Nutrient condition in the microenvironment determines essential
metabolisms of CD8+ T cells for enhanced IFNJJ production by
metformin
12Chao Ruoyu, Mikako Nishida, Nahoko Yamashita, Miho Tokumasu, Zhao Weiyang, Ikuru Kudo,
and Heiichiro Udono

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Department of Immunology, Okayama University Graduate School of Medicine, Dentistry, andPharmaceutical Sciences, Okayama, 700, Japan.

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6Running title; Metformin’s direct effect on CD8+ T cells

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Correspondence:

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Heiichiro Udono MD. Ph.D.

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udono@cc.okayama-u.ac.jp

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Keywords: CD8T cells, IFNJ, glycolysis, FAO, autophagy, glutaminolysis,

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Abstract

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Metformin (Met), a first-line drug for type 2 diabetes, lowers blood glucose levels by suppressing
gluconeogenesis in the liver, presumably through the liver kinase B1-dependent activation of AMPactivated protein kinase (AMPK) after inhibiting respiratory chain complex I. Met is also implicated as adrug to be repurposed for cancers; its mechanism is believed identical to that of gluconeogenesis
inhibition. However, AMPK activation requires high Met concentrations at more than 1 mM, which areunachievable in vivo. The immune-mediated antitumor response might be the case in a low dose Met.
Thus, we proposed activating or expanding tumor-infiltrating CD8+ T cells (CD8TILs) in a mouse modelby orally administering Met in free drinking water. Here we showed that Met, at around 10 PM and aphysiologically relevant concentration, enhanced production of IFNJ,TNFD and expression of CD25 of
CD8+ T cells upon TCR stimulation. Under a glucose-rich condition, glycolysis was exclusively involvedin enhancing IFNJ production. Under a low-glucose condition, fatty acid oxidation or autophagydependent glutaminolysis, or both, was also involved. Moreover, phosphoenolpyruvate carboxykinase 1(PCK1), converting oxaloacetate to phosphoenolpyruvate, became essential. Importantly, the enhanced
IFNJ production was blocked by a mitochondrial ROS scavenger and not by an inhibitor of AMPK. In
addition, IFNJ production by CD8TILs relied on pyruvate translocation to the mitochondria and PCK1.
Our results revealed a direct effect of Met on IFNJ production of CD8+ T cells that was dependent on
differential metabolic pathways and determined by nutrient conditions in the microenvironment. (238words).

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Cancer incidence and mortality are significantly improved in patients with diabetes who take metformin(Met) for a long period compared with those taking other anti-diabetes drugs (1, 2, 3). As a result, theantineoplastic effect of Met has received increasing attention. However, the precise mechanism of the Met-

Introduction

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dependent antineoplastic effect is highly controversial, according to current research. As respiratory chain
complex I is a target of Met (4, 5), the inhibition of oxidative phosphorylation (OxPhos) followed by theactivation of liver kinase B1 (LKB1)/ AMP-activated protein kinase (AMPK) axis may downregulate
mTORC1 in tumor cells (6,7,8). However, this strategy requires treatment with Met at the mM range,
which is practically impossible to achieve in the clinical setting. For example, according to a previous
study, Met concentrations in plasma and tumor are both in the range of 3.2 to12.4 PM when the micereceive 1.25 mg/mL Met in free drinking water (9); the dosage of Met is close to that for the patients withdiabetes receiving Met. Usually, these concentrations of Met do not inhibit tumor growth in vitro.

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Previously, we reported that Met administration via free drinking water rendered the mice to reject once
established, highly immunogenic solid RL1 tumor (10). This phenomenon was mediated by CD8 T+ cells
as the injection of an anti-CD8 monoclonal antibody (mAb) abolished Met’s antitumor effect. A similareffect was observed in other tumor models, such as Renca (renal cell carcinoma), and a smaller effect infour other tumor models (10). Moreover, we recently demonstrated that Met-induced mitochondrial ROS(mtROS) stimulated both Glut-1expression on cell surface of tumor-infiltrating CD8+ T cells (CD8TILs) to
produce IFNJ and activation of Nrf2/mTORC1/p62 axis for CD8TILs to proliferate in tumor (11). In
another study, Met was shown to enhance the effect of the cancer immunotherapy of PD-1 blockade in the
MC38 (colon carcinoma) model; the effect was attributed to the improvement in tumor hypoxia following
the OxPhos inhibition in tumor cells by Met (12). However, the authors did not propose a direct effect of
Met on CD8TILs. In addition to the immune-modulatory effect of Met, there is evidence for the nonimmune-mediated antineoplastic effect by biguanides, including phenformin, in immune-deficient mice
(13,14). Indeed, we also observed Met’s antitumor effect against osteosarcoma in SCID mice; however,
the effect was canceled by the injection of anti-CD11b mAb (15), suggesting the involvement of M1-likemacrophages or NK cells but not T lymphocytes in certain tumor cells. Meanwhile, other studies suggestthat Met’s antineoplastic effect is attributed to the downmodulation of PD-L1 on tumor cells via its
degradation in the endoplasmic reticulum (ER) (16,17,18). Lastly, a recent study suggests that Met
enhances an antitumor vaccine’s effect by reducing the PD-L1 levels on tumor cells (19).

