicated number of cells were introduced via tail
vein injection.
IL-7R stains was RatIgG2a biotin (eBioscience). Intracellular staining for
total cellular protein levels were determined by fixation in 1% paraformaldehyde for 10 min at 37˚C, permeabilization with 100% methanol
on ice for 30 min, and anti-Glut1 Ab (Abcam, Cambridge, MA) or anti–
Bcl-2 Ab (BD Pharmingen, San Diego, CA), followed by a fluorescently
conjugated anti-rabbit secondary (eBioscience). Proliferation was determined by staining T cells with CFSE (Molecular Probes, Eugene, OR)
prior to culture and analyzed flow cytometrically. Survival assays were
performed by propidium iodide (PI) (Molecular Probes) exclusion and flow
cytometry. Flow cytometry was performed on a FACScan or FACSCanto
(BD Biosciences, San Jose, CA) and analyzed with FlowJo software (Tree
Star, Ashland, OR).
Immunoblotting
To probe for Glut1, cells were lysed for 1 h on ice in PBS plus 1% Triton
X-100 and 0.1% SDS containing protease inhibitors (BD Pharmingen) as
previously described (6). When probing for any other protein, cells were
lysed in radio immunoprecipitation assay buffer and quantitated as previously described (22). Equivalent protein concentrations were subjected
to 4–15% or 4–20% SDS-PAGE (Bio-Rad, Hercules, CA). Abs used were
mouse anti-Akt1, rabbit anti–phospho-Akt (S473), rabbit anti–phosphoSTAT5 (Y694) (Cell Signaling Technology, Beverly, MA), rabbit anti–Bcl2, mouse anti-STAT5 (BD Pharmingen), rabbit anti-Mcl-1 (Biolegend, San
Diego, CA), rabbit anti-Glut1 (Abcam), mouse anti-Pim1 (Santa Cruz
Biotechnology, Santa Cruz, CA), or mouse anti-actin (Sigma-Aldrich).
Secondary Abs Alexa Fluor 680 anti-rabbit IgG (Invitrogen, Carlsbad, CA)
and IRDye 800 anti-mouse IgG (LI-COR, Lincoln, NE) were detected
using a LI-COR Odyssey infrared detection system (LI-COR). Contrast
and brightness were adjusted uniformly for each image.
Metabolic assays
Glucose uptake was performed as previously described (19). Glycolytic flux
analysis was also performed as previously described (17). Briefly, glycolysis was determined in freshly isolated 2 3 106 viable T cells by
washing cells in PBS followed by incubation in glucose free Kreb’s buffer
for 30 min prior to addition of 10 mCi of D-[5-3H](N)-glucose (PerkinElmer, Wellesley, MA) and non–radio-labeled glucose to bring total
glucose concentration to 10 mM prior to culture for 1 h. Each reaction was
stopped by addition of an equal volume of 0.2 N HCl. [3H]H20 was separated from [3H]glucose by evaporated equilibrium in a sealed environment. Levels of [3H]H20 produced during glycolysis were measured on
a scintillation counter, and glycolytic flux was determined.
Results
IL-7 and T cell atrophy
Based on the ability of IL-7 to regulate T cell survival (23, 24), size,
and metabolism in vitro (11, 17), we proposed that IL-7 may play an
essential role to sustain T cell metabolism in vivo to prevent T cell
atrophy and apoptosis. To test this hypothesis, purified T cells were
adoptively transferred into IL-7+/+ (wild-type [WT]) or IL-72/2
hosts, and T cells were observed after 7 d. Consistent with prior
studies showing a role for IL-7 in T cell survival, recovery and
survival of transferred T cells was reduced in IL-72/2 hosts (Fig.
T cell purification and culture
Proliferation, survival, and flow cytometry
Cells were stained with fluorescently conjugated Abs against murine CD4,
CD8, CD11c, CD25, CD44, CD62L, CD69, B220, Mac1, Gr1, Nk1.1,
Thy1.1, and Thy1.2 (eBioscience). IL-7R levels were analyzed with anti–
IL-7R conjugated to biotin (clone A7R34 from eBioscience) and a steptavadin PE-Cy5–conjugated secondary (eBioscience). Isotype control for
A
B
8
*
7
330
Mean Foward Scatter
Cell Number (Millions)
T cells were purified from spleen and mesenteric lymph nodes by negative
selection (StemSep, StemCell Technologies, Vancouver, British Columbia,
Canada) and when indicated cultured in RPMI 1640 (Mediatech, Washington, DC) supplemented with 10% FBS (Gemini Bio-Products, Woodland,
CA). Where indicated, IL-7 was supplemented into media at a concentration
of 10 ng/ml (eBioscience, San Diego, CA). Stimulation was accomplished by
culturing T cells on plates coated with anti-CD3ε (clone 145-2C11) and antiCD28 (clone 37.51) (eBioscience) at 5 mg/ml in PBS. Cells were counted,
and cell size was determined on a Coulter Z2 particle counter (Beckman
Coulter, Fullerton, CA).
6
5
4
3
2
1
wt
IL-7 -/-*
320
310
300
290
280
270
260
0
wt
IL-7 -/-
CD4
CD8
FIGURE 1. T cell size and number in the absence of IL-7. A total of 5 3
106 WT T cells were stained with CFSE and adoptively transferred into IL72/2 or WT control animals for 7 d. A, The number of transferred cells
remaining in the spleen was determined by flow cytometric analysis of
CFSE-positive cells. B, Cell size of adoptively transferred cells was determined by mean forward light scatter. Each panel is representative of
three independent experiments. pp , 0.05.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
and localization of IL-7, nor have other cell extrinsic signals been
identified that may play this crucial role in vivo.
