Figure S1

b

00.10.20.30.40.50.60.70.80.9

B10G5free sMICB

a

c

20 10 5 2.5 1.25 0.6250

10

20

30

40

50

60TC2-MICB

TC2-MICB+mIgG

TC2-MICB+ mAb B10G5

Supplement Figure 1. Basic functional characterization of B10G5 antibody. a. B10G5 neutralizing free sMICA in in vitro assay. Various concentration of B10G5 antibody was added to normal human serum containing a given amount (0.8 ng) of sMICA. Free sMICB in the serum was then measured with ELISA. Data shown that B10G5 neutralizing free sMICA. b. Histogram plots demonstrating that B10G5 binding to surface MICB overexpressed on TC2 cells. c. A standard 51Cr-release NK cell cytotocixity assay demonstrating that B10G5 enhance NK cell killing of TC2-MICB cells. Data demonstrate the agonist nature of B10G5 in enhancing NK cell activity against TC2MICB cells.

ng

/ml

TC2-MICB

TC-MICBTC2

Sp

ec

ific

Ly

sis

(%

)

E:T

Figure S2

a

b

G-C

SF

GM

-CSF

IFNy

IL-2

IL-3

IL-4

IL-5

IL-6

IL-7

IL-9

IL-1

0

IL-1

2(p4

0)

IL-1

2(p7

0)IL

-13

IL-1

5IL

-17

MCP-1

M-C

SF

MIP

-1a

MIP

-1b

MIP

-2

CCL5

TNFa

1

10

100

1000

10000

100000

cIgG B10G5 anti-CD4 +B10G5

G-C

SF

GM

-CSF

IFNy

IL-2

IL-3

IL-4

IL-5

IL-6

IL-7

IL-9

IL-1

0

IL-1

2(p4

0)

IL-1

2(p7

0)IL

-13

IL-1

5IL

-17

MCP-1

M-C

SF

MIP

-1a

MIP

-1b

MIP

-2

CCL5

TNFa

1

10

100

1000

10000

100000

cIgG B10G5 anti-NK +B10G5

Ser

m le

vel (

pg/m

l)S

erm

leve

l (pg

/ml)

Supplement Figure 2. Changes in serum levels of cytokines and chemokines with depletion of lymphocyte subsets during B10G5 therapy. (a) With CD4 depletion during B10G5 therapy (n>5 animals per group). (b) with NK depletion during B10G5 therapy (n>6 per group). Data show that serum cytokine “storm” in response to B10G5 therapy was an accumulative effect of activation of multiple cell types. Cytokine level was measured with multiplex assay by Eve Technologies.

Figure S3

a

6.77 4.89NK

1.1

14.1 3.09

spleen

B10G5 cIgG

LN

B10G5 cIgG

V450 dilution

b

0.0

2.0

4.0

6.0

8.0 *

B10G5 cIgGPro

lifer

atio

n of

CD

45.1

+N

K

(sp

leen

)

0

5

10

15

20 *

B10G5 cIgGPro

lifer

atio

n of

CD

45.1

+N

K

(iy

mph

onod

e)

TRAMP/MIC mice

Age 6 16 24 27 32 33 (wks)

Tx (B10G5 or cIgG)

i.v. injection of 5x105 V450-labeled congenic CD45.1 NK

c

Supplement Figure 3. Anti-sMIC mAb B10G5 therapy significantly increase homeostatic proliferation of adoptively transferred NK cells. (a) Depiction of the therapy and adoptive transfer of CD45.1 congenic NK cells. Five days before therapy study end point, mice (CD45.2) received i.v. injection of V450-labeled congenic CD45.1 NK cells (5x105/mouse). (b) Representative graphs of flow cytometry analyses of V450-dilution of CD45.1 NK cells in the spleen and lymph nodes of mice in respective therapy groups. (c) Summary statistics of CD45.1 NK cell proliferation in the spleen and lymph nodes.

a

PBMC

11.4 11.014.4 11.9 26.1 26.9

dLN

B10G5 cIgG

17.3 11.1

spleen

B10G5 cIgG

dLN

B10G5 cIgG

spleen

B10G5 cIgG B10G5 cIgG

CD3

CD

4

b

T-bet

CD

4

Figure S4

Supplemental Figure 4. B10G5 therapy did not impact CD4 T cell homeostasis but increased T-bet expression.