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The synthesis of the transporters for Met, such as organic cation transporter 1(OCT-1), is critical for theeffect of Met. We recently found that protein expressions of OCT-1 and glucose transporter-1 (Glut-1)
were substantially enhanced via the stimulation of TCR using anti-CD3 and anti-CD28 antibodies (Abs),
although these genes were much less expressed in unstimulated splenic CD8+ T cells (11). Therefore, it isnecessary to investigate whether CD8+ T cell functions, such as IFNJ production, are enhanced by Met at alow concentration. The current study revealed the direct effect of Met at 10 PM on IFNJ production by
CD8+ T cells and that the essential metabolism varied under different nutrient conditions in the
microenvironment.

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Results

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2.1

Met at 1 mM or higher downregulates oxygen consumption rate (OCR) while
upregulates ECAR in tumor cells

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First, we explored the dose-response relationship between Met and the metabolism of tumor cells. Murine
MO5 (B16 melanoma expressing ovalbumin) and 3LL (lung adenocarcinoma) cells were cultured withvarious doses of Met at 0–5 mM for 48 h. Then, OCR and ECAR were simultaneously examined using a
Seahorse flux Analyzer. Consistent with previous studies, OCR was inhibited by Met at 1 mM or higher
in both cell lines (Figure 1A, B, E, and F). Surprisingly, ECAR, indicating glycolysis, was elevated by Met
at 1 mM or higher in a dose-dependent manner (Figure 1C, D, G, and H), suggesting an acceleration of
the Warburg effect in the progression of oxidative stress due to Met at 1 mM or higher. In any case,physiologically relevant concentration of Met below 100 PM did not affect the metabolic profile of tumor
cells.

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Next, we examined whether Met affected the IFNJ production by CD8+ T cells upon TCR stimulation.
CD8+T cells isolated from mice spleen cells using magnet beads were stimulated with anti-CD3 Ab andLess than 100 PM Met enhances IFNJ production by CD8+ T cells upon TCR
stimulation

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anti-CD28 Ab for 72 to 120 h in the presence of varying doses of Met at 0–5 mM to examine the IFNJ
production. At 10 PM, Met markedly enhanced IFNJ production during the 72-, 96-, and 120-h
incubations (Figure 2). Although Met at 100 PM also enhanced IFNJ production during the 72- and 96-h
incubations, the enhancement declined at 120 h (Figure 2). Importantly, Met at greater than 1 mM
completely abolished the IFNJ production by CD8+ T cells such that IFNJ levels were even lower thanthose in the cells without Met (Figure 2). Similar results were observed on the expression of CD25, anactivation marker of T cells, on the cell surface (Figure S1A) and on the production of TNFD (Figure S1B)
of CD8+ T cells. The results indicated that Met at 10 PM, a physiologically relevant concentration, could
enhance the IFNJ production by CD8+ T cells upon TCR stimulation and that Met at concentrations highenough to downregulate OCR of tumor cells, hampered the IFNJ production of CD8+ T cells.

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Recently, we observed that the enhanced IFNJ production by the CD8TILs in mice treated with Met wascompletely negated by the co-administration of MitoTEMPO, a mitochondrial ROS (mtROS) scavenger(11). Therefore, we examined the effect of MitoTEMPO on the in vitro IFNJ production by CD8+ T cells
upon TCR stimulation with 10 PM Met. As expected, the enhanced IFNJ production was reduced by
MitoTEMPO to the level of the CD8+ T cells without Met treatment (Figure 3A; upper panel, Figure 3B,
C). However, compound C, an inhibitor for AMPK, did not suppress the IFNJ production of CD8+ T cells(Figure 3A; lower panel, Figure 3B, C), suggesting that Met stimulated IFNJ production of CD8+ T cells ina mtROS but not AMPK-dependent manner. It is of note that both MitoTEMPO and compound C had no
inhibitory effect on IFNJ production of CD8+ T cells upon TCR stimulation without Met (Figure 3D, E).
Concerning mitochondrial biogenesis, we examined the level of PGC1D in the cells. Met at 10PM elevatedthe PGC1D levels in CD8+ T cells in the 96 but not 72-h incubation (Figure 3F). As we previously foundthat autophagy was involved in the proliferation of CD8TILs in mice treated with Met (11), we checked
the level of LC3B, an autophagy marker, in the CD8+ T cells with or without chloroquine (CQ), a blockerof the degradation of LC3B within autophagosomes. The accumulation of LC3B was evident at 72 h post
culture, and its level was more pronounced in the 96-h incubation in the presence of CQ (Figure 3G).
These findings, together with our previous findings (11), confirmed that Met stimulated the production of
mtROS, IFNJ production, PGC1D expression, and autophagy.

