Supplemental Figure legends

Supplemental Fig. 1 Gating strategy for isolation of regulatory T cells. (A) A gate

surrounding CD3-positive cells of spleen cells was utilized for the isolation of T cells

from TILs. The isolated populations were separated from doublets by gating for FSC-A

and FSC-H, and further isolated according to FSC and SSC. The populations were

further enriched as CD4+T cells. The spleen and TILs were recovered from MethA-

bearing mice on day 7 after the tumor challenge. (B) CD4+CD25+T cells were identified

from the populations of (A) and further examined for the expression of Foxp3.

Supplemental Fig. 2 Met-induced reduction of CD4+CD25+Treg cells in TILs but not

in the spleen is caused by increased apoptosis induction.��(A) On days 10 and 13 (or

day14), spleen cells and TILs were recovered from tumor (MCA or RLmale1)-bearing

mice, which were receiving (+) or not receiving (−) Met, and the CD4+CD25+Treg

population was examined by FACS analysis. (B) Annexin V binding to the

CD4+CD25+Treg population in (A) was examined. Increased annexin V binding to the

Treg cells was only observed in TILs but not in the spleen of the mice receiving Met.

Pooled cells from each group (N = 5) were used for the FACS analysis. The results are

representative of four independent experiments.

Supplemental Fig. 3 Met-induced increase in cleaved caspase-3 in CD4+Foxp3+Treg

cells in TILs but not in the spleen. (A) On day 13, TILs were recovered from tumor

(MethA)-bearing mice, treated with (+) or without (−) Met, and the CD4+Foxp3+Treg

and the CD4+Foxp3- conventional T cell population was examined by FACS analysis

for the expression of cleaved caspase-3. Increased cleaved caspase-3 was observed in

CD4+Foxp3+Treg population in Met-treated mice (N=3). (B) On day 13, spleen cells

and TILs were recovered from tumor (MethA)-bearing mice, treated with (+) or without

(−) Met, and the CD4+Foxp3+Treg population was examined by FACS analysis for the

expression of cleaved caspase-3. Increased cleaved caspase-3 was observed in TILs but

not spleens of mice receiving Met. Pooled cells from each group (N = 3) were used for

FACS analysis. The individual results are plotted as a bar graph (N = 3). The results are

representative of two independent experiments. *P < 0.05

Supplemental Fig. 4 Viable Treg cells are reduced in TILs of mice receiving Met

compared with those of mice not taking Met.��B6 mice inoculated with MCA tumor

cells received Met dissolved in free drinking water at a concentration of 5 mg/mL from

day 6 after the tumor challenge (+). On days 6, 10, and 13, TILs were recovered from

each mouse and stained with antibodies to CD3, CD4, CD8, and CD25 to identify Treg

cells. Annexin V binding to these Treg populations was evaluated by FACS analysis. N

= 10 for each group. (A, B, and C) The ratios of CD4+CD25+/CD4+ (A), CD4+CD25+

annexin V+/ CD4+CD25+ (B), CD4+CD25+ annexin V-/CD4+ (C) in each group were

plotted as bar graphs. The ratios of CD4+CD25+ annexin V-/CD4+ on days 10 and 13

were dramatically reduced in the mice receiving Met compared with those in the mice

not taking Met. (D, E) Absolute numbers of CD4+CD25+ annexin V- T cells (D) and

CD8+ annexin V- T cells (E) per tumor volume (mm3) are shown.�(F) The ratios of

CD4+CD25+annexin V-/CD8+ annexin V- T cells are shown.�(G) The tumor (MCA)

growth curve was monitored in the mice receiving (+) or not receiving (−) Met. N = 10

for each group. The results are representative of four independent experiments. *P <

0.05, **P < 0.01.

Supplemental Fig. 5 No Foxp3 expression changed in Met-pretreated nTreg cells in

response to TCR stimulation.

nTreg cells were isolated from spleen cells and pretreated with Met (+) or not (-) along

with or without inhibitors, rapamaycin (RA) or compound C (CC), and were stimulated

with anti-CD3 and anti-CD28 mAbs in the presence of TGF!. No effect was observed

in the nTreg cells. The results are representative of three independent experiments.

