Supplemental Figure 1 (Sato et. al.) A H&E Ck5 · Supplemental Figure 7 (Sato et. al.) A C pTRE...
Transcript of Supplemental Figure 1 (Sato et. al.) A H&E Ck5 · Supplemental Figure 7 (Sato et. al.) A C pTRE...
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MCB6C1526 mutations
MCB6A1524 mutations
Mouse HumanEvidence for functional
relevance
Trp53T122K
TP53T125K
Recurrent in human cancer (Ref 26)
Kdm6aH1146Y
KDM6AH1146Y
Targets Catalytic Activity( Ref 24)
Sf3b1 E873K
SF3B1 E873K
Recurrent in human cancer (Ref 26)
Candidate driver mutations in MCB6A
Supplemental Figure 1 (Sato et. al.)
A
B C
D E
F
H&E Ck5
Supplemental Figure 1: Characterization of the MCB6A organoid line. A. Histologic and immuno-histochemical evaluation of s.c. MCB6A tumor collected 35 days after injection of organoid cells. Scale bar, 500 M. B. Comparison of mutations identified in MCB6A and MCB6C. C. Likely driver mutations in MCB6A. D. s.cgrowth of MCB6A organoids with and without combined CD4+ and CD8+ T-cell depletion. Data plotted as mean diameter +/- s.e.m, n = 4 mice per group. T-cell depletion was initiated on day -3 and repeated weekly throughout the experiment. Comparison is by 2 way ANOVA for repeated measures. E. MCB6A treated with combination ICB starting on day 9 and repeated every 3 days for a total of 6 treatments. Indicated T-cell depletions were initiated on day 7 and repeated weekly. Data plotted +/- s.e.m, n=8-12 mice per group, comparison by 2-way ANOVA for repeated measures. F. Confirmation of T-cell depletion in peripheral blood from a subset of mice from panel E on day 28. NS>0.05,*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Mea
n di
amet
er (
mm
)
CD4+ T
cell
CD8+ T
cell
% o
f Lym
ph
ocyt
es
0 10 20 30 400
5
10
15
20
Days Post Injection
Mea
n di
amet
er (
mm
)
ControlICBICB + CD4ICB + CD8ICB + CD4/ CD8
***
****
**** nsns
A
Supplemental Figure 2: Evaluation of lymphocyte depletion (related to Figure 2). A. Relative CD4+ and CD8+ T-cells in TIL and peripheral blood collected from tumor bearing mice on day 28 and treated with depletion antibodies administered as described in Figure 2A. B. Measurement of lymphocyte depletion on day 20 in peripheral blood of mice from experiment shown if Figure 2C. C. Measurement of lymphocyte depletion on day 20 in peripheral blood of mice from experiment shown in Figure 2D. D. Measurement of lymphocyte depletion in peripheral blood collected on day 28 post-reinjection from experiment in Figure 2F.
Supplemental Figure 2 (Sato et. al.)
D
B C
% o
f me
an o
f con
trol
(per
iph
eral
blo
od)
CD4+ T
-cell
s
CD8+ T
-cell
s
NK cells
0
5
10
15
% o
f Lym
ph
ocyt
es
Control
Combi
Combi + CD4
Combi + CD8
Combi + NK1.1
****
********
% o
f me
an o
f con
trol
(intr
atum
oral
)
CD4+ T
-cell
s
CD8+ T
-cell
s
% o
f Lym
ph
ocyt
es
CD4+T-cell
CD8+ T-cell
0
5
10
15
% o
f Lym
phoc
ytes Control
CD4
CD8
CD4/ CD8
RorT
Gat
a3
T-bet+
RorT+
Gating strategy to detect NK cells, CD8+ T-cells and CD4+ T-cell subsets
CD45Live/Dead
CD4
CD
8
CD4
Fox
p3
T-bet
Lymphocytes
T cellsCD4+
CD8+
Foxp3+
Foxp3-
NK
1.1
SS
C-W
SSC-H
SS
C
FSC FSC-H
FS
C-W
SS
C
SS
C
FSC
SS
C
NK cells
TCR
Gat
a3
Gata3+
Live
T-b
et
CD8
T-bet+
Gating strategy to detect IFN expressing T-cells
CD45Live/Dead
CD4
CD
8
CD4
Fox
p3
Foxp3
IFN
Lymphocytes
T cellsCD4+
CD8+
Foxp3+
Foxp3-
SS
C
SS
C-W
SSC-H
SS
C
FSC FSC-H
FS
C-W
SS
C
SS
C
T-bet
IFN
FSC
SS
C
CD8
IFN
T-bet
IFN
Supplemental Figure 3 (Sato et. al.)
Supplemental Figure 3: Gating strategies for lymphocyte evaluation.
CD4+ IHC CD4+ IHC (Color enhanced)
CD8+ IHC CD8+ IHC (Color enhanced)
Con
trol
ICB
Con
trol
ICB
Supplemental Figure 4 (Sato et. al.)
Supplemental Figure 4: Immunohistochemical analysis of CD4+ or CD8+ T-cells in MCB6C tumors on day 14, five days after ICB initiation. Arrowhead indicates CD8+ T-cell in the tumor. Images on right enhanced with contrast and hue adjustment in Adobe Photoshop such that stained T-cells appear pink. Scale bar = 200 M
TIL
Supplemental Figure 5 (Sato et. al.)