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Next, we searched for the metabolic pathway utilized for the IFNJ production by the CD8+ T cells in vitro
and the CD8TILs ex vivo. The translation of IFNJ in CD8+ T cells depends on the GAPDH in glycolysis(20), while the transcription of the IFNJ gene depends on phosphoenolpyruvate (PEP) (21), a metabolite inglycolysis that is essential in Ca2+ mobilization to promote NFAT translocation into the nucleus before
IFNJ synthesis. The contribution of GAPDH to IFNJ production exclusively depends on glucose.
However, PEP can be generated from either glucose or oxaloacetate in a PCK1-dependent manner (21).
Oxaloacetate, a metabolite in the TCA cycle, may be generated from glucose, fatty acid, andglutamine/glutamate, suggesting that PEP generation, and hence IFNJ production, can depend on
glycolysis, fatty acid oxidation (FAO), and glutaminolysis. These three mechanisms can fuel the TCA
cycle in a process called anaplerosis.

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To identify the metabolic (anaplerotic) pathways involved in IFNJ production, we generated effector CD8+
T cells in vitro upon TCR stimulation using Met. We also generated CD8TILs ex vivo on day 10 from themice treated with Met from day 7 after inoculation with MO5 tumor cells. The in vivo experiment revealed
a significant reduction of MO5 cell growth (Figure 4A). In addition, the metabolic pathways and theinhibition sites likely inhibited by the specific inhibitors were shown (Figure 4B). Thus, we evaluated the
IFNJ production by the CD8+ T cells in vitro and by the CD8TILs ex vivo in the presence of the metabolic
inhibitors.

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We found that only 2DG (a hexokinase inhibitor) blocked the IFNJ production by CD8+ T cells upon TCR
stimulation with Met on day 3 (72 h), and etomoxir (a CPT1 inhibitor blocking FAO) significantly blocked

Enhanced IFNJJ production by Met-treated CD8+ T cells is abolished by amitochondrial ROS scavenger

Differential metabolic pathways required for Met-induced IFNJ production by CD8+ T
cells and CD8TIL

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the IFNJ production by CD8+ T cells on day 4 (96 h) (Figure 4C, D, E), suggesting that on day 3, the CD8+
T cells depended on glycolysis only, while on day 4 they depended on both glycolysis and FAO for IFNJ
production. Furthermore, in the case of the CD8TILs, 2DG and MPC1 (blocking pyruvate entry to
mitochondria), but not etomoxir, significantly blocked IFNJ production, suggesting that pyruvate
generation from glucose and its translocation to the mitochondria are involved in the IFNJ production by
CD8TILs. It is of note that IFNJ production by CD8+ T cells upon TCR stimulation without Met was notsensitive to inhibition of etomoxir (Figure 4G, H), which was in contrast to the results of CD8+ T cells with
Met (Figure 4E).

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We further investigated if autophagy, glutaminolysis, and PCK1-dependent pathways were involved in
IFNJ production. On day 3, we found in CD8+ T cells a weak inhibition by 3MPA (PCK1 inhibitor) and no
inhibition by BPTES (an inhibitor of GLS blocking the glutamine-to-glutamate conversion) and CQ,
suggesting that glutaminolysis and autophagy were not involved in IFNJ production by day 3 in CD8+ T
cells (Figure 5A, B). However, profound inhibition by 3MPA and CQ and a weak but significant inhibition
by BPTES were observed on day 4 in the CD8+ T cells (Figure 5A, C), suggesting that on day 4, CD8+ T
cells depended on the PCK1-dependent PEP production from oxaloacetate and autophagy involving
glutaminolysis for IFNJ production. It is of note that IFNJ production by CD8+ T cells upon TCR
stimulation without Met was not blocked by 3MPA and CQ (Figure 5E, F), in contrast to the results of
CD8+ T cells with Met (Figure 5C). Intriguingly, 3MPA significantly blocked the IFNJ production by
CD8TILs (Figure 5A, D). As CQ and BPTES did not block the IFNJ production by CD8TILs, autophagy
and glutaminolysis were not involved, unlike the results on the CD8+ T cells on day 4. Together with theresults in Figure 4F, CD8TILs appear to depend on both glucose-dependent anaplerosis and PCK1dependent PEP production from oxaloacetate for IFNJ production.

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2.5

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On day 3, the CD8+ T cells exclusively depended on glycolysis for IFNJ production; in contrast, on day 4,
the CD8+ T cells depended on FAO, autophagy, and glutaminolysis in a PCK1-dependent manner. We
wondered the differential metabolic requirement between days 3 and 4 CD8+ T cells was due to thedifference in glucose concentrations in the culture supernatant. Therefore, we monitored the glucose
concentration in the culture supernatant. We detected the glucose concentration at 12 mM on day 0, which
declined to 6 mM on day 3 and 3–4 mM on day 4 (Figure 6A). There was no significant difference in the
glucose concentration in the supernatant of the cells with or without Met (Figure 6A). Therefore, theglucose concentration at 3–4 mM might be a point where the metabolism of CD8+ T cells switched to FAO
for IFNJ production in the presence of Met. If so, glucose supplementation to the culture would keep the
IFNJ production exclusively dependent on glycolysis. Thus, we added glucose back to the culture toincrease the glucose concentration to 12 mM for the final 24 h on day 4, then IFNJ production was
evaluated in the presence of metabolic inhibitors. We found that IFNJ production was decreased by CQ,
etomoxir, BPTES, EGCG (an inhibitor of GDH blocking the glutamate-to-DKG conversion), and PCK1
inhibitor; on the other hand, IFNJ production was completely restored by glucose supplementation (Figure
6B-F). These data suggest that glucose is a critical factor for determining the metabolic pathways required
for IFNJ production by CD8+ T cells in vitro.

