FIGURE 5.

Inhibition of O-glycosylation reverses galectin-1 inhibition of iDC migration and prevents CD43 clustering on iDCs in the presence of galectin-1. A, iDCs were treated with 2 mm Bn-α-GalNAc (iDC + Bn-α-GalNAc) or with buffer control (iDC). Expression of CD43 bearing core 2 O-glycans was analyzed by flow cytometry with the mAb 1D4. Filled histogram is the isotype control. Treatment with Bn-α-GalNAc decreased binding of mAb 1D4. B, in vitro migration assays were performed as described in Fig. 3C. Bn-α-GalNAc-treated and untreated iDCs were migrated across Matrigel^TM plus LECs in the presence or absence of recombinant galectin-1 in Matrigel^TM. Inhibition of O-glycan elongation reversed the inhibitory effect of galectin-1 on iDC migration. Results are shown as % migration in the presence of galectin-1 in Matrigel^TM/% migration in the absence of galectin-1 in Matrigel^TM. Results are representative of three independent experiments. Three replicate samples ± S.D. are shown for each data point, *, p = 0.019. C, iDCs or tDCs were treated with recombinant galectin-1 for 1 h at 37 °C. After fixation, samples were processed for confocal microscopy using antibody against CD43 (mAb DF-T1) and FITC-coupled secondary antibody. Galectin-1 clustered CD43 on iDCs (white arrows) but not on tDCs. Nuclei were visualized with DAPI. Two different images from different experiments are shown for each condition. Scale bar, 5 μm. D, iDCs or tDCs were treated with 2 mm Bn-α-GalNAc before recombinant galectin-1 was added for 1 h at 37 °C. Samples were processed as above. Inhibition of O-glycan elongation with Bn-α-GalNAc abrogated galectin-1-induced clustering of CD43 on iDCs. Two different images from different experiments are shown for each condition. Scale bar, 5 μm.