828830 696

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