Supplemental Fig.6 Relationship between CD4+ CD25high Ti-Treg cells and

CD103+KLRG1+Ti-Treg cells. The same gating strategy was used for the isolation of

Ti-Treg cells as in Supplemental Fig. 1A. The isolated populations were further

determined to be CD4+CD25+ cells (24.7%) and CD4+ CD25high cells (3.8%), and

expression of CD103 and KLRG1 in the two distinct Treg populations was shown as

indicated. Note that CD25high population includes a larger CD103+KLRG1+ population

and that phosphorylated mTOR (p-mTOR) levels are significantly higher in the

population. The results are representative of three independent experiments.

Supplemental Fig.7 Only Met-pretreatment of naïve CD4T cells before TCR

stimulation increased glycolysis. (A) Pre-treatment assay: CD4+CD25− T cells were

treated with Met for 6 hours and washed before TCR stimulation. Post-treatment assay:

CD4+CD25− T cells were treated with culture medium for 6 hours and then treated with

Met during TCR stimulation. Three days after TCR stimulation, the Foxp3 expressions

were examined. The histogram results are representative of three independent

experiments, and the individual data are plotted as a bar graph.

(B) The cells on day 3 culture in (A) were subjected to metabolic analysis. The OCR

and ECAR were assayed using a Seahorse XF96 analyzer by the addition of anti-

mitochondrial reagents, oligomycin, FCCP, rotenone (R), antimycin A, and 2-

deoxyglucose (2-DG). Data in the upper two panels are representative of three

independent experiments. The lower panels show the basal levels of OCR and ECAR a

under each cell culture condition. The ECAR was assayed in Dulbecco's modified

Eagle’s medium without glutamate. The ratio of OCR/ECAR is plotted as a bar graph.

Notably, only Met pretreatment decreased OCR/ECAR in iTregs. The results are

representative of three independent experiments. *P < 0.05; **P < 0.01

Supplemental Fig.8 16S rRNA sequence of the fecal microbiota of tumor-bearing mice

treated or not treated with Met.��Feces were collected from MO5-bearing mice 1 week

after the Met administration. 16S rRNA was PCR-amplified and sequenced by MiSeq.

Phylum (A), class (B), order (C), family (D), and genus (E) levels are shown.

Supplemental Fig. 1

CD3

SSC

FSC-A

FSC

-H

FSC

SSC

CD4

SSC

CD3

SSC

FSC-A

FSC

-H

FSC

SSC

CD4

SSC

A gating strategy

Tumor

Sp

MethA

CD4

CD

25

CD

25

Sp

CD4

CD

25

CD

25

TumorB

Foxp3 Foxp3

14.9 20.6

13.7 86.3

00

9.95 90.0

00

64.1

35.7

75.4

23.7

95.9

4.05

95.7

4.20day 13

day 10

95.2

4.70

95.3

4.67

54.6

45.4

59.5

39.5

CD4

CD

25

10.3

88.9

11.5

87.9

20.0

78.4

11.5

87.5

10.7

88.5

11.9

87.4

66.9

27.9

28.2

69.3

17.8 17.4

15.1 20.2

32.0

1.98

10.1 7.14 16.0

3.75 3.41 13.0

85.5

51.9

41.7

28.3

A

B

Met - +Sp

- +Tumor

day 10

day 14

MCA

RLmale1

Met - + - +Sp Tumor

MCA

day 13

day 10

day 10

day 14

RLmale1

Annexin V

Supplemental Fig. 2

SS

C

Supplemental Fig. 3

CD3

SSC

-A

CD4

SSC

-A

FSC-A

FSC

-H

SSC

-A

FSC-A CD4

Foxp

3

0 5.2 7.9

24.3 36.1 28.7

3.1 00

1.6 6.1 3.6

Sp

Tumor

Met (−)

Met (+)

Met (−)

Met (+)

sample 1 sample 2 sample 3

Foxp

3

cleaved-caspase-3

%cl

eave

d ca

spas

e-3+

in

Foxp

3+C

D4+

cells

Met (−) Met (+)5.81

16.7

4.35

19.5

0.93

21.5

26.356.8

CD4

Foxp

3

cleaved caspase-3

12.375.0

22.1

8.12

26.7 22.2

6.9514.8CD4Fo

xp3

cleaved caspase-3

sample 1 sample 2 sample 3 sample 1 sample 2 sample 3

A

B

50

25

0Sp Tumor

*Met (−)Met (+)

