Stimulation of B-Cell Lymphoproliferations with CpG-Oligonucleotide DSP30 Plus IL-2 Is More Effective than with TPA to Detect Clonal Abnormalities.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1480-1480
Author(s):  
Stéphanie Struski ◽  
Carine Gervais ◽  
Catherine Helias ◽  
Raoul Herbrecht ◽  
Bruno Audhuy ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Cycle Progression ◽  
Chromosome Abnormalities ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Cpg Oligonucleotide ◽  
Chromosomal Changes ◽  
Cell Mitogen ◽  
Stimulation Of

Abstract Conventional cytogenetics (CC) of B-cell lymphoproliferations remains difficult because of low mitotic in vitro activity of the leukemic cells. Therefore, mitogen stimulation of B-cells is required to analyze an adequate number of metaphases. Chromosome abnormalities using CC with TPA can be detected in up to 50% of chronic lymphocytic leukemia (CLL), but the development of FISH techniques has allowed the detection of selected chromosome abnormalities in more than 80% of CLL. However, FISH is restricted, since information is only available for the genes/loci for which probes are used. So, for a comprehensive genetic analysis, CC is essential because it provides an overview of all microscopically visible chromosome abnormalities important as prognostic factors. The use of the immunostimulatory CpG-oligonucleotide DSP30 to effectively induce cell cycle progression of CLL cells in vitro has been reported. This proliferation is markedly enhanced upon addition of Interleukine-2 to cultures. To our knowledge and to date, there has been no direct comparison of classical TPA versus DSP30+IL-2. DSP30+IL-2 stimulation has been successfully tested in CLL but no data are available for other lymphoproliferations. We cultured 132 B-cell lymphoproliferations (80 CLL and 52 other B-cell lymphoid neoplasms (BCLN)) in parallel, in presence of TPA or DSP30+IL-2. The objective of this study was to evaluate the suitability of DSP30+IL-2 as a routine B-cell mitogen for metaphase cytogenetics. CC successfully analyzed 94.9% of CLL and 98.1% of BCNL with more than 80% abnormal karyotypes. For CLL, failures of karyotypes were more frequent in cultures with DSP30+IL-2 (14%) than in those with TPA (4%). The rate of failures were similar for BCLN (6% versus 4%). For CLL, there were significantly fewer metaphases in DSP30+IL-2 than in TPA spreads (mean of 50 versus 72 metaphases per slide respectively, p=0.0007), as well as for BCLN (mean of 50 versus 71 metaphases per slide respectively, p=0.009). However, the proportion of abnormal metaphases was significantly higher in DSP30+IL-2 (mean of 59%) compared to TPA cultures (mean of 26%, p=0.00265) for CLL and for BCLN (mean of 57% versus 33%, p=0.0065). Stimulation with DSP30+IL-2 allowed to detect more abnormalities, more abnormal subclones and more complex karyotypes in CLL and in the majority of BCLN. Though FISH exploration using a large probe panel has yielded valuable results in lymphoproliferative diseases, it underestimates the heterogeneity of chromosomal aberrations. Complexity of chromosomal changes, recently associated to unfavorable outcomes, can only be assessed with CC. In conclusion, our results in both CLL and BCLN indicate that the immunostimulatory oligonucleotide DSP30 in combination with IL-2 is an easy and efficient stimulus in metaphase generation for routine chromosomal banding.

Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: a mechanism for survival

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2973-2979 ◽  
Author(s):  
Anne J. Novak ◽  
Richard J. Bram ◽  
Neil E. Kay ◽  
Diane F. Jelinek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocyte ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Aberrant Expression ◽  
B Lymphocyte Stimulator