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The current study reports for the first time the direct effect of Met on the IFNJ production by CD8+ T cells.
OCT1, a transporter for Met, was shown to be significantly enhanced in CD8+ T cells by TCR stimulationin vitro (11), suggesting that the direct effect of Met was only on activated T cells and not on naive T cells.
This finding is consistent with the in vivo observation that Met administration activates IFNJ production of
CD8TILs but not CD8+T cells in the lymph node and the spleen (11). The suitable concentration of Met
for IFNJ production by CD8+ T cells is less than 100 PM and ideally 10PM, a physiologically relevant
concentration. Our findings may contribute to the understanding of Met’s antineoplastic effect on patientswith diabetes whose plasma concentration of Met is around 10 PM; thus, the effect may be mediated by
the immunosurveillance mechanism. Our findings are consistent with another study, which found that thedirect effect of 10 PM Met on NK cells to produce IFNJ might be involved in immunosurveillance (22).

Glucose prevents the metabolic switch from glycolysis to FAO for IFNJJ production by
CD8+ T cells

Discussion

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Met at 1 mM or higher inhibits oxidative phosphorylation (OxPhos) while stimulating glycolysis oftumor cells. It may imply that Met at 1mM enhances the Warburg effect, which may have a risk to give
preferential effect for the tumor to survive under certain conditions, even if such a high concentration of
Met was possible in vivo.

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T cells almost do not produce IFNJ when the glucose concentration is less than 1 mM due to the GAPDHmediated- inhibition of IFNJ synthesis (20) and the reduced production of PEP, which is essential for thetranscription of the IFNJ gene (21). The glucose concentration in our in vitro experiments was found to be
3–12 mM, more than sufficient for IFNJproduction. However, the metabolic pathways involved are
different for 3mM and 12 mM glucose. Thus, CD8+ T cells shift their dependence on glycolysis to FAO or
autophagy-dependent glutaminolysis or both, followed by PCK1-dependent IFNJ production, as the
glucose concentration declined to 3mM on day 4 from 6mM on day 3. The flexible metabolism of CD8+ T
cells is indeed the case in the presence of Met and is not observed without Met. Met-dependent PGC1D
synthesis and autophagy induction became apparent after 96 h of incubation, coinciding with the metabolic
switch to FAO from glycolysis. Thus, mitochondrial activation by anaplerosis with fatty acids or
glutamine/glutamate or both might be a key step for the metabolic reprogramming in CD8+ T cells.

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Quite surprisingly, the ex vivo IFNJ production by CD8TILs appeared to depend on glycolysis-derived
pyruvate that would be converted to oxaloacetate in the TCA cycle, followed by PCK1-dependent IFNJ
production (Figure 7). This pathway might activate both glycolysis and OxPhos in CD8TILs, enhancing
IFNJ production and causing cell proliferation, respectively. In fact, our previous study identified a novelrole of Met in stimulating the production of mtROS. mtROS stimulates glycolysis to produce IFNJwhile
promoting cell proliferation via the activation of the Nrf2/mTORC1/p62 axis in CD8TILs (11). CD8TILs
metabolism may be unique to Met’s treatment because Met upregulates Glut-1 level on the surface of the
CD8TILs; moreover, it elevates the glucose concentration in a tumor, likely through the downregulation of
the glycolysis of tumor cells in an IFNJ-dependent manner (11). However, CD8TILs metabolism was only
examined in MO5 melanoma cells in this study; it is necessary to investigate this mechanism in othertumor models.

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The conflicting argument that glycolysis and OxPhos are both most important for effector T cells to fightcancer (23-26), may be two sides of the same coin. The effector function, such as IFNJ production of
CD8TILs, is heavily dependent on glycolysis. The analysis of CD8TILs for their IFNJ production revealed
the importance of glycolysis over OxPhos. On the other hand, OxPhos is more important in effector T cells
that will be adoptively transferred to tumor-bearing mice (26, 27) because the transferred T cells must
adapt to a new environment, thus requiring elevated OxPhos in the healthy mitochondria. After ensuring
survival in tumor tissues, the transferred T cells can proliferate while fighting cancer. In this phase,
Oxphos for cell survival and proliferation and glycolysis for T cell function become important.

