To study tumor targeting, mice were grafted with 700,000 B16-HER2 cells i.v., and systemic treatment, as described in Figure 4, was started 10 days after the tumor graft. For BrdU-incorporation, mice were injected i.p. with 1 mg BrdU on day 10, and BrdU was added to the drinking water throughout the whole experiment. Two days after the third injection, mice were sacrificed, lymphocytes were isolated from lungs, spleen, and PBMCs and stained with anti-NK1.1–PE, anti-CD3–FITC, and anti-BrdU–APC. Proliferation of iNKT, NK, and CD3 T cells is shown as percent of BrdU-positive cells in the respective population. (A) Dot plots with numbers indicating the percent of BrdU-positive iNKT, NK, and T cells isolated from lung tissue and (B) graphs of BrdU-incorporation in iNKT, NK, and CD3 T cells from lungs, spleen, and PBMC expressed as fold increase of BrdU-positive cells compared with control group. (C) Biodistribution study with radiolabeled sCD1d molecules. Mice were grafted s.c. on each flank with 1 × 10⁶ of either B16-HER2 or B16 wt cells. On day 8, 2 groups of mice were injected with equimolar amounts of either ¹²⁵I-labeled αGalCer/sCD1d or αGalCer/sCD1d–anti-HER2. Twenty-four hours later, mice were sacrificed and radioactivity was measured in tumors and normal tissues. Results are expressed as percent of injected dose per gram of tissue. Mean ± SD of 3 mice per group. *P < 0.05 for αGalCer/sCD1d–anti-HER2 in B16-HER2 tumors versus B16 wt tumors; **P < 0.02 for αGalCer/sCD1d–anti-HER2 versus αGalCer/sCD1d in B16-HER2 tumors.