International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

extracellular vesicles (eVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers. Human serum is a rich source of readily accessible eVs; however, the separation of eVs from serum proteins and non-eV lipid particles represents a considerable challenge. in this study, we compared the most commonly used isolation techniques, either alone or in combination, for the isolation of EVs from 200 µl of human serum and their separation from non-eV protein and lipid particles present in serum. the size and yield of particles isolated by each method was determined by nanoparticle tracking analysis, with the variation in particle size distribution being used to determine the relative impact of lipoproteins and protein aggregates on the isolated EV population. Purification of eVs from soluble protein was determined by calculating the ratio of eV particle count to protein concentration. finally, lipoprotein particles co-isolated with eVs was determined by Western blot analysis of lipoprotein markers ApoB and Apoe. overall, this study reveals that the choice of eV isolation procedure significantly impacts EV yield from human serum, together with the presence of lipoprotein and protein contaminants. Extracellular vesicles (EVs) were originally identified in reticulocytes as a means of disposing of obsolete membrane proteins such as α4β1 and transferrin receptor during reticulocyte maturation 1-3 , and have since been shown to participate in cell-cell signalling via transfer of proteins, nucleic acids and metabolites 4-6. EVs have been identified in a diverse range of human biofluids including serum, plasma, urine, saliva, breast milk, amniotic fluid, ascites fluid, cerebrospinal fluid and bile 7,8. These EVs are classified into three groups; exosomes, microvesicles and apoptotic bodies depending on their size, biogenesis and method of cellular release. Microvesicles and apoptotic bodies generally range from 100 to 1000 nm and 1-4 µm respectively, and are formed by budding from the plasma membrane 4,9. In contrast, exosomes have a diameter of 30-150 nm and are formed by inward budding of the late endosome lumen to form a multivesicular body (MVB) that is secreted by fusion with the plasma membrane 10. The overlap in exosome and microvesicle size (100-150 nm) and density (1.08-1.19 g/ml) makes it difficult to distinguish the two groups and as a result exosomes are often defined by their content of endosome-associated proteins including tetraspanins CD9, CD63, and CD81. However, since microvesicles from haematopoietic cells are also enriched for endosomal proteins such as CD63 and CD81 11 exosomes and microvesicles <150 nm are collectively referred to as small extracellular vesicles (sEVs) 12. EV secretion has been shown to be elevated in response to inflammation 13 , hypoxia 14,15 and an acidic microenvironment 16,17 and is associated with human diseases such as cancer, where secretion levels have b...

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Triple negative breast cancer (TNBC) is an aggressive subtype with relatively poor clinical outcomes and limited treatment options. Chemotherapy, while killing cancer cells, can result in the generation of highly chemoresistant therapeutic induced senescent (TIS) cells that potentially form stem cell niches resulting in metastases. Intriguingly, senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Our aim was to profile EVs harvested from TIS TNBC cells compared with control cells to identify a potential mechanism by which TIS TNBC cells maintain survival in the face of chemotherapy. TIS was induced and confirmed in Cal51 TNBC cells using the chemotherapeutic paclitaxel (PTX) (Taxol). Mass spectrometry (MS) analysis of EVs harvested from TIS compared with control Cal51 cells was performed using Ingenuity Pathway Analysis and InnateDB programs. We demonstrate that TIS Cal51 cells treated with 75 nM PTX for 7 days became senescent (senescence-associated β-galactosidase (SA-β-Gal) positive, Ki67-negative, increased p21 and p16, G2/M cell cycle arrest) and released significantly more EVs (P=0.0002) and exosomes (P=0.0007) than non-senescent control cells. Moreover, TIS cells displayed an increased expression of the multidrug resistance protein 1/p-glycoprotein. MS analysis demonstrated that EVs derived from senescent Cal51 cells contained 142 proteins with a significant increased fold change compared with control EVs. Key proteins included ATPases, annexins, tubulins, integrins, Rabs and insoluble senescence-associated secretory phenotype (SASP) factors. A fluorescent analogue of PTX (Flutax-2) allowed appreciation of the removal of chemotherapy in EVs from senescent cells. Treatment of TIS cells with the exosome biogenesis inhibitor GW4869 resulted in reduced SA-β-Gal staining (P=0.04). In summary, this study demonstrates that TIS cells release significantly more EVs compared with control cells, containing chemotherapy and key proteins involved in cell proliferation, ATP depletion, apoptosis and the SASP. These findings may partially explain why cancer senescent cells remain viable despite chemotherapeutic challenge.

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein for the G-␣-q and G-␣-i subunits of heterotrimeric Gproteins that turns off signaling by G-protein coupled receptors. RGS18 is highly expressed in platelets. In the present study, we show that the 14-3-3␥ protein binds to phosphorylated serines 49 and 218 of RGS18. Platelet activation by thrombin, thromboxane A2, or ADP stimulates the association of 14-3-3 and RGS18, probably by increasing the phosphorylation of serine 49. In contrast, treatment of platelets with prostacyclin and nitric oxide, which trigger inhibitory cyclic nucleotide signaling involving cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI), induces the phosphorylation of serine 216 of RGS18 and the detachment of 14-3-3. Serine 216 phosphorylation is able to block 14-3-3 binding to RGS18 even in the presence of thrombin, thromboxane A2, or ADP. 14-3-3-deficient RGS18 is more active compared with 14-3-3-bound RGS18, leading to a more pronounced inhibition of thrombin-induced release of calcium ions from intracellular stores. Therefore, PKA-and PKGI-mediated detachment of 14-3-3 activates RGS18 to block Gq-dependent calcium signaling. These findings indicate cross-talk between platelet activation and inhibition pathways at the level of RGS18 and Gq. IntroductionIn healthy vasculature, endothelial cells lining the blood vessels constantly produce and release prostacyclin (PGI 2 ) and nitric oxide (NO) into the vessel lumen. The interaction of endothelial factors with platelets plays a fundamental role in controlling hemostasis and in maintaining platelets in a resting state. Platelet inhibition by both PGI 2 and NO has been well established. The signaling pathways of both molecules result in an elevation of cyclic nucleotides that activate cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI). These in turn phosphorylate an unknown number of substrate proteins, resulting in reduced release of calcium ions (Ca 2ϩ ) from intracellular stores and reduced activation of G-proteins such as Rap1, ultimately leading to a block of platelet adhesion, granule release, and aggregation. PKA and PKGI have overlapping substrate specificities, which may explain the synergistic role of the 2 pathways. Few substrates have been established in platelets, among them Rap1GAP2, a GTPase-activating protein (GAP) of the small G-protein Rap1, as we have shown previously. 1 Other substrates include vasodilator-stimulated phospho-protein (VASP), heatshock protein 27 (HSP27), and LIM and SH3 domain protein (LASP), all of which regulate actin dynamics. 2,3 The IP 3 -receptor and the IP 3 -receptor-associated G-kinase substrate (IRAG) are the only PKA and/or PKGI substrates that have been shown to mediate cAMP/cGMP effects on intracellular Ca 2ϩ release. [2][3][4] Limited data are available on the specific substrates and signaling events that translate PKA/G substrate phosphorylation into platelet inhibition.Conversely, bindin...