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Similarly, Met’s effect on effector T cells may have different aspects. We previously observed thattreatment with Met for a short period of approximately 6 h conferred a better migration or expansion
ability, or both, on tumor-specific, naive CD8+ T cells in the tumor after their adoptive transfer to tumorbearing mice, compared with those of the CD8+ T cells without Met treatment. This effect was blocked by
the treatment of T cells with compound C before their adoptive transfer (10); thus, this effect might be
AMPK-dependent. However, the specificity of compound C is broad beyond AMPK. Met appeared toprotect the transferred T cells from apoptosis after their migration into the tumor in this case. The current
study, however, has revealed the importance of glycolysis and pyruvate-dependent anaplerosis for IFNJ
production by CD8TILs ex vivo; the effect is sensitive to MitoTEMPO but not compound C. Concerning
the involvement of AMPK for T cell effector function, a previous report suggest that LKB1-AMPK axis
negatively regulates T cell function (28), which might be consistent with our results in the current study.
Therefore, the AMPK requirements of effector T cells for antitumor response may vary depending on theexperimental conditions.

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4Materials and Methods

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4.1

Metformin, inhibitors

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Metformin hydrochloride (Tokyo Chemical Industry), 2-deoxy-D-glucose (Sigma-Aldrich), Etomoxir(Selleck Chemicals), ethyl sulfide (BPTES, Sigma-Aldrich), Chloroquine hydrochloride (Sigma-Aldrich),
UK5099 (Sigma-Aldrich), epigallocatechin gallate (Tokyo Chemical Industry), 3MPA (Sigma-Aldrich),
MitoTempo (Sigma-Aldrich), Compound C (Sigma-Aldrich) were purchased.

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4.2

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Female C57BL/6 (B6) mice (SLC, Shizuoka, Japan) were used for all experiments. All mice weremaintained under specific pathogen-free conditions in the animal facility of Okayama University. This
study was approved by The Institutional Animal Care and Use Committee of Okayama University
Graduate School of Medicine.

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4.3

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B6 OVA gene-transduced B16 melanoma MO5 cells were used for tumor assays (Eikawa et al., PNAS).
Meanwhile, 3LL cells (kindly provided by Dr. H. Yagita at the Juntendo University of Medicine in Tokyo,
Japan) were cultured in a 96-well plate with 100 μl of RPMI supplemented with 10% fetal calf serum(FCS), L-glutamine, 2-ME, sodium pyruvate, and NEAA, treated with metformin at 0, 1, 10, 100, 1000, or
5000 μM for 48 h, and used for extracellular flux analysis.

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4.4

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The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of MO5 and 3LL cells
were measured in XF media (nonbuffered RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, and
1 mM sodium pyruvate) under basal conditions and in response to mitochondrial inhibitors, 4 μM
oligomycin (Sigma-Aldrich), 8μM FCCP (Sigma-Aldrich), or 100 mM 2DG (Sigma-Aldrich) only or 1
μM rotenone (Sigma-Aldrich) combined with 1μM antimycin A (Sigma-Aldrich) and 100 mM 2DG(Sigma-Aldrich) using an XFe96 Extracellular Flux Analyzer (Agilent Technologies). Cell count evaluatedfor the metabolism were normalized using a Cytation1 Cell Imaging Multi-Mode Reader (BioTek).

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4.5

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CD8+ T cells were purified from the spleen using magnetic separation (Miltenyi Biotec), cultured with
1μg/ml plate-bound anti-CD3 (eBioscience) and 2 μg/ml soluble anti-CD28 (eBioscience), in a 96-wellplate with 200 μl of RPMI supplemented with 10% FCS, 2mM L-glutamine, 5 x 10-5M 2-mercaptoethanol,
1mM sodium pyruvate, and 0.1 mM non-essential amino acids. 5 x 104 CD8+ T cells were cultured in eachwell. Cell blast formation occurred 24 h after cell culture. Cell numbers became 6.5 x 105 after 72-hourculture and dead cells were observed below 10% under microscope by trypsin staining. For the metformintreated group, cells were treated with metformin at 0, 1, 10, 100, 1000, or 5000 μM. Cells were collected at
48, 96,120 h for extracellular flux analysis or flow cytometry.

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4.6

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For melanoma cell engraftment, 2 × 105 MO5 cells were suspended in 200 μl of RPMI and injectedsubcutaneously into the right side of the back of a wild-type C57BL/6 mouse. Then, 7 days after
engraftment, 5mg/mL metformin was administered perorally. Next, 3 days after metformin treatment,
tumors were collected for FACS analysis. For the tumor volume experiment, the metformin treatment
lasted 30 days. The long (a) and short (b) tumor axes were measured using a pair of Vernier calipers to
calculate the mean diameter, whereas tumor volume (V) was calculated as V = ab2/2.

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4.7

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Tumors were dissected from mice and minced in phosphate-buffered saline (PBS) supplemented with 5
mM EDTA and 2% FCS. Cells were harvested from the minced tumor tissues using the BD Medimachine
system. All cells, including TILs and tumor cells, were stained with fluorescently-labeled antibodies (seebelow) and subjected to flow cytometry.

