A Combination of Cytokines Rescues Highly Purified Leukemic CLL B-Cells from Spontaneous Apoptosis <i>In Vitro</i>

<div><p>B-chronic lymphocytic leukemia (B-CLL), the most common human leukemia, is characterized by predominantly non-dividing malignant mature CD5+ B lymphocytes with an apoptosis defect. Various microenvironmental stimuli confer a growth advantage on these leukemic cells and extend their survival <i>in vivo</i>. Nevertheless, when cultured <i>in vitro</i>, CLL B-cells rapidly die from apoptosis. Certain cytokines may extend the survival capacity of CLL B-cells <i>in vitro</i> and individual anti-apoptotic effects of several cytokines have been reported. The potential cumulative effect of such cytokines has not been studied. We therefore investigated the effects on CLL B-cells survival <i>in vitro</i> of humoral factors, polyclonal lymphocyte activators and a combination of cytokines known for their anti-apoptotic effects. Purified CLL B-cells were cultured in the presence or absence of various soluble molecules and the leukemic cell response was assessed in terms of viability. Apoptotic cell death was detected by flow cytometry using annexinV and 7-amino-actinomycin. The survival of CLL B-cells <i>in vitro</i> was highly variable. When tested separately, cytokines (IL-2, -6, -10, -12, -15, -21, BAFF and APRIL) improved CLL B cell survival moderately; in combination, they significantly enhanced survival of these cells, even up to 7 days of culture. We also report that humoral factors from autologous serum are important for survival of these malignant cells. Our findings support the concept that the CLL microenvironment is critical and suggest that soluble factors may contribute directly to the prolonged survival of CLL B-cells. Therefore, the combination of cytokines we describe as providing strong resistance to apoptosis <i>in vitro</i> might be used to improve the treatment of CLL.</p> </div

CLL B-cells migrate from the peripheral blood to lymph nodes or bone marrow to receive the appropriate signals for their growth and survival.

<p>The peripheral blood of CLL patients contains various cytokines that can protect CLL B-cells from apoptosis. When CLL B-cells travel into lymph nodes or bone marrow, they make contact with various cells in the microenvironment (dendritic cells, stromal cells, T cells and Nurse-like cells), the cytokines produced by them, and various antigens that are able to promote CLL B-cell survival.</p

The pro-survival effect of PMA, IL-4 and a cytokine cocktail is sustained for 7 days of culture.

<p><b>A.</b> Apoptosis was evaluated after 24, 48, 72 and 168 hours of culture by annexin V–PE/7-AAD staining and flow cytometry. The survival of B-CLL cells in the presence of PMA or IL-4 or cytokine cocktail was greater than that of controls for up to 168 h. Lower panel: Cytometry plots from a representative patient at 168 h. Upper panel: The values reported are means ± SEM for 9 independent experiments, each performed in duplicate. <b>B.</b> 10⁶ purified B-CLL cells/well were cultured in 24-well plates in 1 ml of RPMI 1640 complete medium in the presence of PMA or IL-4 or the cytokine cocktail. Changes in viable cell number were assessed (counted in duplicate) by a trypan blue exclusion method after 24, 48, 72 and 168 hours of culture. The values reported are means ± SEM for 9 independent experiments. The significance of differences was calculated with the Wilcoxon test: ^*p<0.05 ^**p<0.01. Red asterisks for PMA versus Medium, clear blue asterisks for Cc versus Medium and indigo asterisks for IL-4 versus Medium.</p

MSCs' soluble factors secretion.

<p>Interleukin-6 (IL-6), IL-8, Monocytes chemotactic protein-1 (MCP-1), Dickkopf-related protein-1 (DKK-1), prostaglandin-E2 (PGE2), and stromal cell-derived factor-1 (SDF-1) levels were measured in supernatants obtained from controls and GD-MSCs (n = 8). Levels were normalized to the number of cells.</p

GD patients characteristics.

<p>SMG, splenomegaly; HMG, hepatomegaly; Hb, hemoglobin; MCV, mean corpuscular volume; WBC, white blood cell; MRI, magnetic resonance imaging; BI, bone infiltration; ERT, enzyme replacement therapy.</p

GBA activity measurement in blood and BM cells.

<p>GBA activity was measured in blood and BM cell subtypes of GD patients (GD) and healthy donors (Ct). Results are expressed as an index defined by the ratio between median fluorescence intensity (MFI) of cells incubated with PFB-FDGlu alone and MFI of cells incubated with CBE and PFB-FDGlu (blood: n = 10 for GD and controls; BM: n = 8 for GD and 6 for controls).</p

GD-MSCs characterization.