B-cell chronic lymphocytic leukemia (B-CLL) is defined by the accumulation of CD5+ B cells in the periphery and bone marrow. This disease is not characterized by highly proliferative cells but rather by the presence of leukemic cells with significant resistance to apoptosis and, therefore, prolonged survival. B-lymphocyte stimulator (BLyS) is a newly identified tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis and it shares significant homology with another TNF superfamily member, APRIL. The striking effects of BLyS on normal B-cell maintenance and survival raises the possibility that it may be involved in pathogenesis and maintenance of hematologic malignancies, including B-CLL. In this study, we investigated the status of APRIL and BLyS expression, as well as their receptors, in this disease. All B-CLL patient cells studied expressed one or more of 3 known receptors for BLyS; however, the pattern of expression was variable. In addition, we demonstrate for the first time that B-CLL cells from a subset of patients aberrantly express BLyS and APRIL mRNA, whereas these molecules were not detectable in normal B cells. Furthermore, we provide in vitro evidence that BLyS protects B-CLL cells from apoptosis and enhances cell survival. Because these molecules are key regulators of B-cell homeostasis and tumor progression, leukemic cell autocrine expression of BLyS and APRIL may be playing an important role in the pathogenesis of this disease.

scholarly journals CD160 signaling mediates PI3K-dependent survival and growth signals in chronic lymphocytic leukemia

Blood ◽  
2010 ◽  
Vol 115 (15) ◽  
pp. 3079-3088 ◽  
Author(s):  
Feng-Ting Liu ◽  
Jerome Giustiniani ◽  
Timothy Farren ◽  
Li Jia ◽  
Armand Bensussan ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Cycle Progression ◽  
Lymphocytic Leukemia ◽  
Cytochrome C Release ◽  
Pi3k Inhibitors ◽  
Phosphoinositide 3 Kinase ◽  
Dose Dependent

Abstract B-cell chronic lymphocytic leukemia (CLL) expresses CD160, a glycosylphosphatidylinositol-linked receptor found on normal natural killer (NK) and T cells, but not B cells. CD160 is a multifunctional molecule in normal lymphocytes, but its role in CLL biology is unknown. In vitro, CLL cells undergo rapid spontaneous apoptosis, which CD160 activation protected against—mean cell viability increased from 67% to 79% (P < .001). This was associated with up-regulation of Bcl-2, Bcl-xL, and Mcl-1, but not Bax. As expected from these changes in Bcl-2/Bax and Bcl-xL/Bax ratios, CD160 triggering reduced mitochondrial membrane potential collapse and cytochrome c release. CD160 stimulation also induced DNA synthesis, cell cycle progression, and proliferation. B-cell antigen receptor (BCR)–induced CLL proliferation was generally greater than with CD160, but marked variation was seen. Both BCR and CD160 signaling led to CLL secretion of interleukin-6 (IL-6) and IL-8, although CD160 induced greater increases of IL-6 (51-fold) and IL-8 (15-fold). Survival and activation signals mediated by CD160 showed dose-dependent suppression by phosphoinositide-3 kinase (PI3K) inhibitors. Thus, in vitro, CLL cells can use the CD160 pathway for survival and activation, mimicking CD160 signaling in normal NK and CD8+ T cells. Establishing the pathophysiologic relevance of these findings may reveal new therapeutic targets.

Detection of chromosomal changes in chronic lymphocytic leukemia using classical cytogenetic methods and FISH: application of rich mitogen mixtures for lymphocyte cultures

2016 ◽  
Vol 64 (4) ◽  
pp. 894-898 ◽  
Author(s):  
Dorota Koczkodaj ◽  
Sylwia Popek ◽  
Szymon Zmorzyński ◽  
Ewa Wąsik-Szczepanek ◽  
Agata A Filip
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mitotic Index ◽  
Cell Cultures ◽  
Lymphocytic Leukemia ◽  
Pokeweed Mitogen ◽  
Structural Aberrations ◽  
Chromosomal Changes

