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◥ are inversely correlated to patient survival.

RESEARCH ARTICLE SUMMARY Necrotic cells release intracellular contents
into the extracellular space. Because the intra-
cellular concentration of potassium is higher
IMMUNOMETABOLISM
than the extracellular compartment (~145 mM
versus ~5 mM), local potassium levels within
T cell stemness and dysfunction in the tumor interstitial space can exceed 40 mM.
We hypothesized that elevated extracellular

tumors are triggered by a potassium found in tumor interstitial fluid

would disrupt the electrochemical gradient that
facilitates T cell nutrient uptake, simultaneously
common mechanism limiting the acquisition of effector programs and
the coincident loss of stemness. In the present
Suman Kumar Vodnala*, Robert Eil*, Rigel J. Kishton, Madhusudhanan Sukumar, work, we sought to explore the impact of high
Tori N. Yamamoto, Ngoc-Han Ha, Ping-Hsien Lee, MinHwa Shin, levels of potassium found in tumors on T cell
Shashank J. Patel, Zhiya Yu, Douglas C. Palmer, Michael J. Kruhlak, Xiaojing Liu, stemness and antitumor capacity.
Jason W. Locasale, Jing Huang, Rahul Roychoudhuri, Toren Finkel,
Christopher A. Klebanoff, Nicholas P. Restifo† RESULTS: We found that elevated extracel-
lular potassium characteristic of the extracel-
lular space within tumors reduced the uptake

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INTRODUCTION: Cancers persist and pro- renewal, multipotency, and persistence. Al- and consumption of local nutrients by anti-
gress despite the presence of tumor-infiltrating though these cells appear to be responsible for tumor T cells, inducing a state of functional
lymphocytes (TILs). A paradox of tumor im- tumor destruction in the setting of successful caloric restriction. A starvation response en-
munology is that tumor antigen–specific TILs immunotherapy, the mechanisms underlying sued, resulting in autophagy, mitochondrially
are dysfunctional in situ and yet can mediate their generation and maintenance are unknown. ◥
dominant metabolism, and
regression of large metastatic tumors after ON OUR WEBSITE a paucity of available cofac-
immune checkpoint blockade or adoptive cell RATIONALE: To progress from a naïve, stem Read the full article tors obligatory for histone
transfer. TILs are predominantly considered cell–like state after activation, T cells rely on at [Link] modification and the epi-
“exhausted” because of chronic antigen ex- the uptake and consumption of extracellular org/10.1126/ genetic remodeling required
posure, but recent studies have revealed that nutrients to enact robust aerobic glycolysis science.aau0135 for progressive differenti-
..................................................
T cells within tumors exist in a continuum and mTOR-driven anabolic growth. In multiple ation. Both nucleocytosolic
of epigenetic, transcriptional, and metabolic organisms, the cellular response to starvation acetyl–coenzyme A (CoA) and methionine inter-
states. A small subset of T cell clonotypes in preserves stemness and enhances organismal mediates were depleted. Depletion of nucleo-
TILs expressing the transcription factor TCF7 longevity. Cellular necrosis is a feature of many cytosolic acetyl-CoA limited the acquisition
possess stem cell–like behaviors including self- solid tumors, and higher densities of necrosis of histone acetylation on the promoters and
enhancers of genes encoding effector mol-
Physiologic condition Tumor microenvironment ecules. Simultaneously, nutrient deprivation
Catabolic
metabolism reduced methionine intermediates, depressing
T cell T cell methylation of histone marks that normally
suppress stemness-associated programs. Treat-
ment of antitumor T cells with elevated extra-
cellular potassium as well as pharmacologic or
TCF1 gene therapies mimicking mechanisms of func-
tional starvation resulted in T cells with retained
th stemness, evidenced by self-renewal and multi-
/d ea
nce potency, thereby enabling the enhanced destruc-
ce
n es tion of large, established tumors.
Se Stemness

CONCLUSION: These data provide a link be-

Anabolic
Therapeutic efficacy metabolism tween tumor-induced immune suppression and
the stem cell–like properties of some anti-
mTOR Differentiation
tumor T cells. Moreover, these findings deepen
Chron
ic
stimu antigen Progressive our understanding of how cancer can progress
lation Proliferative capacity differentiation despite the presence of T cells that continue to
harbor the capacity for its destruction. Finally,
Chron
ic antig we identify new therapeutic strategies to meta-
en stim
ulatio bolically induce stemness programs in antitumor
▪
n Effector
T cells that enhance cancer immunotherapies.
Modified “Waddington valley” depicting T cell differentiation and the role of potassium in
preserving T cell stemness. Left: Physiologic conditions driving T cell differentiation after
activation (blue) to engage anabolic metabolism for acquiring effector functions (red). Right: The list of author affiliations is available in the full article online.
Starvation response resulting from elevated extracellular potassium (↑[K+]e, white snow in valleys) *These authors contributed equally to this work.
†Corresponding author. Email: restifon@[Link]
drives catabolic metabolism and maintains T cell stemness. Height of the valley defines Cite this article as S. K. Vodnala et al., Science 363,
Tcell potential to differentiate; segments of the valley define duration to reach senescence or death. eaau0135 (2019). DOI: 10.1126/science.aau0135

Vodnala et al., Science 363, 1417 (2019) 29 March 2019 1 of 1

R ES E A RC H

◥ (mammalian target of rapamycin) signaling and

RESEARCH ARTICLE heritable changes to the epigenetic landscape
(16–18). This anabolic metabolism requires the
continual uptake and consumption of extracel-
IMMUNOMETABOLISM lular nutrients, facilitated by a transmembrane
electrochemical gradient present in all living

T cell stemness and dysfunction in cells (19). Maintenance of this electrochemical

gradient involves the monovalent cation potas-
sium, which has an intracellular concentration
tumors are triggered by a of ~145 mM, much higher than normally found
in the extracellular space (~5 mM).

common mechanism Despite tumor cell death, a higher density of

necrosis is inversely correlated with patient sur-
vival in some tumor histologies (20, 21). We pre-
Suman Kumar Vodnala1,2*, Robert Eil1,2,3*, Rigel J. Kishton1,2, Madhusudhanan Sukumar1,2, viously reported that necrosis leads to a profound
Tori N. Yamamoto1,2,4, Ngoc-Han Ha5, Ping-Hsien Lee1,2, MinHwa Shin5†, alteration of the potassium ion gradient within
Shashank J. Patel1,2‡, Zhiya Yu1,2, Douglas C. Palmer1,2, Michael J. Kruhlak6, Xiaojing Liu7, the tumor microenvironment (TME) (22). Cellular
Jason W. Locasale7, Jing Huang5, Rahul Roychoudhuri8, Toren Finkel9, necrosis liberates potassium and other intra-
Christopher A. Klebanoff 10,11,12, Nicholas P. Restifo1,2§ cellular contents into the extracellular space, which
contains the tumor interstitial fluid (TIF). Dis-
A paradox of tumor immunology is that tumor-infiltrating lymphocytes are dysfunctional in rupted tumor vasculature and abnormal lymphatic

