Article

The Bone Marrow Protects and Optimizes

Immunological Memory during Dietary Restriction
Graphical Abstract Authors
Nicholas Collins, Seong-Ji Han,
Michel Enamorado, ...,
Dorian B. McGavern,
Pamela L. Schwartzberg,
Yasmine Belkaid

Correspondence
ybelkaid@niaid.nih.gov

In Brief
Calorie restriction triggers memory T cell
homing to the bone marrow to promote
survival and enhanced protective
function.

Highlights
d Dietary restriction promotes memory T cell accumulation
in BM

d BM trophic factors and adipocytes promote memory T cell

accumulation in BM

d Memory T cells display enhanced protective function during

dietary restriction

Collins et al., 2019, Cell 178, 1088–1101

August 22, 2019 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.cell.2019.07.049
 Article

The Bone Marrow Protects and Optimizes

Immunological Memory during Dietary Restriction
Nicholas Collins,1 Seong-Ji Han,1 Michel Enamorado,1 Verena M. Link,1 Bonnie Huang,2 E. Ashley Moseman,3
Rigel J. Kishton,4 John P. Shannon,5 Dhaval Dixit,6 Susan R. Schwab,6 Heather D. Hickman,5 Nicholas P. Restifo,4
Dorian B. McGavern,3 Pamela L. Schwartzberg,2 and Yasmine Belkaid1,7,*
1Metaorganism Immunity Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National

Institutes of Health, Bethesda, MD 20892, USA

2Cell Signaling Section, Laboratory of Immune System Biology, National Institutes of Health, Bethesda, MD 20892, USA
3Viral Immunology and Intravital Imaging Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health,

Bethesda, MD 20892, USA

4Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20814, USA
5Viral Immunity and Pathogenesis Unit, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD

20892, USA
6Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016, USA
7Lead Contact

*Correspondence: ybelkaid@niaid.nih.gov
https://doi.org/10.1016/j.cell.2019.07.049

SUMMARY
Mammals evolved in the face of fluctuating food avail-
ability. How the immune system adapts to transient
nutritional stress remains poorly understood. Here,
we show that memory T cells collapsed in secondary
lymphoid organs in the context of dietary restriction
(DR) but dramatically accumulated within the bone
marrow (BM), where they adopted a state associated
with energy conservation. This response was coordi-
nated by glucocorticoids and associated with a
profound remodeling of the BM compartment, which
included an increase in T cell homing factors,
erythropoiesis, and adipogenesis. Adipocytes, as
well as CXCR4-CXCL12 and S1P-S1P1R interactions,
contributed to enhanced T cell accumulation in BM
during DR. Memory T cell homing to BM during DR
was associated with enhanced protection against in-
fections and tumors. Together, this work uncovers a
fundamental host strategy to sustain and optimize
immunological memory during nutritional challenges
that involved a temporal and spatial reorganization of
the memory pool within ‘‘safe haven’’ compartments.
INTRODUCTION
Host survival depends on the ability to adapt to challenges in a
way that sustains and protects fundamental physiological pro-
cesses. Immunological memory is a cardinal feature of the adap-
tive immune system, which confers a survival advantage by
allowing the host to rapidly and effectively control subsequent
challenges. Such responses rely on the ability of memory
T cells to persist long term, which can be divided into circulating
and resident subsets. Circulating cells include central, effector,
and peripheral memory T cells (TCM, TEM, and TPM) (Gerlach
et al., 2016; Sallusto et al., 1999) that are required for body-
wide immunosurveillance, whereas tissue resident memory cells
(TRM) are essential for initiating and amplifying local responses
(Jameson and Masopust, 2018; Mueller and Mackay, 2016). At
steady state, memory T cell homeostasis is under the control
of various cytokines, transcription factors, and metabolic fuels
(Buck et al., 2016; Cui et al., 2015; Kaech and Cui, 2012; Pan
et al., 2017; Surh and Sprent, 2008). However, these long-lived
cells are faced with numerous challenges throughout the life of
the host, including their persistence and maintenance of protec-
tive function during stress and reduced nutritional availability.
Indeed, food accessibility was and can remain highly contingent
on encounters with distinct environments and climatic condi-
tions. Thus, mechanisms may have evolved to ensure that the
host can adapt and thrive in situations where calories and nutri-
ents are limited. Of interest, caloric restriction or dietary restric-
tion (DR) has been shown to promote various aspects of host
fitness, including the improvement of metabolic profiles, preven-
tion of cellular aging, and reduced incidence of cancer (Nikolich-
Zugich and Messaoudi, 2005; Redman et al., 2018; Robertson
and Mitchell, 2013; Speakman and Mitchell, 2011). However,
the consequence of DR on the memory T cell compartment re-
mains to be addressed.
Due to the importance of memory T cells for host survival,
defined strategies or compensatory mechanisms may be in place
to sustain these cells in the context of nutritional challenges. Of
relevance, we and others have found that white adipose tissue
(WAT) is a reservoir for memory T cells (Han et al., 2017; Masopust
et al., 2001). While WAT is reduced during DR, the bone marrow
(BM) paradoxically shows increased adipogenesis in this context
(Cawthorn et al., 2014; Devlin et al., 2010). These observations
raised the possibility that an alliance between defined tissue com-
partments may serve the purpose of preserving immunological
memory in the face of nutritional challenges.
Here, we show that DR induces a whole-body response, re-
sulting in the collapse of circulating memory T cell populations
in secondary lymphoid organs (SLOs) and blood but enhanced

1088 Cell 178, 1088–1101, August 22, 2019 ª 2019 Elsevier Inc.
 A

B C

F G
E

H I

Figure 1. Memory T Cells Accumulate in BM during DR

(A) Number of CD8+ CD44+ T cells in spleen (spl), cervical lymph node (cLN), blood, and gonadal adipose tissue (GAT) over time during 50% DR.
(B) Number of CD8+ CD44+ T cells in femur BM of mice on DR over time.
(C) Confocal microscopy of CD4+ (magenta) and CD8+ (yellow) T cells in BM after 3 weeks of DR.
(D) Number of CD8+ CD44+ T cells in BM from tibia, skull, thoracic vertebrate, humerus, and ilium of mice on DR for 3–6 weeks.
(E) Mice were infected and 4 weeks later put on DR for 4–6 weeks.
(F and G) (F) Number of memory CD8+ T cells in GAT, spl, and BM specific for the YopE antigen of Yersinia pseudotuberculolsis or (G) the nucleoprotein antigen of
influenza A virus after 4–6 weeks of DR.
(legend continued on next page)

Cell 178, 1088–1101, August 22, 2019 1089

accumulation in BM. Such a response was associated with pro-
found remodeling of the BM compartment, with increases in ad-
ipocytes and T cell trophic factors. The ability of memory T cells
to accumulate in BM not only protected the memory pool from
inhospitable conditions during DR, but also optimized their func-
tion in the face of secondary challenges. Altogether, this work
uncovers a fundamental host strategy to adapt to physiological
nutritional challenges, which are associated with a temporal
and spatial reorganization of the memory pool within ‘‘safe
haven’’ tissue compartments.
RESULTS
Memory T Cells Accumulate in the Bone Marrow during
Dietary Restriction
To assess the fate of memory T cells in the context of a transient
reduction in nutrition, mice were placed on DR, which involved
receiving 50% of their daily food intake. This resulted in approx-
imately 10%–15% weight loss (Figure S1A) and a reduction in fat
mass (Figure S1B) after 1 week, followed by a plateau (Fig-
ures S1A and S1B). DR caused a decrease in SLO cellularity (Fig-
ure S1C), resulting in a decrease in number of antigen-experi-
enced CD8+ and CD4+ T cells (Figures 1A, S1D, and S1E), as
well as regulatory T cells (Treg), natural killer (NK) cells, and
mature B cells (Figures S1D and S1F–S1H). A similar decrease
was observed in blood and WAT (Figures 1A and S1E–S1H).
Thus, DR is associated with a rapid and profound collapse of an-
tigen-experienced T cells in the periphery, raising the possibility
that memory T cells may redistribute to a distinct niche under
these conditions.
In contrast to other compartments examined, antigen-experi-
enced CD8+ and CD4+ T cells were significantly increased in BM
during DR (Figures 1B, 1C, and S1E). Lymphocyte redistribution
occurred by 1 week following the initiation of DR and was stable
for at least 6 weeks (Figure 1B). Accumulation of antigen-experi-
enced CD8+ T cells occurred across the entire BM compartment,
with an increase of T cells observed in BM from the femur, tibia,
skull, vertebrate, humerus, and ilium during DR (Figures 1B–1D).
Such an increase was selective to memory T cells, as the number
of Treg, NK cells, mature B cells, and plasma cells in BM was pre-
served during DR but not increased (Figures S1F–S1I).
To determine whether memory T cells induced following
infection redistributed during DR, we tracked antigen-specific
responses following an oral infection with Yersinia pseudo-
tuberculosis DyopM (Yptb DyopM) (Han et al., 2017) or an intra-
nasal infection with influenza A virus (A/PR/8/34). Following path-
ogen clearance and the establishment of memory (4 weeks), mice
were placed on DR (Figure 1E), showing that these pathogen-spe-
cific memory CD8+ T cells were reduced in spleen and WAT but
found at higher numbers in BM during DR (Figures 1F and 1G).
We next assessed whether a reduction in calories alone was
responsible for memory T cell redistribution during DR or if
defined nutrients played a role. To this end, we designed diets
(H) Number of CD8+ CD44+ T cells in BM and spl of mice fed the indicated diet ad libitum or at 50% restriction for 3 weeks.
(I) Number of CD8+ CD44+ T cells in BM and spl of mice on DR for 3 weeks or on DR for 3 weeks then refed ad libitum for a further 1 or 3 week(s).
Each symbol represents an individual mouse. Data show the mean representative of four (C) or pooled from two to four experiments (A, B, D, and F–I) with two to
five mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; two-tailed unpaired Student’s t test. See also Figure S1.
1090 Cell 178, 1088–1101, August 22, 2019
that when given at 50% restriction would contain normalized
levels of vitamins and minerals (V&M), essential amino acids
(EAA), or total protein. Normalizing V&M, EAA, or total protein still
induced memory T cell redistribution to BM, while reducing cal-
ories alone was sufficient to drive the response (Figure 1H).
Further, accumulation of memory T cells in BM during DR was
reversible, with the steady-state number rapidly restored in BM
and spleen upon refeeding (Figure 1I). Collectively, these results
indicate that memory T cells collapse in SLO and blood but
rapidly and reversibly accumulate in the BM compartment in
response to a reduction in calories.
Circulating Memory T Cells Accumulate in Bone Marrow
but Maintain the Ability to Recirculate during Dietary
Restriction
Several memory T cell populations exist and are characterized
by distinct migratory patterns and functional potential. DR
induced a decrease in the memory T cell subsets found in SLO
and WAT (Figure 2A). In contrast, the number of TCM and TEM
was significantly increased in BM during DR, whereas T cells ex-
pressing a TRM or TPM phenotype were sustained (Figures 2B
and S2A). The expression of the canonical memory markers
CD127 (interleukin [IL]-7Ra), CD122 (IL-2Rb/IL-15Rb), CD25
(IL-2Ra), and CD62L (L-selectin), and transcription factors
T-BET and EOMES, was similar between TCM and TEM from
mice fed ad libitum or on DR (Figures S2B–S2G). Thus, DR pref-
erentially promoted the accumulation of TCM and TEM in the BM.
CD8+ T cells (isolated ex vivo or pre-activated in vitro) intrave-
nously transferred into recipient mice on DR were found at
reduced numbers in the spleen but were increased in BM
compared to mice fed ad libitum (Figures 2C and S2H), support-
ing the idea that BM accumulation was the result of cellular redis-
tribution. In line with this, memory T cells in BM during DR did not
show increased levels of homeostatic proliferation (Figure S2I).
In fact, the rate of proliferation during DR was significantly lower
as compared to mice fed ad libitum (Figure S2I). On the other
hand, memory T cells vigorously proliferated when mice on DR
were re-fed ad libitum, which was particularly pronounced in
the TCM subset (Figure S2I). As such, TCM may be critical to
restore the memory T cell compartment following the restoration
of calories.
The major function of circulating memory T cells is immunosur-
veillance. To assess whether circulating memory T cells perma-
nently accumulated in BM during DR or if they retained the ca-
pacity to migrate, we performed parabiotic surgery on mice
that had been on DR for 1 week prior to surgery and for
the remainder of the experiment or control mice that were fed
ad libitum throughout (Figure 2D). By 3 weeks post surgery,
circulating memory T cell populations in the spleen and BM
were in migratory equilibrium, while TRM cells in BM maintained
their tissue-residency status (Figure 2E). This was the case
both in parabiotic pairs fed ad libitum and those on DR, indi-
cating that circulating memory T cells retained the ability to
 A