One of the research methods of prognostic value in chronic lymphocytic leukemia (CLL) is cytogenetic analysis. This method requires the presence of appropriate B-cell mitogens in cultures in order to obtain a high mitotic index. The aim of our research was to determine the most effective methods of in vitro B-cell stimulation to maximize the number of metaphases from peripheral blood cells of patients with CLL for classical cytogenetic examination, and then to correlate the results with those obtained using fluorescence in situ hybridization (FISH). The study group involved 50 consecutive patients with CLL. Cell cultures were maintained with the basic composition of culture medium and addition of respective stimulators. We used the following stimulators: Pokeweed Mitogen (PWM), 12-O-tetradecanoylphorbol 13-acetate (TPA), ionophore, lipopolysaccharide (LPS), and CpG-oligonucleotide DSP30. We received the highest mitotic index when using the mixture of PWM+TPA+I+DSP30. With classical cytogenetic tests using banding techniques, numerical and structural aberrations of chromosomes were detected in 46 patients, and no change was found in only four patients. Test results clearly confirmed the legitimacy of using cell cultures enriched with the mixture of cell stimulators and combining classical cytogenetic techniques with the FISH technique in later patient diagnosing.

scholarly journals Interleukin-15 promotes the growth of leukemic cells of patients with B- cell chronic lymphoproliferative disorders

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3327-3335 ◽  
Author(s):  
L Trentin ◽  
A Cerutti ◽  
R Zambello ◽  
R Sancretta ◽  
C Tassinari ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Nk Cell ◽  
Lymphoproliferative Disorders ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Interleukin 15

The recently discovered cytokine, interleukin-15 (IL-15), has been demonstrated to share several biologic properties with IL-2 in different cell systems, including T-cell and natural killer (NK) cell compartments. As for B lymphocytes, IL-15 has been shown to provide stimulatory activities in normal preactivated B cells that are mainly transduced through IL- 2 receptor (IL-2R) complex components. Since leukemic B cells from patients with chronic lymphoproliferative disorders (CLD) bear IL-2R and grow in response to IL-2, we investigated whether IL-15 triggers the proliferation of malignant B cells obtained from 12 patients with B-cell chronic lymphocytic leukemia (B-CLL) and five patients with hairy cell leukemia (HCL). Enriched B cells recovered from five healthy subjects were also studied as controls. IL-15 stimulated the proliferation of freshly isolated leukemic B cells, but not resting normal B lymphocytes, the latter being able to grow in the presence of IL-15 only after in vitro preactivation with phorbol myristate acetate. The proliferation elicited by IL-2 on leukemic cells was comparable to that determined by IL-15. Following addition of graded concentrations of IL-15 to low/intermediate-dose IL-2, resting leukemic B cells showed a higher stimulatory rate than that observed using the two cytokines separately. In normal resting B lymphocytes, this cumulative effect was not observed. The role of different IL-2R subunits in IL-15-driven growth of malignant B cells was investigated both by their expression on leukemic cells and by the block of different IL-2R subunits (p55, p75, and p64) with specific monoclonal antibodies (MoAbs). Using flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses we demonstrated that both B-CLL and HCL leukemic B cells express the beta and gamma chains of the IL-2R system. The stimulatory activity achieved by IL-15 decreased significantly, blocking the beta and gamma chains of the IL-2R. Taken together, these findings demonstrate that IL-15 triggers the growth of leukemic B cells through IL-2R system subunits, pointing to the role of this novel cytokine in regulating the neoplastic proliferation in CLD.

scholarly journals Modeling the Leukemia Microenviroment In Vitro

2020 ◽  
Vol 10 ◽  
Author(s):  
Cristina Scielzo ◽  
Paolo Ghia
Keyword(s):  
Drug Resistance ◽  
B Cell ◽  
Current Knowledge ◽  
3D Culture ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
B Cell Malignancies ◽  
Tissue Microenvironment