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4.8

Mice

Tumor cell lines, cell culture

Extracellular flux analysis

CD8+ T cell purification and expansion

Tumor Engraftment and in vivo Metformin treatment

Tumor-infiltrating lymphocytes collection

Flow Cytometry and intracellular Cytokine staining
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For intracellular IFNγ or TNFD measurement, cells were incubated with or without metformin and/or an
inhibitor, thus, 2DG (500 mM), Etomoxir (10PM), BPTES (20PM), Chloroquine(50PM), UK5099(10PM),
EGCG(50PM), 3MPA(500PM), MitoTempo (50PM), Compound C(10PM), in the presence of 1.25ng/mL
Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich), 50nM ionomycin (Sigma-Aldrich) and GolgiStop
the Protein Transport Inhibitor (containing Monensin, BD Bioscience) for 6 h (Etomoxir only for last 3 h).
After incubation, cells were collected for surface staining with CD3 (17A2) and CD8 (53-6.7) Abs in PBS
supplemented with 5 mM EDTA and 2% FCS, in the dark for 30min at 4℃, followed by fixation andpermeabilization using Fixation/Permeabilization kit (BD Biosciences) in the dark for 30min at 4℃. Thenintracellular IFNγ (XMG 1.2) or TNFα (MP6-XT22) Ab probed for cells in the dark for 30min at 4℃. For
LC3B measurement, cells were collected for CD8 and CD3 surface staining, LC3B (Novus Biologicals)
antibodies staining was performed using a Fixation/Permeabilization kit (BD Biosciences). For PGC1αmeasurement, cells were collected for CD8 and CD3 surface staining. Staining for transcription factors
was performed with a Transcription Factor Buffer Set (BD Pharmingen™) with PGC1α (Novus
Biologicals) antibody. For CD25 measurement, cells were collected and performed CD8, CD3 and CD25
(PC61) surface staining in the dark for 30min at 4℃.

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4.9

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Cell culture Supernatants were collected 72, 96, and 120h after culturing CD8+ T cells and diluted
1:20 for analysis. A Glucose Assay Kit-WST (DojinDo Laboratories) was used to measure the glucose
concentrations.

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4.10 Glucose supplement for T cells

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After 72 h CD8+ T cells culture, D-(+)-glucose (Fujifilm Wako Pure Chemical Corporation) in 10μl of
PBS was added to medium to achieve 12mM glucose concentration in culture medium, consisting with theconcentration at 0 h. Meanwhile, 10 μl of PBS was added to the negative control group. Flow Cytometry
and intracellular cytokine staining were performed 24 h after the glucose supplement.

Measurement of Glucose in CD8+ T cells supernatants

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Conflict of Interest

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Author Contribution

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Funding

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Acknowledgements

Authors declare no competing interests.
C.R. performed the experiments and wrote some parts of paper. M.N. designed the all experiments
and supervised the experimental procedures. N.Y. maintained the mice used in the experiments.
M.N., M.T., Z.W., and I.K., contributed to the extensive discussion throughout the entire
experiments and proof reading of the manuscript. H.U. supervised the project, designed theexperiments, and wrote the paper.
This study was supported by grants to H.Udono from JSPS KAKENHI Grant number 18H04033
and 17K19598.
We thank Ms. Tachibana in the animal facility of Okayama University for their kind help tomaintain and expanding the mice colony to support this study.

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References

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28. MacIver NJ, Blagih J, Saucillo DC, Tonelli L, Griss T, Rathmell JC, et al. The liver kinase B1 is acentral regulator of T cell development, activation, and metabolism. J Immunol (2011)187: 4187-4198.
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Figure Legends

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Figure 1. Met at 1 mM or higher downregulates oxygen consumption rate (OCR) while
upregulates ECAR in tumor cells. MO5 and 3LL cells were cultured in vitro with the indicatedconcentrations of Met for 48 h before the extracellular flux analysis. (A–D) The metabolic characteristicsof the MO5 cells showing (A) OCR, (B) basal OCR, (C) ECAR and (D) basal ECAR levels. (E–H) The
metabolic characteristics of the 3LL cells showing (E) OCR, (F) basal OCR, (G) ECAR and (H) basal
ECAR levels. The graphs represented Mean ± SEM of the results of three independent experiments.
Statistical analysis was performed by unpaired, two-tailed Student’s t-test (*P ≤ 0.05; **P ≤ 0.01; ***P
≤ 0.001; ****P ≤ 0.0001; *****P ≤ 0.00001).

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Figure 2. Met below 100 PM enhances IFNγ production by CD8+ T cells upon TCR stimulation.
Splenic CD8+ T cells were cultured with the indicated concentrations of Met for 72, 96, and 120 h. The
resulting cells were stimulated with PMA and ionomycin for 6 h, followed by the staining of intracellular
IFNγ. (A) A representative flow cytometry plot showing the IFNγ levels in the CD8+ T cells. (B–D) The
graph represents the percentage of the IFNγ+ CD8+ T cells at (B) 72 h, (C) 96 h, and (D) 120 h. (E-G) The
graph represents the MFI of the IFNγ+ CD8+ T cells at (E) 72 h, (F) 96 h, and (G) 120 h. The graphs
represent Mean ± SEM of the results of three independent experiments. Statistical analysis was performedby unpaired, two-tailed Student’s t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; *****P≤ 0.00001).

