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. 2020 Feb 25;11:305. doi: 10.3389/fimmu.2020.00305

Figure 1.

Figure 1

Ligand-specific binding of terminal GalNAc glycodendrimers to MGL enhances LPS-induced IL-10 production in human moDCs. (A) Binding of titrated αGalNAc (blue squares) or GalNAcβ1-4Gal (orange triangles) glycodendrimers (x-axis) to the plant lectins Helix pomatia agglutinin (HPA) and Vicia villosa lectin (VVL) (upper panels), and to MGL-Fc and MGL H259T-Fc (lower panels). Binding was measured using an ELISA-based assay and was detected at an optical density of 450 nm. Binding of control glycodendrimers was used as a negative control (black circles). (B) Terminal glycan structures of the generated control, αGalNAc and GalNAcβ1-4Gal glycodendrimers. *The ring form of galactose, used to generate the control glycodendrimers, was opened up during glycodendrimer generation upon reductive amination. Galactose (yellow circle), N-acetyl-D-galactosamine (yellow square). (C) Unstimulated monocyte-derived dendritic cells (moDCs) were analyzed for MGL expression (black line) after 4 days of differentiation using flow cytometry. Isotype control is depicted in gray. One representative of three independent experiments is shown. (D) moDCs were stimulated overnight with control (white), αGalNAc (blue), or GalNAcβ1-4Gal (orange) glycodendrimers in the absence or presence of LPS after which IL-10 production was measured in the supernatants. Relative secretion compared to moDCs stimulated with LPS and control glycodendrimers is depicted (set to 100%). Error bars represent standard deviation of six or nine independent experiments for moDCs and LPS-stimulated moDCs, respectively. (E) moDCs were stimulated overnight with LPS and control (white), αGalNAc (blue), or GalNAcβ1-4Gal (orange) glycodendrimers in the absence or presence of anti-MGL blocking antibodies. IL-10 production was measured in the supernatants. Relative secretion compared to moDCs stimulated with control glycodendrimers is depicted (set to 100%). (F) Monocytes (n = 2) were stimulated overnight with the three different glycodendrimers in the absence or presence of LPS as described for moDCs (D). (nd = not detectable).