Identification of signals that regulate T cell metabolism in vivo
may provide insight into fundamental T cell homeostatic mechanisms and suggest possible new approaches to immunomodulation.
We hypothesized that IL-7 and IL-7R may play essential roles as
homeostatic social control signals in vivo to regulate basal T cell
metabolism and describe in this study a model for inducible deletion of the IL-7R to test this notion. In this conditional transgenemediated rescue system, excision of IL-7R in resting mature T cells
by Cre-recombinase resulted in decreased T cell size, number,
survival, and growth. Importantly, IL-7R–deficient T cells showed
a marked decrease in glycolytic flux. Other hematopoietic cell
lineages were also restored by transgenic expression of IL-7R yet
were unaffected by the acute loss of IL-7R expression, supporting
a T cell intrinsic effect of IL-7R loss on T cell metabolism. This
study establishes for the first time that, in addition to preventing
apoptosis, homeostatic or social control signals are required
in vivo to maintain cell metabolism and identifies IL-7 as an essential in vivo homeostatic signal to prevent atrophy and sustain
the basal rate of metabolism and glycolysis in resting T cells.
IL-7 MAINTAINS T CELL GLYCOLYSIS
The Journal of Immunology
3463
1A). Importantly, the size of surviving CD4 and CD8 cells was also
decreased, suggesting that T cells also require IL-7 to prevent atrophy (Fig. 1B) and possibly to maintain cell metabolism. This
decrease in cell size in vivo mimicked in scale that seen following
growth factor withdrawal in vitro (2). The relationship between cell
size and glucose metabolism established from in vitro work suggested that this decrease in cell size may have been due to a decrease in available energy and glucose uptake or metabolism.
Nevertheless, it was not technically possible to recover sufficient
T cells following adoptive transfer to perform biochemical glucose
uptake assays to test this notion. To address IL-7 regulation of
glucose metabolism in vivo more directly, an inducible IL-7R Cre/
Lox transgenic system was produced to allow isolation of large
numbers of T cells deprived IL-7 in vivo.
Model for conditional expression of IL-7R in vivo
A
CD4
C
IL-7Rα
loxP
loxP
floxed IL-7R tg
wt
IL-7Rflox
-/IL-7R
isotype
% of Max
Lck
CD8
-/-
X IL-7R
X
IL-7R
IL7-R
B
null
floxed IL-7R tg
-/Cre:ER, IL-7R
+ Tamoxifen
floxed IL-7R tg excised
-/Cre:ER, IL-7R
IL-7R
D
WT KO Tg non Tg non
Thymus
F
CD4
wt
-/IL-7R
flox
IL-7R
160
140
120
100
80
60
40
20
0
*
*
Thymus
E
CD8
10
4
10
3
10
2
80.1
4.97
0
10
4
10
3
10
2
10
10
Spleen
CD8
CD25
wt
6.04
101
10
10
0
8.84
4
10
1
10
2
10
3
10
4
IL-7R
4
3
10
3
10
2
10
2
10
1
10
1
10
0
10
0
4
10
4.65
4
10
3
10
2
1
10
0
10
79.7
4.31
10
0
11.4
10
1
10
2
0
10
1
10
2
10
3
10
3
10
4
4
10
3
10
2
1
10
1
0
10
0
1.31
IL-7R
5.43
10
15.9
10
flox
10
10
10
12.2
Spleen
-/-
81.1
3.06
10
0
10.4
10
1
10
2
4
10
0
10
1
10
2
10
3
3
10
CD44
G
15.3
10
4
10
0
10
1
10
2
10
3
10
B220
CD11c
CD69
Mac1
1.13
0.86
2.4
% of Max
CD62L
2.7
2.55
13.7
9.11
8.42
35.2
59.7
50.7
7.09
wt
IL-7Rflox
-/IL-7R
Nk1.1
5.0
4.92
12.9
Gr1
4
7.54
0.92
10
10
CD4
% of Max
FIGURE 2. An inducible IL-7R knockout system.
A, Schematic of the IL-7Rflox transgene, rescue of IL7R2/2 with the transgene, and incorporation of Cre:
ER to allow for in vivo excision. B, DNA extracted
from animal tail snips was subjected to PCR and
agarose gel electrophoresis to amplify a portion of
the IL-7R transgene. C, IL-7R expression on CD4
and CD8 splenic T cells was determined by flow
cytometry. D, Numbers of thymocytes and splenocytes from WT, IL-7R2/2, and IL-7Rflox animals
were determined. E, CD4 and CD8 expression was
determined flow cytometrically in thymocytes and
splenocytes. F, CD4 and CD8 T cells were measured
for expression levels of CD25, CD44, CD62L, and
CD69 flow cytometrically in splenocytes. G, B220,
Mac1, Nk1.1, CD11c, and Gr1 expression were determined flow cytometrically in splenocytes. Each
panel is representative of two or three independent
experiments. pp , 0.01.
flox
Cre:ER
-/IL-7R
Cell Number (millions)
floxed IL-7R tg
-/IL-7R
wt
IL-7Rflox
IL-7R-/-
4
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
T cells have been previously deprived IL-7 in vivo through adoptive
transfer assays or by treatment with IL-7 neutralizing Ab (23–26). Due
to the altered lymphoid environment and whole animal loss of IL-7,
however, these studies did not prove a T cell intrinsic requirement for
IL-7. Nor, due to the low number of recoverable cells after adoptive
transfer, did they allow detailed biochemical and metabolic analyses
to determine the metabolic state of T cells deprived of IL-7 in vivo.