B C

D E

F G

Figure 2. Circulating Memory T Cells Accumulate in BM during DR

(A) Number of CD8+ TCM (CD44+ CD62L+ CD69 ), TEM (CD44+ CD62L CD69 ), and TRM (CD44+ CD62L CD69+) in the indicated tissue of mice on DR for
3–4 weeks.
(B) Plot showing CD62L and CD69 expression by CD8+ CD44+ cells in BM. Number of the indicated subset in BM from mice on DR for 3–4 weeks.
(C) Purified CD8+ CD44+ T cells were transferred into hosts fed ad libitum or on DR. Number of transferred cells in BM and spl after 1 week is shown.
(D) Congenically distinct mice were put on DR for 1 week and joined to form parabiotic pairs then maintained on DR for 3 weeks. Control pairs were fed ad libitum
throughout.
(E) Frequency of host-derived memory T cells in spl and BM from (D).
(F) In vitro activated OT-I mTomato+ T cells (pseudocolored green) were transferred into hosts fed ad libitum or on DR for 3 weeks and skull BM was imaged
1 week later. Images show representative frames. Bottom images show track displacement length from 0–25 or more mm.
(legend continued on next page)

Cell 178, 1088–1101, August 22, 2019 1091

migrate in the context of DR and that the increase in number of
T cells within the BM was associated with transient, rather than
permanent, accumulation.
We next assessed the motility of individual T cells in BM by
intravital imaging. To this end, we transferred in vitro activated
mTomato+ OT-I cells and performed two-photon imaging on
skull BM. More T cells were observed within the BM of mice on
DR compared to mice fed ad libitum (Figure 2F). The behavior
of T cells was impacted by DR, evidenced by significantly
reduced speed and track displacement length compared to
T cells in BM of mice fed ad libitum (Figures 2F and 2G;
Video S1). Collectively, these results support the idea that circu-
lating memory T cells maintain their ability to migrate in the
context of DR but display enhanced dwell time within the BM
compartment.
Glucocorticoids Promote the Accumulation of
Circulating Memory T cells in Bone Marrow during
Dietary Restriction
The accumulation of memory T cells in BM suggested that this
niche provided a survival advantage in the context of DR. DR is
associated with responses aimed at restoring energy balance,
a process dominantly coordinated by glucocorticoids (GCs)
(Cain and Cidlowski, 2017). However, heightened levels of GC
can promote T cell death (Fujita et al., 2002; Wing et al., 1988).
We found that while GCs were elevated in the blood of mice on
DR compared to those fed ad libitum, the concentration of GC
in BM was significantly lower both at baseline and during DR
(Figure 3A). In agreement, the frequency of dead (DAPI+) memory
T cells was increased in the spleen of mice early post DR but was
lower in BM both at steady state and during DR (Figure 3B).
Circulating memory T cells in the BM also expressed higher
levels of the anti-apoptotic factor BCL-2 during DR (Figures 3C
and S3A), suggesting that this compartment can promote mem-
ory T cell survival in the context of nutritional stress.
To directly address a role for GC in orchestrating memory
T cell redistribution during DR, we placed mice lacking adrenal
glands (adrenalectomy or ADX) on DR (Fujita et al., 2002; Wing
et al., 1988). Under this setting, memory T cells did not collapse
in SLO, nor did they accumulate in BM (Figures 3D and S3B).
Furthermore, administration of the synthetic GC dexamethasone
(Dex) reproduced the phenotype seen during DR, with a reduc-
tion of TCM in the spleen and an increase in BM (Figure 3E). While
Dex treatment resulted in increased expression of BCL-2 by both
subsets in BM (Figures 3F and S3C), its impact in terms of redis-
tribution was more pronounced on TCM than TEM (Figures 3E and
S3D), suggesting that these cells may have different levels of
sensitivity to GCs.
We next transferred CD8+ T cells from wild-type (WT) mice or
those lacking the Nr3c1 gene that encodes the GC receptor
(Mittelstadt et al., 2012) into mice fed ad libitum or on DR.
Approximately half the number of WT cells could be recovered
1 week post transfer in the spleen of mice on DR when compared
(G) Speed and track displacement length of OT-I cells in skull BM of mice on DR.
Each symbol represents an individual mouse except for (F), in which each symbol represents an individual cell. Data show the mean pooled from two (A, C, and E)
or three experiments (B and G) or representative of three experiments (F). **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. Two-tailed unpaired Student’s
t test. ND, not detected. See also Figure S2 and Video S1.
1092 Cell 178, 1088–1101, August 22, 2019
to the number of WT cells in the spleen of mice fed ad libitum
(Figure 3G). In contrast, the number of Nr3c1 / T cells was
not reduced in the spleen of mice on DR compared to the num-
ber in the spleen of mice fed ad libitum (Figure 3G). On the other
hand, WT and Nr3c1 / T cells were both able to accumulate in
the BM to a similar degree during DR (Figure 3G). Together,
these results support the idea that a direct interaction between
GCs and the GC receptor on T cells promoted their peripheral
collapse during DR, while GCs induce T cell accumulation in
the BM indirectly.
Bone Marrow Remodeling during Dietary Restriction
Promotes Enhanced Recruitment and Retention of
Memory T Cells
Our results supported the idea that the BM milieu was impacted
in a way that promoted the accumulation and survival of memory
T cells during DR. Whole-tissue RNA sequencing revealed that
the BM was profoundly remodeled during DR, with 3,981 genes
significantly upregulated and 4,173 genes downregulated
compared to mice fed ad libitum (Figure 4A). The most prominent
changes involved the upregulation of genes associated with adi-
pogenesis, whereas those involved in B cell development were
downregulated (Figure 4A). Of note, cytokines involved in mem-
ory T cell homeostasis (IL-7 and IL-15) were not differentially
expressed (Figure S4A).
In regard to T cell homing, several chemokines were differen-
tially expressed in the BM during DR (Figure 4A). This included
Cxcl12, previously shown to promote T cell migration to the
BM (Mazo et al., 2005), which was abundantly expressed at
baseline and modestly increased during DR (Figures 4A and
S4B). Of interest, CXCR4, the receptor for CXCL12, was
increased on the surface of TCM and TEM during DR (Figures
4B and S4C). To assess a role for this axis in promoting T cell
migration to BM during DR, pre-activated WT CD8+ T cells or
those lacking Cxcr4 were adoptively transferred into mice fed
ad libitum or on DR. This showed that CD8+ T cells lacking
Cxcr4 had a reduced ability to home to the BM compared to
WT cells (Figure 4C). Furthermore, memory T cells were reduced
in number in BM during DR following treatment with an inhibitor
of CXCR4 (AMD3100) (Figure S4D). Thus, enhanced T cell accu-
mulation within the BM (via recruitment and/or retention) during
DR was controlled by a CXCR4-CXCL12 axis.
A striking component of the BM remodeling during DR was an
increase in red blood cells (RBCs), which we observed macro-
scopically and by confocal imaging (Figure 4D). This was
confirmed by flow cytometry, which revealed an increase in
both precursor (TER-119+ CD71+) and mature (TER-119+
CD71 ) RBCs (Figure 4E). In agreement with previous work (Fly-
gare et al., 2011; Zhang et al., 2013), mice fed ad libitum that
were treated with Dex showed enhanced erythropoiesis (Fig-
ure S4E). Mature RBCs are the major source of sphingosine-1-
phosphate (S1P) in the blood (Hänel et al., 2007; Pappu et al.,
2007), a molecule that plays a critical role in controlling T cell
A B C Figure 3. GCs Drive Memory T Cells into BM
D E
G Graphs show transferred cells recovered in spl and
F BM, expressed as the fold-change in DR mice over
migration (Cyster and Schwab, 2012). RBCs can also store and
release chemokines and cytokines (Karsten et al., 2018a; Kars-
ten et al., 2018b), suggesting that increased numbers of RBCs
may contribute to T cell accumulation in the BM during DR.
To determine a role for S1P-S1P1R (S1P receptor 1) in retaining
memory T cells in BM during DR, mice were treated with FTY720,
which prevents migration toward S1P (Matloubian et al., 2004).
This treatment resulted in reduced T cell numbers in BM of both
control and DR mice (Figures 4F and S4F). As FTY720 promotes
accumulation of T cells in lymph node (LN) (Mandala et al.,
2002), we could not exclude that reduced accumulation in BM
was due to impaired egress from LN. To circumvent this, mice
were treated with both FTY720 and an anti-CD62L antibody, the
latter restricting access to LN (Harp et al., 2010). Anti-CD62L treat-
ment alone resulted in an increase in T cell numbers in BM (Fig-
ure 4F), presumably due to an inhibition of LN entry. In mice on
DR (but not in mice fed ad libitum) treated with both FTY720
and the anti-CD62L antibody, the number of TCM in BM was signif-
icantly reduced compared to vehicle treated DR mice (Figure 4F).
Further, transfer of pre-activated CD8+ T cells lacking the S1P1R
showed reduced accumulation in BM of mice on DR (Figure 4G).
Together, these results supported the idea that CXCR4-CXCL12
and S1P-S1P1R contributed to increased recruitment and/or
retention of circulating memory T cells in BM during DR.
DR is associated with increased adipogenesis in the BM (Caw-
thorn et al., 2014; Devlin et al., 2010). This was confirmed by an
increase in cells expressing PERILIPIN-1, a marker of lipid drop-
lets, in BM of mice on DR, as well as increased expression of the
during DR
(A) Concentration of corticosterone in serum and the
extracellular environment of BM in mice on DR for
3 weeks.
(B) Frequency of DAPI+ cells in spl and BM of mice
on DR for 1 week.
(C) BCL-2 expression by BM TCM in mice fed ad
libitum (blue) or on DR (red) for 3 weeks.
(D) Mice received sham surgery or an adrenalec-
tomy (ADX) 1 week prior to being put on DR for
1 week. Graphs show number of TCM in spl and BM.
(E and F) (E) Number of TCM in spl and BM and (F)
BCL-2 expression by these cells in mice fed ad
libitum that were administered veh (blue) or dex (red)
daily for 2 weeks.
(G) WT or Nr3c1fl/fl x Lck-Cre mice (Cre ; WT, Cre+;
Nr3c1 / ) CD8+ CD44+ T cells were transferred into
mice fed ad libitum or on DR for 3 weeks. Cells were
enumerated 1 week post transfer.
the average number found in mice fed ad libitum.
Each symbol represents an individual mouse. Data
show the mean, pooled from 3 (A) or 2 (B–G) ex-
periments. **p < 0.01, ***p < 0.001, ****p < 0.0001,
ns; not significant. Two-tailed unpaired Student’s t
test. See also Figure S3.
adipocyte gene Fabp4 (Figures 4A and
4H). To determine if BM adipocytes
contributed to memory T cell survival or
accumulation, we generated mice in which adipocytes could
be inducibly deleted (Adipoq-CreERT2 3 Rosa26-DTA). These
mice were placed on DR for 2 weeks before tamoxifen treatment
then maintained on DR for an additional 3 weeks. A reduction in
BM adipocytes was confirmed by reduced expression of Fabp4
in whole-BM samples (Figure S4G). In mice with reduced adipo-
cytes, TCM were unable to efficiently persist in BM during DR,
suggesting a direct role for BM adipocytes in promoting memory
T cell retention or survival in this compartment (Figure 4I). Previ-
ous work suggested that memory T cell survival depends on
long-chain fatty acids (LC-FAs) to fuel mitochondrial fatty-acid
oxidation (FAO) (O’Sullivan et al., 2014; Pan et al., 2017; Pearce
et al., 2009). However, memory T cells in the BM of mice on DR
were comparable in their ability to take up and store lipids
compared to cells in the BM of mice fed ad libitum (Figures
S4H and S4I).
Collectively, these data show that DR is associated with a
complex set of alterations within the BM, which is associated
with an increase in chemokines, RBC, and adipocytes, all of
which have the potential to promote circulating memory T cell
accumulation within the BM niche.
Memory T Cells Are in a State of Energy Conservation
during Dietary Restriction
We next assessed whether memory T cells had undergone
intrinsic adaptations to maximize long-term survival during DR.
Principal-component analysis (PCA) of mRNA expression by
TCM from BM and spleen revealed that the majority of the
Cell 178, 1088–1101, August 22, 2019 1093
 A

