Fig. 2. APC migration from the skin to the dLN in the naïve and melanoma contexts.

CD86 and CD206 signal (A and B) in CD11c⁺DEC205⁺ cells of the skin or tumor and dLN of naïve and day 10 B16F10-bearing animals, with the MFI and number of cells expressing CD86 and CD206 quantified, both normalized to values in the naïve skin/LNs. (C) IVIS (in vivo imaging system) imaging showing a site of 500-nm injection (white arrow) and LN drainage (yellow circle), demonstrating specific accumulation in dLNs after 72 hours (positive pixels as collected by IVIS imaging are red-yellow, negative background is gray scale). (D) Accumulation of 500-nm tracer⁺ cells in the dLN over time, analyzed flow cytometrically. Five hundred–nanometer accumulation among CD45⁺ cells in LNs draining skin or day 7 B16F10 melanomas over time, assessed flow cytometrically (E). Fold change in the area under the curve (AUC) (0 to 72 hours) of number of cells containing MPs within the dLN relative to naïve accumulation (F). MFI of MP fluorescent signal (G) in LNs draining the MP-injected skin or B16F10 melanomas over time of all MP-containing cells, and as fold change of AUC (0 to 72 hours) within individual cell subtypes relative to naïve tissues at 4, 24, and 72 hours (H). Representative flow cytometry plots of 500-nm tracer uptake and Ag presentation, as assessed by 25D1.16 staining for H-2K^b:SIINFEKL 72 hours after Ag-MP injection into naïve skin or day 7 B16F10 melanomas (I). Number (J) and MFI (K) of cells presenting SIINFEKL Ag in the TdLN relative to the naïve skin dLN at 72 hours after Ag-MP administration as determined by 25D1.16 staining for H-2K^b:SIINFEKL. (L) MP and H-2K^b:SIINFEKL (SIINFEKL) positivity among dLN B cells and dDCs in naïve and tumor contexts 72 hours after Ag-MP administration. * indicates significance by one-way ANOVA; n = 5 to 6 animals (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.005, **** indicates P < 0.0001); (D) to (H) are representative of at least two independent studies.