To overcome these limitations, we generated a genetic model for
conditional IL-7R expression in mature resting T cells. In this model
(Fig. 2A), an IL-7R transgene flanked by loxP sites (floxed IL-7R tg)
was expressed (under control of 3.5 bp of the Lck promoter along
with the CD2 enhancer) and used to rescue IL-7R expression on an
IL-7R2/2 background (IL-7Rflox). This promoter construct has been
previously used to express transgenes specifically in T cells (19, 27),
although some leakiness in expression was possible. Animals were
genotyped by PCR amplification with primers within the transgene to
demonstrate the presence of transgenic IL-7Rflox DNA (Fig. 2B).
Expression of IL-7R by the IL-7Rflox transgene on an IL-7R2/2
background was reduced compared with a nontransgenic WT mouse,
but was significantly higher than background staining observed on
IL-7R2/2 T cells or isotype control (Fig. 2C). Expression of a tamoxifen-regulated Cre-recombinase transgene (Cre:ER) allowed for
excision of the floxed IL-7R transgene in vivo to produce IL-7R–
deficient T cells (IL-7Rnull). This transgenic rescue system provided
a rapid means to generate large numbers of acutely IL-7R–deficient
T cells to determine the intrinsic role of IL-7 in resting mature T cells.
It was first important to establish if expression of the IL-7Rflox
transgene reconstituted development and function of T cells that
otherwise lacked endogenous IL-7R. As expected, the number of
cells present in the thymus and spleen of IL-7R2/2 mice was
significantly reduced compared with WT, and expression of the
IL-7Rflox transgene resulted in an effective restoration of cell
numbers in these tissues compared with IL-7R2/2 (Fig. 2D). The
somewhat lower cell number in the thymus and spleen of IL-7Rflox
compared with WT animals may have been due in part to lower
IL-7R expression level in IL-7Rflox relative to WT. Nevertheless, T
cell CD4 and CD8 percentages in IL-7Rflox animals resembled
those of WT in both the thymus and the spleen (Fig. 2E),
3464
IL-7 MAINTAINS T CELL GLYCOLYSIS
IL-7Rflox is efficiently excised
We evaluated the T cell intrinsic role for IL-7R by treatment of IL7Rflox transgenic mice with tamoxifen to activate Cre:ER and excise the IL-7Rflox transgene. Three days postinjection, IL-7Rflox
was excised in ∼80–90% of CD4 and CD8 T cells, whereas no loss
of IL-7R expression was detected on IL-7Rflox T cells that did not
express the Cre:ER transgene (Fig. 4A). To better characterize the
deletion of IL-7Rflox transgene, surface IL-7R levels were measured
over time posttreatment with tamoxifen (Fig. 4B). One day after
tamoxifen injection, IL-7R levels were decreased, with the most
significant loss of IL-7R expression on days 2 and 3. By day 7
posttreatment, IL-7R+ T cells that likely avoided deletion of the
transgene appeared to outgrow, and IL-7R+ T cells were readily
detected. To most efficiently isolate T cells rendered IL-7Rnull
in vivo, therefore, animals were examined 3 d after tamoxifen
treatment in the following experiments unless otherwise noted. As
acute loss of IL-7R and IL-7–induced signals may affect multiple
aspects of T cell phenotype and physiology, it was important for
comparison of survival and metabolic characteristics of IL-7Rflox
and IL-7Rnull T cells to determine if IL-7Rnull T cells retained their
immunologic phenotype at this time point. T cell activation markers
were examined, and expression of CD25, CD44, CD62L, and CD69
was found unaltered in both CD4 and CD8 T cells following IL7Rflox excision (Fig. 4C). In addition, although B cell numbers were
largely rescued by expression of the IL-7Rflox transgene on an IL7R2/2 background (Table I), they were not affected by deletion of
IL-7Rflox and appeared to retain their numbers over time (Fig. 4D
and data not shown). Acute loss of IL-7R, therefore, did not appear
to alter T cell activation phenotype or provide an immediate selective advantage to a particular subset, although it is possible such
a selective advantage may occur over longer time periods, and other
hematopoietic lineages appeared unaffected.
Decreased IL-7R expression after tamoxifen treatment also
resulted in a functional loss of IL-7R. Phosphorylation of STAT5 and
induction of the STAT5 target gene Pim1 were reduced in IL-7–
treated IL-7Rnull T cells (Fig. 5A), and IL-7Rnull T cells failed to
upregulate Bcl-2 and Mcl-1 antiapoptotic proteins when cultured in
IL-7 (Fig. 5B). Culture of purified IL-7Rflox and IL-7Rnull T cells in
the presence IL-7 revealed that IL-7Rflox T cells exhibited an ∼70%
survival rate during 3 d of culture, whereas IL-7Rnull T cells underwent more rapid apoptosis (Fig. 5C). IL-7Rnull cells cultured in
the absence of cytokine also demonstrated an increased rate of
death compared with IL-7Rflox cells (Fig. 5C), suggesting that loss
of IL-7R in vivo primed cells for a more rapid death. However,
T cells isolated from tamoxifen-treated IL-7Rflox mice and cultured
in IL-7 had marginally improved survival over IL-7Rflox T cells
cultured in the absence of cytokine that was likely due to the small
remaining population of IL-7R+ cells. Together, these data demonstrated efficient excision of the transgenic IL-7R and inhibition
of T cell responses to endogenous IL-7.
