DCs matured for 2 days with LPS express high levels of O-linked polysialic acid.

(A) mRNA levels of sialyltransferase genes of iDCs or mDCs triggered for 1 day with LPS were determined by quantitative RT-PCR. Averages of 6 donors are depicted. (B) Expression levels of ST8Sia IV in myeloid cells as measured by quantitative RT-PCR (N = 3). (C) Time course of ST8Sia IV expression after LPS triggering, as analyzed by quantitative RT-PCR. One representative donor is shown. (D) Expression levels of polySia by iDCs (dotted line) and DCs matured for 2 days with LPS (bold line) or 1 day with LPS (thin line), as measured by flow cytometry with moAb 735. Results are representative of 5 independent experiments. (E) Flow cytometric analysis of the glycosylation of mDCs treated with kifunensine to block N-glycan synthesis, or with benzyl-α-GalNAc to block O-glycan synthesis using the lectins Con-A (high-mannose structures) or HPA (Tn antigen). Depicted are the relative fluorescence values of kifunensine or benzyl-α-GalNAc treated cells compared to untreated cells. Relative fold increase in expression of polySia after maturation (MFI mDC/MFI iDC) was analysed with mAb 735 (polySia). One representative donor is shown. (F, G) 50% of PolySia expressed by mDCs is removed with neuraminidase and Endo-neuraminidase treatment as analyzed by flow cytometry with moAb 735. One out of 3 independent experiments is shown.