The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation

AbstractInitiation of the development of the anterior–posterior axis in the mouse embryo has been thought to take place only when the anterior visceral endoderm (AVE) emerges and starts its asymmetric migration. However, expression of Lefty1, a marker of the AVE, was recently found to initiate before embryo implantation. This finding has raised two important questions: are the cells that show such early, preimplantation expression of this AVE marker the real precursors of the AVE and, if so, how does this contribute to the establishment of the AVE? Here, we address both of these questions. First, we show that the expression of another AVE marker, Cer1, also commences before implantation and its expression becomes consolidated in the subset of ICM cells that comprise the primitive endoderm. Second, to determine whether the cells showing this early Cer1 expression are true precursors of the AVE, we set up conditions to trace these cells in time-lapse studies from early periimplantation stages until the AVE emerges and becomes asymmetrically displaced. We found that Cer1-expressing cells are asymmetrically located after implantation and, as the embryo grows, they become dispersed into two or three clusters. The expression of Cer1 in the proximal domain is progressively diminished, whilst it is reinforced in the distal–lateral domain. Our time-lapse studies demonstrate that this distal–lateral domain is incorporated into the AVE together with cells in which Cer1 expression begins only after implantation. Thus, the AVE is formed from both part of an ancestral population of Cerl-expressing cells and cells that acquire Cer1 expression later. Finally, we demonstrate that when the AVE shifts asymmetrically to establish the anterior pole, this occurs towards the region where the earlier postimplantation expression of Cer1 was strongest. Together, these results suggest that the orientation of the anterior–posterior axis is already anticipated before AVE migration

CD62L expression identifies a unique subset of polyfunctional CD56dim NK cells

Human natural killer (NK) cells comprise 2 main subsets, CD56 bright and CD56dim cells, that differ in function, phenotype, and tissue localization. To further dissect the heterogeneity of CD56dim cells, we have performed transcriptome analysis and functional ex vivo characterization of human NK-cell subsets according to the expression of markers related to differentiation, migration or competence. Here, we show for the first time that the ability to respond to cytokines or to activating receptors is mutually exclusive in almost all NK cells with the exception of CD56dim CD62L+ cells. Indeed, only these cells combine the ability to produce interferon-γ after cytokines and proliferate in vivo during viral infection with the capacity to kill and produce cytokines upon engagement of activating receptors. Therefore, CD56 dim CD62L+ cells represent a unique subset of polyfunctional NK cells. Ex vivo analysis of their function, phenotype, telomere length, frequencies during ageing as well as transfer experiments of NK-cell subsets into immunodeficient mice suggest that CD56dim CD62L + cells represent an intermediate stage of NK-cell maturation, which after restimulation can accomplish multiple tasks and further develop into terminally differentiated effectors. © 2010 by The American Society of Hematology

RORγt+ Innate Lymphoid Cells Acquire a Proinflammatory Program upon Engagement of the Activating Receptor NKp44

SummaryRORγt+ innate lymphoid cells (ILCs) are crucial players of innate immune responses and represent a major source of interleukin-22 (IL-22), which has an important role in mucosal homeostasis. The signals required by RORγt+ ILCs to express IL-22 and other cytokines have been elucidated only partially. Here we showed that RORγt+ ILCs can directly sense the environment by the engagement of the activating receptor NKp44. NKp44 triggering in RORγt+ ILCs selectively activated a coordinated proinflammatory program, including tumor necrosis factor (TNF), whereas cytokine stimulation preferentially induced IL-22 expression. However, combined engagement of NKp44 and cytokine receptors resulted in a strong synergistic effect. These data support the concept that NKp44+ RORγt+ ILCs can be activated without cytokines and are able to switch between IL-22 or TNF production, depending on the triggering stimulus

Proficiency Testing to Assess Technical Performance for CTC-Processing and Detection Methods in CANCER-ID

Abstract Background Multiple technologies are available for detection of circulating tumor cells (CTCs), but standards to evaluate their technical performance are still lacking. This limits the applicability of CTC analysis in clinic routine. Therefore, in the context of the CANCER-ID consortium, we established a platform to assess technical validity of CTC detection methods in a European multi-center setting using non-small cell lung cancer (NSCLC) as a model. Methods We characterized multiple NSCLC cell lines to define cellular models distinct in their phenotype and molecular characteristics. Standardized tumor-cell-bearing blood samples were prepared at a central laboratory and sent to multiple European laboratories for processing according to standard operating procedures. The data were submitted via an online tool and centrally evaluated. Five CTC-enrichment technologies were tested. Results We could identify 2 cytokeratin expressing cell lines with distinct levels of EpCAM expression: NCI-H441 (EpCAMhigh, CKpos) and NCI-H1563 (EpCAMlow, CKpos). Both spiked tumor cell lines were detected by all technologies except for the CellSearch system that failed to enrich EpCAMlow NCI-H1563 cells. Mean recovery rates ranged between 49% and 75% for NCI-H411 and 32% and 76% for NCI-H1563 and significant differences were observed between the tested methods. Conclusions This multi-national proficiency testing of CTC-enrichment technologies has importance in the establishment of guidelines for clinically applicable (pre)analytical workflows and the definition of minimal performance qualification requirements prior to clinical validation of technologies. It will remain in operation beyond the funding period of CANCER-ID in the context of the European Liquid Biopsy Society (ELBS). </jats:sec