We have previously deleted both endogenous copies of the clathrin heavy-chain gene in the chicken pre B-cell-line DT40 and replaced them with clathrin under the control of a tetracycline-regulatable promoter (Tet-Off). The originally derived cell-line DKO-S underwent apoptosis when clathrin expression was repressed. We have also described a cell-line DKO-R derived from DKO-S cells that was less sensitive to clathrin-depletion. Here we show that the restriction of transferrin uptake, resulting in iron deprivation, is responsible for the lethal consequence of clathrin-depletion. We further show that the DKO-R cells have up-regulated an anti-apoptotic survival pathway based on the chemokine SDF-1 and its receptor CXCR4. Our work clarifies several puzzling features of clathrin-depleted DT40 cells and reveals an example of how SDF-1/CXCR4 signalling can abrogate pro-apoptotic pathways and increase cell survival. We propose that the phenomenon described here has implications for the therapeutic approach to a variety of cancers.

Cross-presenting CD8-+ conventional dendritic cells (cDCs) are important for the eradication of cancers and viral infections due to their ability to induce cytotoxic T lymphocyte (CTL) responses. Cross presenting DCs are a very rare subset and in the past, studies of these cells have been limited by their scarcity of specific cell surface markers. Therefore, methods for the detection and isolation of these cells were commonly based on a multitude of immunophenotypic criteria, such as the expression of CD11c and CD8- and the absence of CD3, CD4, SIRP-- and CD11b. Recently, it was demonstrated that crosspresenting cDCs in lymphoid and non-lymphoid tissues specifically express XCR1, which correlates with the ability to take up and cross-present exogenous antigens. Combining our recombinant REAfinity™ Anti-XCR1 mouse mAb with MACS® Technology based on UltraPure MicroBeads, we developed a new method for the fast and easy isolation of cross-presenting DCs. Using this method XCR1+ DCs can be routinely enriched with high recovery and purity without the need for time-consuming laborious flow sorting. This will facilitate the study of cross-presenting DC subset, which will ultimately help to develop new therapeutic strategies employing DCs in the future.

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.