30.4 9.7

Figure S5

dLN spleen TIL

CD8TRAMP/MICB +B10G5

TRAMP/MICB +cIgG

TRAMP

Supplement Figure S5. Undetectable endogenous SV40TAg epitope I-specific CD8 T cells shown by lack of positive staining by H-2Db-restrcited TAg epitope I-specific tetramer (Db/I-Tetramer).

Db/I-Tetramer

Figure S6

Tx(Day 20)

*

Days post tumor inoculation

Tum

or v

olum

e (m

m3)

8 12 15 19 22 25 28

0

200

400

600

800

1000

1200

1400

1600cIgG

B10G5

a

e

0.00.20.40.60.81.01.21.4

IFNg

+ C

D8

T in

the

LN

cIgG B10G5

*

d

Brd

U+%

NK

(S

plee

n)

0.02.04.06.08.0

10.012.0

cIgG B10G5

*

0.0

5.0

10.0

15.0

20.0

25.0

30.0

B

rdU

+%

NK

(bon

e m

arro

w)

cIgG B10G5 *

c

0.00.20.40.60.81.01.21.4

NK

% in

LN

cIgG B10G5

*

NK

% in

spl

enoc

ytes

cIgG B10G50.0

1.0

2.0

3.0

4.0

5.0

6.0 *

0.0

0.5

1.0

1.5

2.0

NK

% in

Tum

or in

filtr

ates *

cIgG B10G5

f

0.0

0.2

0.4

0.6

0.8

IFNg

+ C

D4

T in

the

LN

cIgG B10G5

*

0.01.02.03.04.05.06.0

CD

8 T

% in

Tum

or in

filtr

ates

cIgG B10G5 *

g

Supplement Figure 6. Anti-sMIC mAb B10G5 therapy inhibits the growth of transplantable TRAMP-sMICB tumors. sMIC-expressing prostate tumor cell line TRAMP-C2 (designated as TRAMP-C2-sMICB) was implanted subcutaneously (s.c.) into syngeneic B6.MICB transgenic mice (5x105 cells/mouse). 20 days post-tumor inoculation, animals were randomized into two treatment groups (Tx, n=12 per group): control IgG (cIgG) or B10G5. Animals receive respective treatment i.p. twice weekly at the dose of 4 mg/Kg body weight. (a and b) Tumor growth curve (a) and Kaplan-Meier survival curve (b) demonstrating that B10G5 significantly inhibited tumor growth and increase survival. Survival end point defined as tumor volume reaches 1500 mm3. (c) B10G5 therapy significantly increased NK cell frequency in the periphery. (d) B10G5 therapy significantly enhanced the ability of NK cell renewal in tumor host shown by marked increase in BrdU uptake. (e, f) B10G5 therapy significantly potentiated CD8 and CD4 T cell function as shown by IFNg production in response to PMA/ionomycin re-stimulation. (g, h) B10G5 therapy significantly increased cytotoxic NK and CD8 T cells to tumor parenchyma. Data in c-h were obtained from 6 animals from B10G5 or cIgG treatment group at day 8 after treatment initiation. . * p < 0.05.

h

b.