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situ, yet are capable of stem cell–like behavior including self-renewal, expansion, and drainage lead to TIF stasis, causing tumors to be
multipotency, resulting in the eradication of large metastatic tumors. We find that the deprived of oxygen and crucial extracellular nu-
overabundance of potassium in the tumor microenvironment underlies this dichotomy, trients, whereas the concentration of TIF potassium
triggering suppression of T cell effector function while preserving stemness. High levels of remains elevated. The increased extracellular
extracellular potassium constrain T cell effector programs by limiting nutrient uptake, potassium of the TIF acutely suppresses T cell
thereby inducing autophagy and reduction of histone acetylation at effector and effector function after TCR ligation, limiting T cell
exhaustion loci, which in turn produces CD8+ T cells with improved in vivo persistence, activation in a nonredundant manner to both
multipotency, and tumor clearance. This mechanistic knowledge advances our PD-L1 and CTLA-4 immune checkpoints (22). Yet
understanding of T cell dysfunction and may lead to novel approaches that enable the the impact of high levels of extracellular potassium
development of enhanced T cell strategies for cancer immunotherapy. (↑[K+]e) on nutrient uptake, metabolism, epigenetics,
and cellular differentiation has not been explored.
Here, we report experiments in which ↑[K+]e

T
umor-infiltrating lymphocytes (TILs) are The existence of stem cell systems in mature diminished the uptake and consumption of local
dysfunctional and can be frequently ob- organisms is now well established (6, 7), yet it has nutrients despite adequate extracellular resources,
served in a quiescent state next to growing been less appreciated that some post-thymic a state we define as functional caloric restriction.
tumor cells. Nonetheless, the T cells of some T cells also harbor stem cell–like properties. Elevated [K+]e subsequently triggered a T cell
patients maintain the capacity to prolif- However, like other adult stem cells in the gut, starvation response leading to autophagy, which
erate, persist, and mediate the complete de- skin, and olfactory systems, stem cell–like T cells drove metabolic and epigenetic reprogramming,
struction of large tumor deposits (1). This is best do not function like highly differentiated lym- thereby limiting the acquisition of effector func-
illustrated by immunotherapy using immune phocytes with regard to the production of ef- tions and preserving Tcf7 and functional stemness.
checkpoint blockade or adoptive cell transfer fector cytokines and cytotoxicity (8–10). Each This epigenetic reprogramming was mediated by
(ACT), which together represent some of the T cell specificity (clonotype) can retain key hall- the depletion of nucleocytosolic acetyl–coenzyme
most important advances in cancer treatment marks of stem cells such as self-renewal, prolifera- A (AcCoA). Reductions in available AcCoA de-
in the past decade (2–5). tion, and multipotency. For example, TILs can creased histone acetylation, which in turn limited
be expanded ex vivo (>1010 cells) and readminis- the activation of effector genes. Thus, we find that
1
Surgery Branch, Center for Cancer Research, National tered to cancer patients, resulting in clonal re- ↑[K+]e characteristic of the TME attenuates AcCoA
Cancer Institute, Bethesda, MD 20892, USA. 2Center for population and the durable destruction of large processing, generating antitumor T cells with
Cell-Based Therapy, National Cancer Institute, Bethesda, MD
20892, USA. 3Department of Surgery, Memorial Sloan
metastatic solid tumors (2, 3). We and others improved persistence, engraftment, self-renewal,
Kettering Cancer Center, New York, NY 10065, USA. have previously identified the transcription factor multipotency, and tumor clearance—all charac-
4
Immunology Graduate Group, University of Pennsylvania, Tcf7 as being critically involved in the retention teristics of T cell stemness (23). These data help
Philadelphia, PA 19104, USA. 5Laboratory of Cancer Biology of stem cell–like attributes in antigen-experienced to resolve the current immunotherapy paradox
and Genetics, National Cancer Institute, Bethesda, MD
20892, USA. 6Experimental Immunology Branch, National
T cells (9, 11). Although TILs have historically as to why cancers progress despite the presence
Cancer Institute, Bethesda, MD 20892, USA. 7Department of been thought to be terminally differentiated, TCF7 of T cells that harbor the capacity for rejuvena-
Pharmacology and Cancer Biology, Duke University School of expression has defined a TIL subset harboring tion and tumor clearance.
Medicine, Duke University, Durham, NC 27710, USA. stem cell–like properties (8, 9, 11–15). However,
8
Babraham Institute, Babraham Research Campus,
the mechanisms that maintain this TCF7-positive Elevated [K+]e triggers functional
Cambridge, UK. 9Aging Institute, Department of Medicine, caloric restriction in T cells
University of Pittsburgh School of Medicine, Pittsburgh, PA TIL population are minimally understood.
15261, USA. 10Parker Institute for Cancer Immunotherapy, The differentiation of naïve T cells to memory We previously reported that ↑[K+]e is charac-
New York, NY 10065, USA. 11Center for Cell Engineering and and effector states is largely driven by the strength teristic of the TME (an additional 40 mM on
Department of Medicine, Memorial Sloan Kettering Cancer
Center, New York, NY 10065, USA. 12Weill Cornell Medical
of the signals they receive, primarily via the T cell average) and acutely silences T cell effector func-
College, Cornell University, New York, NY 10065, USA. receptor (TCR). This maturation process can be tion (22), but the impact of ↑[K+]e on the trans-
*These authors contributed equally to this work. †Present address: subverted by many inhibitory signals that disrupt membrane electrochemical gradient and its
Department of Biochemistry and Molecular Biology, Korea Univer- the signaling cascade after TCR ligation. The consequences on T cell nutrient uptake and
sity College of Medicine, Seoul, Korea. ‡Present address:
Department of Cancer Immunology and Immune Modulation,
progression of T cell differentiation toward full cellular metabolism remain unclear. We per-
Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT, USA. effector function is metabolically driven in part formed comprehensive metabolite analysis using
§Corresponding author. Email: restifon@[Link] by an anabolic program involving Akt-mTOR liquid chromatography and mass spectrometry

