1. Supplementary Figure 1. [ 18 F]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 [ 18 F]FHBG. After a 1hr uptake, mice were sacrificed and tumors were harvested. The following measurements were made: (A, D) amount of [ 18 F]FHBG retained in cells, (B, E) amount of [ 18 F]FHBG phosphorylated, (C, F) amount of [ 18 F]FHBG incorporated in DNA after 4hr. A.D. B. C. E. F.

2. 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 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 10 1.3 0.3 %ID/g Day 12 Day 15 Day 17 Day 19 Day 21 1.3 0.3 %ID/g T TT TTTTT TTT TTT T TTTT TTTT BB B B B B B B B BB B B B A B A A B B AA A A A A C C C C Mouse 1 Mouse 2 Mouse 3 C C C B B A

3. 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 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/g B B T T T T T T T TT T T T T A A A A A B B B B B T TT TT T T 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 B T T T B B B CC C

4. Mouse 7 Day 1 Day 3 Day 5 Day 8 Day 10 1.3 0.3 %ID/g B B B B T B T Day 12 Day 15 Day 17 Day 19 Day 21 1.3 0.3 %ID/g TT T T B Supplementary Figure 2. Longitudinal changes in T cell trafficking. Seven mice were imaged with microPET/CT 60 min after i.v. injection with [ 18 F]FHBG. Representative 3-D microPET/CT images of the same mouse on different days are displayed. Red arrows indicate days that mice received gp100 25-33 /DC vaccine and high dose IL-2. A, axillary LN; B, brachial LN; C, cervical LN; T, tumor

5. Supplementary Figure 3. Detection of sr39tk + T-cells over the nonspecific accumulation of [ 18 F]FHBG in B16 tumors. (A) C57BL/6 mice were implanted s.c. with 1 x 10 5 B16 cells in the right shoulder on day 1. Mice with tumors >1cm in diameter were imaged with [ 18 F]FHBG microPET/CT on day 8. Immediately after imaging, 5x10 6 sr39tk+ T-cells were injected intra- tumorally. After 30 min, a second dose of [ 18 F]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 [ 18 F]FHBG in B16 tumors alone and the same B16 tumors with sr39tk+ T-cells. A. B. 1.7 0.1 %ID/g 1.7 0.1 1.7 0.1 %ID/g Mouse #1 Mouse #2 Mouse #3 B16 alone B16 + sr39tk + T-cell B16 melanoma bearing mouse [ 18 F]FHBG PET Intratumoral injection of sr39tk + T-cells [ 18 F]FHBG PET C. T T T T T T