B C

D E F

G H I

Figure 4. BM Remodeling during DR Promotes Memory T Cell Recruitment and Retention

(A) Scatterplot showing gene expression of whole-BM samples from mice fed ad libitum or on DR for 3 weeks. TPR, transcripts per million.
(B) CXCR4 expression by TCM in BM and spl of mice fed ad libitum (blue) or on DR (red).
(C) CD8+ T cells from Cxcr4fl/fl 3 UBC-CreERT2 (Cre ; WT, Cre+; Cxcr4 / ) mice treated with tamoxifen were in vitro activated and transferred into mice fed
ad libitum or on DR for 3 weeks. Analysis was performed 1 week post transfer. Graphs expressed in the same way as 3G.
(D) Images of BM from mice on DR for 3 weeks, showing TEr-119 (green) and DAPI (blue).
(E) Number of RBC (CD71+ precursors and CD71 mature cells) in BM of mice on DR for 3 weeks.
(F) Number of CD8+ TCM in BM after 1 week of DR treated with FTY720 (FTY), an anti-CD62L antibody, or a combination of both. Treatment commenced at same
time as initiation of DR.
(G) CD8+ T cells from S1pr1fl/fl x UBC-CreERT2 (Cre ; WT, Cre+; S1pr1 / ) mice that had been treated with tamoxifen were activated in vitro and transferred into
mice fed ad libitum or on DR for 3 weeks. Graph expressed in same way as 3G.
(H) Image showing BM of mice on DR for 3 weeks, staining for adipocytes by PERILIPIN-1 (cyan), CD8+ T cells (red), and DAPI (blue). Graph shows Fabp4 gene
expression in whole BM as determined by qPCR.
(I) Adipoq-CreERT2 3 Rosa26-DTA mice were put on DR for 2 weeks, treated with tamoxifen, and then maintained on DR for 3 weeks. Graph shows the number of
CD8+ TCM in BM.
Each symbol represents an individual mouse except for (A), in which each symbol represents an individual gene. Data show the mean representative of at least
two (D, F, and H) or pooled from two (C, E, and I) or three (A, B, and G) experiments with three to five mice per group. *p < 0.05, **p < 0.01, ***p < 0.001,
****p < 0.0001; two-tailed unpaired Student’s t test. ns, not significant. See also Figure S4.

variance could be explained by the nutritional status of the host (GSEA) revealed an enrichment for pathways associated with
(PC1, 24.29%), while effects of the tissue also had an effect on heat-shock protein chaperone binding and regulation of protein
gene expression (PC2, 12.87%) (Figure S5A). Pathway analysis folding in TCM from both BM and spleen of mice on DR (Figures
by gene ontogeny (GO) terms and gene set enrichment analysis 5A and 5B). In agreement with unaltered fatty-acid metabolism in

1094 Cell 178, 1088–1101, August 22, 2019

 A

C D

Figure 5. Memory T Cells Are in a State of Energy Conservation during DR

(A and B) CD8+ TCM were sorted from spl and BM of mice fed ad libitum or on DR for 3 weeks for RNA sequencing. (A) GO terms and (B) GSEA from genes
upregulated in TCM from BM (blue) or spl (green) of mice on DR.
(C and D) (C) OCR and (D) SRC in TCM from SLO and BM of mice on DR for 3 weeks.
(E) Expression of phosphorylated mTOR2448, S6240/244, and AKT473 in TCM from spl and BM of mice on DR for 3 weeks.
Each symbol represents an individual mouse except for (C), which is pooled from 10–15 mice. Data are expressed as mean, or mean ± SD, pooled from
three biological replicates with five mice per group (A and B), representative of 3 (C), or pooled from three to four experiments (D and E). *p < 0.05, ***p < 0.001,
****p < 0.0001; ns, not significant. Two-tailed unpaired Student’s t test. See also Figure S5.

the context of DR (Figures S4H and S4I), we did not observe an
enrichment in pathways associated with lipid metabolism (Fig-
ures 5A and 5B). Of interest, genes associated with amino-acid
deprivation and the cellular response to rapamycin, a negative
regulator of the mechanistic target of rapamycin (mTOR), fell
within the protein folding module (Figures 5A and 5B), supporting
the idea that TCM may have reduced levels of mTOR signaling
during DR.
mTOR is a protein kinase that functions in larger multipro-
tein complexes known as mTOR complex 1 (mTORC1) and
mTOR complex 2 (mTORC2) (Saxton and Sabatini, 2017).
Reduced mTOR signaling promotes survival during nutrient
deprivation by decreasing anabolic processes and promoting
those that are catabolic (Aramburu et al., 2014). In agreement,
TCM in both the BM and SLO of mice on DR showed reduced
oxygen consumption rate (OCR) (Figure 5C) and spare respi-
ratory capacity (SRC) (Figure 5D), suggesting that memory
T cells were in a state of reduced metabolic activity or quies-
cence during DR. Examination of mTOR in TCM from BM of
mice on DR showed reduced phosphorylation at serine 2448
(Figure 5E), indicating a reduction in mTOR activity. Further,
TCM from the BM of mice on DR showed reduced phosphory-
lation of ribosomal protein S6 (pS6) at serine 240/244 and AKT
at serine 473, downstream targets of mTORC1 and mTORC2,
respectively (Figure 5E). Of note, mTOR downregulation was
more pronounced in TCM from BM compared to those in the
spleen in the context of DR (Figure 5E), supporting the idea
that circulating memory T cells may integrate local cues
when in BM to adopt a program compatible with energy con-
servation or quiescence.

