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Supplemental Figure legends (revision-2) · PDF file Supplemental Figure legends Supplemental Fig. 1 Gating strategy for isolation of regulatory T cells. (A) A gate surrounding CD3-positive

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  • 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

    SS C

    FSC-A

    FS C

    -H

    FSC

    SS C

    CD4

    SS C

    CD3

    SS C

    FSC-A

    FS C

    -H

    FSC

    SS C

    CD4

    SS C

    A gating strategy

    Tumor

    Sp

    MethA

    CD4

    C D

    25

    C D

    25

    Sp

    CD4

    C D

    25

    C D

    25

    Tumor B

    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

    4.20 day 13

    day 10

    95.2

    4.70

    95.3

    4.67

    54.6

    45.4

    59.5

    39.5

    CD4

    C D

    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

    S S

    C

  • Supplemental Fig. 3

    CD3

    SS C

    -A

    CD4

    SS C

    -A

    FSC-A

    FS C

    -H

    SS C

    -A

    FSC-A CD4

    Fo xp

    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

    Fo xp

    3

    cleaved-caspase-3

    % cl

    ea ve

    d ca

    sp as

    e- 3+

    in

    Fo xp

    3+ C

    D 4+

    ce lls

    Met (−) Met (+)5.81

    16.7

    4.35

    19.5

    0.93

    21.5

    26.3 56.8

    CD4

    Fo xp

    3

    cleaved caspase-3

    12.3 75.0

    22.1

    8.12

    26.7 22.2

    6.9514.8CD4 Fo

    xp 3

    cleaved caspase-3

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

    A

    B

    50

    25

    0 Sp Tumor

    * Met (−) Met (+)

  • B

    0

    20

    40

    60

    80

    100 *

    *

    (%)

    CD4+CD25+Annexin V+ /CD4+ CD25+ T cells

    0

    10

    20

    30 ** ** (%)

    C CD4+CD25+Annexin V-

    /CD4+ T cells

    D (x103)

    0

    5

    10

    15

    20

    25

    CD4+CD25+Annexin V-

    F

    0

    5

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