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

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

Table <b>1.</b> Baseline characteristics of patients.

<p>Table <b>1.</b> Baseline characteristics of patients.</p

Demographic, clinical and biological characteristics of patients with myeloid and lymphoid SM-AHNMD patients at inclusion.

1<p>C-findings according to WHO classification.</p>2<p>including fatigue, headache, flushes, fever, hypotension, choc, syncope, WHO; world health organization, CM; cutaneous mastocytosis, AHNMD; associated clonal hematologic non-mast cell lineage disease, UP; urticaria pigmentosa, TEMP; telengietasia eruptive macularis persistans, DCM; diffuse cutaneous mastocytosis, BMD; bone mineral density.</p