Influence of NK cell magnetic bead isolation methods on phenotype and function of murine NK cells.

Natural killer (NK) cells are a promising tool for cell therapy due to their capacity to lyse tumor cells without prior activation and their influence on the innate as well as the adaptive immunity. To characterize distinct NK cell populations, it is important to find a reliable isolation method. We investigated separation methods for NK cell isolation by magnetic bead labeling. There are three commonly used different MACS protocols to isolate NK cells from murine spleen: CD49b (DX5) MicroBeads, the NK Cell Isolation Kit and a separation method which is based on a positive selection for NKp46 expressing cells. Interestingly, we found a significant difference of NK cell purities depending on the mouse strains and the purification protocol used. We observed a significantly higher level of purity and yield of NK cells by preparations from Balb/c splenocytes. In this study, we modified the negative selection protocol and adapted it to C57Bl/6 mice to obtain equal purity, viability and yields of NK cells in the different mouse strains. Moreover, we optimized the NKp46 NK cell selection method by addition of a B cell depletion step. To our knowledge, this is the first report that has directly compared and essentially modified the different NK cell purification strategies in parallel, both in C57Bl/6 and Balb/c mouse strains. Altogether, these results are a basic prerequisite for the further development of NK cell therapy protocols in murine in vivo models.

bZIP-Type transcription factors CREB and OASIS bind and stimulate the promoter of the mammalian transcription factor GCMa/Gcm1 in trophoblast cells.

One of the master regulators of placental cell fusion in mammals leading to multi-nucleated syncytiotrophoblasts is the transcription factor GCMa. Recently, we proved that the cAMP-driven protein kinase A signaling pathway is fundamental for up-regulation of GCMa transcript levels and protein stability. Here, we show that Transducer of Regulated CREB activity (TORC1), the human co-activator of cAMP response element-binding protein (CREB), but not a dominant-negative CREB mutant, significantly up-regulates the GCMa promoter. We identified potential cAMP response element (CRE)-binding sites within the GCMa promoter upstream of the transcriptional start site. Only the CRE site at -1337 interacted strongly with CREB in promoter mapping experiments. The characterization of GCMa promoter mutants and additional bZIP-type family members demonstrated that also old astrocyte specifically-induced substance (OASIS) is able to stimulate GCMa transcription. Knockdown of endogenous CREB or OASIS in BeWo cells decreased endogenous GCMa mRNA level and activity. Overexpression of TORC1 or OASIS in choriocarcinoma cells led to placental cell fusion, accompanied by placental expression of gap junction forming protein connexin-43. Further, we show that CREB expression is replaced by OASIS expression around E12.5 suggesting that a sequential order of bZIP-type family members ensures a high rate of GCMa transcription throughout placentation.