A B
C D
dLN
dLN dLN
G H
TIL
dLN
E F
dLN dLN
CD4+ T-cells
CD8+ T-cells
0
10
20
30
40
50
% o
f Lym
phoc
ytes
Control
PD-1
CTLA-4
PD-1 + CTLA-4
T-bet
+
Gata3
+
RorT+
Foxp3
+
% o
f Lym
ph
ocyt
es
Contro
l
PD-1
CTLA-4
PD-1 +
CTLA
-40
20
40
60
% K
i67
+ o
f T-b
et+
CD
4+ T
-cel
ls
****
NS
CD62L+CD44-
CD62L+CD44+
CD62L-CD44+
0
20
40
60
80%
in T
-bet
+ C
D4+
T-c
ells Control
PD-1
CTLA-4
PD-1 + CTLA-4
** *
Contro
l
PD-1
CTLA-4
PD-1 +
CTLA
-40
20
40
60 *****
*
% in
Fo
xp3+
CD
4+ T
-cel
ls
NKCD4
CD80
10
20
30
40
50
% o
f Lym
ph
ocyt
es
Control
PD-1
CTLA-4
PD-1 + CTLA-4
**
****
NS
T-bet
+
Gata3
+
RorT+
Foxp3
+0
5
10
15
20
25
% o
f Lym
ph
ocyt
es
Control
PD-1
CTLA-4
PD-1 + CTLA-4
****
NS NS
NS
Supplemental Figure 5: ICB monotherapy effects on T-cell subsets in TIL and dLN. A. CD4+ and CD8+ T-cells in dLN plotted as a percent of lymphocytes on day 14. ICB was initiated on day 9 and repeated on day 12. Data are shown as mean ± s.d, n=5 mice. B. Same as A, but showing indicated CD4+ T-cell subsets. C. Same as A, but showing percent Ki67 positive cells in CD4+ T-bet+ population. D. Same as A, but showing proportion of T-bet+ CD4+ T cells with each of the indicated phenotypes. E. Same as A, but showing percent Ki67 positive cells in CD4+ Foxp3+ population. F. Same as A, showing proportion of Foxp3+ CD4+ T-cells with each of the indicated phenotypes. G. T-cell subsets in TIL on day 14, mean ± s.d, n=4 mice. I. CD4+ T-cell subsets in TIL on day 14, mean +/- s.d. All statistical analysis by Student’s t-test. NS>0.05,*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Control (Day 14) Combi ICB (Day 14)
Combi ICB (Day 14 high mag)
Supplemental Figure 6: Histologic evaluation of ICB treated tumors. A. H&E staining of MCB6C tumor sections obtained on day 14, five days after initiation of combination ICB. Images obtained with NanoZoomer-XR (10X). Scale bar 200 M. B. Similar to A, but 20X and white arrows indicate giant cells, scale bar 200 M. C. CD31 IHC staining of MCB6C tumor sections obtained on day 14, five days after initiation of combination ICB. Images obtained with NanoZoomer-XR (5X), scale bar 500 M.
Supplemental Figure 6 (Sato et. al.)
A
B
C Control (CD31 staining) Combination ICB (CD31 staining)
+DOX
No DOX
MCB6C (Parent)
dsRED
EpCAM+ / CD45‐ cells
Supplemental Figure 7 (Sato et. al.)
A
C
FLAG Mouse IFN DSREDP2ApTRE PuroR rTreRT2ApPGK
Dox‐inducible‐IFN vectorB
Supplemental Figure 7: IFN mediates ICB activity and is sufficient to inhibit tumor growth (related to Figure 5). A. Ck5 staining of MCB6C tumor sections obtained 5 days after initiation of combination ICB with and without TNF neutralization. TNF neutralization antibody was administered on days 8 and 11 post MCB6C injection. Quantification and comparison of Ck5 staining as described in figure 5B. B. dsRED expression as a surrogate for rIFN expression in epithelial compartment of tumors described in Figure 5D. For flow analysis, day 34 tumors from the IFN neutralizing groups were used so that adequate epithelial cells were available for evaluation. dsRED in tumors cells from 4 mice in each group is compared to background signal from non-transduced MCB6C tumor cells. C. Tumor cell mass calculated by multiplying total mass by %Ck5 positivity (see Figure 5E, F). Comparison by Student’s t-test.
Control Combi + Isotype
Combi +aTNFa
0
20
40
60
80
Ck5
are
a (
%)
*******
Nor
mal
ized
Tum
orC
ell M
ass
A
B
Supplemental Figure 8 (Sato et. al.)
Supplemental Figure 8: Evaluation of intratumoral myeloid cells. A. Gating strategies for myeloid cell evaluation. B. Quantification of myeloid lineages in tumor as proportion of total leukocytes
Gating strategy for Myeloid cell subsets
CD45Live/Dead
CD11b
CD
11
c
MHC-II
SS
C
Ly6C
F4/
80
Eosinophils
Gr-MDSCsMHC-II+
Ly6G
SS
C-W
SSC-H
SS
C
FSC FSC-H
FS
C-W
SS
C
SS
C
MHC-II
CD
20
6
FSC
SS
C
CD11b
MH
C-I
I
CD11b
CD24
MHC-II-
F4/
80
Macrophages
DCs
CD103+ DCs
CD11b+ DCs
Mo-MDSCs
CD206+
CD206-
Macrophages DCs