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Figure 3. Met-induced enhancement of IFNJ, PGC1D, and LC3B of CD8+ T cells and the effects ofinhibitors. Splenic CD8+ T cells were cultured with anti-CD3 and anti-CD28 Abs in the presence orabsence of 10 μM Met for 48, 72 or 96 h. (A-E) The resulting cells were stimulated with PMA andionomycin in the presence or absence of Met and inhibitors as indicated for 6h. (A) A representative flow
cytometry plot showing IFNγ levels in the CD8+ T cells treated with or without MitoTempo (upper
panels), and Compound C (lower panels). (B-E) The graph represents the MFI of IFNγ levels at (B, D) 72
h and (C, E) 96 h. (F) A representative flow cytometry plot showing PGC1α levels in the CD8+ T cells thatwere cultured for 72 or 96 h with or without Met. (G) A representative flow cytometry plot showing LC3B
levels in the CD8+ T cells that were cultured for 48,72, and 96h with or without treatment by CQ for last
6h. The graphs represent Mean ± SEM of the results of three independent experiments. Statistical analysis
was performed by unpaired, two-tailed Student’s t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P≤ 0.0001; *****P ≤ 0.00001).

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Figure 4. FAO is essential for IFNJ production by CD8+ T cells cultured for 96 but not 72 hours
For in vitro activated CD8+ T cell preparation, splenic CD8+ T cells were cultured as in Figure 3. For
CD8TILs preparation, mice were treated with Met from day 7 post tumor inoculation, and on day 10,
tumors were collected for flow cytometry analysis. The in vitro activated CD8+ T cells and CD8TIL were
stimulated with PMA and ionomycin in the presence or absence of MPC1i (UK5099, a MPC1inhibitor) for
6h, and Etomoxir (Eto) and 2DG for 3h. (A) MO5 tumor growth curve of mice treated with or without
Met. (B) The inhibition sites by the specific inhibitors. (C) The levels of IFNJ of in vitro activated CD8T
cells (72h, 96h) and CD8TIL treated with or without inhibitors as indicated. (D-H) The graph represents
the MFI of IFNγ levels in vitro activated CD8T at (D, G) 72h, (E, H) 96 h, and CD8TILs (F). The graphs
represent the mean ± SEM of the results of three independent experiments. Statistical analysis was
performed by unpaired, two-tailed Student’s t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001;*****P ≤ 0.00001).

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Figure 5. Differential metabolisms required for IFNJ production by in vitro activated CD8T cells
and CD8TILs
The in vitro activated CD8+ T cells and CD8TILs were prepared as in Figure 4. The cells were stimulatedwith PMA and ionomycin in the presence or absence of 3MPA (PCK1i), chloroquine (CQ), and BPTES
for 6h. (A) The levels of IFNJ of in vitro activated CD8T cells (72h, 96h) and CD8TIL treated with orwithout inhibitors as indicated. (B-F) The graph represents the MFI of IFNγ levels in vitro activated CD8+
T cells at (B, E) 72h, (C, F) 96 h, and CD8TILs (D). The graphs represent the mean ± SEM of the results

10Running Title455
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of three independent experiments. Statistical analysis was performed by unpaired, two-tailed Student’s ttest (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; *****P ≤ 0.00001).

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Figure 6. Glucose concentration determines metabolism required for IFNJJ production by in vitro
activated CD8+ T cells(A) The graphs represent the glucose concentrations in the supernatant of CD8+ T cell cultured with orwithout Met. (B–F) In vitro activated CD8+ T cells at 72h were further incubated with or without
supplementation of glucose for additional 24 hours and the resulting cells were treated with PMA andionomycin in the presence or absence of inhibitors, as indicated in CQ (B), Etomoxir (C), BPTES (D),epigallocatechin gallate (EGCG) (E), and 3MPA(PCL1i) (F). A representative flow cytometry plot
showing the in vitro IFNγ levels in the CD8+ T cells at 96 h. The graphs represent the mean ± SEM.

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Figure 7. Metabolic preference of in vitro activated CD8+ T cells and CD8TILs for Met-induced
enhancement of IFNJ production(A) In vitro activated CD8+ T cells at 72 h exclusively depends on glycolysis.
(B) In vitro activated CD8+ T cells at 96 h depends on FAO, glutaminolysis/autophagy, and PCK1.
(C) CD8TILs depends on anaplerosis from glucose metabolism and PCK1.