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Fig. 1. Increased [K+]e limits nutrient
uptake and triggers functional caloric
restriction in T cells. (A) Experimental
setup for activation of CD8+ T cells in the
indicated conditions: control (regular media;
5 mM K+) or ↑[K+]e (an additional 40 mM K+).
(B and C) Quantification of glycolysis
metabolites (B) and amino acids (C) by liquid
chromatography and mass spectrometry in
the indicated conditions (data shown are
means ± SEM of six replicates per condition).
(D) 2-NBDG uptake in the indicated
conditions with representative histograms
and quantification. (E) Representative fluorescence-activated cell sorting subset of lipid species enriched involving autophagy flux. Data are means ± SEM
(FACS) histogram, quantification, and confocal images showing reduced of six replicates per conditionper condition. (H) Volcano plot representing
BODIPY FLC16 uptake in ↑[K+]e versus control T cells. In (D) and (E), decreased glycolysis metabolites (black) and enrichment of Kennedy inter-
values indicate average geometric mean fluorescence intensity; data are mediates (red). Metabolite abundance is represented as relative change
means ± SEM and are representative of two independent experiments. (x axis) versus significance (y axis). (I) Schematic of the molecular pathways
See fig. S1B for confocal quantifications. (F) Schematic of necrotic tumors (pink) involved in autophagy and the role of Kennedy pathway components in LC3
liberating intracellular potassium into the tumor interstitial fluid. This disrupts lipidation for phagophore formation. (J) GSEA of ↑[K+]e versus control
the electrochemical gradient and limits the ability of T cells to take up nutrients transcriptional profile compared with wild-type versus Atg7 KO datasets
(blue, purple, and red symbols), resulting in functional caloric restriction. (GSE57047). NES, normalized enrichment score. All metabolomic data are
(G) Left: Heat map shows relative abundance of profiled metabolites in means ± SEM of six replicates per condition. **P < 0.01, ***P < 0.001,
T cells under control and ↑[K+]e conditions. Right: Quantifications highlight the ****P < 0.0001 (two-tailed t tests).

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of T cells conditioned in ↑[K+]e or control media fig. S2B). Concurrent flow cytometric analysis AcCoA, a molecule that enables carbons derived
containing ~5 mM K+. This analysis revealed confirmed that ↑[K+]e resulted in a greater propor- from carbohydrates, fats, and proteins to be uti-
that ↑[K+]e exposure resulted in a significant re- tion of mCherry+GFP– cells, suggesting ongoing lized in the citric acid cycle for oxidative phos-
duction of glycolytic intermediates and essential autophagic flux (Fig. 2D). These dynamic changes phorylation. AcCoA also serves as a substrate
amino acids within T cells (Fig. 1, A to C, and fig. in fluorescence were not attributable to artifac- for histone and non-histone protein acetylation
S1A). These alterations were attributable to a tual spectral overlap, as the T cell surface marker (31, 32). Because the mitochondrial membrane
reduction in electrochemically dependent nutrient Thy1.1 was used as an independent validation of is impermeable to AcCoA, there are two dis-
uptake, because T cells conditioned with ↑[K+]e transduction, reporter persistence, and increased tinct pools of AcCoA in cells: mitochondrial
or treated with BaCl2, a nonselective inhibitor of autophagy in ↑[K+]e-treated cells (fig. S2C). and nucleocytosolic (the latter because cytosolic
potassium channels in the plasma membrane, The relative abundance of the autophagosome AcCoA can move freely across the nuclear pore
had diminished uptake of fluorescently labeled proteins LC3b-I and the PE-conjugated form, complex) (Fig. 3A) (31). The quantity of AcCoA
glucose and lipid analogs, 2-NBDG and BODIPY LC3b-II, were examined by immunoblot. We in the mitochondria is estimated to be several
FLC16, respectively (Fig. 1, D and E, and fig. S1, B found that the LC3b-II/LC3b-I ratio was elevated times that in the nucleocytosolic compartment.
and C). Neither T cell survival nor proliferative after ↑[K+]e in both mouse and human T cells, Thus, total cellular AcCoA is a surrogate for mito-
potential after restimulation were compromised consistent with increased autophagosome for- chondrial AcCoA (33). In the setting of ↑[K+]e,
by ↑[K+]e-mediated reduction of nutrient uptake mation (Fig. 2, E and F) (30). Collectively, these we observed an elevation in total cellular AcCoA,
(fig. S1, D to F). We therefore propose that re- data demonstrate that antitumor T cells exposed a finding consistent with mitochondrially oriented
duced levels of intracellular anabolic metabolites to ↑[K+]e acquire a metabolic state defined by metabolism (Fig. 3B).
in the setting of unchanged extracellular nutrient functional caloric restriction and ongoing auto- Despite higher levels of total AcCoA, we mea-
abundance induced by ↑[K+]e result in a state phagy. To investigate whether autophagy induced sured a reduction in nucleocytosolic AcCoA and
of functional caloric restriction. These findings by ↑[K+]e was related to a starvation response its precursor, citrate, in T cells after ↑[K+]e condi-

