CD6. 2. Llo. CD8. +. T. Ce lls. % CD4. 4hi. CD6. 2. Lhi. CD8. +. T. Ce lls. Naive. Teff or TEM. TCM. % C. D. 2. 7hi. CD1. 1 bhi. NK Ce lls. % CD2. 7lo. CD1. 1 bhi.
Figure S1
MFI: 82.4, 1118 %+: 6.5%, 99.3%
% Max
% Max PD-L1
-IFNg +IFNg Isotype
TGFbRII
MFI: 53.8, 52.9 %+: 16.1%, 14.7%
1
10
0.01
C.
100 80
20
60 40
10
20 0
0 0 10 20 30 40 50 60 70 80 90 100
Time (m)
1
10
100 1000
Concentration (µM)
D.
SMAD2/3 Phosphorylation 30
0.1
% Max
% PD-L1+
100 1000
% SMAD2/3 +
-IFNg +IFNg Isotype
0.1
Concentration (µM)
1.5
1.0
0.5
0.0
2.0 1.5 1.0 0.5 0.0
M7824
TGFbRII
PD-L1
0.01
PD-L1 Isotype
PD-L1
MUT
100 1000
0
PBS
10
Concentration (µM)
20
p-SMAD1/5 / SMAD5 Fold Change ∆PBS)
1
0
PD-L1 Isotype
PD-L1
M7824
B.
0.1
20
NT PD-L1 MUT M7824
40
MUT
0.01
PD-L1 Isotype
NT PD-L1 M7824 MUT
40
60
PBS
0
PD-L1
60
gMFI: 444 % Pos: 98.1%
80
p-SMAD1/5 / SMAD1 Fold Change ∆PBS)
20
gMFI: 1267 % Pos: 98%
80
NT MUT
100
% Max
NT PD-L1 MUT M7824
40
% Max
% PD-L1+
60
NT MUT
100 gMFI: 916 % Pos: 98.4%
80
MC38 (Colon)
4T1-pSMAD2-luc (Breast)
NT MUT
% p-SMAD2/3 +
4T1 (Breast) 100
% PD-L1+
A.
Figure S1. (A) M7824 binds cell surface PD-L1 on murine tumor cell lines in vitro. 4T1, 4T1-pSMAD2-luc, and MC38 tumor cells were exposed to 100nM IFNg for 24 hours followed by treatment with nothing (no treatment-NT), anti-PD-L1 (PD-L1), M7824mut (MUT), or M7824 for 30 minutes prior to analysis of surface PD-L1 expression by flow cytometry. Data represent 3 independent experiments. (B,C) 4T1-pSMAD-luc cells express PD-L1 and TGFbRII and activate TGFb signaling pathways upon TGFb1 stimulation. 4T1-pSMAD2-luc tumor cells were left untreated or treated with100nM IFNg for 24 hours. (B) PD-L1 and TGFbRII expression were determined by flow cytometry. (C) 4T1-pSMAD2-luc tumor cells were exposed to 2.5ng/ml TGFb1 and level of total and phosphorylated SMAD2/3 was determined by flow cytometry. Data represent 3 independent experiments. (D) M7824 does not affect intratumoral SMAD1 or SMAD5 activation. EMT6 tumor cells were implanted as in Figure 1. When tumor volumes reached 500mm3, mice were treated at days 17, 19, and 21 with MUT or M7824 i.p. 6 hours after the last treatment, phosphorylation and total level of SMAD1 and SMAD5 were determined by capillary Western blot. Data combined from 2 independent experiments, n=2-5 mice per experiment.
Figure S2
0.0
5
4.0×106
PBS MUT M7824
%CD4+ Treg
1×106
40
20
0
PBS MUT M7824
1×106
*
4 3 2 1 0
PBS MUT M7824
LYMPH NODES
8.0×106
2×106
0
60
**
5
**
3×106
PBS MUT M7824
% CD4+ T Cells
# CD4+ T Cells
10
0
PBS MUT M7824
*
15
**
*
PBS MUT M7824 5
***
8×105
%CD4+ Treg
5.0×106
20
# CD4+ Treg
1.0×107
0.0
4×106
SPLEEN
1.5×107
1.2×107
Treg
25
% CD4+ T Cells
SPLEEN
# CD4+ T Cells
2.0×107
LYMPH NODES
B.
CD4+ T Cells
# CD4+ Treg
A.
6×105 4×105 2×105
*
*
4 3 2 1 0
0
PBS MUT M7824
PBS MUT M7824
Figure S2. M7824 increases CD4+ T cell and Treg numbers in non-tumor-bearing mice. Naïve Balb/c mice received 3 doses of MUT or M7824 on day 0, 2, and 4. Immune populations in the spleen and lymph nodes 3 days after the last treatment were analyzed by flow cytometry. Graphs show frequency (of total live cells) and number of CD4+ T cells (A) and CD4+ Treg (B) 3 days post-treatment. All graphs show mean±SD. Data combined from 2 independent experiments, n=3-5 mice per experiment.
Figure S3
Day 24
Primary Tumor 1000
PBS M7824
600
*
400 200 0
0
10
B.
20
30
Day Post-Tumor Implant
800
p=0.0265
600 400
3000 2000
p=0.0257
1000
200 0
C. 4000
# Lung Mets
800
Tumor Volume (mm3)
Tumor Volume (mm3)
A.
PBS
M7824
0
PBS
M7824
Number of Mets in Lung ≥ 2000 ≤ 1000 ≤ 500 ≤ 300 ≤ 100 ≤ 50 ≤ 10 0 Median Mean
Figure S3. M7824 decreases 4T1 breast tumor cell metastasis. 5x104 4T1 tumor cells were orthotopically implanted into female Balb/c mice. Mice received 2 doses of 492µg M7824 i.p. at days 7 and 9 post-tumor implant. Twenty-four days after tumor implant, lungs were harvested and single-cell suspensions were plated with 6-TG for 14 days to visualize lung metastases. (A) Primary tumor growth curves (left panel) and tumor volumes of individual mice at day 24 (right panel) show mean±SD. Number of lung metastases in individual mice (mean±SD) shown in (B). Table showing the distribution of number of lung metastasis in (C). Data from 1 independent experiment, n=11-14 mice. Statistics in A (right panel) and B determined by a two-tailed t test.
