Supplementary Figure 1 - cloudfront.net
the indicated doses of IL-18+IL-12 and p-ERK1/2 was analyzed by flow cytometry. (D) Splenic cells from WT or B2m-/- Ncr1+/gfp mice were stimulated for 30’ with the indicated doses of IL-2 or H9 and p-ERK1/2 was analyzed by flow cytometry. (E-F) 13 days after injection of RMA-S cells, mice were treated or not with 100 ng of IL-12 and IL-18 and the responsiveness of tumor infiltrating NK cells (E) was assessed as described in the legend of Figure 3. Expression of CD25 (F) was determined by flow cytometry. In Panel A and E and F NK cells were gated as indicated in the legend of Figure 3, whereas in Panel B-D NK cells were GFP+. Bars represent means ± s.d. The experiments included at least 4 mice/group and were performed 3 times with similar results. Statistical analyses were performed with unpaired Student’s t-tests.
Supplementary Figure 1: Representative FACS plots for CD11b/CD27 or MHC class I specific inhibitory receptors stainings are shown for NK cells infiltrating RMA and RMA-S tumors.
Supplementary Figure 2: (A-B) Ratios of Tregs or MDSCs to NK cells were calculated in RMA or RMA-S derived tumors. NK cells were gated as viable-CD3CD19-Ter119-NKp46+ cells. Tregs were gated as viable-CD19-Ter119-NKp46CD3+CD4+FoxP3+ cells and MDSCs were gated as viable-CD3-CD19-Ter119NKp46-CD11b+ cells. Several repetitions of each experiment are shown. Statistical analyses were performed with the two-tailed unpaired Student’s t-test.
45
Analysis of pooled results in panel A or in panel B revealed no statistically significant differences.
Supplementary Figure 3: Active TGF-β1 protein was detected by ELISA in the tumor bed (A) or in the serum (B) of RMA or RMA-S tumor bearing mice.
Supplementary Table 1. List of primers used for qPCR analysis
18S
β-actin
GAPDH
IL-10
Forward
Reverse
5’-
5’-
GTCTGTGATGCCCTTAG
AGCTTATGACCCGCACTT
ATG-3’
AC-3’
5’-
5’-
AGAGGGAAATCGTGCGT
CAATAGTGATGACCTGGC
GAC-3’
CGT-3’
5’-
5’-
TGTGTCCGTCGTGGATC
TTGCTGTTGAAGTCGCAG
TGA-3’
GAG-3’
5’-
5’-
46
Supplementary Material
Supplementary Table 1. List of primers used for qPCR analysis Forward
Reverse
5’-
5’-
GTCTGTGATGCCCTTAG
AGCTTATGACCCGCACTT
ATG-3’
AC-3’
5’-
5’-
AGAGGGAAATCGTGCGT
CAATAGTGATGACCTGGC
GAC-3’
CGT-3’
5’-
5’-
TGTGTCCGTCGTGGATC
TTGCTGTTGAAGTCGCAG
TGA-3’
GAG-3’
5’-
5’-
CACTGCCTTGCTCTTATT
CCCTGGGTGAGAAGCTG
TTCACA-3’
AAG-3’
IL-
5’-
5’-
12p40
GGAAGCACGGCAGCAG
AACTTGAGGGAGAAGTA
18S
β-actin
GAPDH
IL-10
IL-18
TGF-β
AATA-3’
GGAATGG-3’
5’-
5’-
CAGGCCTGACATCTTCT
TCTGACATGGCAGCCATT
GCAA-3’
GT-3’
5’-
5’-
CAATTCCTGGCGTTACC
GTATTCCGTCTCCTTGGT
T-3’
T-3’
A NK cells from:
RMA 8.4
10 5
CD27
RMA-S 55.1
10 5
10 4
10 4
10 3
10 3
10 2
10 2
0
0
33.1
3.56 0 10
2
10
3
10
4
10
10.7
49.2
5.16
34.8 0 10 2
5
10 3
10 4
10 5
CD11b
B NK cells from: RMA! RMA-S!
0
102
103
104
Ly49A!
105
0 102
103
104
Ly49C!
105
0
103
104
NKG2A!
105
0
103
104
Ly49G2!
105
0 102
103
104
105
Ly49I!