B

0

20

40

60

80

100*

*

(%)

CD4+CD25+Annexin V+

/CD4+ CD25+ T cells

0

10

20

30 ** **(%)

CCD4+CD25+Annexin V-

/CD4+ T cells

D(x103)

0

5

10

15

20

25

CD4+CD25+Annexin V-

F

0

5

10

15

20

25***

(%)CD4⁺CD25+Annexin V-

/CD8+ Annexin V-

G(x102)

treatment

0

2

4

6

8

10

12

0 5 10 15 20 25 30

****

**

****

**

****

**

*

day after inoculation

Mea

n tu

mor

vol

ume

(mm

3 )

TIL

popu

latio

n

A

0

10

20

30

40

50*

*

(%)

CD4+CD25+T cells/CD4+ T cells

E

0

2

4

6

8

10

*

(x103)CD8+Annexin V-

Supplemental Fig. 4

cell

num

ber

cell

num

ber

− ＋

d10− ＋

d13−d6

− ＋

d10− ＋

d13−d6

− ＋

d10− ＋

d13−d6

− ＋

d10− ＋

d13−d6

− ＋

d10− ＋

d13−d6

− ＋

d10− ＋

d13−d6

noneMet

Supplemental Fig. 5

no inhibitor Rapamycin Compound C

Foxp3

1 μM5 μM

Met (+)

Met (−)

CD25high 3.8

CD25 24.7

CD3

SSC

CD4SS

C-A

FSC-A

FSC

-H

FSC-A

SSC

-A

CD4

CD

25

CD25 CD25high

KLRG1

CD

103

Supplemental Fig. 6

12.3 39.0CD25high CD103+KLRG1+

pmTOR

Met (−)Met (+)

50

OC

R (p

mol

/min

)

ECAR

(mpH

/min

)

25

0

20

10

0

oligo FCCP R&A oligo 2-DG

9 2718Time (min)

preMetformin

− −+−−+

+ +

post

9 2718

Foxp3

Cel

l cou

nt

OC

R (p

mol

/min

)

pre −− +

−−+ +

+post

40

30

20

10

0

*

ECAR

(mpH

/min

)

pre −− +

−−+ +

+post

20

15

10

5

0

**

OC

R/E

CAR

pre −− +

−−+ +

+post

4

3

2

1

0

**

50.753.117.838.7

80

60

40

20

0pre −

− +−

−+ +

+post

***

Supplemental Fig. 7

A

B

0% 20% 40% 60% 80% 100%

BacteroidetesFirmicutesProteobacteriaActinobacteriaTenericutesDeferribacteresOthersUnclassfied

0% 20% 40% 60% 80% 100%

BacteroidiaClostridiaDeltaproteobacteriaBacilliUnclassified BacteroidetesActinobacteriaMollicutesCoriobacteriaOthersUnclassified

0% 20% 40% 60% 80% 100%

BacteroidalesClostridialesDesulfovibrionalesTuricibacteralesUnclassified BacteroidetesBifidobacterialesLactobacillalesCoriobacterialesDeferribacteralesAnaeroplasmatalesEnterobacterialesRF39ErysipelotrichalesBurkholderialesFlavobacterialesOthersUnclassified

0% 20% 40% 60% 80% 100%

S24-7BacteroidaceaeLachnospiraceaeRuminococcaceaeUnclassified ClostridialesParaprevotellaceaePrevotellaceaeUnclassified ClostridialesDesulfovibrionaceaeRikenellaceaePorphyromonadaceaeRF39Unclassified BacteroidetesBifidobacteriaceaeUnclassified BacteroidaleLactobacillaceaeOthresUnclassified

0% 20% 40% 60% 80% 100%

Unclassified S24-7gBacteroidesUnclassified LachnospiraceaeUnclassified ClostridialesPrevotella (Paraprevotellaceae)Prevotella (Prevotellacea)RuminococcusUnclassified ClostridialesOscillospiraUnclassified LachnospiraceaeUnclassified DesulfovibrionaceaUnclassified RikenellaceaeParabacteroidesTuricibacterCoprococcusOthersUnclassified

A

B

C

D

E

Supplemental Fig. 8

Met (+)

Met (ｰ)

Met (+)

Met (ｰ)

Met (+)

Met (ｰ)

Met (+)

Met (ｰ)

Met (+)

Met (ｰ)