Supplementary Figure 1. [18F]FHBG is not retained, phosphorylated, or incorporated into the DNA of B16 cells. (A-C) For the in vitro studies a panel of cell lines was used: B16 melanoma cells, BaF3 wt and BaF3 with sr39tk over-expressed, primary activated pmel T-cells and activated pmel cells transduced with sr39tk. (D-F) For the in vivo studies B16 and BaF3-sr39tk cells were subcutaneously injected into mice 1 week prior to i.v. injection of [18F]FHBG. After a 1hr uptake, mice were sacrificed and tumors were harvested. The following measurements were made: (A, D) amount of [18F]FHBG retained in cells, (B, E) amount of [18F]FHBG phosphorylated, (C, F) amount of [18F]FHBG incorporated in DNA after 4hr.

B16BaF

3

BaF3-

sr39

tkpm

el

pmel-

sr39

tk0

50

100

150

200

[18F

]FH

BG

(nC

i)

B16BaF

3

BaF3-

sr39

tkpm

el

pmel-

sr39

tk0

50

100

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200

250

Pho

spho

ryla

ted

[18F

]FH

BG

(nC

i)

B16

BaF3-

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BaF3-

sr39

tkpm

el

pmel-

sr39

tk0

510

20

30 0 hr4 hr

[18F

]FH

BG

(nC

i)

B16

BaF3-

sr39

tk0

50

100

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[18F

]FH

BG

(x1

e6 nC

i)re

tain

ed/ c

ell

B16

BaF3-

sr39

tk0

1

2

3

4

5

Pho

spho

ryla

ted

[18F

]FH

BG

(nC

i)

B16

BaF3-

sr39

tk0.0

0.5

1.0

1.5

[18F

]FH

BG

(nC

i)

A. D.

B.

C.

E.

F.

Day 1 Day 3 Day 5 Day 8 Day 10

1.3

0.3

%ID

/g

Day 12 Day 15 Day 17 Day 19 Day 21

1.3

0.3

%ID

/g

Day 1 Day 3 Day 5 Day 8 Day 101.3

0.3

%ID

/g

Day 12 Day 15 Day 17 Day 19 Day 21

1.3

0.3

%ID

/g

Day 1 Day 3 Day 5 Day 8 Day 101.3

0.3

%ID

/g

Day 12 Day 15 Day 17 Day 19 Day 21

1.3

0.3

%ID

/gT T T

T TT T T

TTT

T T T T

T T T T

T T T T

B B B

BBB

B B

B B B

BB B

A B

A A

B B

A A

A

A

A

A

C

C C C

Mouse 1

Mouse 2

Mouse 3

C C C

B

BA

Day 1 Day 3 Day 5 Day 8 Day 101.3

0.3

%ID

/g

Day 12 Day 15 Day 17 Day 19 Day 21

1.3

0.3

%ID

/g

Day 1 Day 3 Day 5 Day 8 Day 10

1.3

0.3

%ID

/g

Day 12 Day 15 Day 17 Day 19 Day 21

1.3

0.3

%ID

/g

Day 12 Day 15 Day 17 Day 19 Day 21

1.3

0.3

%ID

/gB

B

TTT

TT

T TTT

TTTT

A

A A

AA

BB

B

B B

T T T

TTTT A

B

B

Mouse 5

Mouse 6

Mouse 4 Day 1 Day 3 Day 5 Day 8 Day 10

1.3

0.3%

ID/g

B BT T TBBB

CC C

Mouse 7 Day 1 Day 3 Day 5 Day 8 Day 10

1.3

0.3

%ID

/gB B B BT B T

Day 12 Day 15 Day 17 Day 19 Day 21

1.3

0.3

%ID

/g

T T TT B

Supplementary Figure 2. Longitudinal changes in T cell trafficking. Seven mice were imaged with microPET/CT 60 min after i.v. injection with [18F]FHBG. Representative 3-D microPET/CT images of the same mouse on different days are displayed. Red arrows indicate days that mice received gp10025-33/DC vaccine and high dose IL-2. A, axillary LN; B, brachial LN; C, cervical LN; T, tumor

Supplementary Figure 3. Detection of sr39tk+ T-cells over the nonspecific accumulation of [18F]FHBG in B16 tumors. (A) C57BL/6 mice were implanted s.c. with 1 x 105 B16 cells in the right shoulder on day 1. Mice with tumors >1cm in diameter were imaged with [18F]FHBG microPET/CT on day 8. Immediately after imaging, 5x106 sr39tk+ T-cells were injected intra-tumorally. After 30 min, a second dose of [18F]FHBG was injected i.v.. Following a 1hr uptake of the probe mice were again imaged by microPET/CT. (B) Representative 3-D microPET/CT images of three mice with B16 tumors alone (left panels) and the same B16 tumors with sr39tk+ T-cells (right panels). T, tumor. (C) Quantitation of [18F]FHBG in B16 tumors alone and the same B16 tumors with sr39tk+ T-cells.

mou

se #

1

mou

se #

2

mou

se #

30

20

40

60

80

100

B16B16 + pmel-sr39tk

%ID

of [

18F

]FH

BG

A.

B.

1.7

0.1%

ID/g

1.7

0.1

1.7

0.1

%ID

/g%

ID/g

Mouse #1

Mouse #2

Mouse #3

B16 aloneB16 +

sr39tk+ T-cell

B16 melanoma bearing mouse

[18F]FHBG PET

Intratumoral injection of

sr39tk+ T-cells

[18F]FHBG PET

C.

T T

TT

T T