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are consistent with those of previous studies in after depletion of intracellular nutrients, we mea- tioning (Fig. 3C). These findings, along with a
nonimmune cells demonstrating that alterations sured the activity of the Akt-mTOR and AMPK depletion of glycolytic products, suggested pref-
to the transmembrane potassium concentration (adenosine monophosphate–activated protein erential generation and utilization of AcCoA
and membrane potential (Vmem) can attenuate kinase) nutrient-sensing signaling cascades in within the mitochondria for use in oxidative
nutrient uptake (24–27) (Fig. 1F). both mouse and human CD8+ T cells. Increased phosphorylation. To determine the effect of
Although most intracellular metabolites were ↑[K+]e mediated reduced phosphorylation of Akt ↑[K+]e conditioning and consequent depression
depressed globally, we noted enrichment in a (Ser473), mTOR (Ser2448) (fig. S2, D to F), and two of nucleocytosolic AcCoA, we measured total
subset of phospholipid species (Fig. 1G). These modulators of ribosomal activity, S6 (Ser235/6) acetylated Lys levels in mouse and human T cells
included glycerophosphoethanolamine, glycero- (Fig. 2G and fig. S2, G and H) and 4E-BP1 (Thr37/46) (Fig. 3D and fig. S4A). We found a notable re-
phosphocholine, phosphoethanolamine, and CDP- (fig. S2I). Moreover, immunoblot analysis revealed duction in the setting of ↑[K+]e characteristic
ethanolamine (Fig. 1H). These compounds are increased activity of the energy sensor AMPK (fig. of the TME.
hallmark metabolites of the Kennedy pathway, S2J). Similar findings were observed in human The acetylation of histone proteins acts to
a metabolic pathway involved in the de novo bio- CD8+ T cells, as evidenced by hypophosphoryla- disrupt DNA-histone salt bridges, allowing for
synthesis of phosphatidylethanolamine (PE), an tion of S6 (Ser235/6) (Fig. 2H), suggesting induc- a euchromatin structural organization and in-
early initiator of autophagosome fusion and auto- tion of nutrient conservation (17). creased local gene transcription (34). We sought
phagy (Fig. 1I) (28). Analysis of human CD8+ We next sought to measure the consequences to assess the potential epigenetic consequences
T cells after ↑[K+]e conditioning also revealed of this starvation response for the metabolic fitness related to the limited nuclear availability of
enrichment in Kennedy cycle intermediates of T lymphocytes expanded in ↑[K+]e conditions. AcCoA in the setting of ↑[K+]e. Therefore, we
(fig. S1G). Additionally, RNA sequencing (RNA- After ↑[K+]e, T cells exhibited robust mitochondrial performed chromatin immunoprecipitation and
seq) analysis of primary T cells cultured in the function, as evidenced by marked spare respiratory polymerase chain reaction (ChIP-PCR) of acetylated
presence or absence of ↑[K+]e demonstrated sig- capacity (Fig. 2I). ↑[K+]e preserved the oxygen Lys9 and Lys27 of histone H3 (H3K9; H3K27Ac)
nificant enrichment in autophagy-associated consumption capacity of human TILs after re- and found decreased acetylation at both residues
transcripts (Fig. 1J) (29). These data indicated that activation and proliferation, as well as that of for Ifng regulatory loci (Fig. 3E). To obtain a whole-
levels of ↑[K+]e characteristic of the TME result in TCR-engineered T cells (Fig. 2J and fig. S3A). genome map of histone acetylation (H3K9Ac)
a selective accumulation of metabolites required Consistent with the induction of a mitochon- loci and consequent chromatin accessibility, we
for autophagosome maturation and a gene ex- drially dominant program of cellular metabolism, performed ChIP sequencing (ChIP-seq). We ob-
pression signature indicative of ongoing autophagy. we found that ↑[K+]e-conditioned human CD8+ served reduced acetylation of the promoter and
T cells maintained a lower mitochondrial mem- enhancer loci of Ifng, as well as loci associated
Functional caloric restriction mediated brane potential (Dym) (fig. S3B). Scanning elec- with T cell activation and exhaustion, including
by increased [K+]e promotes autophagy tron microscopy of T cells expanded in ↑[K+]e Pdcd1 (PD1), Cd244 (2B4), Havcr2 (Tim-3), and
and metabolic reprogramming conditions revealed an increase in mitochondrial Klrg1 (Fig. 3, F and G, and fig. S4, B and C). We
To evaluate the impact of ↑[K+]e on autophagic number and quality (fig. S3C), which suggests that then performed RNA-seq under the same condi-
flux in live T cells, we used an eGFP (enhanced T cells maintained mitochondrial integrity and tions to measure the functional consequences of
green fluorescent protein)–mCherry-LC3b fusion the capacity for marked on-demand oxygen con- H3K9 acetylation, with concordant results.
reporter system (29) (Fig. 2, A and B, and fig. S2A). sumption. From these data, we conclude that cells To visualize patterns of changes to H3K9Ac at
Microtubule-associated protein I light chain beta subjected to ↑[K+]e had limited ongoing con- a whole-genome level, we plotted relative change
(LC3) is hydrolyzed to expose Gly120 to create sumption of extracellular energy resources and versus P value for differentially acetylated loci of
LC3b-I (Fig. 1I). This molecule is in turn con- instead existed in a metabolic state of functional effector genes (Fig. 3H, left, and fig. S5A). We
jugated with PE to generate LC3b-II, enabling starvation, with a resultant induction in autophagy found a notable reduction in activating H3K9Ac
phagophore expansion to create autophagosomes and mitochondrially driven energy production. histone marks on regulatory elements associated
(30). An autophagy-incompetent construct con- with T cell effector and exhaustion programs.
taining a Gly120 → Ala substitution (G120A) served Functional caloric restriction depletes Quantification by gene set enrichment analysis
as a negative control (fig. S2A). Confocal micros- nucleocytosolic AcCoA to suppress (GSEA) revealed that H3K9Ac acetylation was
copy demonstrated loss of GFP fluorescence in effector programs indeed lost at sites responsible for activation of
mCherry puncta in the presence of ↑[K+]e, indicat- The enhanced mitochondrial function of T lym- exhaustion (Tim3+ versus CXCR5+ UP) and ef-
ing autophagosome-lysosome fusion (Fig. 2C and phocytes exposed to ↑[K+]e led us to quantify fector transcriptional programs (Fig. 3H, right).

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Fig. 2. T cells exposed to increased [K+]e recycle nutrients via autophagy for LC3b-I and LC3b-II in control or ↑[K+]e conditions. Quantification of auto-
during functional caloric restriction. (A) Schematic for monitoring phagy flux is represented by ratio of LCb3-II/LC3b-I intensities. Data are means
autophagy flux using retroviral mCherry-eGFP-LC3 construct. (B) Experimental ± SEM of three independent Western blots. (G and H) Immunoblot densitometry
setup of in vitro culture conditions for activation of pmel CD8+ T cells with of CD8+ T cells obtained from mouse or human for phosphor-S6 (Ser235/236)
gp100, followed by retroviral transduction with mCherry-GFP-LC3 construct in control or ↑[K+]e conditions. (I and J) Representative O2 consumption rates
at indicated time points. (C) Representative live-cell confocal images defining (OCR) of CD8+ pmel transgenic T cells and CD8+ T cells obtained from fresh
the GFP and mCherry puncta in cells treated under the indicated conditions. tumor digest of a melanoma patient cultured in control or ↑[K+]e measured in
See fig. S2B for quantification of confocal images. (D) Representative flow real time under basal conditions in response to the mitochondrial inhibitors
cytometry plot and quantification of autophagy flux in the indicated conditions oligomycin, FCCP [carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone],
by measuring the loss of GFP in mCherry populations. Data are means ± and R&A (rotenone and antimycin). For all relevant panels here and in later
SEM and are representative of two independent experiments. (E and figures, center values and error bars represent means ± SEM. **P < 0.01,
F) Immunoblot densitometries and quantification of mouse or human CD8+ Tcells ***P < 0.001, ****P < 0.0001 (two-tailed t tests).

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Fig. 3. Elevated [K+]e-

mediated metabolic
programming depletes
cytoplasmic AcCoA to
preserve epigenetic
stemness. (A) Schematic
of the molecular pathways
involving compartmentali-
zation of mitochondrial
and cytoplasmic AcCoA.
Model depicts how reduc-
tion in cytoplasmic AcCoA
inhibits acquisition of effec-
tor functions through epi-
genetic changes. (B and
C) Quantifications of total
cellular AcCoA, cyto-
plasmic citrate, and
nucleocytosolic AcCoA