IL-7Rflox transgene largely rescues IL-7R2/2 phenotype
The rescue of normal T cell numbers and phenotypes by expression
of the IL7Rflox transgene suggests that IL-7R signaling and function
were reconstituted on an IL-7R2/2 background. To test this, the
ability of the IL-7Rflox transgene to promote phosphorylation of the
transcription factor STAT5, induction of the STAT5 target gene
Pim1, and activation of the PI3K/Akt pathway were determined
(11). Similar to WT T cells, addition of IL-7 to purified IL-7Rflox
transgenic T cells led to phosphorylation of STAT5 and induction of
the STAT5 target gene Pim1 (Fig. 3A). The PI3K/Akt pathway was
also activated by the IL-7Rflox transgene, although to a lesser extent
than that observed in WT T cells. Each of these signaling pathways
can regulate cell metabolism (6, 11, 30), and the glycolytic rates of
resting WT and IL-7Rflox T cells were tested and found to be indistinguishable (Fig. 3B). Additionally, IL-7Rflox T cells exhibited
a similar level of glucose uptake ex vivo and maintenance of glucose uptake in response to IL-7 compared with WT (Fig. 3C). The
IL-7Rflox transgene also promoted cell survival similar to WT cells.
Endogenous and IL-7Rflox rescued T cells cultured in the presence
of IL-7 maintained an ∼80% survival rate, whereas without IL-7,
only 10% survival was achieved in both WT and IL-7Rflox cells
after 3 d (Fig. 3D). Together, these data suggest that expression of
the IL-7Rflox transgene rescued the T cell developmental defect of
IL-7R2/2 animals and allowed generation of normal mature T cells
capable of inducing canonical IL-7 signaling and survival.
Table I.
Hematopoietic phenotype of IL-7Rflox mice
Cell Number (Millions)
WT
B220+
Mac1+
NK1.1+
CD11c+
Gr1+
49.9
3.5
2.8
1.6
0.7
6
6
6
6
6
IL-7R
10.7
3.0
0.4
ND
0.1
31.6
2.7
1.8
1.3
0.6
flox
6
6
6
6
6
4.0
2.1
0.9
ND
0.3
IL-7R Expression (MFI)
IL-7R
3.8
1.5
0.7
1.0
0.2
6
6
6
6
6
2/2
3.5
1.6
0.4
ND
0.1
IL-7Rflox
WT
23.4
80.1
230
33.2
233
6
6
6
6
6
5.9
45.3
85.8
ND
136
23.7
81
136
33.2
112
6
6
6
6
6
0.2
40.0
76.0
ND
37.2
IL-7R2/2
28.6
34.6
18.5
27.4
35.2
6
6
6
6
6
3.7
13.0
6.5
ND
7.2
Number of positive splenocytes for a given stain for WT, IL-7Rflox, and IL-7R2/2 animals is shown on the left in millions.
Mean fluorescence intensity for IL-7R is indicated for each stain.
MFI, mean fluorescence intensity; ND, not determined.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
suggesting that although numbers were modestly reduced, expression of the IL-7R under an ectopic promoter did not negatively impact T cell development (28, 29). In addition, the surface
expression levels of activation markers CD25, CD44, CD62L, and
CD69 were similar between WT and IL-7Rflox when compared
with IL-7R2/2 CD4 or CD8 T cells (Fig. 2F). As the transgene
promoter was intended to be T cell specific and should result in
restored IL-7R expression levels in T cells alone, the effects on the
levels of other immune cells were investigated. Interestingly, the
splenic numbers and phenotype of B cells, macrophages, NK cells,
granulocytes, and dendritic cells more closely resembled WT in IL7Rflox animals than IL-7R2/2 (Fig. 2G, Table I). Some expression
of IL-7R was detectable on these cells in IL-7Rflox animals (Table
I). It is unclear if this restoration of numbers of other hematopoietic
lineages was due to leaky expression of the transgene within these
cell types or indirect effects through the presence of T cells. It is
important to note, however, that this phenotype was observed in
multiple transgenic founder mice (data not shown), demonstrating
that rescue of these hematopoietic lineages was not due to an insertional effect of the transgene. Together, these data demonstrate
that although not wholly restored to WT, expression of the IL7Rflox transgene effectively rescued the T cell developmental defect
of IL-7R2/2 animals and allowed generation of mature T cells and
other hematopoietic cell lineages that appeared normal.
