Gangliosides inhibit the development from monocytes to dendritic cells

Abstract

Dendritic cell (DC) development and function is critical in the initiation phase of any antigen-specific immune response against tumours. Impaired function of DC is one explanation as to how tumours escape immunosurveillance. In the presence of various soluble tumour-related factors DC precursors lose their ability to differentiate into mature DC and to activate T cells. Gangliosides are glycosphingolipids shed by tumours of neuroectodermal origin such as melanoma and neuroblastoma. In this investigation we address the question of whether gangliosides suppress the development and function of monocyte-derived DC in vitro. In the presence of gangliosides, the monocytic DC precursors showed increased adherence, cell spreading and a reduced number of dendrites. The expression of MHC class II molecules, co-stimulatory molecules and the GM-CSF receptor (CD116) on the ganglioside-treated DC was significantly reduced. Furthermore, the function of ganglioside-treated DC was impaired as observed in endocytosis, chemotactic and T cell proliferation assays. In contrast to monocytic DC precursors, mature DC were unaffected even when higher doses of gangliosides were added to the culture. With regard to their carbohydrate structure, five different gangliosides (GM2, GM3, GD2, GD3, GT1b), which are typically shed by melanoma and neuroblastoma, were tested for their ability to suppress DC development and function. Suppression was induced by GM2, but not by the other gangliosides. These data suggest that certain gangliosides impair DC precursors, implying a possible mechanism for tumour escape.

Fig. 1

Ganglioside-mediated changes in the morphology and adherence of monocytic DC-precursors. Monocytes were isolated from PBMC and incubated in X-vivo/0·5% autologous serum supplemented with IL-4 and GM-CSF as described in the Methods. Gangliosides were added on day 1 of culture and remained in culture. Photos (200×) were taken at different time-points. (a) Control culture on day 2 without gangliosides; (b) culture on day 2, 20 h after incubation with a ganglioside mixture (40 µg/ml); (c) culture on day 2, 20 h after incubation with GD2 (20 µm); (d) culture on day 2, 20 h after incubation with GM2 (20 µm); (e) crystal violet staining of adherent cells after 20 h of incubation without gangliosides; (f) crystal violet staining of adherent cells after 20 h of incubation with gangliosides (40 µg/ml); (g) adherence assay with crystal violet staining after 20 h of incubation with or without gangliosides. O.D., optical density.

Fig. 2

Surface expression of mature DC with and without ganglioside pretreatment. Dendritic cells were cultured with (black line) or without gangliosides (40 µg/ml) (grey filled curve) on day 1, stimulated with inflammatory cytokines on day 7 and analysed by flowcytometry on day 9 of culture. Median MFI of 12 experiments for HLA-DR was 330 ± 109 versus 210 ± 72*, for CD80 22 ± 12 versus 18 ± 12 (77% versus 50% pos. cells*), for CD86 72 ± 20 versus 59 ± 20, for CD116 113 ± 41 versus 63 ± 28*, for CD40 77 ± 24 versus 50 ± 14, for CD1a 38 ± 25 versus 28 ± 2 (48·5% versus 26% pos. cells), for CD83 18 ± 7 versus 17 ± 10 (53% versus 42% pos. cells, P = 0·068). * Indicates statistical significance (P = 0·001). Viability was >90% in both groups. The graphs represent one of 12 independent experiments.

Fig. 3

Presence of gangliosides during early DC differentiation results in an impaired ability to stimulate T cells. DC were cultured with and without gangliosides from day 1 onwards. Immature (7-day culture) or mature (9-day culture with cytokine-induced maturation) DC were used as stimulator cells and coincubated with 2 × 10⁵ allogeneic CD4⁺ T cells for 5 days. (a,b) Immature (a) or mature (b) DC treated either with the ganglioside mixture (40 µg/ml) (○), with GM2 (20 µm)(n) or without gangliosides (▪). (♦), ceramide control (20 µm). (c) Mature DC treated with gangliosides (40 µg/ml) either on day 1 of culture (○) or on day 8 of culture (▴). (▪), untreated control. Background activity for T cells alone and DC alone was below 1000 cpm. The data represent one of four independent experiments.

Fig. 4

Ganglioside-treated DC have a reduced endocytotic activity. Cells were incubated with gangliosides on day 1 of DC culture (40 µg/ml). On day 6, immature DC were harvested and washed. 2 × 10⁵ DC were incubated with FITC-dextran (MW 30000) for 30–210 min at 37°. Controls were incubated on ice. Cells were washed and fixed on ice before analysis by flowcytometry. •, DC at 37°, gangliosides added on day 1; ▪, control DC at 37° without ganglioside pretreatment; s, control DC at 0°, gangliosides added on day 1; □, control DC at 0°, without gangliosides. The data represent one of five experiments.

Fig. 5

Ganglioside-treated DC show a reduced response to MIP-1α. DC were cultured with GM-CSF and IL-4. On days 6–7 immature DC were harvested and washed. 1 × 10⁵ DC were incubated in the upper chamber of a transwell system with 100 ng/ml MIP-1α in the lower chamber. After 4 h the inserts were removed, and the cells in the lower chamber were harvested completely and absolute cell counts were determined by flowcytometry using fluorescent microspheres as described. (a) Left bar: control DC without pretreatment of gangliosides; middle: DC with ganglioside pretreatment (40 µg/ml) on day 1; right bar: spontaneously migrated DC without MIP-1α. (b) Untreated DC were harvested on day 6 and incubated with different concentrations of the ganglioside mixture in the upper chamber of the transwell system as indicated. All groups were performed and analysed in duplicate, and absolute cell counts were calculated and normalized to 10⁵ input cells as described in the Methods. The data represent one of three independent experiments.