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from cells treated in the
indicated conditions. Data
are means ± SEM and are
representative of two
independent experiments
with at least three culture
replicates. (D) Representa-
tive immunoblot showing
reduced protein acetylation
on Lys residues in the indi-
cated conditions, with
quantification at right. Data
are means ± SEM and are
representative of two
independent experiments.
(E) Drosophila DNA spike-
in normalized quantifica-
tions of H3K9Ac and
H3K27Ac enrichment by
ChIP-PCR at the Ifng locus.
Data are means ± SEM and
are representative of two
independent experiments
with at least three culture
replicates. (F) Representa-
tive genomic alignments of
H3K9Ac ChIP-seq and
RNA-seq measurements
showing reduced deposi-
tion of acetylation or tran-
scripts at the Ifng locus
of CD8+ T cells treated in
the indicated conditions.
Black bars under the tracks
represent the called com-
mon peaks in ↑[K+]e
and control. Black boxes
are sites of acetylation
at transcription start site
(TSS) and enhancer loci. (G) Representative genomic alignments of H3K9Ac from normalized read counts of the peaks annotated to nearest TSS) versus
deposition on the inhibitory receptor gene loci Pdcd1 (CD279), CD244 significance ( y axis). (I) Volcano plots representing reduced H3K9Ac
(2B4), Havcr2 (Tim-3), and Klrg1. (H) Left: Volcano plot depicting a subset deposition of effector genes that showed differential chromatin accessibility in
of effector genes and inhibitory receptors with reduced H3K9Ac deposition ATAC-seq datasets (GSE86797) of the indicated conditions. Abundance is
in ↑[K+]e relative to control T cells. Right: GSEA with statistical analysis of represented as relative change (x axis) versus significance ( y axis). ChIP-seq
effector genes and gene set associated with loss of stemness (Tim3+ versus experiments for each condition were performed with two independent
CXCR5+ UP). Abundance is represented as relative change (x axis, calculated cultures. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t tests).

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By contrast, we did not observe differences in T cell conditioning in ↑[K+]e thus had the dual noma and other solid cancers of the colon, lung,
H3K9Ac enrichment at constitutively expressed effect of reducing chromatin activation and gene and ovary were subjected to whole-exome se-
loci in T cells, such as Cd3e, Actb, or B2M (fig. S4, expression of T cell effector functions and pre- quencing to identify nonsynonymous mutations
D to G), or differences in total levels of H3K9Ac serving a T cell gene signature with augmented that result in the creation of potential neoantigens.
or H3K27Ac in response to ↑[K+]e conditioning stemness. This was confirmed by quantifying The precise antigens recognized by each patient’s
(fig. S4H). Our data are consistent with the ob- the preservation of Tcf7 transcript and protein TIL were identified (table S1). We measured in-
servation that histone acetyltransferase enzymatic (Fig. 4C and fig. S5D). We found that CD8+ T cells creases in the percentages of cells expressing
activity and selectivity are modulated by levels exposed to ↑[K+]e characteristic of the TME or CD62L in these human TILs (Fig. 4, F and G, fig.
of AcCoA. It is likely that existing acetyl groups treated with BaCl2 maintained relatively higher S7D, and table S1). These findings were surprising,
have higher priority for AcCoA than those in- expression of the lymphoid homing marker given that these T cell clonotypes had likely been
volved in the addition of new acetyl groups, CD62L (Fig. 4D and fig. S5, E and F) and the chronically exposed to antigen.
like those associated with T cell differentiation costimulatory marker CD27 (Fig. 4E) and retained To evaluate the effects of ↑[K+]e on antitumor
(35, 36). the capacity to robustly produce interleukin-2 T cell function in vivo, we used the adoptive
To assess whether H3K9Ac correlated with (IL-2) (fig. S5, G and H). These changes were co- transfer of pmel T cells after activation in ↑[K+]e
chromatin accessibility, we compared our incident with reduced acquisition of effector func- to treat mice bearing established B16 melanoma.
observed differential enrichment against a pre- tion, expression of co-inhibitory surface markers, We found that conditioning with ↑[K+]e improved
viously reported gene list of effector and exhaus- and the acute suppression of T cell target cyto- T cell persistence after adoptive transfer within
tion datasets generated by ATAC-seq analysis in lysis (fig. S5, I to K). ↑[K+]e conditioning also secondary lymphoid organs and the tumor site
T cells after chronic antigen stimulation (37). We delayed the acquisition of effector function even (fig. S8, A to C). Furthermore, antitumor T cells
found that H3K9Ac was negatively enriched at after robust T cell priming (fig. S5L). exposed to ↑[K+]e harbored retained multipotency,
loci regulating the transcription of these genes To understand the epigenetic basis underlying demonstrating a comparatively less differentiated
state (CXCR5+Tim3–) within the tumor and spleen

