Synthesis of SulfoCy5‐ and SulfoCy7‐αGalCer Probes as Chemical Tools for Investigating the Uptake of Liposomal αGalCer Conjugates by Antigen Presenting Cells

Abstract α‐Galactosylceramide (αGalCer) is a synthetic glycolipid that, when loaded into the CD1d receptor of antigen presenting cells, activates iNKT cells to coordinate the generation of both innate and adaptive immune responses. In vaccines, the αGalCer adjuvant is generally delivered in liposomes, but interestingly, little is known about the intracellular fate of αGalCer‐antigen conjugates resulting in the absence of clear “design rules” for this type of compounds and their formulations. To unravel the uptake mechanism of liposomal αGalCer conjugates and understand the fate of its components, we synthesized sulfoCy7‐αGalCer 1 and sulfoCy5‐αGalCer 2 . The fluorescent probes were obtained from a linker‐functionalized αGalCer, which was then modified with sulfoCy7/sulfoCy5. After formulation in liposomes, the cellular presentation was studied in a murine dendritic cell line using fluorescence imaging and flow cytometry. Imaging indicates that liposomes loaded with sulfoCy5‐αGalCer are taken up by DC2.4 cells in a heterogeneous and concentration‐dependent manner. Additionally, fluorescence antibody staining confirms that αGalCer is ultimately presented by CD1d receptors, but importantly is cleaved from sulfoCy5 before reaching the cell surface. These results validate our synthetic probes as useful in mechanistic studies of the uptake of αGalCer‐antigen conjugates as shown here for DC2.4 cells.