Search

In this report, we describe the fluorescent labeling of bacterial polysaccharides (Escherichia coli O86:B7, Escherichia coli O19ab, Pseudomonas aeruginosa O10a10b, and Shigella flexneri 2b) at the "natural" amino group of their phosphoethanolamine moiety. Two protocols for labeling are compared: 1) on a scale of a few mg of the polysaccharide, with a dialysis procedure for purification from excessive reagents; and 2) on a scale of 0.1 mg of the polysaccharide, with a simple precipitation procedure instead of dialysis. The microscale version is sufficient for comfortable cytofluorometric analysis. The resulting probes were found to specifically bind to human dendritic cells in a dose-dependent manner. The used limited set of polysaccharides did not allow us even to get close to understanding which dendritic cell-associated lectins and which cognate polysaccharide epitopes are involved in recognition, but the proposed microscale protocol allows to generate a library of fluorescent probes for further mapping of the polysaccharide specificity of the dendritic cells.

Modification of vaccine carriers by decoration with glycans can enhance binding to and even targeting of dendritic cells (DCs), thus augmenting vaccine efficacy. To find a specific glycan-"vector" it is necessary to know glycan-binding profile of DCs. This task is not trivial; the small number of circulating blood DCs available for isolation hinders screening and therefore advancement of the profiling. It would be more convenient to employ long-term cell cultures or even primary DCs from murine blood. We therefore examined whether THP-1 (human monocyte cell line) and DC2.4 (immature murine DC-like cell line) could serve as a model for human DCs. These cells were probed with a set of glycans previously identified as binding to circulating human CD14 low/- CD16 + CD83 + DCs. In addition, we tested a subpopulation of murine CD14 low/- CD80 + СD11c + CD16 + cells reported as relating to the human CD14 low/- CD16 + CD83 + cells. Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ bound to both the cell lines and the murine CD14 low/- CD80 + СD11c + CD16 + cells. Primary cells, but not the cell cultures, were capable of binding GalNAcα1-3Galβ (A di ), the most potent ligand for binding to human circulating DCs. In conclusion, not one of the studied cell lines proved an adequate model for DCs processes involving lectin binding. Although the glycan-binding profile of BYRB-Rb (8.17)1Iem mouse DCs could prove useful for assessing human DCs, important glycan interactions were missing, a situation which was aggravated when employing cells from the BALB/c strain. Accordingly, one must treat results from murine work with caution when seeking vaccine targeting of human DCs, and certainly should avoid cell lines such as THP-1 and DC2.4 cells.

Dendritic cells (DCs) play crucial roles in innate and adaptive immune response, for which reason targeting antigen to these cells is an important strategy for improvement of vaccine development. To this end, we explored recognition of DCs lectins by glycans. For selection of the glycan "vector", a library of 229 fluorescent glycoprobes was employed to assess interaction with the CD14 low/- CD16 + CD83 + blood mononuclear cell population containing the DCs known for their importance in antigen presentation to T-lymphocytes. It was found that: 1) the glycan-binding profiles of this CD14 low/- CD16 + CD83 + subpopulation were similar but not identical to DCs of monocyte origin (moDCs); 2) the highest percentage of probe-positive cells in this CD14 low/- CD16 + CD83 + subpopulation was observed for GalNAcα1-2Galβ (A di ), (Neu5Acα) 3 and three mannose-reach glycans; 3) subpopulation of CD14 low/- CD16 + cells preferentially bound 4'-O-Su-LacdiNAc. Considering the published data on specificity of DCs binding, the glycans showing particular selectivity for the CD14 low/- CD16 + CD83 + cells are likely interacting with macrophage galactose binding lectin (MGL), siglec-7 and dectin-2. In contrast, DC-SIGN is not apparently involved, even in case of mannose-rich glycans. Taking into consideration potential in vivo competition between glycan "vectors" and glycans within glycocalyx, attempting to target vaccine to DCs glycan-binding receptors should focus on A di and (Neu5Acα) 3 as the most promising vectors.