The Journal of Immunology
3465
B
A
STAT5
flox
wt flox wt flox
p-STAT5
Akt
p-Akt
Pim1
Actin
2.5
2
1.5
1
0.5
0
IL-7Rflox
wt
C
D
6000
wt
flox
IL-7R
5000
4000
*
3000
*
2000
1000
Loss of IL-7R effects cell survival
In previous studies, IL-7 signaling has been shown to play a critical
role in survival of resting mature T cells, but it has not been possible
to establish this dependence as T cell intrinsic due to whole-animal
loss or inhibition of IL-7 (23–26). Our model also results in wholeanimal genetic loss of IL-7R, although all hematopoietic lineages
appear phenotypically normal prior to IL-7Rflox deletion and did
not undergo rapid cell death or appear to change phenotype after IL7Rflox deletion. To test the T cell dependence on IL-7, IL-7Rflox
mice with or without expression of Cre:ER were treated with tamoxifen, and T cell numbers were observed (Fig. 6A). Three days
posttreatment, recovery of viable CD4 and CD8 T cells was reduced approximately one third, suggesting that T cell viability required intrinsic expression of IL-7R. Although loss of IL-7R in vivo
appeared to result in T cell death, IL-7Rflox and IL-7Rnull T cells
exhibited only slight decreases in Mcl-1 or Bcl-2 levels ex vivo
(Fig. 6B, 6C). Nevertheless, IL-7Rnull cells were prone to rapid
apoptosis and demonstrated an increased rate of cell death compared with IL-7Rflox cells in culture in the absence of cytokine (Fig.
5C). IL-7 can regulate Bcl-2 and Mcl-1 expression (15, 31), and it is
possible that T cells that lose expression of these antiapoptotic
Bcl-2 family proteins undergo rapid cell death in vivo and were thus
not able to be isolated for inclusion in these analyses.
To directly measure cell survival upon acute loss of IL-7R in an
otherwise normal lymphoid environment, mature resting T cells
from IL-7Rflox animals or WT animals were adoptively transferred
into allelically marked normal hosts. All recipients were treated
with tamoxifen, and the percentage of transferred cells remaining
was measured over a 2-wk period in both spleen (Fig. 6D) and
intestinal lymph nodes (Fig. 6E), where the percentage of IL-7Rnull
T cells declined more rapidly than WT-transferred T cells. After
2 wk, only 20% of the originally transferred IL-7Rnull T cells remained in both tissues, whereas WT T cell numbers remained
constant in the spleen and decreased only 40% in lymph nodes.
These data demonstrate that IL-7 is required for naive T cell survival in a normal immune environment in a T cell-intrinsic manner.
IL-7R and T cell atrophy in vivo
In addition to cell survival, T cells require cell extrinsic signals to
prevent atrophy, which is characterized by decreased cell size and
rate of growth. Treatment with rIL-7 can prevent T cell atrophy
-IL-7
+IL-7
wt
-IL-7
IL-7R flox -IL-7
wt
+IL-7
IL-7R flox +IL-7
Day 1
Day 2
Day 3
in vitro (17), and consistent with a role for IL-7 in regulation of
T cell atrophy, excision of the IL-7Rflox transgene in vivo resulted in
a significant decrease in T cell size (Fig. 7A) that was not due to
increased isolation and measurement of dead cells, as viability of
purified T cells was equivalently high regardless of IL-7R status
(Fig. 7B). Interestingly, this cell size decrease was similar in scale
to that observed following ectopic expression of a Bcl-xL transgene, which prevents apoptosis of growth factor-deprived T cells
and results in accumulation of atrophic T cells in vivo (2). To determine if IL-7Rnull T cells also had a reduced ability to grow when
stimulated, as described for other atrophic cells (2, 32), IL-7Rflox
and IL-7Rnull T cells were stimulated with anti-CD3 and antiCD28, and cell size and proliferation were observed. At early time
points, IL-7Rnull T cells remained smaller than IL-7Rflox T cells, as
shown by size measurement by flow cytometry and particle size
analyzer (Fig. 7C). However, IL-7Rnull T cells ultimately grew to
the same extent (Fig. 7D), demonstrating that the small cell size at
early time points was due to a decreased rate of growth rather than
diminished capacity for growth. Additionally, the decrease in cell
growth was associated with delayed cell division, as loss of IL-7R
in vivo prior to stimulation resulted in decreased proliferation as
measured by CFSE dilution (Fig. 7E). Loss of IL-7 signaling
in vivo, therefore, results in atrophy and an impaired ability of
T cells to grow and proliferate when stimulated.
Glycolysis is regulated by IL-7 signaling in vivo
T cell atrophy in vitro results in decreased glucose uptake and metabolism and ability to survive and grow (2). Conversely, transgenic
expression of Glut1 to elevate glucose uptake can promote increased
T cell size and activity (19). Cellular atrophy and delayed growth of
IL-7Rnull T cells, therefore, suggested that IL-7R may have an essential role as a homeostatic control signal to maintain basal resting
T cell glucose metabolism. Indeed, IL-7R signaling has the capacity
to sustain T cell glucose uptake and glycolysis in vitro (11, 17, 18). It
remained unclear if IL-7 was essential to maintain cell metabolism
in vivo. Loss of IL-7 signals could potentially affect glucose metabolism at the level of the glucose transporter, rate of glucose uptake,
or flux through glycolysis. Unexpectedly, purified IL-7Rnull T cells
had higher total levels of Glut1 protein 3 d postexcision compared
with IL-7Rflox T cells, although 7 d postexcision, Glut1 protein levels
returned to starting IL-7Rflox levels (Fig. 8A, 8B). Nevertheless, loss
of IL-7R in vivo did not affect glucose transport even at day 3
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
0
fresh
90
80
70
60
50
40
30
20
10
0
% Live Cells
Glucose Uptake (CPM)
FIGURE 3. The IL-7R transgene responds to IL7. A, T cells from WT and IL-7Rflox animals were
cultured in the presence or absence of IL-7 for 18 h
prior to lysis and immunoblotting of 40 mg protein
with the indicated Abs. B, Glycolytic flux was measured in WT and IL-7Rflox-purified resting T cells. C,
T cells purified from WT and IL-7Rflox animals were
analyzed for ability to uptake glucose when resting or
following culture in the presence or absence of IL-7 for
24 h. D, Purified mature T cells from WT and IL-7Rflox
animals were cultured in the presence or absence of
IL-7 and analyzed for cell survival at indicated times.
