Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application

Dendritic Cell (DC)-based vaccination approaches in man require a reproduci ble DC generation method that can be performed in conformity with GMP (Good Manufacturing Practice) guidelines and that circumvents the need for multi ple blood drawings to generate DC. To this end we modified our previously d escribed method to generate mature DC from CD14+ monocytes by a two step me thod (priming in GM-SF + IL-4 followed by maturation in monocyte conditione d medium) for use with leukapheresis products as a starting population. Sev eral adaptions were necessary. We established, for example, a modified adhe rence step to reliably enrich CD14+ DC precursors from apheresis mononuclea r cells. The addition of GM-CSF + IL-4 at the onset of culture proved disad vantageous and was, therefore, delayed for 24 h. DC development from aphere sis cells occurred faster than from fresh blood or buffy coat, and was comp lete after 7 days. Monocyte conditioned medium when added on day 6 resulted in fully mature and stable DC (veiled, highly migratory and T cell sensiti zing cells with a characteristic phenotype such as 85% CD83+, p55/fascin+, CD115/M-CSF-R-, CD86+) already after 24 h. The mature DC progeny were shown to remain stable and viable if cultured for another 1-2 days in the absenc e of cytokines, and to be resistant to inhibitory effects of IL-10. Freezin g conditions were established to generate DC from frozen aliquots of PBMC o r to freeze mature DC themselves for later use. The approach yields large n umbers of standardized DC (5-10 X 10(8) mature CD83+ DC/leukapheresis) that are suitable for performing sound DC-based vaccination trials that can be compared with each other. (C) 1999 Elsevier Science B.V. All rights reserve d.