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after ↑[K+]e (Fig. 3I and fig. S5, B and C). Col- the sustained expression of stemness genes, we
lectively, these data suggest that ↑[K+]e leads to further examined chromatin structure by mea- after adoptive transfer (Fig. 4H and fig. S8, D and
depletion of nucleocytosolic AcCoA, triggering suring trimethylation of Lys27 on histone H3 E) and maintaining higher autophagic flux (fig. S8,
reduced histone acetylation and limiting the (H3K27me3), which recruits the polycomb rep- F and G). Both tumor regression and survival were
epigenetic imprints of effector and exhaustion ressive complex with subsequent compaction significantly improved in mice receiving ↑[K+]e-
programs. of chromatin and suppression of transcription. conditioned CD8+ T cells (Fig. 4I). Similar re-
Metabolite analysis after ↑[K+]e revealed deple- sults were also observed in ↑[K+]e-conditioned
Elevated [K+]e maintains CD8+ tion of methionine and members of the methio- antitumor CD4+ T cells (fig. S9F).
T cell stemness nine cycle, including S-adenosylmethionine (SAM) To further assess the capacity of ↑[K+]e-
T cell stemness has generally been defined as the and S-adenosylhomocysteine (SAH) (Fig. 1C and conditioned T cells to retain the epigenetic im-
capacity for engraftment, persistence, clonal re- fig. S6A), without significant effect on the total print of stemness in vivo, we re-isolated CD90.1+
population, self-renewal, and multipotency, in- abundance of H3K27me3 (fig. S6B). To evaluate TILs from established tumors and retransferred
cluding the capacity for their progeny to acquire the impact on histone methylation and cellular them to separately congenically distinguishable
typical effector functions such as cytotoxicity and function, we assessed H3K27me3 deposition by CD90.2+ hosts with concurrent antigen-specific
the production of high levels of interferon-g ChIP-seq. Comparative analysis of whole-genome vaccination. We found that ↑[K+]e-conditioned
(IFN-g) (2, 8, 23). These characteristics exist as H3K27me3 enrichment revealed a lack of these T cells harbored greater capacity for recall re-
a spectrum and coordinately change as T cells repressive marks at the regulatory loci of stemness- sponse to antigen (Fig. 4J), were maintained at
mature from naïve to exhausted states (11, 38). defining transcription factors, including Bach2, a comparatively less differentiated state, and dis-
Multiple independent preclinical and clinical Tcf7, Klf2, and Id3 (fig. S6C). Consistent with ↑[K+]e- played greater persistence and self-renewal after
datasets have identified these “stem-like” fea- mediated disruption of methionine abundance antigen rechallenge (fig. S9, A to E). We surmise
tures to be highly favorable for cancer immuno- driving epigenetic preservation of T cell stemness, that ↑[K+]e mediates a preservation of cellular
therapy in the settings of immune checkpoint we found an inverse enrichment for H3K27me3 function and phenotype characteristic of T cell
blockade and ACT (11, 13, 14). We have previously repression within naïve associated genes (fig. S6, stemness despite exposure to the dysregulatory
identified stem cell–like T cells as a subpopula- D and E). These findings indicate that ↑[K+]e de- environment of the TME.
tion that has not yet acquired effector program- pletes metabolites required for suppression of
ing but expresses the transcription factor Tcf7, stemness-associated loci during T cell differentia- Increased [K+]e preserves stemness
the lymphoid homing marker CD62L (Sell, tion (39). We found that ionic control of meta- by mediating AcCoA abundance
L-selectin), and the costimulatory molecule bolic and epigenetic programs was specific to and autophagy
CD27 (8, 9, 23). changes in extracellular potassium, because T cells conditioned by ↑[K+]e experienced a star-
We found that ↑[K+]e conditioning triggered changes to sodium concentration, tonicity, and vation response that resulted in the lack of
functional caloric restriction in T cells, driving inert osmolytes did not have a similar effect on acquisition of effector function and the preserva-
a starvation response, thereby altering both cel- T cell phenotype (fig. S6, F and G). The mod- tion of stemness. Although this provides correla-
lular metabolism and the epigenetic landscape of ulatory effects of ↑[K+]e were not restricted to tion between these changes and the depression
effector and stem-associated loci. To understand CD8+ T cells, and acquisition of effector programs of nucleocytosolic AcCoA, we sought to determine
the impact of ↑[K+]e on the function and pheno- was similarly delayed in CD4+ T cells, with de- whether these associations were causal. To this
type of antitumor T cells, we performed RNA-seq creased IFN-g and preserved IL-2 production end, we supplemented T cells with exogenous
and found that the mRNAs for key markers of in the setting of ↑[K+]e (fig. S7, A to C). acetate, the immediate precursor of AcCoA, or
T cell memory formation and stemness, includ- Given the impact of conditioning T cells in directly electroporated AcCoA into ↑[K+]e-treated
ing Bach2, Il7r, Bcl6, and Klf2, were compara- ↑[K+]e on chromatin structure and gene expres- cells (40, 41). We confirmed that these interven-
tively enriched after exposure to ↑[K+]e (Fig. 4A). sion, we sought to measure its impact on clinically tions restored nucleocytosolic AcCoA (Fig. 5A)
GSEA revealed that ↑[K+]e conditioning resulted relevant human tumor-specific T cells, especially and subsequently observed a reduction in CD62L
in a transcriptional profile that parallels the sub- those specific for neoantigens. We therefore ob- expression (Fig. 5, B and C) and autophagy (Fig.
set of CD8+ T cells recently identified as retaining tained tumor-infiltrating T cells isolated from 5D) in the setting of ↑[K+]e. Similarly, AcCoA or
stemness and the capacity to respond to PD-1 patient tumors and expanded them in the pres- acetate provision restored T cell IFN-g pro-
therapy in the setting of chronic antigen ex- ence or absence of ↑[K+]e. Tumor samples from duction despite prior conditioning with ↑[K+]e
posure (15) (Fig. 4B). patients with diverse cancers including mela- (Fig. 5, E and F, and fig. S10, A and B). Thus,

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Fig. 4. Exposure to
increased [K+]e limits
terminal effector differ-
entiation in murine and
human T cells, enhancing
persistence and anti-
tumor function. (A) Vol-
cano plot representing
differentially expressed
genes analyzed by RNA-seq of CD8+ T cells cultured in ↑[K+]e versus
control. Genes highlighted on the positive axis show enrichment
of memory gene signature in ↑[K+]e; effector genes with decreased transfer into mice bearing established B16 melanoma tumors. Represen-
expression are shown on the negative axis. Abundance is represented tative FACS plots show percentages and absolute numbers of pmel
as relative change (x axis) versus significance (y axis). (B) GSEA of (CD90.1), CXCR5+, and CXCR5+ Tim3– T cells present in tumors 10 days
↑[K+]e versus control comparing with genes enriched in CXCR5+ after adoptive transfer. Data are means ± SEM and are representative
versus Tim3+high dataset. (C) Quantification (left) of Tcf7 mRNA of two independent experiments with at least five mice per group.
transcripts relative to actin as determined by reverse transcription PCR. (I) Tumor growth curves (left) and survival rates (right) of mice bearing B16
(D) Quantification of CD62L+. (E) CD27+ cells in pmel CD8+ T cells tumors treated with pmel T cells cultured in control or ↑[K+]e. In vivo data
cultured in the indicated conditions. Data are means ± SEM and are shown are representative of three independent experiments with n = 5 to
representative of two (C) or three [(D) and (E)] independent 10 mice per [Link] measurements were plotted as mean ± SEM for each
experiments. (F) Representative FACS plots showing the starting data point; tumor treatment curves were compared by Wilcoxon rank sum
population phenotype of CD8+ TILs obtained from a fresh tumor digest test; animal survival was assessed by log-rank test. (J) Schematic of adoptively
of a melanoma patient, with schematic of in vitro culture conditions. transferred CD90.1 TIL isolation from B16 tumors and secondary transfer to
(G) Quantifications of relative CD8+ CD62L+ yields of neoantigen-specific CD90.2 mice. Recall response of transferred TILs in CD90.2 host was assessed
and CD8+ TILs from various tumor types by culturing in ↑[K+]e conditions. by challenging with recombinant vaccinia virus expressing gp100. Absolute
Data are means of culture replicates per patient sample. (H) Schematic numbers are from day 5 after viral challenge. Data are means ± SEM with n ≥
of control or ↑[K+]e T cell culture conditions and adoptive T cell 5 mice per group. *P < 0.05, ***P < 0.001, ****P < 0.0001 (two-tailed t tests).