<p>(A) Histograms represents CFU-F quantification per 10⁵ mononuclear cells (left panel) and mean cell number per CFU-F (right panel) obtained after culture of control (Ct) or GD BM samples (n = 10 and 7 for GD patients and controls respectively). (B) MSCs growth kinetics: cumulative population doubling (CPD) are shown for Ct and GD-MSCs (n = 5 and 10 respectively). (C) Cell cycle analysis was performed by flow cytometry on Ct and GD-MSCs. The percentage of cells in each phase of the cell cycle was determined by gating G0/G1, S and G2/M cells (n = 8 and 6 respectively). (D) Representative FSC/SSC flow cytometry profile of Ct and GD-MSCs. (E) Ct and GD-MSCs were stained with anti-alpha-tubulin (red) and with Hoechst for nuclear staining (blue). Typical non-round nuclei (angled panels) and large nuclei (arrows) were observed in GD-MSCs. Scale bars represent 50 µm. A and C: * indicates <i>P</i><0.05.</p

Hematopoiesis in GD.

<p>(A) Relative number of CD34⁺ cells was measured in GD (n = 7) and controls (n = 10) BM samples according to ISHAGE Guidelines <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069293#pone.0069293-Enquist1" target="_blank">[34]</a>. (B) CD34⁺ cells clonogenic potential. Granulocyte–macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and megakaryocyte colony-forming units (CFU-Mk) were scored at day 14 by direct visualization using an inverted microscope (n = 8 and 10 for GD patients and controls, respectively). (C) BM-CD34⁺ cells <i>ex-vivo</i> expansion. Total nucleated cell (TNCs) number was determined after 6 and 12 days culture (n = 6 and 10 for GD and controls respectively). Results are presented as TNCs fold expansion. (D) Primitive hematopoiesis was evaluated by LTC-IC assay. GD patients and controls CD34⁺ cells were cultured on healthy donors MSCs during a period of 5 weeks. All experiments were performed in triplicate (n = 4 and 8 for GD patients and controls, respectively). (E) Four- to 8-week-old NSG mice were transplanted by retro-orbital injection with 1.10⁵ BM CD34⁺ cells from GD patients or controls. The presence of human CD45⁺ cells was assessed by flow cytometry 7 weeks later (n = 5 mice engrafted with CD34⁺ cells from 2 healthy donors and 4 mice engrafted with CD34⁺ cells from 2 GD patients).</p

DFX/VD association induces myeloid differentiation and increases overall survival in elderly AML patients.

<p>(A) Kaplan-Meier estimated OS in DFX/VD and BSC treated patients (B) Multivariate analysis. Forest plot of the odds ratio. (C) OS within subgroups presenting normal VD levels (≥50 nmol/L) or VD deficiency (≤50 nmol/L). (D) Monocytes numbers in VD/DFX treated patients (F) Creatinine levels in treated patients.</p

Identification of SLAMF3 (CD229) as an Inhibitor of Hepatocellular Carcinoma Cell Proliferation and Tumour Progression

<div><p>Although hepatocellular carcinoma (HCC) is one of the most common malignancies and constitutes the third leading cause of cancer-related deaths, the underlying molecular mechanisms are not fully understood. In the present study, we demonstrate for the first time that hepatocytes express signalling lymphocytic activation molecule family member 3 (SLAMF3/CD229) but not other SLAMF members. We provide evidence to show that SLAMF3 is involved in the control of hepatocyte proliferation and in hepatocellular carcinogenesis. SLAMF3 expression is significantly lower in primary human HCC samples and HCC cell lines than in human healthy primary hepatocytes. In HCC cell lines, the restoration of high levels of SLAMF3 expression inhibited cell proliferation and migration and enhanced apoptosis. Furthermore, SLAMF3 expression was associated with inhibition of HCC xenograft progression in the nude mouse model. The restoration of SLAMF3 expression levels also decreased the phosphorylation of MAPK ERK1/2, JNK and mTOR. In samples from resected HCC patients, SLAMF3 expression levels were significantly lower in tumorous tissues than in peritumoral tissues. Our results identify SLAMF3 as a specific marker of normal hepatocytes and provide evidence for its potential role in the control of proliferation of HCC cells.</p></div