advances.sciencemag.org/cgi/content/full/3/5/e1602133/DC1

Supplementary Materials for

Antibody-mediated neutralization of soluble MIC significantly enhances

CTLA4 blockade therapy

Jingyu Zhang, Dai Liu, Guangfu Li, Kevin F. Staveley-O’Carroll, Julie N. Graff, Zihai Li, Jennifer D. Wu

Published 17 May 2017, Sci. Adv. 3, e1602133 (2017)

DOI: 10.1126/sciadv.1602133

This PDF file includes:

fig. S1. Induction of subclinical colitis in sMIChi TRAMP/MIC mice in response

to anti-CTLA4 therapy.

fig. S2. Recapitulation of the negative effect of sMIC on anti-CTLA4 therapy in a

transplantable tumor model.

fig. S3. Antibody neutralizing sMIC eliminates colitis in TRAMP/MICB mice

that received anti-CTLA4 therapy.

fig. S4. Detection of anti-sMIC autoantibody in the sera of a small cohort of

prostate cancer patients who have metastatic disease and enrolled in a clinical trial

(NCT01498978) of ipilimumab in combination with hormone suppression at the

Knight Cancer Institute.

fig. S5. Representative graphs of flow cytometry analyses demonstrate that

combination therapy of anti-sMIC and anti-CTLA4 antibody remarkably

increases CD8 T cell population, activation, and functional potential in tumor-

draining lymph nodes and tumor beds.

fig. S6. Marginal response of CD8 T cells to anti-CTLA4 therapy in TRAMP

mice.

fig. S7. Circulating sMIC or anti-sMIC autoantibody affects response to anti-

CTLA4 therapy in TRAMP/MICB mice compared to MIC-negative TRAMP

mice.

fig. S8. Representative graphs of flow cytometry analyses demonstrate that

combination therapy of anti-sMIC antibody and anti-CTLA4 antibody

cooperatively enhances antigen-specific CD8 T cell anti-tumor responses.

fig. S9. Therapy has no effect on the activation or costimulatory molecule on DCs

in the spleen or non–tumor-dLNs.

S1. Induction of subclinical colitis in sMIChi TRAMP/MIC mice in response to

anti-CTLA4 (αCTLA4) therapy. (A) Representative gross morphology of colon. (B)

Summary of colon length. *, p<0.05. (C) Representative histology of colon. (D)

Representative RT-PCR demonstrating tissue specific MICB expression in male

MICB/B6 or TRAMP/MICB transgenic mice; no MICB expression was detected in colon.

(E) Representative histology demonstrating normal colon histology in male MICB/B6

transgenic mice.

fig.

A.

αCTLA4cIgG

B.

Colo

n le

ngth

(cm

)

TRAMP/MICB

0

2

4

6

8

10

12

survivor

non-survivor

*

αCTLA4cIgG

survivor

sMIClo

non-survivor

sMIChi

survivor

sMIClo

non-survivor

sMIChi

C.αCTLA4

survivor

sMIClo

Non-survivor

sMIChi

cIgG

survivor

sMIClo

non-survivor

sMIChi

MICB

D.E.

MICB/B6

GAPDH

TP/MICB mouse

Pro

sta

te

Sple

en

Lung

Liv

er

Colo

n

Pro

sta

te

Sple

en

Lung

Liv

er

Colo

n

MICB/B6 mouse

fig. S2. Recapitulation of the negative effect of sMIC on anti-CTLA4 therapy in a

transplantable tumor model. (A) Schematic depiction of the transplantable tumor model and

therapy. (B) Tumor growth curve. (C) Representative histology of the colon.

fig. S3. Antibody neutralizing sMIC eliminates colitis in TRAMP/MICB mice that received

anti-CTLA4 therapy.(A) Summary of colon length. (B) Representative histology of the colon.

fig. S4. Detection of anti-sMIC autoantibody in the sera of a small cohort of prostate cancer

patients who have metastatic disease and enrolled in a clinical trial (NCT01498978) of

ipilimumab in combination with hormone suppression at the Knight Cancer Center. The

study was approved by OHSU Institutional IRB and Exampt from the IRB of Medical University

of South Carolina as the sample coded and received de-ID’ed for ELISA assay. Serum

autoantibody was detected by direct ELISA against antigen sMIC and anti-human Fc as

detecting agent. Patient 5254-8 has presented durable response as measured by serum PSA. No

colitis has detected in 5254-8 although he has developed hypoadrenolism.

an

ti-s

MIC

au

toa

ntib

od

y(p

g/m

l)

0

20000

40000

60000

80000

100000

120000

A

CD

3 25.623.216.1

CD8

αCTLA4

+B10G5αCTLA4B10G5cIgG

spln

dLN

TILs

B

NKG2D

20.27.8

B10G5

10.2

cIgG

7.2

3.8 4.9 10.86.2

9.36 17.55.34.6

spln

dLN

TILs

αCTLA4αCTLA4

+B10G5

C

24.5

19.324.2

44

31.2

22.0 32.8

28.7 23.0 37.317.5

18.3

CD44

spln

dLN

TILs

B10G5cIgG αCTLA4αCTLA4

+B10G5

D

IFN-γ

spln

dLN

TILs

B10G5cIgG

5.3 12.93.8

5.7 6.39.5 15.1

8.5 4.33.4

8.7

CD

8

13.3

αCTLA4αCTLA4

+B10G5

S5. Representative graphs of flow cytometry analyses demonstrate that

combination therapy of anti-sMIC and anti-CTLA4 antibody remarkably increases

CD8 T cell population, activation, and functional potential in tumor draining lymph

nodes and tumor beds.

fig.