Cell 178, 1088–1101, August 22, 2019 1095

Memory T Cells Mediate Enhanced Protection against
Secondary Challenges during Dietary Restriction
The low metabolic activity of memory T cells during DR and their
BM tropism begged the question of whether mice on DR could
rapidly respond to secondary challenges. To address this, we first
employed a model of acute oral infection with Yptb DyopM in
which rapid clearance of the microbe is associated with the induc-
tion of a robust population of memory CD8+ T cells (Han et al.,
2017). To investigate circulating memory T cells, naive or previ-
ously infected mice were first challenged intravenously (i.v.) with
the virulent WT strain of Yptb. At 2 days post primary infection,
no difference was observed in bacterial burden between naive
mice fed ad libitum or on DR (Figure 6A, columns A versus B).
As expected, previously infected mice fed ad libitum showed
enhanced protection compared to naive mice 2 days post sec-
ondary challenge (Han et al., 2017) (Figure 6A, columns A versus
C). Antibody depletion in mice fed ad libitum revealed that memory
CD8+ T cells played a role in mediating protection (Figure 6A, col-
umns C versus E), whereas mice depleted of CD4+ T cells trended
toward having defective protection, although this did not reach
statistical significance (Figure 6A, columns C versus G). Next,
mice received a primary infection and developed memory while
fed ad libitum before being placed on DR for 3–4 weeks and
then were challenged i.v. with WT Yptb. Remarkably, these mice
on DR during the secondary challenge had an enhanced ability
to control the infection, with approximately 500-fold fewer col-
ony-forming units (CFUs) of Yptb per gram of spleen compared
to previously infected mice fed ad libitum throughout (Figure 6A,
columns C versus D). Enhanced protection was also observed
in mice on DR during an oral secondary challenge with WT Yptb
(Figure 6B). Thus, DR initiated post the establishment of memory
resulted in significantly enhanced protective responses. On the
other hand, mice on DR throughout both the primary and second-
ary infections displayed a similar ability to control a secondary
infection as mice fed ad libitum (Figure S6A). Thus, the timing of
DR was critical for the enhancement of secondary responses.
Increased protection was associated with an enhanced
breadth of effector responses. Indeed, depletion of CD8+ (Fig-
ure 6A, columns E versus F) or CD4+ (Figure 6A, columns G
versus H) T cells alone did not revert the enhanced protection
afforded by DR. We found that enhanced protection was only
reverted by the simultaneous depletion of both T cell subsets
(Figure 6A, columns I versus J). This indicates that both subsets
contribute to enhanced secondary responses during DR and are
able to effectively compensate for each other in this context.
Further, Yptb-specific memory CD8+ T cells isolated from the
spleen had a greater potential to produce interferon (IFN)g during
DR (Figure 6C). Altogether, this indicates that memory T cells
have enhanced functional capacity and ability to control second-
ary infections during DR.
We next assessed whether appropriate homing during DR
contributed to the optimization of immunological memory. We
previously showed that short-term treatment with FTY720
reduced the accumulation of T cells in BM (Figures 4F and
S4F). Mice that had developed memory to Yptb were placed
on DR for 2 weeks then treated over the course of 1 week with
FTY720 followed by a period of 2 weeks before challenge (Fig-
ure 6D). Mice fed ad libitum throughout and treated with
1096 Cell 178, 1088–1101, August 22, 2019
FTY720 had a similar bacterial burden to mice treated with
vehicle (Figure 6D). In contrast, mice on DR that were treated
with FTY720 no longer displayed enhanced protection (Fig-
ure 6D), suggesting that enhanced protection to secondary
infections was lost during DR if memory T cells were even
transiently restricted from gaining access to the BM.
To determine if enhanced memory T cell function during DR
extended to other settings, we employed a model in which virally
induced T cells provide control of melanoma (Overwijk et al.,
2003). Mice received naive transgenic CD8+ pmel-1 cells that
are specific for an epitope derived from the melanoma-associ-
ated antigen gp100 before being infected with a recombinant
vaccinia virus that expresses human gp100 (VV-hgp100) (Fig-
ure 6E). 1 month later, a time at which gp-100-specific memory
CD8+ T cells had formed, DR was initiated for 3 weeks, and
mice were inoculated with B16 melanoma cells. All mice fed ad
libitum without pmel-1 cells reached the experimental endpoint
(15 mm 3 15 mm) 26 days post tumor inoculation (Figure 6E,
black line), and consistent with previous reports (Kalaany and
Sabatini, 2009), DR alone provided a survival benefit (Figure 6E,
blue). As previously shown (Xiao et al., 2011), infection with
VV-hgp100 alone, without pmel-1 cells, did not provide addi-
tional benefit compared to controls (Figure 6E, magenta and
green), whereas mice fed ad libitum that received both pmel-1
cells and VV-hgp100 had a survival benefit (Figure 6E, red),
although the endpoint was reached by day 35 (Figure 6E, red).
Critically, mice on DR that received both pmel-1 cells and VV-
hgp100 had the greatest survival rate (Figure 6E, orange), with
approximately 15% of mice from this group being completely tu-
mor free after more than 2 months (Figure 6E). These results indi-
cate that DR may enhance the protective function of memory
T cells in the context of both anti-bacterial and tumor immunity.
DISCUSSION
Host fitness depends on the ability to survive and adapt to
changing environments. In these contexts, the long-term persis-
tence of memory T cells is of the utmost importance. How the
host responds in a way that prioritizes and protects defined
branches of the immune system during nutritional stress remains
poorly understood. Previous work supported the idea that the
anorexic response following acute primary infections could pro-
mote host survival in defined contexts (Hart, 1988; Wang et al.,
2016) and that a reduction in vitamin A results in decreased
adaptive immunity but a compensatory increase in innate re-
sponses aimed at protecting barrier sites (Spencer et al.,
2014). Consistent with a recent report (Contreras et al., 2018),
we found that a physiological decrease in calories was associ-
ated with a complex set of alterations that resulted in the stra-
tegic protection of memory T cells within the BM (Figure 6F).
Our results supported the idea that this response was domi-
nantly coordinated by the steroid hormones GCs, which play a
critical role in the regulation of energy balance (Cain and Cidlow-
ski, 2017). Here, we show that one of the consequences of
GC-mediated accumulation of T cells in BM was to protect these
cells from the detrimental effects of GCs themselves. While
circulating memory T cells were particularly enriched within the
BM, other key subsets, such as Treg cells, NK cells, and plasma
 ****
ns
ns
A ns ** B C Spleen
****
ns **** Ctrl ****

WT Yptb CFU/g mLN

****

IFN + of YopE69-77+(%)
**** * * * 9
WT Yptb CFU/g spl

9
10 DR 10 ns Ctrl 100 **
108 Oral Oral DR
108 80
Yptb ΔyopM 1° Yptb WT 2° ***
107 107
106 60
50% DR 106
105 40
CFU 105
104 4wk 3wk 3d
10 4 20
103
102 103 0
CD8 - - - - + + - - + + Naive Memory

trl
R
D
CD4 - - - - - - + + + +

C
A B C D E F G H I J
Naive Memory *** ns
D Oral i.v. 109
ns ** Veh.

WT Yptb CFU/g spl

i.v. Oral i.v.
Yptb ΔyopM 1° Yptb WT 2° 108 FTY
Yptb WT Yptb ΔyopM 1° Yptb WT 2°
107
50% DR 50% DR 50% DR 106
CFU 105
CFU CFU 4wk 2wk 2wk 2d
3wk 2d 4wk 3wk 2d 104
FTYx4
every 2nd day 103
102
Ctrl DR
100 Ctrl (n=33)
E

****
Pmel-1 + n
DR (n=34)
80 n s
VV-hgp100 s.c. B16 Ctrl + VV-hgp100 (n=9)

*
s
Survival (%)

****
60 DR + VV-hgp100 (n=10)
50% DR Ctrl + VV-hgp100 + pmel-1 (n=22)

****
40 DR + VV-hgp100 + pmel-1 (n=21)
4wk 3wk Measure
20
0
F 0 10 20 30 40 50 60 70 80
Ad Dietary Days post tumour inoculation
Libitum Restriction
Adipocyte
Blood/ RBC
SLO GC Memory T cell
RBC
Pathogen
Cxcr4 Cxcr4 Cxcr4
Bcl-2
mTor
Cxcl12 Cxcl12

Bone
Marrow

Figure 6. Memory T Cells Mediate Enhanced Protection against Secondary Challenges during DR
(A) Mice that were naive or previously infected with the Yptb DyopM orally were put on DR for 3 weeks and challenged i.v. with the more virulent WT strain of Yptb.
Bacterial burden was assessed in spl 2 days after challenge. CD4+ or CD8+ T cells were depleted prior to challenge in the indicated groups.
(B) Mice were infected with Yptb DyopM orally then 4 weeks later put on DR for 3 weeks. During DR, mice were challenged orally with WT Yptb. Bacterial burden
was assessed in mesenteric lymph nodes 3 days after challenge.
(C) IFNg production following PMA/ionomycin stimulation by Yptb-specific splenic memory CD8+ T cells at day 2 after i.v. challenge with WT Yptb.
(D) Mice previously infected with Yptb DyopM orally were put on DR for approximately 5 weeks. Two weeks into DR, mice were treated with FTY720 four times
every other day followed by a 2-week period before i.v. challenge with WT Yptb. Bacterial burden was assessed in spl 2 days after challenge.
(E) Mice received naive pmel cells followed by an i.v. infection with vaccinia-hgp100. 1 month later, mice were put on 50% DR for 3 weeks and inoculated with B16
cells subcutaneously (s.c.). Mice were euthanized when tumors reached 15 mm 3 15 mm.
(F) Circulating GCs are increased during DR, inducing memory T cell recruitment and retention in BM, a site with low levels of GCs. The BM is drastically
remodeled during DR to contain increased adipocytes, RBCs, and CXCL12, which have the potential to maintain memory T cells in this niche. Once in BM during
DR, memory T cells express optimal levels of BCL-2 and mTOR to promote survival. Upon secondary challenges, memory T cells have an enhanced ability to
mediate protection during DR.
Each symbol represents an individual mouse. Data show the mean pooled from at least two (A, B, and E), 4 (C), or 3 (D) experiments. *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001; ns, not significant. Two-tailed unpaired Student’s t test except for (E), which was performed by a log-rank (Mantel-Cox) test. See also
Figure S6.

