Lewis X oligosaccharides targeting to DC-SIGN enhanced antigen-specific immune response

Abstract

Dendritic cell-specific intercellular-adhesion-molecule-grabbing non-integrin (DC-SIGN) is a potential target receptor for vaccination purposes. In the present study, we employed Lewis X (Le(x)) oligosaccharides, which mimic natural ligands, to target ovalbumin (OVA) to human dendritic cells (DCs) via DC-SIGN, to investigate the effect of this DC-SIGN-targeting strategy on the OVA-specific immune response. We demonstrated that Le(x) oligosaccharides could enhance the OVA-specific immune response as determined by enzyme-linked immunospot assay (ELISPOT), intracellular interferon-gamma staining and (51)Cr-release assay. An almost 300-fold lower dose of Le(x)-OVA induced balanced interferon-gamma-secreting cells compared to OVA alone. Furthermore, secretion of interleukin-10, a reported mediator of immune suppression related to DC-SIGN, was not increased by Le(x)-OVA, either alone or together with sCD40L-stimulated groups. A blocking antibody against DC-SIGN (12507) reduced the numbers of interferon-gamma-secreting cells during Le(x)-OVA stimulation, yet it did not prevent Le(x) oligosaccharides from promoting the secretion of interleukin-10 that was induced by ultra-pure lipopolysaccharide. These results suggested that the strategy of DC-SIGN targeting mediated by Le(x) oligosaccharides could promote a T-cell response. This DC-targeting may imply a novel vaccination strategy.

Figure 1

Preparation of Le^x-OVA. (a) OVA, SA and SA-OVA run on SDS–PAGE. The electrophoretic behaviour of the protein (SA-OVA) treated with a chemical cross-linking reagent had changed and displayed a smear band in contrast to the native OVA. Residual OVA (runs a little faster than the native OVA) and SA, in the lane marked SA-OVA, were observed. (b) Immunoblotting with anti-OVA antibody to detect OVA in the SA-OVA conjugates. (c) Immunoblotting with anti-SA antibody to detect SA in the SA-OVA conjugates. (d) The standard curve used for quantification of SA-OVA conjugates in the Bradford micro-assay. (e) SA-OVA conjugates, SA (positive control) and OVA (negative control) were used to detect the specific biotin-binding capacity. (f) Capacity of SA-OVA conjugates to bind Le^x-PAA-biotin.

Figure 2

Le^x-OVA was specifically targeted to DC-SIGN. (a) Left panel: expression of DC-SIGN-EGFP (green) fusion protein was assayed on Cos-7 cell lines by confocal microscopy. The fusion protein was stained with anti-DC-SIGN(Cy3). Right panel: expression of DC-SIGN-EGFP (green) fusion protein on K562 cell lines was detected by double colour in a fluorescence-activated cell sorter assay. The phycoerythrin (PE) signal was from anti-DC-SIGN or anti-CD33 (a molecule expressed on the K562 cell lines used to gate). (b) Le^x-PAA-biotin uptake by K562-ED. The cell was stained with streptvidin-Cy3 for localization of Le^x. (c) Le^x-OVA uptake by K562-ED. The cell was stained with rabbit-derived anti-OVA and goat anti-rabbit IgG(Cy3) for localization of OVA. (d) Le^x-OVA was not taken up by K562 cell lines expressing EGFP in 15 min. (e) Le^x-OVA was not taken up by K562 cell lines in 15 min. (f) Le^x-OVA uptake by the primary monocyte-derived DCs. The expression of DC-SIGN on the DCs is also shown.

Figure 3

Antigen-specific IFN-γ-producing cells increased after DC-SIGN targeting via Le^x oligosaccharides. (a) Effector T cells were generated from in vitro sensitization for 14 days and were used to detect the numbers of IFN-γ-secreting cells. DCs loaded with OVA and matured with sCD40L were added to wells to be a specific target; 40 μg/ml anti-DC-SIGN was used for blockade in the 10 μg/ml Le^x-OVA stimulation group. Data are representative of three independent experiments performed in triplicate with similar results. (b) Effector T cells were stimulated and assayed as above, except without specific target cells added into the ELISPOT assay wells. Controls (not shown): lane 1, positive control (autologous PBMC were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml)): 140 ± 47·15 spots/10⁵ PBMC; lane 2, negative control (autologous PBMC): 0–2 spots/10⁵ PBMC; lane 3 negative control (autologous PBMC with OVA 20 μg): 4–6 spots/10⁵ PBMC. Data are representative of three independent experiments performed in triplicate with similar results. (c) The antigen-specific IFN-γ-producing CD8⁺ T cells were observed. Effector T cells were stimulated as above; 40 μg/ml anti-DC-SIGN was used for blockade in the 10 μg/ml Le^x-OVA stimulation group. Data are representative of four independent experiments performed with similar results. Statistical significance of the differences (P < 0·01) is between 0·01 and 1 μg/ml of Le^x-OVA and other controls (SA-OVA and OVA alone) stimulation groups. Asterisks indicated statistical significance (P < 0·01, anova).

Figure 4

Le^x-OVA enhanced antigen-specific cytotoxic response. (a) The expression of OVA was displayed after transfection with OVA gene plasmid for 24 hr in the MCF-7. (b and c) Specific lysis was measured with ⁵¹Cr-release assay. Effector T cells were stimulated with autologous DCs (irradiated) loaded with various antigens at doses of 10 μg/ml for 4 weeks. Then the effector T cells were used to assay antigen-specific lysis. Data are representative of three independent experiments performed in triplicate with similar results. Asterisks indicate statistical significance (P < 0·01, anova).

Figure 5

Le^x-OVA together with ultra-pure LPS instead of sCD40L promoted the secretion of IL-10. (a) Le^x-OVA (10 μg/ml) together with LPS promoted the secretion of IL-10 on DCs. The supernatants of DCs treated differently were used to assay levels of IL-10 and IFN-γ. Data are representative of three experiments. (b) Secretion of IL-10 was examined on the supernatants of DCs treated by Le^x-OVA together with graded doses of ultra-pure LPS. The block antibody (40 μg/ml) could not prevent but enhanced the secretion of IL-10. Data are representative of three independent experiments in triplicate. (c) As above, secretion of IL-6 was assayed. Data are representative of three independent experiments in triplicate. (d) Le^x oligosaccharides monomers (10 μg/ml) together with ultra-pure LPS (10 ng/ml) could not increase the level of IL-10. However, the blocking antibody (20 μg/ml) still enhanced the secretion of IL-10. Data are representative of three independent experiments. (e) Le^x-OVA engagement to DC-SIGN did not decrease the expression of CD86 by flow cytometry. Data are representative of two experiments. Asterisks indicate statistical significance (P < 0·01, Student's t-test).