Column-free magnetic enrichment of pDCs yields significant contamination with DNA-containing dead cells that affect the activation status of the isolated cells

Abstract

Abstract Despite their role in the induction of adaptive immune responses, plasmacytoid dendritic cells (pDC) play also a crucial role in innate immune defense by recognizing pathogen derived molecular patterns. Unmethylated CpG motifs of bacterial DNA bind specifically endosomal and lysosomal TLR9 in pDCs, thereby inducing secretion of type I IFN or proinflammatory cytokines and classical DC maturation, respectively. However, TLR9 also recognizes CpG motifs in vertebrate DNA of damaged cells as danger-associated molecular patterns (DAMPs) that activate pDCs. For in-vitro research pDCs may be isolated by indirect magnetic cell separation using either Miltenyi Biotec’s column-based or a column-free system from other suppliers. Here we describe the influence of the isolation method on the functionality of the purified pDCs. In addition to the significantly better separation performance, our column-based method enriched 10- and 30-fold less DNA-containing dead cells and debris, respectively. Although both methods yielded pDCs with a comparable phenotype instantly after isolation, after 24h of culture without stimulation pDCs isolated with the column-free system appeared pre-activated as demonstrated by elevated expression of CD80, −83, −86 and HLA-DR and by significantly higher basal IFN-α secretion. Moreover, upon stimulation with CpG-B pDCs isolated with the column-free method induced extraordinary high amounts of IFN-α, whereas pDCs isolated with our system exhibited normal responses to CpG-A and CpG-B. These results clearly demonstrate that unspecific enrichment of dead cells and debris may significantly influence the activation status of the isolated pDCs and therefore affect the results of downstream applications.