SUMMARYWe have studied the effect of mycophenolate mofetil (MMF), a new drug used in prevention of transplant rejection, on differentiation, maturation and allostimulatory activity of human monocyte-derived dendritic cells (MDDC). MDDC were generated in vitro with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 in the presence or absence of MMF. MMF reduced the number of immature MDDC in culture, dose-dependently, by inducing apoptosis and inhibited their stimulatory activity on allogeneic lymphocytes. These changes correlated with down-regulation of costimulatory and adhesion molecules such as CD40, CD54, CD80 and CD86. No differences were observed in mannose receptor (MR)-mediated endocytosis, measured by the uptake of fluorescein isothiocyanate (FITC)-dextran. MDDC differentiated in the presence of MMF showed significantly reduced maturation upon stimulation with lipopolysaccharide, as judged by lower expresson of CD83 and co-stimulatory molecules, lower production of tumour necrosis factor (TNF)-a , IL-10, IL-12 and IL-18 as well as lower stimulation of alloreactive T cells including naive CD4 + CD45RA + T cells. In contrast, MDDC matured in the presence of MMF showed a more marked decrease in the FITC-dextran uptake than mature MDDC cultivated without MMF and the phenomenon correlated with downregulation of the MR expression. These results suggest that MMF impairs differentiation, maturation and function of human MDDC in vitro , which is an additional mechanism of its immunosuppressive effect.

Several laboratories have developed culture systems that allow the generation of large numbers of human dendritic cells (DC) from monocytes using granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-4 (IL-4). In this work we provided evidence that GM-CSF (100 ng/ml) in combination with a low concentration of IL-4 (5 ng/ml) was efficient in the generation of immature, non-adherent, monocyte-derived DC as the same concentration of GM-CSF, and ten times higher concentration of IL-4 (50 ng/ml). This conclusion was based on the similar phenotype profile of DC, such as the expression of CD1a, CD80, CD86, and HLA-DR, down-regulation of CD14, and the absence of CD83, as well as on their similar allostimulatory activity for T cells. A higher number of cells remained adherent in cultures with lower concentrations of IL-4 than in cultures with higher concentrations of the cytokine. However, most of these adherent cells down-regulated CD14 and stimulated the proliferation of alloreactive T cells. In contrast, adherent cells cultivated with GM-CSF alone were predominantly macrophages, as judged by the expression of CD14 and the inefficiency to stimulate alloreactive T cells. DC generated in the presence of lower concentrations of IL-4 had higher proapoptotic potential for the Jurkat cell line than DC differentiated with higher concentrations of IL-4, suggesting their stronger cytotoxic, anti-tumor effect.

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