Supplemental Figure legends (revision-2) · Supplemental Figure legends Supplemental Fig. 1 Gating...
Transcript of Supplemental Figure legends (revision-2) · Supplemental Figure legends Supplemental Fig. 1 Gating...
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)