pDC recruitment to B16 melanoma after Aldara treatment is CCR6-dependent. (A) B16 melanoma tumors of WT and CCR6-deficient mice were treated daily with Aldara or vehicle cream for 6 consecutive days. At day 7, harvested tumors after purification of CD45⁺ immune cells by MACS sorting were analyzed for the frequency of B220⁺ BST2⁺ pDC infiltration after gating on viable (DAPI⁻) and CD11c⁺/CD11b⁻ DCs (see supplemental Figure 2B). The numbers of CD11c⁺ CD11b⁻ BST2⁺ B220⁺ pDCs among total viable tumor cells were compared using R2.13.2 software (R foundation for Statistical Computing, Vienna, Austria, url:http://www.R-project.org) between WT animals and CCR6-deficient animals treated with Aldara or vehicle cream (n = 9 animals). (B) pDC-enriched cells from in vivo Flt3-L-expanded splenocytes of B6 donors were analyzed for the presence of pDCs (CD11c⁺ CD11b⁻ BST2⁺ B220⁺). (C) pDC-enriched cells from WT Flt3-L-expanded splenocytes were labeled with CellTrace Violet and intravenously injected into recipients. The occurrence of donor pDCs in the recipient's spleen (top) and B16 tumor (bottom) preparation was analyzed 18 hours later (gate on CellTrace violet⁺ cells). (B-C) Data shown are representative for 5 animals of 2 independent experiments. (D) pDC-enriched cells from splenocytes of Flt3-L–treated WT or CCR6-deficient mice were labeled with CellTrace Violet and CFSE, respectively, for the pool CCR6^−/−/WT (black bars). As a control, pDC-enriched cells from splenocytes of Flt3-L–treated B6 were labeled either with CellTrace Violet or CFSE, respectively, for the pool WT/WT (white bars). Cells were adjusted to equal number of pDCs and injected at a ratio 1:1 in B16-carrying B6 recipients that were previously treated with Aldara as described in panel A. After 18 hours, recipients were killed, tumors were processed as described in panel A, and the ratio of donor B6 (violet) and CCR6-deficient (CFSE) pDCs or donor B6 (violet) and donor B6 (CFSE) pDCs was analyzed in the recipient's spleen and tumors (mean ± SD; n = 5 recipients). Statistical significance was obtained with a nonparametric Wilcoxon test.