Cell 178, 1088–1101, August 22, 2019 1097

cells, were preserved at this site during DR, supporting the idea
that our findings may broadly apply to many keystone popula-
tions. In addition to its fundamental role in hematopoiesis, the
BM is a site that circulating memory T cells traffic through at
steady state (Becker et al., 2005; Chaix et al., 2014; Di Rosa
and Gebhardt, 2016; Klonowski et al., 2004; Mazo et al., 2005;
Pabst et al., 1986; Parretta et al., 2005). Under resting condi-
tions, CXCR4-CXCL12 interactions contribute to the homing of
memory T cells to BM (Mazo et al., 2005). Of relevance, GCs
can upregulate CXCR4 expression by T cells (Besedovsky
et al., 2014a, 2014b; Ghosh et al., 2009; Shimba et al., 2018),
while reduced mTORC2 signaling also results in increased
CXCR4 expression and sequestration of naive T cells in BM
(Arojo et al., 2018). As such, our results support the idea that
the CXCR4-CXCL12 axis dominantly contributes to T cell reten-
tion and/or accumulation within the BM during DR.
Protection of memory T cells within the BM was not associated
with permanent sequestration but rather increased dwell time in
a privileged site, a response that may result from the dramatic in-
crease in erythropoiesis observed during DR. RBCs are the ma-
jor source of S1P in blood (Hänel et al., 2007; Pappu et al., 2007),
a molecule critical in orchestrating T cell migration (Cyster and
Schwab, 2012). As such, an increase of RBC in BM may impact
the local concentration of S1P within the BM, delaying the ability
of T cells to re-acquire S1P1R and re-enter the circulation
(Maeda et al., 2010). Furthermore, S1P may play an important
role in the survival of T cells within the BM compartment during
DR (Mendoza et al., 2017).
The role of GCs in coordinating these responses also supports
the idea that our observation may be relevant to numerous forms
of stress, promoting cell survival in the privileged environment of
the BM. Conversely, this adaptive response may have detri-
mental consequences. Notably, in the context of glioblastoma,
T cells were severely depleted from the periphery and tumor
site but found at high numbers in BM, and as in our settings,
S1P-S1P1R interactions contributed to this response (Chongsa-
thidkiet et al., 2018). Thus, what may have evolved as a means to
adapt to physiological alterations in food intake or stress may
also be co-opted in the setting of cancer, depriving the tumor
microenvironment of the adaptive immune repertoire.
We and others have shown that WAT is a hub for memory
T cells (Han et al., 2017; Masopust et al., 2001). However, this
compartment rapidly declines during DR, whereas the BM para-
doxically becomes enriched in adipocytes (Cawthorn et al.,
2014; Devlin et al., 2010). Under these settings, the BM acts as
an endocrine organ that is critical for compensating for the loss
of WAT in the periphery (Cawthorn et al., 2014). Further in align-
ment with WAT being a privileged site for memory T cells, abla-
tion of adipocytes during DR reduced the ability of memory
T cells to accumulate in BM. Of note, increased adipogenesis
in BM occurs in several settings, such as irradiation and chemo-
therapy (Zhou et al., 2017), suggesting that this response could
represent a general response following stress aimed at preser-
ving physiological processes, including immunological memory.
An intriguing question that remains to be addressed is the na-
ture of the adipocytes and adipocyte-derived fuels that develop
in BM during DR. BM adipocytes express a complex gene
signature associated with both white and brown adipocytes
1098 Cell 178, 1088–1101, August 22, 2019
(Scheller et al., 2015, 2016). Reports indicate that memory
T cells critically require FAO to fuel their metabolism and long-
term survival (O’Sullivan et al., 2014; Pearce et al., 2009; van
der Windt et al., 2012), although whether this is the case in vivo
is unclear (Raud et al., 2018a, 2018b). The import of extracellular
glycerol has been shown to be critical for this process in circu-
lating memory T cells (Buck et al., 2016; Cui et al., 2015; O’Sulli-
van et al., 2014; van der Windt et al., 2012), while the survival of
skin TRM requires uptake of exogenous lipids (Pan et al., 2017).
We found that memory T cells in BM during DR did not have
an enhanced ability to uptake, store, or process fatty acids.
Indeed, unlike white adipocytes that undergo high rates of lipol-
ysis, BM adipocytes undergo only minimal amounts of lipolysis
(Scheller et al., 2019). As such, a key question that remains to
be addressed is how BM adipocytes contribute to T cell survival
and/or persistence in the BM niche during DR.
Our work supports the idea that memory T cells from hosts on
DR had entered a state of energy conservation or quiescence
that was associated with reduced mTOR signaling. When nutri-
ents are abundant, mTORC1 is activated to stimulate anabolic
processes that supports cell growth, whereas when these fac-
tors are reduced, mTORC1 signaling is suppressed, stimulating
catabolic processes (Saxton and Sabatini, 2017). This places
mTOR as a key rheostat that balances the metabolic profile of
cells to align with the level of nutrients or stress within the host
(Aramburu et al., 2014). Restricting the downregulation of
mTOR under conditions of nutrient deprivation in vitro is detri-
mental for cell survival and function (Choo et al., 2010). Of rele-
vance to our observation, reduced mTOR activity enhances
memory T cell development, maintenance, and function
(Araki et al., 2009). Consistent with this, mTOR inhibitors show
promise in the clinic by enhancing vaccine responses and anti-
viral immunity (Mannick et al., 2018). As such, this phenomenon
may provide a mechanistic link for our observation of enhanced
protective responses in the context of DR.
Together, our work raises intriguing questions regarding the
optimal state for immunological memory preservation and func-
tion. Access to a constant level of calories and nutrients is only a
recent occurrence in high-income countries, and all animals
have evolved in settings of variable food availability. As such,
fluctuations in nutrition or intermittent fasting may represent
the ideal state for supporting the function of memory T cells.
Furthermore, understanding the nature of the factors within the
BM milieu that potentially optimizes immunological memory
could be utilized to improve and protect fundamental arms of
the immune system in the context of disease.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d LEAD CONTACT AND MATERIALS AVAILABILITY
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Mice
B Cell lines
B Bacteria and virus strains
 d METHOD DETAILS
B Dietary restriction
B Surgery
B Tissue processing and flow cytometry
B Confocal microscopy
+ J. Immunol. 174, 1269–1273.
B In vitro activation of CD8 T cells and adoptive transfer
B In vivo treatments
B Ex vivo assays
B RNA extraction, cDNA synthesis and Quantitative PCR
B RNA Sequencing
B ELISA
B 2-photon imaging and analysis
B Oxygen consumption flux evaluation
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND CODE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
cell.2019.07.049.
ACKNOWLEDGMENTS
Y.B. is supported by the Division of Intramural Research of NIAID (NIAID; ZIA-
AI001132, ZIA-AI001133), NIH. N.C. and Y.B. were supported by the Office of
Dietary Supplements Research Scholar program, NIH. Work in the Schwab lab
was supported by NIH grants R01AI085166 and R01AI123308, and a Vilcek
Scholar Award to D.D. We thank the Belkaid lab for their suggestions, support,
and critical reading of the manuscript and J. Kehr and N. Bouladoux for man-
aging the program of the laboratory. We thank K. Beacht, E. Lewis, and J. Le-
Grand for technical assistance; the NIAID animal facility staff; T. Hawley (NIAID
flow cytometry facility); O. Schwartz (NIAID biological imaging facility); and B.
Tran and J. Sheti (NCI sequencing core facility).
AUTHOR CONTRIBUTIONS
N.C. and Y.B. designed the study and wrote the manuscript. N.C. performed
experiments and analyzed the data. S.-J.H., M.E., V.M.L., B.H., E.A.M.,
J.P.S., R.J.K., and D.D. participated in performing experiments, provided intel-
lectual expertise, and helped to interpret experimental results. S.R.S., N.P.R.,
H.D.H., D.B.M., and P.L.S. provided reagents and intellectual expertise and
helped to interpret experimental results.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: December 18, 2018
Revised: May 28, 2019
Accepted: July 29, 2019
Published: August 22, 2019
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Cell 178, 1088–1101, August 22, 2019 1101
STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Anti-mouse B220, PE-CF594 (RA3-6B2) BD
Anti-mouse BrDU, FITC BD
Anti-mouse CD4, BV605 (RM4-5) Biolegend
Anti-mouse CD8b, PE (H35-17.2) eBioscience
Anti-mouse CD8b, BV650 (H35-17.2) BD
Anti-mouse CD16/32, purified (2.4G2) Bio-X-Cell
Anti-mouse CD25, AF488 (PC61.5) eBioscience
Anti-mouse CD31, AF647 (MEC13.3) Biolegend
Anti-mouse CD44, PE-Cy7 (IM7) eBioscience
Anti-mouse CD44, AF700 (IM7) eBioscience
Anti-mouse CD45, APC-eFluor 780 (30-F11) eBioscience
Anti-mouse CD45.1, FITC (A20) eBioscience
Anti-mouse CD45.2, APC-eFluor 780 (104) eBioscience
Anti-mouse CD62L, FITC (MEL-14) eBioscience
Anti-mouse CD69, PE (H1.2F3) eBioscience
Anti-mouse CD71, APC (R17217) eBioscience
Anti-mouse CD90.2, BV785 (30-H12) Biolegend
Anti-mouse CD122, PerCP-eFluor 710 (TM-b1) eBioscience
Anti-mouse CD127, BV605 (A7R34) Biolegend
Anti-mouse CXCR4, PE (2B11) eBioscience
Anti-mouse CX3CR1, PE (SA001F11) Biolegend
Anti-mouse EOMES, eFluor450 (Dan11mag) eBioscience
Anti-mouse FOXP3, PerCP (FJK-16S) eBioscience
Anti-mouse NK1.1, PeCy7 eBioscience
Anti-mouse TCRgd, PE-CF594 (eBioGL3) BD
Anti-mouse TER-119, eFluor450 (TER-119) eBioscience
Anti-mouse Va2, PE (B20.1) eBioscience
Anti-mouse BCL-2, eFluor450 (10C4) eBioscience
Anti-mouse IFN-g, eFluor450 (XMG1.2) eBioscience
Anti-human/mouse T-BET, BV421 (eBio4B10) Biolegend
Anti-mouse TCRb, PerCP-Cy5.5 (H57-597) eBioscience
Anti-mouse AKT473, PeCy7 (MRRBY) eBioscience
Anti-mouse mTOR2448, PE (SDRNR) eBioscience
Anti-mouse pS6240/244, AF488 (D68F8) Cell signaling technologies
Anti-mouse PERILIPIN1, purified Abcam
Normal Goat Serum Jackson ImmunoResearch
Laboratories
Rat Gamma Globulin Jackson ImmunoResearch
Laboratories
Goat anti-rabbit AF647 Life Technologies
Purified anti-mouse CD3 BD
Purified anti-mouse CD28 BD
Cat# 562290; RRID:AB_11151901
557891
BioLegend Cat# 100548; RRID:AB_2563054
Cat# 12-0083-82; RRID:AB_657767
Cat# 740552; RRID:AB_2740253
Cat# CUS-HB-197; RRID:AB_2687830
Cat# 53-0251-82; RRID:AB_763472
Cat# 102516; RRID:AB_2161029
Cat# 25-0441-82; RRID:AB_469623
Cat# 56-0441-82; RRID:AB_494011
Cat# 47-0451-82; RRID:AB_1548781
Cat# 11-0453-85; RRID:AB_465059
Cat# 47-0454-82; RRID:AB_1272175
Cat# 11-0621-85; RRID:AB_465110
Cat# 12-0691-83; RRID:AB_465733
Cat# 17-0711-82; RRID:AB_1834355
Cat# 105331; RRID:AB_2562900
Cat# 46-1222-82; RRID:AB_11064442
Cat# 135041; RRID:AB_2572047
Cat# 12-9991-81; RRID:AB_891393
Cat# 149005; RRID:AB_2564314
Cat# 48-4875-82; RRID:AB_2574062
Cat# 45-5773-82; RRID:AB_914351
Cat# 25-5941-82; RRID:AB_469665
Cat# 563532; RRID:AB_2661844
Cat# 48-5921-82; RRID:AB_1518808
Cat# 12-5812-82; RRID:AB_465949
Cat# 48-6992-42; RRID:AB_2574099
Cat# 48-7311-82; RRID:AB_1834366
Cat# 644816; RRID:AB_10959653
Cat# 45-5961-82; RRID:AB_925763
Cat# 12-9715-42; RRID:AB_2637101
Cat# 25-9718-42; RRID:AB_2573550
Cat# 5018; RRID:AB_10695861
Cat# ab3526; RRID:AB_2167274
Cat# 005-000-121; RRID:AB_2336990
Cat# 012-000-002; RRID:AB_2337135
Cat# A27040; RRID:AB_2536101
Cat# 553057; RRID:AB_394590
Cat# 553294; RRID:AB_394763
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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Purified anti-mouse CD62L (Mel-14) Bio-X-Cell Cat# BE0021; RRID:AB_1107665
Purified anti-mouse CD8 (2.43) Bio-X-Cell Cat# BE0061; RRID:AB_1125541
Purified anti-mouse CD4 (GK1.5) Bio-X-Cell Cat# BE0003-1; RRID:AB_1107636
Bacterial and Virus Strains
Yersinia pseudotuberculosis (32777) Laboratory of Y.B. N/A
Influenza A Virus, A/Puerto Rico/8/1934 (PR8), Laboratory of H.D.H, originally N/A
strain H1N1 from Mount Sinai School
of Medicine
Recombinant vaccinia-virus expressing Laboratory of N.P.R N/A
human gp100
Chemicals, Peptides, and Recombinant Proteins
2-Mercaptoethanol Sigma-Aldrich M3148-25ML
Brefeldin A (GolgiPlug) BD Biosciences 555029
BSA Sigma-Aldrich A3059-500G
DAPI Sigma-Aldrich D9542
DNase I Sigma-Aldrich DN25-5G
DMSO Sigma-Aldrich D2650
EDTA (0.5M) Corning 46-034-Cl
FBS Hyclone SH30070.03
L-Glutamine Corning 25-005-Cl
HEPES Corning 25-060-Cl
Ionomycin Sigma-Aldrich I0634-5MG
Bodipy FL C16 Life Technologies D-3821
BrdU Thermo Fisher or BD B23151 or 550891
Biosciences
Liberase TL Roche 5401020001
Sodium Pyruvate (100X) Corning 25-000-Cl
SIINFEKL peptide Genscript N/A
Triton X Sigma-Aldrich T9284
Liberase TL Roche 05401020001
MEM Non-essential Amino Acids (100X) Corning 25-025-Cl
Paraformaldehyde Electron Microscopy 15714-S
Sciences
Pennicillin-Streptomycin (100X) Corning 30-002-Cl
Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich P8139-10MG
ProLong Gold Antifade Mountant Molecular Probes P36930
RPMI 1640 medium Corning 10-040-CV
Methanol JT Baker 9093-03
Bodipy FL-C16 Life Technologies D-3821
LipidTOX Life Technologies H34476
DNase I Sigma DN25-5G
Liberase TL Roche 5401020001
Trizol Thermo Fisher 15596018
OCT Fisher 23-730-571
Recombinant human IL-2 Biolegend 589106
Recombinant human IL-7 Biolegend 581906
Dexamethasone Sigma-Aldrich D1756-100MG
Tamoxifen Sigma-Aldrich T5648-5G
Corn oil Sigma-Aldrich C8267-500ML
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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
AMD3100 Sigma-Aldrich 239820-5MG
FTY720 Sigma-Aldrich SML0700-25MG
Glutamine Sigma-Aldrich 1294808-100MG
Glucose Sigma-Aldrich G8270-100G
Oligomycin Sigma-Aldrich 75351-5MG
FCCP Sigma-Aldrich C2920-10MG
Rotenone Sigma-Aldrich R8875-5G
Antimycin A Sigma-Aldrich A8674-100MG
Fatty-acid free BSA Sigma-Aldrich A8806
2X YT medium Sigma-Aldrich Y1003
Regular mouse chow Lab Diet Advanced 5V75
Protocol
Control isocaloric diet Envigo TD.160179
Adequate vitamins and minerals diet Envigo TD.190106
Adequate essential amino acids diet Envigo TD.190107
Adequate protein diet Envigo TD.190108
Diet in which only calories at 50% Envigo TD.190105
Critical Commercial Assays
Anti-PE microbeads Miltenyi Biotec 130-048-801
Arcturus PicoPure RNA Isolation kit Thermo Fisher KIT0204
FITC BrdU Flow kit BD Biosciences 557891
BD Cytofix/Cytoperm BD Biosciences 554722
BD Perm/Wash BD Biosciences 554723
Foxp3 / Transcription Factor Staining Buffer Set eBioscience 00-5523-00
High Sensitivity D1000 ScreenTape Agilent 5067-5584
LIVE/DEAD Fixable Blue Dead Cell Staining Kit Life Technologies L23105
MACS Cell Separation Column LS Miltenyi Biotec 130-042-401
Omniscript RT kit QIAGEN 205111
Taq PCR Master Mix Life Technologies K0171
Mouse corticosterone ELISA Thermofisher EIACORT
Nextera XT DNA Library Prep Kit Illumina FC-131-1096
SMARTer Ultra Low Input RNA Kit Clonetech 634946
Deposited Data
Raw RNA-Seq data This manuscript GEO: GSE124063
Experimental Models: Cell Lines
B16 melanoma The laboratory of N.P.R. N/A
Experimental Models: Organisms/Strains
Mouse: C57BL/6 Taconic Farms Mouse strain: C57BL/6NTac
Mouse: B6.SJL-Ptprca/BoyAiTac (CD45.1) Taconic Farms – NIAID exchange Mouse strain: Tac 8478
/
Mouse: OT-I CD45.1 RAG Taconic Farms – NIAID exchange Mouse strain: Tac 300
Mouse: C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I) Jackson laboratories Mouse strain: Jax, 003831
Mouse: B6.129(Cg)-Gt(ROSA)26Sortm4 Jackson laboratories Mouse strain: Jax, 007676
(ACTB-tdTomato,-EGFP)Luo/J (mTomato)
Mouse: mTomato x OT-I Laboratory of D.B.M N/A
Mouse: C57BL/6-Tg(Adipoq-cre/ERT2)1Soff/J Jackson laboratories Mouse strain: Jax, 025124
Mouse: B6.129P2-Gt(ROSA)26Sortm1(DTA)Lky/J Jackson laboratories Mouse strain: Jax, 009669
Mouse: Adipoq-CreERT2 x Rosa26-DTA Laboratory of Y.B. N/A
Mouse: Cxcr4fl/fl x UBC-CREERT2 Laboratory of S.R.S N/A
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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
fl/fl ERT2
Mouse: S1pr1 x UBC-CRE Laboratory of S.R.S N/A
Mouse: B6Cg.Thy1a/Cy Tg(TcraTcrb)8Rest/J Jackson laboratories Mouse strain: Jax, 005023
Oligonucleotides
Hprt Thermofisher 4331182 (Mm03024075_m1)
Fabp4 Thermofisher 4331182 (Mm00445878_m1)
Software and Algorithms
Flowjo software Treestar RRID: SCR_008520
Imaris software Bitplane RRID: SCR_007370
Prism software Graphpad RRID: SCR_002798
ImageJ https://imagej.net/ RRID: SCR_003070
STAR aligner Dobin et al., 2013 RRID: SCR_015899
HOMER http://homer.ucsd.edu/ RRID: SCR_010881
DESeq2 https://bioconductor.org/packages/ RRID: SCR_015687
release/bioc/html/DESeq2.html
Metascape http://metascape.org/gp/index.html#/ RRID: SCR_016620
main/step1
GSEA http://www.broadinstitute.org/gsea/ RRID: SCR_003199
FastQC Babraham Bioinformatics RRID: SCR_014583
R https://www.r-project.org/ N/A

