RFX transcription factors control a miR-150/PDAP1 axis that restrains the proliferation of human T cells.

Within the immune system, microRNAs (miRNAs) exert key regulatory functions. However, what are the mRNA targets regulated by miRNAs and how miRNAs are transcriptionally regulated themselves remain for the most part unknown. We found that in primary human memory T helper lymphocytes, miR-150 was the most abundantly expressed miRNA, and its expression decreased drastically upon activation, suggesting regulatory roles. Constitutive MIR150 gene expression required the RFX family of transcription factors, and its activation-induced down-regulation was linked to their reduced expression. By performing miRNA pull-down and sequencing experiments, we identified PDGFA-associated protein 1 (PDAP1) as one main target of miR-150 in human T lymphocytes. PDAP1 acted as an RNA-binding protein (RBP), and its CRISPR/Cas-9-mediated deletion revealed that it prominently contributed to the regulation of T-cell proliferation. Overall, using an integrated approach involving quantitative analysis, unbiased genomics, and genome editing, we identified RFX factors, miR-150, and the PDAP1 RBP as the components of a regulatory axis that restrains proliferation of primary human T lymphocytes.

Cell-intrinsic mechanisms to restrain inflammatory responses in T lymphocytes.

A mechanistic understanding of the regulatory circuits that control the effector responses of memory T helper lymphocytes, and in particular their ability to produce pro-inflammatory cytokines, may lead to effective therapeutic interventions in all immune-related diseases. Activation of T lymphocytes induces robust immune responses that in most cases lead to the complete eradication of invading pathogens or tumor cells. At the same time, however, such responses must be both highly controlled in magnitude and limited in time to avoid unnecessary damage. To achieve such sophisticated level of control, T lymphocytes have at their disposal an array of transcriptional and post-transcriptional regulatory mechanisms that ensure the acquisition of a phenotype that is tailored to the incoming stimulus while restraining unwarranted activation, eventually leading to the resolution of the inflammatory response. Here, we will discuss some of these cell-intrinsic mechanisms that control T cell responses and involve transcription factors, microRNAs, and RNA-binding proteins. We will also explore how the same mechanisms can be involved both in anti-tumor responses and in autoimmunity.

Fluorescence-Activated Cell Sorting Is More Potent to Fish Intervertebral Disk Progenitor Cells Than Magnetic and Beads-Based Methods.

Low back pain related to intervertebral disk (IVD) degeneration has a major socioeconomic impact on our aging society. Therefore, stem cell therapy to activate self-repair of the IVD remains an exciting treatment strategy. In this respect, tissue-specific progenitors may play a crucial role in IVD regeneration, as these cells are perfectly adapted to this niche. Such a rare progenitor cell population residing in the nucleus pulposus (NP) (NP progenitor cells [NPPCs]) was found positive for the angiopoietin-1 receptor (Tie2+), and was demonstrated to possess self-renewal capacity and in vitro multipotency. Here, we compared three sorting protocols; that is, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and a mesh-based label-free cell sorting system (pluriSelect), with respect to cell yield, potential to form colonies (colony-forming units), and in vitro functional differentiation assays for tripotency. The aim of this study was to demonstrate the efficiency of three widespread cell sorting methods for picking rare cells (<5%) and how these isolated cells then behave in downstream functional differentiation in adipogenesis, osteogenesis, and chondrogenesis. The cell yields among the isolation methods differed widely, with FACS presenting the highest yield (5.0% ± 4.0%), followed by MACS (1.6% ± 2.9%) and pluriSelect (1.1% ± 1.0%). The number of colonies formed was not significantly different between Tie2+ and Tie2- NPPCs. Only FACS was able to separate into two functionally different populations that showed trilineage multipotency, while MACS and pluriSelect failed to maintain a clear separation between Tie2+ and Tie2- populations in differentiation assays. To conclude, the isolation of NPPCs was possible with all three sorting methods, while FACS was the preferred technique for separation of functional Tie2+ cells. Impact Statement Tissue-specific progenitor cells such as nucleus pulposus progenitor cells of the IVD could become an ultimate cell source for tissue engineering strategies as these cells are presumably best adapted to the tissue's microenvironment. Fluorescence-activated cell sorting seemed to outcompete magnetic-activated cell sorting and pluriSelect concerning selecting a rare cell population from IVD tissue as could be demonstrated by improved cell yield and functional differentiation assays.