Each panel is representative of two or three independent experiments. pp , 0.02.
+IL-7
Glycolysis (nmol/million*hours)
-IL-7
3466
IL-7 MAINTAINS T CELL GLYCOLYSIS
96.2%
16.1%
IL-7R
flox
IL-7R
IL-7Rnull
CD8
96.8%
29.6%
95.4%
70
1 day IL-7R
IL-7Rflox -IL-7
IL-7Rflox +IL-7
IL-7Rnull -IL-7
IL-7Rnull +IL-7
50
40
30
null
2 days IL-7R
0
24hrs
3 days IL-7R
16.0%
15.0%
24.2%
28.8%
% of Max
7 days IL-7R
80
48hrs
72hrs
FIGURE 5. Loss of IL-7 responses. A and B, T cells cultured in the presence or absence of IL-7 for 18 h were lysed, and 35 or 40 mg, respectively,
were immunoblotted with the indicated Abs. Quantified protein levels indicated are normalized to actin. C, PI exclusion was used to determine cell
survival at 24 h intervals of cells cultured in the presence or absence of IL-7.
Each panel is representative of two or three independent experiments.
inducible IL-7R knockout system, we identified IL-7 as a homeostatic control signal for the regulation of metabolism via maintenance of glycolysis. Additionally, our data support a T cell-intrinsic
dependence on IL-7 to maintain T cell survival and metabolism in an
% of Max
posttreatment, as ex vivo glucose uptake was unchanged in IL-7Rnull
T cells compared with IL-7Rflox T cells (Fig. 8C). This apparent
discrepancy may reflect a decreased proportion of Glut1 protein on
the cell surface due to loss of IL-7R signals and Akt activation (6, 11),
yet a compensatory increase in Glut1 protein may have resulted in
maintenance of overall glucose uptake. In addition to glucose uptake,
glucose metabolism is regulated by a number of proteins and additional control mechanisms, including hexokinase and phosphofructokinase (33, 34) that may have affected metabolism despite
maintenance of glucose uptake in the absence of IL-7R. Consistent
with these additional control mechanisms and in support of a key
metabolic role for IL-7, IL-7Rnull T cells failed to maintain a basal
rate of glycolytic flux compared with IL-7Rflox T cells after 3 d invivo lacking IL-7 signals (Fig. 8D). This finding suggests that IL-7
provides a key homeostatic signal for T cell metabolism, and, following the loss of IL-7R, a decrease in glycolytic flux may result in
an inability of T cells to produce the energy required to prevent atrophy and maintain survival.
Discussion
Together, these data show that mature resting T cells have an intrinsic
requirement for IL-7R signaling to maintain cell survival, growth
rates, and glucose metabolism. Through the development of an
A
Cell Number (millions)
B
*
20
18
16
14
12
10
8
6
4
2
0
flox
IL7R
null
IL7R
flox null
Mcl1
Bcl2
Actin
*
Mcl1/Actin
Bcl2/Actin
CD4
1
1
0.85
0.88
CD8
C
D
wt
null
IL-7R
1.6
flox
% of Max
B220
FIGURE 4. The IL-7R
is efficiently excised. IL-7Rflox animals both
with and without the Cre:ER transgene were treated with tamoxifen, and
splenocytes were stained for CD4, CD8 and IL-7R and analyzed by flow
cytometry after 3 d (A) or the indicated time (B). Numbers indicate percentage of cells in the shown gate. C, IL-7Rflox animals both with and
without the Cre:ER transgene were treated with tamoxifen, and 3 d posttreatment splenocytes were stained for CD4, CD8, CD25, CD44, CD62L,
and CD69 and analyzed by flow cytometry. D, IL-7Rflox animals both with
and without the Cre:ER transgene were treated with tamoxifen, and 3 d
posttreatment splenocytes were stained for B220. Each panel is representative of two independent experiments.