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supplementation of AcCoA functionally reversed setting of 2-HC treatment again restored IFN-g ity for metabolic reprogramming is a prerequisite
T cell autophagy and stemness programs, enabling production and ablated markers of autophagy for CD8+ T cell response to ↑[K+]e.
the acquisition of effector programs that are other- (Fig. 5L and fig. S12, A to D), indicating that
wise suppressed by conditioning T cells in ↑[K+]e. modulation of AcCoA abundance that does not Increased [K+]e promotes selective
To examine whether these changes had an involve ↑[K+]e can act primarily to determine induction of mitochondrial AcCoA
epigenetic basis, we provided acetate to T cells T cell fate and function. synthetase 1 (Acss1)
expanded under ↑[K+]e conditions and measured The impact of 2-HC on T cell persistence in vivo Enzymes responsible for AcCoA metabolism in
H3K9Ac at the Ifng promoter and Pdcd1 enhancer was assessed by adoptively transferring con- the mitochondria and cytoplasm include acyl-
loci (Fig. 5G). We found that provision of acetate genically marked pmel TCR transgenic CD8+ CoA synthetase short-chain family members Acss1
enabled acetylation of these hallmark chromatin T cells treated with 2-HC and challenging the and Acss2 (Fig. 6A). Prior investigations have
sites associated with effector and exhaustion pro- mice with vaccinia virus expressing hgp100. An found these enzymes to be induced under nu-
grams. We also clearly visualized that CD62L, a increased persistence of 2-HC–treated cells was trient restriction as a starvation response, allow-
phenotypic marker of stemness, was lost upon found 7 days after ACT, in comparison to con- ing for enhanced conversion of acetate to AcCoA
electroporation of AcCoA. These findings reflect trol conditions (Fig. 5M and fig. S12E). After ACT to support cell survival (41, 43). Given our prior
the capacity for AcCoA replenishment to restore for the treatment of both large established sub- findings, we sought to delineate changes in these
effector programs across multiple time points cutaneous tumors and pulmonary metastases, enzymes after exposure to ↑[K+]e. Although we
collected throughout T cell activation (fig. S10, C 2-HC treatment of pmel T cells resulted in im- found that transcripts for both enzymes were
to H). Thus, this metabolic intervention enabled proved tumor treatment and host survival (Fig. 5, higher under ↑[K+]e conditioning, protein levels
the liberation of an effector phenotype in T cells N and O, and fig. S12F). These data demonstrate were elevated only for Acss1 (Fig. 6, B and C).
grown under the influence of ↑[K+]e conditioning, that multiple independent routes for attenuating We investigated whether expression of Acss1 or
regardless of prior priming. Furthermore, these AcCoA abundance have common epigenetic and Acss2 could mediate metabolic reprogramming,

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results indicated that restoration of AcCoA was metabolic mechanisms that can determine T cell and found that enforced expression of Acss1, but
sufficient to normalize histone acetylation of function. not Acss2, enhanced CD8+ T cell basal and maximal
effector programs even in conditions of ↑[K+]e. These findings led us to further examine the oxygen consumption (Fig. 6D), consistent with the
To assess the causal relationship between role of autophagy across T cell differentiation view that ↑[K+]e induces mitochondrially oriented
T cell differentiation and AcCoA abundance, states. We observed that after ↑[K+]e, those cells metabolism. On the basis of these observations,
we used alternate means to manipulate AcCoA that harbored ongoing autophagy were more we explored whether Acss1 could metabolically
levels. Nucleocytosolic AcCoA can be generated likely to express CD62L (Fig. 5P). To directly reprogram T cells, including those used in thera-
by the conversion of citrate to AcCoA and oxalo- address whether intact autophagy was a require- peutic settings. We retrovirally transduced tumor-
acetate by the enzyme ATP citrate lyase (ACLY) ment for T cell stemness after ↑[K+]e condition- reactive CD8+ T cells to enable their constitutive
(Fig. 5H). The compound 2-hydroxycitrate (2-HC) ing of T cells, we employed CRISPR/Cas9-mediated expression of Acss1, and confirmed its localiza-
inhibits ACLY, resulting in reduced nucleocyto- disruption of the Atg7 locus, a gene obligate for tion to mitochondria (Fig. 6E). Metabolite anal-
solic AcCoA (42). We found that T cell exposure intact autophagy (29). We generated multiple ysis of Acss1-overexpressing cells revealed an
to 5 mM 2-HC induced accumulation of cyto- single guide RNAs for this purpose and validated increase in mitochondrial AcCoA (as measured
plasmic citrate and depleted nucleocytosolic their disruption of Atg7 at the protein level (fig. by total cellular AcCoA) without any differences in
AcCoA (Fig. 5, H and I). Similar to ↑[K+]e con- S13, A and B). We found that after Atg7 knockout nucleocytosolic AcCoA or cytoplasmic citrate levels
ditioning, 2-HC reduced CD8+ T cell H3K9Ac (KO), ↑[K+]e failed to enrich CD62L surface ex- (fig. S13, D and E). We observed increased autoph-
enrichment at the Ifng promoter (Fig. 5, J and pression, indicating that autophagy was indeed agic flux upon enforced expression of Acss1 (Fig.
K), increased CD62L surface expression, en- required for retained stemness in this setting 6F) as well as reduced p4E-BP1 and pS6 in cells
hanced autophagic flux, and limited apoptosis (Fig. 5Q and fig. S13C). Taken together, these overexpressing Acss1 (Fig. 6, G and H). Our findings
and the acquisition of effector programs with- data support a mechanistic relationship among au- are consistent with Acss1 induction of autophagic
out compromise to cellular expansion (fig. S11, tophagy, AcCoA metabolism, and ↑[K+]e-mediated flux and mitochondrially oriented metabolism,
A to G). Provision of exogenous acetate in the retention of stemness and suggest that the capac- the metabolic features of memory T cells.

Fig. 5. Increased [K+]e-mediated metabolic reprogramming is culture replicates per experiment. (K) Representative FACS plots of autophagy
mediated by AcCoA subcellular abundance and autophagy. flux in the indicated conditions by measuring the loss of GFP in mCherry+
(A) Rescue of ↑[K+]e effects on cytoplasmic AcCoA by external acetate populations. (L) Representative FACS plots showing effect of external acetate
(5 mM) supplementation. (B and C) Representative FACS plot supplementation on CD62L expression in 2-HC–treated cells. Data are
quantifications of effects of external acetate (B) and electroporated means ± SEM and are representative of two independent experiments
AcCoA(50 mM) (C) on CD62L cell surface expression in CD8+ T cells in with n = 3 culture replicates per experiment. (M) Representative flow
the indicated conditions. Data are means ± SEM and are representative of cytometry analysis (left) and absolute number quantification (right) of
two (A) or three [(B) and (C)] independent experiments. (D) Immunoblot transferred Ly5.1+ CD8+ T cells in the spleen of tumor-bearing mice on
showing reduction of autophagy in ↑[K+]e by provision of external day 7 in the indicated conditions. Data are representative of two independent
acetate. (E and F) Representative FACS plots and quantification showing experiments with at least n = 5 mice per group. (N and O) Antitumor efficacy
augmentation of IFN-g cytokine expression by electroporation of external (left) and survival rates in mice (right) bearing B16 tumors treated with
AcCoA (50 mM). Data are means ± SEM and are representative of two pmel T cells cultured in control (n = 10) or 2-HC (n = 10). Tumor
independent experiments. (G) Quantifications of H3K9Ac enrichment measurements were plotted as mean ± SEM for each data point; tumor
by ChIP-PCR at the Ifng and Pdcd1 enhancer locus in the indicated conditions. treatment curves were compared by Wilcoxon rank sum test; animal
Data are means ± SEM from at least three culture replicates. (H) Schematic survival was assessed by log-rank test. Data are representative of two
of the molecular pathways, substrates, and associated enzymes involved in independent experiments. (P) Representative flow cytometry analysis and
the generation of cytoplasmic AcCoA pools. CTP, citrate transporter; 2-HC, quantifications showing reduced autophagic flux in CD62L– populations.
ACLY inhibitor (5 mM hydroxycitrate). (I) Quantification of cytoplasmic Data are means ± SEM and are representative of two independent
citrate and cytoplasmic AcCoA from the cells treated in the indicated experiments with n = 3 culture replicates per experiment. (Q) Representative
conditions. (J) ChIP-PCR quantification showing reduced H3K9Ac deposition flow cytometry quantification showing abrogation of increased CD62L
at the Ifng locus in 2-HC–treated cells. Data are means ± SEM and are expression driven by ↑[K+]e upon genetic perturbation of Atg7 (highlighted
representative of three (I) or two (J) independent experiments with n = 3 in gray). *P < 0.05, **P < 0.01, ****P < 0.0001 (two-tailed t tests).