Supplementary Figure 1
Treg/NK cell ratio
A RMA RMA-S
2.0
p=0.03
1.5 1.0 0.5 0.0
Experiment:
#1
#2
#3
MDSC/NK cell ratio
B 80 60
RMA RMA-S
40 20 0
Experiment:
#1
#2
#3
#4
#5
#6
Supplementary Figure 2
Serum
400
active TGF-β1 (pg/ml)
active TGF-β1 (pg/ml)
Tumor bed
300 200 100 0
1500 1000 500 0
RMA
RMA-S
Supplementary Figure 3
Control RMA mice
RMA-S
Supplementary Figure 1: Representative FACS plots for CD11b/CD27 or MHC class I specific inhibitory receptors stainings are shown for NK cells infiltrating RMA and RMA-S tumors.
Supplementary Figure 2: (A-B) Ratios of Tregs or MDSCs to NK cells were calculated in RMA or RMA-S derived tumors. NK cells were gated as viable-CD3CD19-Ter119-NKp46+ cells. Tregs were gated as viable-CD19-Ter119-NKp46CD3+CD4+FoxP3+ cells and MDSCs were gated as viable-CD3-CD19-Ter119NKp46-CD11b+ cells. Several repetitions of each experiment are shown. Statistical analyses were performed with the two-tailed unpaired Student’s t-test.
45
Analysis of pooled results in panel A or in panel B revealed no statistically significant differences.
Supplementary Figure 3: Active TGF-β1 protein was detected by ELISA in the tumor bed (A) or in the serum (B) of RMA or RMA-S tumor bearing mice.
Supplementary Table 1. List of primers used for qPCR analysis
18S
β-actin
GAPDH
IL-10
Forward
Reverse
5’-
5’-
GTCTGTGATGCCCTTAG
AGCTTATGACCCGCACTT
ATG-3’
AC-3’
5’-
5’-
AGAGGGAAATCGTGCGT
CAATAGTGATGACCTGGC
GAC-3’
CGT-3’
5’-
5’-
TGTGTCCGTCGTGGATC
TTGCTGTTGAAGTCGCAG
TGA-3’
GAG-3’
5’-
5’-
46
Supplementary Material
Supplementary Table 1. List of primers used for qPCR analysis Forward
Reverse
5’-
5’-
GTCTGTGATGCCCTTAG
AGCTTATGACCCGCACTT
ATG-3’
AC-3’
5’-
5’-
AGAGGGAAATCGTGCGT
CAATAGTGATGACCTGGC
GAC-3’
CGT-3’
5’-
5’-
TGTGTCCGTCGTGGATC
TTGCTGTTGAAGTCGCAG
TGA-3’
GAG-3’
5’-
5’-
CACTGCCTTGCTCTTATT
CCCTGGGTGAGAAGCTG
TTCACA-3’
AAG-3’
IL-
5’-
5’-
12p40
GGAAGCACGGCAGCAG
AACTTGAGGGAGAAGTA
18S
β-actin
GAPDH
IL-10
IL-18
TGF-β
AATA-3’
GGAATGG-3’
5’-
5’-
CAGGCCTGACATCTTCT
TCTGACATGGCAGCCATT
GCAA-3’
GT-3’
5’-
5’-
CAATTCCTGGCGTTACC
GTATTCCGTCTCCTTGGT
T-3’
T-3’
A NK cells from:
RMA 8.4
10 5
CD27
RMA-S 55.1
10 5
10 4
10 4
10 3
10 3
10 2
10 2
0
0
33.1
3.56 0 10
2
10
3
10
4
10
10.7
49.2
5.16
34.8 0 10 2
5
10 3
10 4
10 5
CD11b
B NK cells from: RMA! RMA-S!
0
102
103
104
Ly49A!
105
0 102
103
104
Ly49C!
105
0
103
104
NKG2A!
105
0
103
104
Ly49G2!
105
0 102
103
104
105
Ly49I!
Supplementary Figure 1
Treg/NK cell ratio
A RMA RMA-S
2.0
p=0.03
1.5 1.0 0.5 0.0
Experiment:
#1
#2
#3
MDSC/NK cell ratio
B 80 60
RMA RMA-S
40 20 0
Experiment:
#1
#2
#3
#4
#5
#6
Supplementary Figure 2
Serum
400
active TGF-β1 (pg/ml)
active TGF-β1 (pg/ml)
Tumor bed
300 200 100 0
1500 1000 500 0
RMA
RMA-S
Supplementary Figure 3
Control RMA mice
RMA-S