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Yasmine
Belkaid (ybelkaid@niaid.nih.gov).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice
C57BL/6NTac mice were purchased from Taconic Farms. B6.SJL-Ptprca/BoyAiTac (CD45.1), OT-I CD45.1 RAG / (OT-I) were
obtained through the NIAID Taconic exchange program. C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I) and B6.129(Cg)-Gt(ROSA)26Sort-
m4(ACTB-tdTomato,-EGFP)Luo/J (mTomato) mice were purchased from the Jackson Laboratories and crossed to generate
mTomato OT-I mice. C57BL/6-Tg(Adipoq-cre/ERT2)1Soff/J and B6.129P2-Gt(ROSA)26Sortm1(DTA)Lky/J were purchased from Jack-
son laboratories and crossed to generate Adipoq-CreERT2 x Rosa26-DTA mice. B6Cg.Thy1a/Cy Tg(TcraTcrb)8Rest/J (pmel) were
from the laboratory of N.P.R. All experiments involved female mice 6–23 weeks of age. Mice were bred and maintained under specific
pathogen-free conditions at an American Association for the Accreditation of Laboratory Animal Care (AAALAC)-accredited animal
facility at the NIAID and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals.
Cxcr4fl/fl x UBC-CREERT2 and S1pr1fl/fl x UBC-CREERT2 mice were housed at the Skirball institute animal facility. Experiments were
performed under an animal study proposal (LISB-18E, 19E and 20E) approved by the NIAID Animal Care and Use Committee.