Over the last decade, the active role of the microenvironment in the pathogenesis, development and drug resistance of B cell malignancies has been clearly established. It is known that the tissue microenvironment promotes proliferation and drug resistance of leukemic cells suggesting that successful treatments of B cell malignancies must target the leukemic cells within these compartments. However, the cross-talk occurring between cancer cells and the tissue microenvironment still needs to be fully elucidated. In solid tumors, this lack of knowledge has led to the development of new and more complex in vitro models able to successfully mimic the in vivo settings, while only a few simplified models are available for haematological cancers, commonly relying only on the co-culture with stabilized stromal cells and/or the addition of limited cocktails of cytokines. Here, we will review the known cellular and molecular interactions occurring between monoclonal B lymphocytes and their tissue microenvironment and the current literature describing innovative in vitro models developed in particular to study chronic lymphocytic leukemia (CLL). We will also elaborate on the possibility to further improve such systems based on the current knowledge of the key molecules/signals present in the microenvironment. In particular, we think that future models should be developed as 3D culture systems with a higher level of cellular and molecular complexity, to replicate microenvironmental-induced signaling. We believe that innovative 3D-models may therefore improve the knowledge on pathogenic mechanisms leading to the dissemination and homing of leukemia cells and consequently the identification of therapeutic targets.

scholarly journals IN VITRO STIMULATION OF B-CELL INDUCES DIFFERENTIATION AND NOT PROLIFERATION

1989 ◽  
Vol 72 (1) ◽  
pp. 113-113 ◽  
Author(s):  
David Barnett ◽  
Jackie Storr ◽  
George A. Buckley ◽  
John T. Reilly
Keyword(s):  
B Cell ◽  
Stimulation Of

Chronic lymphocytic leukemia: revelations from the B-cell receptor

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4389-4395 ◽  
Author(s):  
Freda K. Stevenson ◽  
Federico Caligaris-Cappio
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Of Origin ◽  
Regulatory B Cell ◽  
V Genes

Abstract The finding that chronic lymphocytic leukemia (CLL) consists of 2 clinical subsets, distinguished by the incidence of somatic mutations in the immunoglobulin (Ig) variable region (V) genes, has clearly linked prognosis to biology. Antigen encounter by the cell of origin is indicated in both subsets by selective but distinct expression of V genes, with evidence for continuing stimulation after transformation. The key to distinctive tumor behavior likely relates to the differential ability of the B-cell receptor (BCR) to respond. Both subsets may be undergoing low-level signaling in vivo, although analysis of blood cells limits knowledge of critical events in the tissue microenvironment. Analysis of signal competence in vitro reveals that unmutated CLL generally continues to respond, whereas mutated CLL is anergized. Differential responsiveness may reflect the increased ability of post-germinal center B cells to be triggered by antigen, leading to long-term anergy. This could minimize cell division in mutated CLL and account for prognostic differences. Unifying features of CLL include low responsiveness, expression of CD25, and production of immunosuppressive cytokines. These properties are reminiscent of regulatory T cells and suggest that the cell of origin of CLL might be a regulatory B cell. Continuing regulatory activity, mediated via autoantigen, could suppress Ig production and lead to disease-associated hypogammaglobulinemia. (Blood. 2004;103:4389-4395)

scholarly journals Low molecular weight B cell growth factor induces proliferation of human B cell precursor acute lymphoblastic leukemias

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 132-138 ◽  
Author(s):  
B Wormann ◽  
SR Mehta ◽  
AL Maizel ◽  
TW LeBien
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Growth Factor ◽  
Cell Growth ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Cell Precursor ◽  
B Cell Growth Factor ◽  
B Cell Precursors

Experiments were conducted to determine the effect of low mol wt B cell growth factor (L-BCGF) on B cell precursor acute lymphoblastic leukemia (ALL). L-BCGF induced a significant increase in 3H-TdR incorporation in 28 of 37 bone marrow aspirates from patients with B cell precursor ALL, with stimulation indices ranging from 2 to 129. Fluorescence-activated cell sorting confirmed that in five of seven patients the common acute lymphoblastic leukemia antigen (CALLA)/CD10 positive leukemic cells were responding directly to L-BCGF. L-BCGF was capable of inducing, in some patients, an increase in absolute viable cells and could also induce colony formation in vitro. The response of B cell precursor ALL was not attributable to beta IL 1, IL 2, or gamma interferon. These results indicate that the majority of B cell precursor ALL undergo a proliferative response to L-BCGF, suggesting a regulatory role for this lymphokine in the growth of B cell precursors.

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