Moribund Blood Monocytes: Apoptosis Limits the Yield of Monocyte Derived Dentritic Cells from Apheresis Samples.

Cells of the monocyte/macrophage/dendritic cell (DC) lineage play a central role in inflammation and form the bridge between innate and acquired immunity. In recent years monocytes also found special attention as source of precursor cells for the generation of monocyte derived dendritic cells for immunotherapy. However, monocytes circulate in the blood stream only temporarily with a half life of about 24 h and die spontaneously from apoptosis in the absence of specific survival signals. When we investigated the survival of blood monocytes isolated by CD14 positive selection from apheresis samples and cultivated in the presence of GM-CSF/IL4, we observed a loss of viability of about 66% of the cells within 48h. This would be consistent with the view that about 80% of the monocytes are bound to die at the time of isolation. Detailed flow cytometric analysis revealed that loss of vital cells is accompanied by the appearance of a CD14-negative population, which stained positive for annexin V. To analyse the fate of these cell populations CD14-positive and CD14-negative cells were separated by cell sorting at day 1 and day 2. Flow cytometry and electron microscopy identified predominantly dying cells in the CD14-negative population, whereas a large fraction of the CD14-positive population survived. The CD14-positive cells can be differentiated with an efficiency of 64%–91% to mature DC after cultivation under DC generating conditions; these include the presence of GM-CSF and IL4 and addition of a maturation mix (TNFa, IL1b, IL6 and PGE2) on day 5. DC generated from sorted CD14-positive cell populations were qualitatively and quantitatively comparable to DC from unsorted monocytes. To study the genes involved we analyzed the transcriptome on each cultivation day using Affymetrix HG-U133A DNA-Arrays. During the first 24h, the expressions of about 4700 genes were regulated. About 3000 genes were up regulated and about 1700 genes were down regulated. Especially, we focused on the expression of genes involved in the apoptosis pathway during the early phase of cultivation. Among apoptosis related genes caspase 3, a central player in natural apoptosis of monocytes, was up regulated on day 1. In summary these data suggest that apoptosis limits the yield of monocyte derived DC and that cultivation in the presence of caspase inhibitors or survival signals could improve the efficiency of generation of monocyte derived dendritic cells.