Cell lines
B16 melanoma cells (National Cancer Institute tumor repository) were cultured at 37 C, 5% CO2 in complete media DMEM (GIBCO)
with 10% FBS, 1% glutamine and 1% penicillin-streptomycin.

Bacteria and virus strains

Wild-type or mutant Yersinia pseudotuberculosis (32777) strains were grown from bacterial culture in 2X YT media at 25 degrees
overnight. Mice were infected with 1 3 107 colony forming units (CFU) of Yptb DyopM via oral gavage (mice were fasted for
12 h prior). For secondary infections, mice were injected intravenously with 2 3 102 CFU WT Yptb and bacterial burden was as-
sessed in the spleen 48 h later, or for an oral challenge, mice were gavaged with 5 3 109 CFU WT Yptb and bacterial burden was
assessed in the mesenteric lymph node after 72 h. Bacterial burden was determined by serial plating on MacConkey plates and
colonies were counted after incubation at room temperature (RT) for 48 h.

Cell 178, 1088–1101.e1–e7, August 22, 2019 e4

 For influenza infections, A/Puerto Rico/8/1934 (PR8) (originally obtained from Mount Sinai School of Medicine, strain H1N1) was
grown in allantoic cavities of 10-day-old specific pathogen free (SPF) embryonated chicken eggs. Allantoic fluid was collected 48 h
post infection and purified by differential centrifugation. Viral titers were determined by 50% tissue culture infective dose (TCID50) on
MDCK cells using the Reed-Muench method. Mice were anesthetized using isoflurane and intra-nasally infected with PR8 at 5
TCID50 in 25 mL sterile saline solution supplemented with 0.1% BSA.
For experiments involving recombinant vaccinia virus expressing human-gp100 followed by a B16 challenge, mice initially received
3 3 105 naive enriched pmel cells then were vaccinated intravenously with 1 3 107 plaque-forming units of this virus. After one month,
mice were randomly assigned to undergo DR for the remainder of the experiment or to remain being fed ad libitum. Following 3 weeks
of DR, mice were inoculated subcutaneously with 7.5 3 105 B16 cells and tumor burden was monitored over time. Mice were eutha-
nized when tumors reached a size of 15 mm x 15 mm.

METHOD DETAILS

Dietary restriction
The daily intake of regular chow (LabDiet Advanced Protocol PicoLab Verified – 75 IF; 20% protein, 5% fat) by individually caged mice
was determined by giving a defined amount of food and weighing the remainder daily for 2 weeks. From this, we found that individual
mice consumed approximately 2.75 g of food per day, consistent with previous reports (Acosta-Rodrı́guez et al., 2017). This equates
to an intake of roughly 11.4 kcal per day. As such, we provided 1.375 g of food to mice daily (5.7 kcal per day) to ensure 50% DR. Mice
on DR consumed all the food provided. Diets in which specific factors were normalized were designed in consultation with a nutri-
tionist (Envigo). These included the CR diet (only calories at 50%), in which everything (macromolecules, V & M, EAA) except for
calories was doubled. Thus, when given at 50% restriction everything is normalized except calories. Normalized V & M diet, in which
V & M were doubled. Thus, vitamins and minerals are normalized when given at 50% restriction. Normalized EAA diet, in which EAA
were doubled. Thus, EAA are normalized when given at 50% restriction. Normalized total protein diet, in which total protein was
doubled. Thus, total protein levels are normalized when given at 50% restriction.

Surgery
For experiments involving parabiotic surgery, mice were put on DR 1 week prior to surgery and then maintained on it for a further
3 weeks while conjoined. For the surgery, mice were anaesthetized with ketamine and xylazine and longitudinal incisions were
made from the elbow to the knee joint of each mouse, which were then connected at the joints. The skin of the two animals was
then sutured together. Adrenalectomy surgery was performed by Jackson laboratories.

Tissue processing and flow cytometry

Mice were euthanized with CO2, perfused via the left ventricle of the heart with 10 mL PBS before tissues were harvested and placed
in cold PBS. Bone marrow was flushed using cold PBS and processed through a 70 mM filter. Spleen and lymph nodes were pro-
cessed through a 70 mM filter. Blood was collected in heparinized tubes. White adipose tissue was chopped in digestion media con-
taining DMEM with 1% low fatty acid BSA, 50 mM HEPES, Liberase TL (0.05 mg/mL) (Roche) and DNase I (0.25 mg/mL) (Sigma) and
incubated for 25 min at 37 C with agitation. The tissue was then filtered through a 70 mm filter. Cells were stained for flow cytometry
for 15–30 min in PBS on ice. Antibodies for flow cytometry were purchased from eBioscience, BD Pharmingen or Biolegend and were
conjugated to Pacific Blue, BV605, BV785, BV510, BV650, eFluor 450, APC, AlexaFluor 647, Pe-Texas Red, PE-CF594, FITC, PE,
PerCPCy5.5, PECy7, APCCy7, APCe780, PerCPe710, BV421 or Alexa Flour 700. DAPI or Live/Dead fixable stain (Life Technologies)
was used to identify dead cells in all experiments. Intracellular staining for transcription factors and cytokine producing cells was per-
formed using BD intracellular staining kit or the eBiosciences FOXp3 staining kit. To determine cytokine production, cells were stim-
ulated with phorbol myristate acetate (PMA) (50 ng/mL), ionomycin (1 mM), and BD GolgiPlug (BFA) (1 mL/mL) for 2.5 h at 37 C before
staining for flow cytometry. The following antibodies were used for staining of murine cells: CD4 (RM4-5), CD8b (H35-17.2), CD25
(PC61.5) T-BET (4B10), FEXP3 (FJK-16S), EOMES (Dan11mag), CXCR4 (2B11), TER-119 (TER-119), CD71 (R17217), BCL-2
(10C4), IFN-g (XMG1.2), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), CD69 (H1.2F3), CD127 (A7R34), CD90.2 (30-H12), TCRb
(H57-597), CD62L (MEL-14), CD44 (IM7), NK1.1 (PK136), gdTCR (GL3), B220 (RA3-6B2), CD122 (TMb1). Phosphostaining was per-
formed using either the eBiosciences kit (for AKT473 and mTOR2448 - eBiosciences) or by methanol fixation (for pS6240/244 - Cell
signaling technologies). For the latter, cells were first stained with surface antibodies, washed then treated with 4% PFA at RT for
10 min in PBS. Cells were then incubated with methanol at 20 C for 1 h or at 4 C overnight, followed by phospho-staining for
1 h on ice in the dark. BrdU staining was performed according to the manufacturers protocol (FITC BrdU flow kit - BD Biosciences).
Flow cytometric data were acquired on an LSR II or LSR Fortessa (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Confocal microscopy
To image whole-mounted bone marrow tissue, one side of the bone (femur or tibia) was cut open, fixed for 8-16 h in PLP-buffer at 4 C
and left in 30% sucrose for another 10-16 h. Fixed bones were embedded in optical cutting temperature compound (OCT) over dry
ice and stored at 80 C. For staining, bones were trimmed 60-100 mm on a cryostat and collected by melting the OCT. Exposed
bones were fixed again in 4% PFA for 30 min, blocked for 1 h at RT with 1% BSA, 0.25% Triton X-100, 1% Fc-Block. If rabbit

e5 Cell 178, 1088–1101.e1–e7, August 22, 2019

anti-PERILIPIN-1 antibody (Abcam) was used, 10% normal goat serum was added in the blocking buffer. Antibodies (TER-119, CD8,
CD4 are the same as used for flow cytometry) or nuclei stain (DAPI) were diluted in blocking buffer and tissues were stained for 1 h at
RT, washed 3x in PBS and then imaged directly, or if PERILIPIN-1 was used, bones were incubated for 1 h at RT with the secondary
antibody AF647 goat anti-rabbit IgG. Bones were imaged in a PBS filled 4-well chambered cover glass using an upright inverted
confocal microscope from Leica (Leica TCS SP8). Imaris software (Bitplane) was used for all confocal image processing.

In vitro activation of CD8+ T cells and adoptive transfer

Spleen and lymph nodes from wild type and knock out mice were processed through a 70 mm filter, stained with an anti-CD8b
antibody conjugated to PE then positively enriched using anti-PE microbeads (Miltenyi) and a magnetic column (Miltenyi). Cells
were cultured in a 24-well plate coated with 5 mg anti-CD3 and anti-CD28 antibodies in complete media with 10 ng/mL IL-2. Cells
were split on days 2 and 3, with 10 ng/mL IL-7 added for days 4 and 5 of culture. OT-I cells were isolated from spleen of OT-I
mice (CD45.1 or mTomato+) background and activated with SIINFEKL peptide. 2–5 million cells were transferred intravenously via
the retroorbital vein.

In vivo treatments
Dexamethasone (Sigma) was administered to mice fed ad libitum at a dose of 2 mg/kg i.p. every day for 2 weeks. For inducible gene
deletion, 75 mg/kg tamoxifen (Sigma) in 90% corn oil: 10% ethanol for 5 consecutive days i.p. Experiments were performed 3 weeks
after the final treatment. FTY720 (Sigma) was administered i.p. four times every other day at a dose of 1 mg/kg, with or without 0.5 mg
of an anti-CD62L antibody (Mel-14, BioXcell). For AMD3100 (Sigma) experiments, mice were put on DR for 3 weeks then injected with
10 mg/kg subcutaneously for 4 consecutive days. 1 mg/kg BrdU (BD) was administered i.p. every day for 7 days prior to the exper-
iment. Anti-CD4 (GK1.5, BioXcell) and anti-CD8 (2.34, BioXcell) was administered i.p. at a dose of 0.2 mg for three consecutive days
prior to infection.

Ex vivo assays
Bodipy-C16 uptake assays were performed as previously described (Pan et al., 2017). LipidTOX (Thermofisher) staining was
performed on BM cells after surface staining according the manufactures instructions.