flox
IL-7R
IL-7Rnull
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Bcl2
Day2
E
Day4
Day7
Day14
wt
IL-7Rnull
1.2
% Transferred Cells
% of Max
flox
IL-7R
IL-7Rnull
Mcl1/Actin 1.00 0.85 1.53 0.80
Bcl2/Actin 1.00 0.85 1.45 0.82
1.00 0.16
10
13.8%
IL-7R
D
0.00
1
0.8
0.6
0.4
0.2
0
Day2
Day4
Day7
Day14
FIGURE 6. Loss of IL-7R signaling and cell survival. A, The number of
purified CD4 and CD8 T cells 3 d after tamoxifen treatment was determined from IL-7Rflox and IL-7Rnull spleens. B, Purified T cells were
lysed ex vivo, and 40 mg was immunoblotted as indicated. Quantified
protein levels shown are normalized to actin. C, Purified T cells were
stained intracellularly for Bcl-2 and analyzed by flow cytometry. D and E,
A total of 4.5 million cells from IL-7Rflox or WT animals were adoptively
transferred in Thy1.1 congenic mice. Twelve hours following transfer,
recipient animals were tamoxifen treated, and percentage of transferred
cells remaining was determined by staining of splenocyte (D) or lymph
node (E) suspension with Thy1.1 and Th1.2 fluorescently conjugated Abs
and flow cytometry. Each panel is representative of two to six independent
experiments. pp , 0.01.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
CD62L
Mcl1
Bcl2
Actin
60
23.0%
flox null flox null
20
17.2%
null
CD44
null flox null
STAT5
p-STAT5
Pim1
Actin
C
null
CD25
CD69
flox
-IL-7 +IL-7
B
-IL-7 +IL-7
A
10.0%
p-STAT5/Actin 0.00
null
C
CD8
% Transferred Cells
0
CD4
14.8%
% Live Cells
% of Max
95.4%
12.4%
CD4
B
CD8
isotype
CD4
IL-7Rflox Cre ER-
A
The Journal of Immunology
3467
A
*
B
145
10 4
140
10 3
135
10
2
130
10 1
PI
Cell Size (fL)
150
125
IL-7Rnull
IL-7Rflox
Mean Forward Scatter
330
310
290
270
250
230
210
190
170
150
IL-7R flox
IL-7R null
*
10 0
0
200
400
400
600
800
1000
0
200
400
800
1000
*
flox
IL-7R
null
IL-7R
600
*
300
250
200
100
CD3
CD3 + CD28
no stimulation
CD3
CD3 + CD28
flox
Mean Forward Scatter
no stimulation
1.86%
2.08%
CD3
CD3 + CD28
CD3
72.8%
67.1%
CD3 + CD28
83.4%
71.9%
IL-7Rflox
IL-7Rnull
CFSE
otherwise apparently normal lymphoid compartment. However, the
mechanism of cell survival maintenance remains elusive, as levels of
Bcl-2 family members appeared unchanged in vivo, possibly due to
death of cells with decreased levels of protein. Additionally, IL-7
signaling was important prior to T cell activation, as the ability of
A
B
% of Max
flox
IL-7R
Day 3 IL-7Rnull
null
Day 7 IL-7R
flox null
Glut1
Actin
Glucose Uptake (CPM)
C
D
4500
4000
3500
3000
2500
2000
1500
1000
500
0
IL-7R
flox
IL-7R
null
Glycolysis (nmol/million*hours)
Glut1
*
8
7
6
5
4
3
2
1
0
IL-7Rflox
flox
IL-7R null
FIGURE 8. Loss of IL-7R and glycolysis. IL-7R – and IL-7Rnull–
purified T cells were permeabilized and stained with anti-Glut1 Ab followed by flow cytometry 3 or 7 d post tamoxifen treatment (A) or lysed and
10 mg immunoblotted 3 d post tamoxifen treatment (B). IL-7Rflox– and IL7Rnull–purified resting T cells were analyzed for ability to uptake glucose
3 d post tamoxifen treatment (C) or exposed to [3H]glucose to measure
glycolytic flux (D). Each panel is representative of two to six independent
experiments. pp , 0.001.
cells to stimulate was decreased in the absence of IL-7 signaling.
Most critically, IL-7R was found to be essential to maintain homeostatic control over T cell metabolism and glycolytic flux, with
significantly decreased glycolysis in T cells lacking IL-7R in vivo,
despite myriad other microenvironmental signals. Taken together,
this work identifies a novel feature of IL-7 control of naive T cell
metabolism that may have broad import for control of T cell homeostasis and survival.
The transgenic model for conditional deletion of IL-7R employed
in this study led to rescue of multiple hematopoietic cell types, yet
showed an apparent specific role for IL-7R in resting mature T cells.
It is not clear why numbers of non-T cells were near normal in IL7Rflox mice, but this phenotype was observed in several transgenic
founder lines, and it is likely that leaky transgene expression may
have rescued IL-7–dependent developmental checkpoints. Nevertheless, treatment of mice with tamoxifen did not appear to lead to
significant loss of mature cells of these lineages. T cell development
and phenotype also appeared normal in this model despite constitutive transgene-driven IL-7R expression, and loss of IL-7R did not
detectably alter T cell phenotype or the presence of distinct T cell
subsets. It appears, therefore, that resting mature T cells have an
intrinsic dependence on IL-7R signaling for metabolism and survival, although a subtle shift in T cell development and status or
a secondary effect on T cell metabolism of altered signaling and IL7R deficiency due to somewhat reduced numbers or phenotype of
other cell types cannot be formally excluded.