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Figure 1.
10μM

100μM

1000μM

B

5000μM

OCR (pmol min-1 103 cells)

12

Oligomycin

FCCP

20

40

R/A

2DG

10
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ECAR (mpH min-1 103 cells)

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8
7
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E

0μM

60

Met
1uM
Time
(min)

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Met 100uM
Met 1mM
Oligomycin
R/A
FCCP

Met 5mM

20

16
OCR (pmol min-1 103 cells)

Control

1μM

Oligomycin

40

100μM

FCCP

R/A

1000μM

2DG

10
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6
4
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0

ECAR (mpH min-1 103 cells)

G
5

Control

60
80
Met
1uM
Time
(min) Met 10uM

Met 100uM

Met 1mM

Oligomycin

40

FCCP

R/A

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Met 5mM

2DG

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Met

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Time (min)

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100

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Basal OCR (pmol min-1 103 cells)

0

C

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1μM

Basal ECAR (mpH min-1 103 cells)

0μM

14

H

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*****

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*****
*****

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*****
*****

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Basal ECAR (mpH min-1 103 cells)

A

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10

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*****
*****

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1

Figure 2.
A

Iso

Met(+)

Met(-)

Met 1μM

Met 10μM

Met 1000μM

Met 5000μM

120h

96h

72h

Met 100μM

IFNγ
72h

B

96h

C

**
**

**

**

*

**

***

***

**

**

120h

D

***

****

***

**
*

*

***

****

72h

E

Met

96h

F

**
*

**

G

**

*

***

0.08

*

μM

120h
**

**
*

Met

μM

Met

*

μM

Met

μM

Figure 3.
A

B

**

Iso
Met(-)
Met(+)
Met+C.C.
Met+M.T.

72h

C

72h
**

96h
**

**

**

96h

E

72h

D

96h

IFNγ

F

G
Iso

Met(-)

Iso

Met(-)

48h

96h

CQ(-)

72h

Met(+)

CQ(+)

PGC1α

LC3B

Met(+)

72h

96h

**

Figure 4.
B

2000

Glucose
HK
G6P

2DG

Fatty
acids

PEP UK5099

1500
1000

VehicleVehicleMetMet500

Pyruvate

PCK1

3MPA

*

Tumor Volume (mm3)

A

MPC1

CPT1

Pyruvate
FAO

Acetyl-CoA
Oxaloacetate

CQ

CitrateAutophagy

TCA cycle

0
1

5

9

13 17 21 26 30
D-KG

Time (day)GDH

Glutamate

72h-CD8+ T

GLS

Glutamine

BPTES

EGCG

C

Eto

96h-CD8+ T

CD8+ TIL

Met+Eto
Met+MPC1i
Met+2DG
Met(+)
Met(-)IFNγ
D

E

72h-CD8+ T**

CD8+ TIL
*

**

*****
***

F

96h-CD8+ T
***

****

****

**

**

**

****

**

0.08

G

72h-CD8+ T

H

96h-CD8+ T

Figure 5.
A

72h-CD8+ T

96h-CD8+ T

CD8+ TIL

Met+PCK1i
Met+CQ
Met+BPTES
Met(+)
Met(-)

IFNγ
72h-CD8+ T

B

**

****

*

*****

96h-CD8+ T

C

*

*****
****
***

**
**

*****
****

***

E

72h-CD8+ T

F

96h-CD8+ T

CD8+ TIL

D***

Figure 6.
A
14

Glucose (mM)

12
10
8

Met(-)

6

Met(+)
Met(-)

4

Met(+)

2
0
0

B

2
3
Time (day)IsoMet(-)
Met(+)
Met+CQ
Met+CQ+Glc

4

C

IFNγ

E

Iso
Met(-)
Met(+)
Met+EGCG
Met+EGCG+Glc

IFNγ

Iso
Met(-)
Met(+)
Met+Eto
Met+Eto+Glc

IFNγ

F

Iso
Met(-)
Met(+)
Met+PCK1i
Met+PCK1i+Glc

IFNγ

D

Iso
Met(-)
Met+BPTESIFNγMet(+)
Met+BPTES+Glc

Figure 7.
B

GlucoseGlucose

G3P

G3P

G3P

1, 3-BPG

1, 3-BPG

1, 3-BPG

2-PG

2-PG

2-PG

PEP

NFAT

IFNγ

PEP

Acetyl-CoA

Acetyl-CoA

FAO

citrateoxaloacetate

NFAT

72h-CD8+ T

Glutamine

IFNγ

Pyruvate

FAO

citrateoxaloacetate

TCA Cycle

Glutamate

NFAT

Acetyl-CoA

FAO

TCA Cycle

Autophagy
α-KG

PEP

citrateoxaloacetate

TCA Cycle

IFNγ

Pyruvate

PCK1

Pyruvate

PCK1

C

GlucosePCK1AAutophagy
α-KG

96h-CD8+ T

Glutamate

Glutamine

Autophagy
α-KG

CD8+ TIL

Glutamate

GlutamineFigure S1.
A

Iso

Met(-)

Met(+)

Met 10μM

Met 100μM

Met 1000μM

Met 5000μM

Met 10μM

Met 100μM

Met 1000μM

Met 5000μM

96h

72h

Met 1μM

CD25

B

Iso

Met(-)96h

72h

Met 1μM

Met(+)TNFα

Figure S1. Less than 100 μM Met enhances CD25 expression and TNFD production by CD8+ T cells upon TCR stimulation.
Splenic CD8+ T cells were cultured with anti-CD3 and anti-CD28 Abs in the presence or absence of indicated concentrations ofMet for 72 or 96 h. (A) A flow cytometry histogram showing CD25 levels on cell surface at 72 h and 96 h. (B) A flow cytometry
histogram showing intracellular TNFD levels at 72 h and 96 h.