RNA extraction, cDNA synthesis and Quantitative PCR

Whole bone marrow from femur or tibia was flushed in Trizol (Thermofisher). Total RNA extraction was performed with RNeasy mini kit
(QIAGEN). DNase-treated total RNA (100 ng) was reverse transcribed into cDNA with Omniscript RT kit (QIAGEN) following
manufacturer’s instructions. qPCR was performed using the Taq PCR Master Mix (Life Technologies) on a Quantstudio 6 Flex
Real-Time PCR (Life Technologies).

RNA Sequencing
CD8+ TCM were sorted from the bone marrow and spleen of mice fed ad libitum or on DR. RNA was extracted using an Arcturus
PicoPure RNA Isolation Kit (Applied Biosystems), as per manufacturer’s instructions. cDNA was synthesized with the Ultralow V4
kit (Clonetech) and sequencing libraries were subsequently prepared with the Nextera XT DNA prep kit (Illumina). RNA-seq reads
were mapped with STAR (Dobin et al., 2013) to the mm10 reference genome with default parameters. Read counts were assessed
with HOMER’s (Heinz et al., 2010) analyzeRepeats with the option rna and parameters -noadj -condenseGenes and -count exons
for three replicates per condition. Differential gene expression was assessed with DESeq2 (Love et al., 2014) using HOMER’s
getDiffExpression.pl using a false discovery rate (FDR) of 5% and a fold change of 1.5. Metascape (Tripathi et al., 2015) was used
for analyzing enriched GO terms, and GSEA was used for gene set enrichment analysis. PCA was performed in R with prcomp,
center = TRUE, scale. = TRUE. GEO accession number is GSE124063.

ELISA
Mouse corticosterone ELISA was performed according to the instructions of the manufacturer (Thermofisher). To obtain bone
marrow extracellular fluid, femurs were flushed in approximately 2 mL of PBS, centrifuged and the supernatant was taken.

2-photon imaging and analysis

Mice fed ad libitum or on DR for 3 weeks received 20 3 106 mTomato+ OT-I cells i.v. and were imaged 1 week later. To image, mice
were anesthetized with ketamine (85 mg/kg), xylazine (13 mg/kg), and acepromazine (2 mg/kg) in PBS and maintained at a core
temperature of 37 C. 20 mg of an anti-CD31 AF647 antibody (clone EMC13.3, Biolegend) was injected i.v. to visualize vessels
immediately before imaging. Two-photon imaging through a surgically thinned skull was adapted from methods previously
described (Manglani and McGavern, 2018). Briefly, a metal bracket was glued to the skull such that the imaging window contained
the frontal bone sutures. The skull was thinned to leave 25 mm of bone above the bone marrow. 3D time lapses were captured
using a Leica SP8 two-photon microscope equipped with an 8,000-Hz resonant scanner, a 25 3 color corrected water-dipping
objective (1.0 NA), a quad HyD external detector array, a Mai Tai HP DeepSee Laser (Spectra-Physics) tuned to 905nm and an
Insight DS laser (Spectra-Physics) tuned to 1050nm. The following dichroic mirrors were used for imaging studies: 495nm-LP,

Cell 178, 1088–1101.e1–e7, August 22, 2019 e6

562nm-LP, and 624-LP. Z-stacks were acquired at 8.5 sec intervals and consisted of 7 optical slices (3.5mm step size). A second
harmonic signal was used to visualize skull bone. Cell speed and track displacement length of mTomato OT-I cells were obtained
using Imaris software (Bitplane). ImageJ software was used to generate the supplemental movies.

Oxygen consumption flux evaluation

CD8+ CD44+ T cells were enriched via negative selection and sorted from secondary lymphoid organs or bone marrow of mice fed
ad libitum or on DR. Real-time oxygen-consumption rate (OCR) in T cells were determined with an XF-96e Extracellular Flux
Analyzer (Seahorse Bioscience); 3 3 105 cells/well were used for each condition. Cell were previously attached by using Cell
Tak (25 ug/mL). The assay was performed in XF DMEM pH = 7.4 supplemented with 2 mM glutamine, 25 mM glucose and
1mM pyruvate. Three consecutive measurements were performed under basal conditions and after the sequential addition of
the following ETC inhibitors: 1 mM oligomycin, 1 mM FCCP, 1 mM rotenone and 1 mM antimycin A. Basal respiration rate (BRR)
was defined as last rate measurement before the first injection. Maximal respiration rate (MRR) was defined as the maximal
rate measurement after FCCP injection. Spare respiration capacity (SRC) was defined as the difference between MRR and BRR.

QUANTIFICATION AND STATISTICAL ANALYSIS

Groups were compared with Prism V8 software (Graphpad) using a two-tailed unpaired Student’s t test or Log-rank (Mantel-Cox) test
where appropriate. Differences were considered to be statistically significant when p < 0.05. Data are expressed as mean, or mean ±
SD. Refer to figure legends for the specific statistical tests used in each experiment. Sample sizes were determined based on
previous experience with similar experiments.

DATA AND CODE AVAILABILITY

The datasets generated during this study can be found using the GEO accession number GEO: GSE124063.

e7 Cell 178, 1088–1101.e1–e7, August 22, 2019

Supplemental Figures

Figure S1. Memory T Cells Accumulate in BM during DR, Related to Figure 1

(A) Percentage weight change of mice on 50% DR for up to 6 weeks. (B) Weight (grams) of gonadal adipose tissue (GAT) in mice on DR. (C) Total hematopoietic
cellularity of spl, cervical LN (cLN) and inguinal LN (iLN) after 3-4 wk of DR. (D) Gating strategy of BM populations analyzed during DR. (E) Number of CD4+ CD44+

(legend continued on next page)

T cells in spl, cLN, blood, GAT, and BM over time during DR. (F) Number of regulatory T cells (G) NK1.1+ cells and (H) B220high B cells in GAT, spl and BM after 5-6
wk of DR. (I) Number of B220- CD138+ plasma cells in BM after 5-6 wk of DR. Each symbol represents an individual mouse, except A, which is the mean of 5 mice.
Data shows the mean, or mean ± SD, representative of at least 5 (A, D), or pooled from 2–3 (B, C, E–I) experiments with 3–5 mice per group. *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001, ns; not significant. Two-tailed unpaired Student’s t test.
Figure S2. Phenotypic Analysis of Memory T Cells during DR, Related to Figure 2
(A) Number of CD44+ CX3CR1int. TPM in BM of mice fed ad libitum or on DR for 3 wk. (B–G) Expression of various surface markers and transcription factors in TCM
and TEM from BM and spl of mice fed ad libitum or on DR for 3–4 weeks. (H) OT-I cells were in vitro activated with peptide and transferred into hosts fed ad libitum
(legend continued on next page)
or on DR for 3 wk. Graphs show number of transferred OT-I cells recovered from BM and spl 1 wk after transfer. (I) BrdU uptake (over 1 wk) by memory T cells in
BM and spl of mice fed ad libitum, on DR for 3 wk, or on DR for 3 wk then re-fed ad libitum for 1 wk. Gated on CD8+ CD44+ cells. Each symbol represents an
individual mouse. Data shows the mean, pooled from 2–3 experiments (A, I) or representative of at least 2 (B–H) experiments. *p < 0.05, **p < 0.01, ***p < 0.001,
****p < 0.0001, ns; not significant. Two-tailed unpaired Student’s t test.
 A B

C D

Figure S3. GC Drive Memory T Cells into BM during DR, Related to Figure 3
(A) BCL-2 expression by TEM in BM of mice fed ad libitum (blue) or on DR (red) for 3 wk. (B) Mice received sham surgery or an adrenalectomy (ADX) 1 wk prior to
being put on DR for 1 wk. Graphs show number of TEM in spl and BM. (C) BCL-2 expression in BM and (D) number of TEM in spl and BM of mice fed ad libitum that
were treated with veh (blue) or dex (red) daily for 2 wk. Each symbol represents an individual mouse. Data shows the mean, pooled from 2 experiments. *p < 0.05,
**p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant. Two-tailed unpaired Student’s t test.
Figure S4. BM Remodeling during DR Promotes Memory T Cell Recruitment and Retention, Related to Figure 4
(A) Scatterplot showing the Il7 and Il15 genes in whole BM samples from mice fed ad libitum or on DR for 3 wk. (B) Cxcl12 gene expression in whole BM of mice fed
ad libitum or on DR for 3 wk, determined by qPCR. (C) CXCR4 surface expression by TEM in the BM and spl of mice fed ad libitum (blue) or on DR for 3 wk (red). (D)
Mice on DR for 3 wk were treated with AMD3100 subcutaneously for 4 consecutive days prior to analysis. Graph shows number of CD8+ TCM in BM. (E) Number of
RBC in BM of mice fed ad libitum treated with veh or dex daily for 2 wk. (F) Mice that had been on DR for 3 wk were treated with FTY720 4 times every other day. (G)
Fabp4 gene expression in whole BM samples from tamoxifen-treated Adipoq-CreERT2 x Rosa26-DTA mice fed ad libitum or on DR for 5–6 wk determined by
qPCR. (H) Uptake of Bodipy-C16 ex vivo by TCM from BM of mice fed ad libitum or on DR. (I) Intracellular lipid content of TCM from BM of mice fed ad libitum or on
DR. Each symbol represents an individual mouse, except for (A), in which each symbol represents an individual gene. Data shows the mean, representative of at
least 2 experiments (C, E) or pooled from 2–3 (A, B, D, F–I) experiments with 3–6 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not
significant. Two-tailed unpaired Student’s t test.
Figure S5. Memory T Cells Are in a State of Energy Conservation during DR, Related to Figure 5
(A) Principle component analysis of mRNA expression by TCM sorted from the spl and BM of mice fed ad libitum or on DR for 3 wk. Each symbol represents a
biological replicate pooled from 5 mice.
Figure S6. Similar Protection to a Secondary Infection When on DR throughout Primary and Secondary Infections, Related to Figure 6
(A) Mice on DR for 3 wk were infected with Yptb DyopM orally, then challenged 7 wk later i.v. with WT Yptb. Bacterial burden was assessed in the spl 2 days after
challenge. Each symbol represents an individual mouse. Data shows the mean, pooled from 2 experiments with 5 mice per group. ns; not significant. Two-tailed
unpaired Student’s t test.