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Notes: Functional dendritic cells (DC) are professional antigen-presenting cells (APC) and can be generated in vitro from healthy as well as from leukaemic cells from AML patients giving rise to APC of leukaemic origin presenting leukaemic antigens. In a comparative methodological analysis of 50 AML samples, we could already show that leukaemia-derived DC can regularly be generated under serum-free culture conditions. In this study, we describe the generation and characterization of DC from different mononuclear cell (MNC) fractions from 24 myelodysplastic syndrome (MDS) patients under those different serum-free culture conditions, determine the optimal culture conditions and compare the results with that from 23 healthy donors. In parallel cultures, we compared DC harvests after 7- or 14-day culture, with total or adherent MNC or T-cell-depleted MNC or PB or BM–MNC, thawn or fresh MNC, in Xvivo or CellGro serum-free media, ±10% autologous plasma or ±FL. In detail, we could show that MDS–DC harvests compared to healthy DC were higher after 10- to 14-day culture; total or adherent PB or BM–MNC fractions yield comparable DC counts; however, from MACS-depleted MNC fractions or thawn MNC lower DC counts can be generated. Whereas the addition of FL increases the DC harvest, the addition of autologous plasma in many cases has inhibitory influence on DC maturation, CellGro and Xvivo media yield comparable DC counts. Optimal harvest of vital and mature DC from MDS samples was obtained with a GM-CSF, IL-4, FL and TNF-α containing serum-free Xvivo medium after 10–14 days of culture (18/26% DC; 54/64% vital DC; 59/51% mature DC were generated from MDS/healthy MNC samples). Surface marker profiles (e.g. costimulatory antigen expression) of DC obtained from MDS samples were comparable with that of healthy DC. The leukaemic derivation of MDS–DC was demonstrated by the persistence of the clonal cytogenetic aberration in the DC or by coexpression of leukaemic antigens on DC. Autologous T-cell activation of leukaemia-derived DC was demonstrated in cases with MDS. Autologous T cells proliferate and upregulate DC-contact-relevant antigens. We are the first who demonstrate that the generation of leukaemia-derived DC is feasible not only in AML but also in MDS under serum-free culture conditions giving rise to DC with comparable characteristics as healthy DC and offering an antileukaemia-directed immunotherapeutical vaccination strategy in AML and MDS.

Notes: Functional dendritic cells (DC) are professional antigen-presenting cells (APC) and can be generated in vitro from healthy as well as from leukaemic cells from acute myeloid leukemia (AML) patients giving rise to APC of leukaemic origin-presenting leukaemic antigens. We describe the generation and characterization of DC from different mononuclear cell (MNC) fractions from 50 AML patients under different serum-free culture conditions, determine the optimal culture conditions and compare the results with that from 23 healthy donors. In parallel cultures, we compared DC harvests after 7- or 14-day culture, with total or adherent MNC or T-cell depleted MNC or peripheral blood (PB) or bone marrow-MNC (BM–MNC), thawn or fresh MNC, in Xvivo or CellGro serum-free media, ±10% autologous plasma or ±FL. In detail, we could show that AML–DC harvests were higher after 10–14 days culture (healthy DC: 7 days); total or adherent PB or BM–MNC fractions yield comparable DC counts, however, from magnetic cell sorting (MACS)-depleted MNC fractions or thawn MNC lower DC counts can be generated. Whereas the addition of FL increases the DC harvest, the addition of autologous plasma in many cases has inhibitory influence on DC maturation. CellGro and Xvivo media yield comparable DC counts. Optimal harvest of vital and mature DC from AML samples was obtained with a granulocyte/macrophage-colony stimulating factor, interleukin-4, FL and tumour necrosis factor-α-containing serum-free Xvivo medium after 10–14 days of culture (36/26% DC; 38/64% vital DC; 46/51% mature DC were generated from AML/healthy MNC samples). Surface marker profiles (e.g. costimulatory antigen expressing) of DC obtained from AML samples were comparable with that of healthy DC. The leukaemic derivation of AML–DC was demonstrated by the persistence of the clonal cytogenetic aberration in the DC or by coexpression of leukaemic antigens on DC. Autologous T-cell activation of leukaemia-derived DC was demonstrated in cases with AML. Autologous T cells proliferate and upregulate DC-contact-relevant antigens. We demonstrate that the generation of leukaemia-derived DC is feasable in AML under serum-free culture conditions giving rise to DC with comparable characteristics as healthy DC and offering an anti-leukaemia-directed immunotherapeutical vaccination strategy in AML.