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Because enforced expression of Acss1 resembled important, we found that pmel CD8+ T cells ↑[K + ] e and that enforced expression of Acss1
↑[K+]e-conditioned T cells in many respects, engineered to express Acss1 mediated improved can metabolically reprogram antitumor T cells
we sought to probe their function in vivo. We tumor treatment and survival of mice bearing by driving enhanced oxygen utilization and au-
found that CD8+ T cells engineered to express established B16 tumors (Fig. 6I). Taken together, tophagy (Fig. 6J), improving T cell persistence
Acss1 demonstrated markedly improved per- these data show that the Acss1 protein is en- as well as tumor clearance and survival in tumor-
sistence after viral infection (fig. S13F). Most riched upon nutrient restriction induced by bearing mice.

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Fig. 6. Acss1 promotes mitochondrial metabolism to augment T cell overexpression conditions. Data are means ± SEM of three independent
persistence and antitumor activity. (A) Schematic of the molecular immunoblots. (G and H) Immunoblot showing reduced phosphorylation of
pathways, substrates, and associated enzymes involved in the generation the nutrient-sensing kinase mTOR substrates p-4EBP and phosphor-S6
of mitochondrial and cytoplasmic AcCoA pools. (B) Quantifications (S235/236) in Acss1-overexpressing CD8+ T cells. (I) Tumor growth (left) and
showing the normalized mRNA transcripts of AcCoA synthetase (Acss1 animal survival curves (right) of sublethally irradiated mice bearing B16
and Acss2). RPKM, reads per kilobase per million mapped reads. tumors treated with T cells transduced with retrovirus control (n = 10) or
(C) Immunoblot showing protein expression of Acss1 and Acss2 in control Acss1-transduced T cells (n = 10). Tumor measurements were plotted
or ↑[K+]e T cells. (D) OCRs of control or Acss1- or Acss2-transduced as mean ± SEM for each data point; tumor treatment curves were
CD8+ pmel T cells were measured in real time under basal conditions in compared by Wilcoxon rank sum test; animal survival was assessed by
response to the indicated mitochondrial inhibitors. Data are means ± SEM log-rank test. In vivo data shown are representative of two independent
and are representative of two independent experiments. (E) Representa- experiments. (J) Illustration showing dual role of potassium in limiting the
tive confocal images defining the localization of Acss1 in mitochondria. acquisition of effector functions and preserving the stemness of T cells.
(F) Immunoblot densitometry of LC3b-I and LC3b-II in RV-control or Acss1 *P < 0.05, ***P < 0.001, ****P < 0.0001 (two-tailed t tests).

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Vodnala et al., Science 363, eaau0135 (2019) 29 March 2019 11 of 12

R ES E A RC H | R E S EA R C H A R T I C LE

help related to AcCoA measurements, and J. Shetty and B. Tran C.A.K. provided intellectual but not experimental input in SUPPLEMENTARY MATERIALS
of the NCI Sequencing Facility. Funding: Supported by the reporting the data; S.K.V., R.E., C.A.K., and N.P.R. wrote the [Link]/content/363/6434/eaau0135/suppl/DC1
Intramural Research Program of the NCI, the Cancer Moonshot manuscript; and N.P.R. supervised the project. Competing Materials and Methods
Program for the Center for Cell-Based Therapy at the NCI, interests: Authors declare no competing interests. Data and Figs. S1 to S14
NIH, and the Milstein Family Foundation. Author contributions: materials availability: Datasets described in the paper Tables S1 to S5
S.K.V., R.E., and N.P.R. conceived the project and designed are deposited in Gene Expression Omnibus (accession number References (45–48)
the experiments; S.K.V., R.E., and R.J.K. performed experiments GSE122156). S.K.V., R.E., R.J.K., and N.P.R. have submitted
and interpreted data; M.S., T.N.Y., N.-H.H., P.-H.L., M.H.S., U.S. Provisional Patent Application 62/661,941, filed 24 April 2018, 27 April 2018; resubmitted 6 November 2018
S.J.P., Z.Y., D.C.P., M.J.K., J.H., X.L., J.W.L., and T.F. contributed for the use of hydroxycitrate on T cells to improve efficacy of Accepted 15 February 2019
in performing experiments and edited the manuscript; T.F. and adoptive cell therapy. 10.1126/science.aau0135

Downloaded from [Link] on March 30, 2019

Vodnala et al., Science 363, eaau0135 (2019) 29 March 2019 12 of 12

T cell stemness and dysfunction in tumors are triggered by a common mechanism
Suman Kumar Vodnala, Robert Eil, Rigel J. Kishton, Madhusudhanan Sukumar, Tori N. Yamamoto, Ngoc-Han Ha, Ping-Hsien
Lee, MinHwa Shin, Shashank J. Patel, Zhiya Yu, Douglas C. Palmer, Michael J. Kruhlak, Xiaojing Liu, Jason W. Locasale, Jing
Huang, Rahul Roychoudhuri, Toren Finkel, Christopher A. Klebanoff and Nicholas P. Restifo

Science 363 (6434), eaau0135.

DOI: 10.1126/science.aau0135

Stemness against adversity

T lymphocytes are powerful immune cells that can destroy tumors, but cancers have developed tricks to evade
killing. Vodnala et al. found that potassium ions in the tumor microenvironment serve a dual role of influencing T cell

Downloaded from [Link] on March 30, 2019

effector function and stemness (see the Perspective by Baixauli Celda et al.). Increased potassium impairs T cell
metabolism and nutrient uptake, resulting in a starvation state known as autophagy. The increased potassium can also
preserve T cells in a stem-like state where they retain the capacity to divide. These seemingly divergent processes are
linked to the cellular distribution of acetyl−coenzyme A, which, when manipulated, can restore the ability of human T cells
to eliminate tumors in mice.
Science, this issue p. eaau0135; see also p. 1395

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