18.4

10.6 13.4 7.8 14.6

14.1 23.2 18.5 30.2

A.

C. D.

Spln

dLN

TIL

20.5 23.9

24.9 25.4

21.2 22.4

CD44

αCTLA4cIgG

B.

NKG2D

SPL

dLN

TIL

αCTLA4cIgG

6.0 7.5

8.5 9.2

4.7 5.1

SPL

dLN

TIL

αCTLA4cIgG

17.8 18.1

11.1 12.4

19.2 20.5

CD8

CD

3

αCTLA

4

cIgG

5.2 6.2

Spln

dLN

TIL

5.84.8

6.55.4

IFN-γ

CD

8

S6. Marginal response of CD8 T cells to anti-CTLA4 therapy .

Data representatively show flow cytometry graphs of CD8 T cell population (A), NKG2D

expression on CD8 T cells (B), IFNg production in response to ex vivo PMA/I re-

stimulation (C), and memory-like/activation status (D). Spln, spleen. dLN, tumor-draining

lymph nodes. TIL, tumor infiltrates. cIgG, control IgG.

fig. in TRAMP mice

S7. Circulating sMIC or anti-sMIC autoantibody response to anti-CTLA4

therapy in TRAMP/MICB mice compare MIC-negative TRAMP mice. Data shown are

representatives of NK and CD8 T cell responses in tumor draining lymph nodes. (A) High levels of

circulating sMIC reduce CD8 T function, whereas autoantibody revamps the function of CD8 T cells

in responses to anti-CTLA4 therapy in TRAMP/MICB mice. The impact on the number of CD8 T

cells is marginal. (B) High levels of circulating sMIC reduce NK cell number and function, whereas

autoantibody revamps the number and function of NK cells in response to anti-CTLA4 therapy.

fig. affects

d to

IFNγ

B.

4.419.94

4.52 0.12

IFN-γ

count

CD

319.3

CD8

TRAMP + αCTLA4

(no sMIC)

(n=9)

16.0 23.311.8

TRAMP/MICB + αCTLA4

sMIClo

(n=5)sMIChi

(n=5)

anti-sMIC

autoantibody

(n=1)

A.

0.93

CD

3

3.24count

TRAMP + αCTLA4

(no sMIC)

(n=9)

0.842.10 7.79

NK1.1

0.35 1.270.80

TRAMP/MICB + αCTLA4

sMIClo

(n=5)sMIChi

(n=5)

anti-sMIC

autoantibody

(n=1)

IFN-γ

A B

Spln

CFSE

Gated on Db/I-tetramer+ CD8 T cells

dLN

TIL

6.7 4.0 13.6

3.3

CFSElo

10.05.5

14.03.3

CFSElo

7.5

3.8

CFSElo

3.9

3.7

CFSEhi

1.3 1.1 1.2 1.0

CFSEhi

1.0 1.0 1.3 0.9

CFSEhi

0.8 0.9 1.2 1.0

αCTLA4

+ B10G5αCTLA4B10G5cIgG

dLN

TILCD

8

Db/I- tetramer

Spln

αCTLA4

+ B10G5αCTLA4B10G5cIgG

2.50.5 0.7

1.04.40.6

4.7

7.7

0.7 5.23.1 0.9

C

dLN

Spln

CD44

TIL

35.719.0

29.710.7

28.412.820.3

25.0

22.0

17.0

11.0

9.66

αCTLA4

+B10G5αCTLA4B10G5cIgG

Gated on Db/I-tetramer+ CD8 T cells

D

dLN

TIL

Spln

IFN-γ

Db/I-t

etr

am

er

(gate

d o

n C

D8

+D

b/I-t

etr

am

er+

)

αCTLA4

+B10G5αCTLA4B10G5cIgG

1.8 3.3 1.3 6.0

7.51.52.40.6

2.62.4 8.56.6

Representative graphs of flow cytometry analyses demonstrate that combination

therapy of anti-sMIC antibody and anti-CTLA4 antibody cooperatively enhances

antigen-specific CD8 T cell anti-tumor responses.

fig. S8.

Therapy has no effect on the activation or costimulatory molecule on DCs in the spleenfig. S9.

or non-tumor dLN (A) Representative graphs of Flow Cytometry analyses. (B) Summary

data of the mean fluorescence intensity (MFI) of respective molecules.

s.

A. In Spleen

CD80 CD86 CD40

MHCII

CD

11c

1.85

In non-dLNs

αCTLA4

αCTLA4

+ B10G5

B10G5

cIgG

CD86 CD40

0

100

200

300

400

500

600

MF

I of

CD

86

(in n

on-d

LN

)

0

200

400

600

800

1000

1200

1400

1600

1800

MF

I of

CD

80

(in n

on-d

LN

)

0

500

1000

1500

2000

2500

MF

I of

CD

40

(in n

on-d

LN

)

P = 0.43

P = 0.092

P=0.45

P = 0.48

P = 0.291

P = 0.175

0

100

200

300

400

500

MF

I of

CD

86

(in s

ple

en )

αCTLA4

+B10G5αCTLA4B10G5

0

500

1000

1500

2000

2500

3000

MF

I of

CD

80

(in s

ple

en )

MF

I of

CD

40

(in s

ple

en )

0

500

1000

1500

2000

2500

3000

P = 0.09

nsP = 0.09

P = 0.07

P = 0.220

P = 0.217B.

αCTLA4

+B10G5αCTLA4B10G5

B10G5 αCTLA4

+B10G5

αCTLA4 B10G5 αCTLA4

+B10G5

αCTLA4 B10G5 αCTLA4

+B10G5

αCTLA4

B10G5 αCTLA4

+B10G5

αCTLA4

CD80