Several glycoconjugates are involved in the immune response. Sialic acid is frequently the glycan terminal sugar and it may modulate immune interactions. Dendritic cells (DCs) are antigen-presenting cells with high endocytic capacity and a central role in immune regulation. On this basis, DCs derived from monocytes (mo-DC) are utilised in immunotherapy, though many features are ignored and their use is still limited. We analyzed the surface sialylated glycans expressed during human mo-DC generation. This was monitored by lectin binding and analysis of sialyltransferases (ST) at the mRNA level and by specific enzymatic assays. We showed that alpha 2-3-sialylated O-glycans and alpha 2-6- and alpha 2-3-sialylated N-glycans are present in monocytes and their expression increases during mo-DC differentiation. Three main ST genes are committed with this rearrangement: ST6Gal1 is specifically involved in the augmented alpha 2-6-sialylated N-glycans; ST3Gal1 contributes for the alpha2-3-sialylation of O-glycans, particularly T antigens; and ST3Gal4 may contribute for the increased alpha2-3-sialylated N-glycans. Upon mo-DC maturation, ST6Gal1 and ST3Gal4 are downregulated and ST3Gal1 is altered in a stimulus-dependent manner. We also observed that removing surface sialic acid of immature mo-DC by neuraminidase significantly decreased its endocytic capacity, while it increased in monocytes. Our results indicate the STs expression modulates the increased expression of surface sialylated structures during mo-DC generation, which is probably related with changes in cell mechanisms. The ST downregulation after mo-DC maturation probably results in a decreased sialylation or sialylated glycoconjugates involved in the endocytosis, contributing to the downregulation of one or more antigen-uptake mechanisms specific of mo-DC.

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SUMMARYDendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric ow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic pro®le and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II /lineage À were identi®ed on the basis of their distinct phenotypic characteristics: one DC subset was CD33 strong and CD123 dim (0´16 6 0´06% of the PB nucleated cells and 55´9 6 11´9% of all PB DC) and the other, CD33 dim and CD123 strong (0´12 6 0´04% of PB nucleated cells and 44´53 6 11´5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor a-chain (CD126) and for CD38. In contrast, the CD123 strong /CD33 dim DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33 strong /CD123 dim DC are able to produce signi®cant amounts of in¯ammatory cytokines, such as IL-1b (97 6 5% of positive cells), IL-6 (96 6 1´1% of positive cells), IL-12 (81´5 6 15´5% of positive cells) and tumour necrosis factor-alpha (TNF-a) (84 6 22´1% of positive cells) as well as chemokines such as IL-8 (99 6 1% of positive cells), the functional ability of the CD123 strong /CD33 dim DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in their immunophenotype but also in their functionality, as regards cytokine production.

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