Although regulation of glucose metabolism suggests a novel means
by which IL-7R may affect cell fate, several questions remain. It is
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
IL-7R
IL-7Rnull
no stimulation
% of Max
91.5
10 1
150
500
450
400
350
300
250
200
150
100
50
0
E
10 2
86.7
350
no stimulation
D
10 3
FSC
*
IL-7Rnull
10 4
10 0
Cell Size (fL)
C
FIGURE 7. Loss of IL-7R and cell growth. A, Purified T cells expressing or lacking expression of IL-7R
were examined for cell size by particle size analyzer.
pp , 0.01. B, Percentage of live cells was determined
following purification by PI exclusion and flow cytometry. C, IL-7Rflox and IL-7Rnull purified T cells were
cultured on control or plates coated with 5 mg/ml antiCD3 with or without 5 mg/ml anti-CD28 Abs and assayed for cell size by mean forward scatter after 18 h in
culture (left panel) (pp , 0.05), by particle size analyzer after 20 h in culture (right panel) (pp , 0.05), or
by mean forward scatter after 48 h in culture (D). E,
Proliferation was measured after 48 h by CFSE dilution.
Each panel is representative of two to six independent
experiments.
IL-7Rflox
3468
the first time that T cells require IL-7–induced signals in vivo to
maintain basal resting glucose metabolism. Maintenance of T cell
homeostasis is critical for proper immunity, and these findings
show that IL-7 provides a homeostatic signal essential for T cell
glycolytic flux. This control of cell metabolism likely influences
a wide array of cell phenotypes. It will be important in future work
to further characterize and establish how this plasticity in basal
T cell glycolytic flux influences T cell size, survival, and growth in
normal immunity as well as in immune pathology.
Acknowledgments
We thank Brian Altman for critical evaluation of this manuscript as well as
the Duke Transgenic Mouse Facility and the Duke University Flow Cytometry Shared Resource. We also thank Eric Brown and Motonari Kondo for
the generous donation of animals.
Disclosures
The authors have no financial conflicts of interest.
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unclear how IL-7 regulation of Bcl-2 family members contributes to
cell survival relative to effects on cell metabolism. Cells must maintain
a minimal rate of glucose metabolism to survive (19, 35), yet Bcl-2
family proteins can slow or prevent cell death even in very low
glucose conditions (36). IL-7 may prevent cell death by simultaneously promoting Bcl-2 and Mcl-1 expression and stimulating
glycolysis. Thus, loss of IL-7 or IL-7 deficiency may lead to decreased expression of antiapoptotic Bcl-2 family proteins and decreased rates of glycolysis, resulting in a cumulative sensitization of
T cells to apoptosis. Nevertheless, we failed to detect large decreases
in Bcl-2 or Mcl-1 protein levels postexcision of the IL-7R in vivo.
These proteins may be maintained through the action of other growth
factors present in a normal lymphoid environment, and IL-7 is potentially not essential in vivo for their expression. Alternatively, decreased levels of these proteins may lead to rapid cell death,
preventing isolation of Bcl-2 and Mcl-1 low-expressing cells for ex
vivo analysis. It is also unclear how IL-7R signals regulate glycolysis.
Both STAT5 and PI3K/Akt signaling pathways can promote glucose
uptake (11), and IL-7 can promote both pathways, although the
PI3K/Akt pathway is poorly activated by IL-7 relative to other cytokines. It will be important in future work to clarify the role of these
signaling molecules in the control of cell metabolism.
Glucose metabolism is controlled through coordinated regulation
of glucose uptake and glycolytic flux. Glucose uptake is limiting in
T cell activation (19) and must be tightly regulated, yet, surprisingly, IL-7R was not essential to maintain glucose uptake in resting
T cells. This may have been due to the balanced effects of decreased
Glut1 trafficking to the cell surface and compensatory increase in
Glut1 protein level. Similarly, glycolysis can be controlled at many
levels, and it is likely IL-7–mediated regulation of glycolytic flux
occurs downstream of glucose uptake. For example, Akt has been
implicated in the control of phosphofructokinase-2 to maintain
glycolytic flux and cell survival (37), as well as the localization of
hexokinase to the mitochondria (38). Alternative splice variants of
glycolytic enzymes have also been shown to regulate glucose metabolism, such as the M2 variant of pyruvate kinase, which increases aerobic glycolysis (39), and similar regulations may occur
as a consequence of IL-7R signaling. As glucose uptake is not altered by the loss of IL-7R, it is possible that these alternate regulations of metabolism may mediate IL-7–dependent glycolysis.
IL-7 regulation of glycolysis may be a critical component of IL7–dependent cell survival in vivo. This may arise by providing
energy to prevent metabolic stress or by metabolic regulation of
apoptotic proteins. In developing thymocytes, deprivation from
Notch signaling decreased glucose uptake and resulted in rapid
apoptosis (35), and expression of a constitutively active Akt can
maintain glucose uptake to prevent cell death in thymocytes and
other cell types (10). Furthermore, directly increased glucose metabolism due to Glut1 overexpression can confer a cell survival
advantage via glycogen synthase kinase 3-dependent maintenance
of Mcl-1 following growth factor withdrawal (22). Conversely, the
proapoptotic proteins Puma and Bim are induced when cells are
deprived of sufficient glucose (40). Glucose metabolism in hematopoietic cells is, therefore, capable of regulating Bcl-2 family
proteins and cell survival, and IL-7 may regulate T cell homeostasis
in part through control of glycolysis and similar signaling pathways. An important implication of this finding is that modulation of
T cell metabolism either directly or through control of metabolic
regulatory mechanisms may provide a useful means to control
T cell homeostasis and trophic state.
The role of the social control model and cell extrinsic signals to
maintain cell viability and function has been widely accepted (1),
yet evidence for how such extrinsic signals impact cell physiology
has been limited. Findings presented in this study demonstrate for
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