Su

rviv

al %

P<0.01

days since Rx

0 10 20 30 400

50

100B10G5

cIgG

H-2Kb/OVA257-264 tetramer

CD

8

B10G5cIgG

a. TIL, gated on CD45+CD3+CD8+

0.15 4.15

0

2

4

6

OV

A25

7-26

4 t

etra

mer

+

(o

f C

D8)

B10G5cIg

G

*

0.6 3.2

CD

8

B10G5cIgG

IFNg

b. TIL, gated on CD45+CD3+CD8+

Figure S7

Supplement Figure 7. Anti-sMIC therapy enhanced antigen-specific CD8 T cellresponses in the transplantable TRAMP-C2-sMICB-OVA tumor model. Although TRAMP-C2 (TC2) cell line was derived from tumors of TRAMP mice, SV40TAg was lost during. To address antigen-specific responses in the transplantable TC2-sMICB model, we overexpressed OVA in TC2-sMICB-TC2 cells. B10G5 therapeutic experiment was performed similarly to which in Supplement Figure 4. At the study end point, tumor infiltrated OVA peptide-specific CD8 T cells were detected with H-2Kb-restricted OVA257-264 tetramer (with H-2Kb/OVA257-264 tetramer). (a) Frequency of H-2Kb/OVA257-264 tetramer+ CD8 T cells in the tumor infiltrates (TILs). (b) Response of CD8 T cells in TILs to OVA257-264 peptide stimulation. Bulked tumor single cell suspension was re-stimulated with OVA257-264 peptide for 6h.

0

2

4

6

IFNg

+ o

f C

D8

*

B10G5cIg

G

Figure S8

Supplementary Figure 8. Therapeutic effect of B10G5 against well-differentiated TRAMP/MICB tumors. Cohorts of 20 week TRAMP/MIC were treated with B10G5 or cIgG (4.0 mg/KG BW) twice a week for an 8-week duration. a, prostate weight. b, Representative micrograph of tumor antigen SV40T staining showing micrometastatic deposits in the lung of animal in control group, but not in the B10G5 treated group. c-f, B10G5 therapy increases total splenic NK cell number (c), the ability of NK cell to self-renew (d), function of NK cell shown by increased IFNg production in response to PMA/I re-stimulation (e) and granzyme B production against TC2-MICB cells (f). g and h, B10G5 therapy potentiates CD8 T cell function and increases CD44hi CD8 T cells in tumor-draining LN. i and j, B10G5 therapy potentiates CD4 T cells to Th1 responses and increases CD44hi CD4 T cells in tumor-draining LN. All data were collected at study end point. *p < 0.05. **p<0.01.

B10G5

B10G5

B10G5

0

2

4

6

8

10

12

IFNg

+C

D8

%

(d

LN

)

cIgG

*

0

5

10

15

20

25

CD

44h

i CD

8 %

(dL

N)

cIgG

*

0

1

2

3

4

IFNg

+C

D4

%

(d

LN

)

cIgGB10G5

**

0

10

20

30

40

50

Brd

U+N

K %

(s

ple

en

)

cIgG

**

0

5

10

15

20

Tota

l sp

len

ic N

K

(X10

5)

cIgGB10G5

**

0

5

10

15

20

25

Gra

nzy

me

+N

K %

(s

ple

en

)

cIgGB10G5

**

0

5

10

15

20

25

30

IFNg

+N

K %

(s

ple

en

)

cIgGB10G5

**

CD

44h

i CD

4 %

(dL

N)

0

5

10

15

20

25

cIgGB10G5

*

0.0

0.5

1.0

1.5

2.0

2.5

Pro

sta

te W

eig

ht

(g)

cIgG B10G5

cIgG B10G5

SV

40T

a. b.

c. d. e. f.

g. h. i. j.

Figure S9

spleen

LN

T-bet

CD

4

29.215.4

20.013.8

Supplementary Figure S9. Representative flow cytometry analyses demonstrating that depletion of NK cells during B10G5 therapy impairs CD4 T cell Th1 responses. (a) Representative dot plot demonstrating that NK depletion during B10G5 therapy impairs CD4 T cell IFNg production in response to PMA and ionomycin re-stimulation. (b) Representative dot plots demonstrating that NK depletion during B10G5 therapy reduces transcriptional factor T-bet expression on CD4 T cells.

a b

spleen

LN

CD

4

5.63.4

3.71.0

IFNg

anti-MIC + aNK1.1

anti-MIC + mIgG

anti-MIC + aNK1.1

anti-MIC + mIgG