Assessment of haematopoietic progenitor cell counting with the Sysmex® XN-1000 to guide timing of apheresis of peripheral blood stem cells.

BACKGROUND: Successful peripheral blood stem cell (PBSC) collection depends on optimal timing of apheresis, as usually determined by flow cytometry CD34-positive (+) cell count in peripheral blood (PB). Since this method is costly and labour-intensive, we evaluated the use of the Hematopoietic Progenitor Cell count programme on a Sysmex® XN haematologic analyser (XN-HPC) as a rapid and inexpensive alternative for predicting CD34+ cell count in PB samples. MATERIALS AND METHODS: Haematopoietic progenitor cell and CD34+ cell counts were compared using 273 PB samples collected from 78 healthy donors and 72 patients who underwent PBSC transplantation. We assessed the effectiveness of the XN-HPC in safely predicting pre-harvest CD34+ counts. The most efficient cut-off values of XNHPC were identified. We also evaluated the imprecision (coefficient of variation, CV) and functional sensitivity. RESULTS: Imprecision of the XN-HPC count was <6.3% on daily measurement of three levels of quality control material. Functional sensitivity was 8.9×106/L. A cut-off value of ≥62×106/L XN-HPC for multiple myeloma (MM) patients and ≥30×106/L for all other subjects had both 100% specificity and 100% positive predictive value for identifying samples with CD34+ cells ≥20×106/L. An XN-HPC threshold of <13×106/L identified preharvest CD34+ cell count <10×106/L with 100% sensitivity and 100% negative predictive value. DISCUSSION: The XN-HPC is a fast, easy and inexpensive test that can safely improve apheresis workflow thus possibly replacing other more expensive CD34 counts currently performed and promoting optimal timing of PBSC collection.

Inhibition of Notch Signaling Enhances Chemosensitivity in B-cell Precursor Acute Lymphoblastic Leukemia.

Notch3 and Notch4 support survival of primary B-cell acute lymphoblastic leukemia (B-ALL) cells, suggesting a role for Notch signaling in drug response. Here we used in vitro, in silico, and in vivo mouse xenograft model-based approaches to define the role of the Notch pathway in B-ALL chemosensitivity. We observed significant Notch receptor and ligand expression in B-ALL primary cells and cell lines. Primary leukemia cells from high-risk patients overexpressed Notch3, Notch4, and Jagged2 while displaying a reduction in expression levels of Notch1-4 following chemotherapy. We then analyzed in vitro cell survival of B-ALL cells treated with conventional chemotherapeutic agents alone or in combination with Notch signaling inhibitors. Gamma-secretase inhibitors (GSI) and anti-Notch4 were all capable of potentiating drug-induced cell death in B-ALL cells by upregulating intracellular levels of reactive oxygen species, which in turn modulated mTOR, NF-κB, and ERK expression. In NOG-mouse-based xenograft models of B-ALL, co-administration of the Notch inhibitor GSI-XII with the chemotherapeutic agent Ara-C lowered bone marrow leukemic burden compared with DMSO or Ara-C alone, thus prolonging mouse survival. Overall, our results support the potential effectiveness of Notch inhibitors in patients with B-ALL.Significance: Inhibition of Notch signaling enhances the chemosensitivity of B-ALL cells, suggesting Notch inhibition as a potential therapeutic strategy to improve the outcome of patients with B-ALL.

Characterization of a new B-ALL cell line with constitutional defect of the Notch signaling pathway.

Notch signaling contribution to B-cell acute lymphoblastic leukemia (B-ALL) development is still under investigation. The serendipitous onset of B-ALL in a patient affected by the germinal Notch mutation-dependent Alagille syndrome allowed us to establish a B-ALL cell line (VR-ALL) bearing a genetic loss of function in components of Notch signaling. VR-ALL is a common-type B-ALL cell line, grows in conventional culture medium supplemented with 10% serum, and gives rise, once injected into immunodeficient NOG mice, to a mouse xenograft model of B-ALL. Exome sequencing revealed deleterious mutations in some components of Notch signaling, including Jagged1, Notch1, and Notch2. In addition, VR-ALL is sensitive both in vitro and in vivo to γ-secretase inhibitors (GSIs) as well as conventional anti-leukemic drugs. For all these reasons, VR-ALL may help to gain more insights into the role of Notch signaling in B-ALL.

Differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on T, B and NK cell functions.

Mesenchymal stromal cells (MSCs) are multipotent cells, immunomodulatory stem cells that are currently used for regenerative medicine and treatment of a number of inflammatory diseases, thanks to their ability to significantly influence tissue microenvironments through the secretion of large variety of soluble factors. Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease. Here, we used a standardized methodological approach to dissect the immunomodulatory effects exerted by MSC-derived EVs on unfractionated peripheral blood mononuclear cells and purified T, B and NK cells. We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division. Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases.

Notch signalling drives bone marrow stromal cell-mediated chemoresistance in acute myeloid leukemia.

Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB.These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML.