Fusion-gene cloning and plasmid constructions. (A) Genes for mature sequences of murine MIP3α or the viral chemokine MC148 were fused in-frame with DNA encoding either human gp100 or mouse OFA-iLRP. Control constructs encoded gp100 alone or fused with the mutant nonactive chemokines (MC148-D-hgp100 and MC148-D-mOFA). To enable purification and detection, c-myc and His peptide tags were fused to fusion constructs (Tag). A spacer fragment was inserted between chemoattractant and antigen moieties to enable proper folding of the protein. (B) OFA and gp100 fusion proteins with MIP3α-mOFA and MIP3α-gp100 (50 μg/mL) are bound with CCR6 on the surface of iDCs at 4°C and are induced to internalize at 37°C. Histograms of gated cells (R1 in forward scatter/side scatter plot) are presented. Representative data yielded similar results in 3 independent experiments. (C) Optical z-sections (0.4 μm) were taken of B6/129 macrophages loaded with MIP3α-gp100 at 4°C for 30 minutes and then transferred to 37°C for 0 or 10 minutes before fixation. Cells were stained with either rabbit anticlathrin (H-300) or antiproteasome 20S mAb (red) and mouse antimyc mAb (green) to detect fusion proteins with overlay of both channels showing colocalization in yellow. No significant binding was detected when cells were incubated with mutant chemokine fusion (MC148D-gp100; data not shown). This experiment was repeated 4 times with similar results and a representative experiment is shown.