Different proliferative and survival capacity of CLL-cells in a newly established in vitro model for pseudofollicles

Plander, Márk; Seegers, Silvia; Ugocsai, Peter; Diermeier-Daucher, Simone; Ivanyi, J; Schmitz, Gerd; Hofstädter, Ferdinand; Schwarz, Stephan; Orsó, Evelyn; Knüchel, Ruth; Brockhoff, Gero

doi:10.1038/leu.2009.145

Cited by 58 publications

(41 citation statements)

References 48 publications

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“…Recently, correlations between functional features of CD40 ligation and IGHV gene mutational status have been reported in some instances. 42,45 This discrepancy could be simply explained by the different number of cases tested and/or by differences in the experimental models used, as CD40 stimulation was studied together with additional stimuli like cytokines 45 or Toll-like receptors ligands. 42 A final answer will be only obtained with a prospective study involving hundreds of cases to eliminate any selection bias.…”

Section: Discussionmentioning

confidence: 99%

The functional in vitro response to CD40 ligation reflects a different clinical outcome in patients with chronic lymphocytic leukemia

et al. 2011

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Malignant B lymphocytes from chronic lymphocytic leukemia (CLL) patients maintain the capacity to respond to CD40 ligation, among other microenvironmental stimuli. In this study, we show that (i) leukemic CLL cells stimulated with the soluble form of CD40L in vitro show differential responses in terms of upregulation of surface markers (CD95 and CD80) and induction of chemokines (CCL22 and CCL17) expression/secretion, and that (ii) these changes are mirrored by a distinct activation of intracellular signalling pathways including increase in IKKalpha/beta phosphorylation and upregulation of antiapoptotic proteins (BCL-2 and MCL-1). CLL patients can then be segregated into two distinct functional subsets. We defined the responsive subset of cases CD40L dependent, considering the capacity to respond as a sign of persistent need of this stimulation for the leukemic expansion. Conversely, we named the unresponsive cases CD40L independent, considering them less dependent on this microenvironmental signal, presumably because of a higher autonomous proliferative and survival potential. Importantly, we report that (iii) the two functional subsets show an opposite clinical outcome, with CD40L-independent cases having a shorter time to progression. This indicates that the functional differences observed in vitro may reflect a different leukemic potential in vivo likely responsible for a distinct clinical course.

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Section: Discussionmentioning

confidence: 99%

The functional in vitro response to CD40 ligation reflects a different clinical outcome in patients with chronic lymphocytic leukemia

et al. 2011

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“…39,40 This is because microenvironmental signals, including interactions between CLL cells and CD40L-expressing T cells, stromal and nurse-like cells are known to increase the apoptotic threshold of CLL cells. 41,42 CD40-CD40L interactions have been shown to augment the antigen-presenting capacity of CLL cells due to increased surface expression of adhesion molecules (CD54) and co-stimulatory molecules such as CD80, CD86 and CD70 leading to CLL cell activation and protection from fludarabine-mediated killing. 18,[43][44][45] Importantly, in this study we showed that CLL cells receiving activation signals through CD40-CD40L interactions in a co-culture model system remained susceptible to blinatumomab-mediated killing.…”

Section: © F E R R a T A S T O R T I F O U N D A T I O Nmentioning

confidence: 99%

Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

et al. 2013

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© F e r r a t a S t o r t i F o u n d a t i o n 2 0 1 3of CLL activation and pro-survival signals. Given these data, this approach represents a novel and promising therapeutic strategy for the treatment of CLL and, potentially, other lymphoid malignancies. Methods Blood samples from patients with chronic lymphocytic leukemiaTwenty-eight CLL patients (aged 53-83 years) were studied with full ethical approval. All blood samples from patients were obtained with informed consent (see Online Supplementary Methods). The majority of the assays were performed on fresh samples from treatment-naïve patients, except those for which the results are shown in Figure 1: in these assays, samples from patients who had received standard chemotherapy treatment were also tested. Antibodies and flow cytometryA full list of the antibodies used can be found in the Online Supplementary Methods. Blinatumomab (MT103, AMG103) was provided by Amgen Inc. (Munich, Germany). T cells were identified using CD4 and CD8 markers, while CLL cells were CD5 + CD4-CD8 -(>99% CD19 + ). Cell culturesPeripheral blood mononuclear cells (PBMC) were incubated in RPMI 1640 supplemented with 5% AB serum (AB media) for 3-7 days. Blinatumomab was added to PBMC cultures at a concentration of 10 or 100 ng/mL. Human T-cell activator CD3/CD28 dynabeads (Invitrogen) were added at 1x10 5 beads/1x10 6 PBMC as a positive control for T-cell activation. For absolute counts of T cells and CLL cells, an anti-CD3 antibody (Invitrogen) at a dose of 27.7 or 277 ng/mL was used as a positive control (same molar concentration as 10 or 100 ng/mL blinatumomab). Intracellular Ki67 stainingPBMC were labeled with antibodies against CD4, CD8, CCR7 and CD45RA, fixed and permeabilized (Fix and Perm with 1% NP40, Caltag-Medsystems, Buckingham, UK) before staining with a Ki67-FITC antibody for flow cytometric analysis. Cytokine secretion assayCytokines in tissue culture supernatants were measured using a Human Th1/Th2 11plex RTU Flowcytomix kit (eBioscience) for simultaneous detection of 11 cytokines. Intracellular cytokine staining and the determination of CD107 and granzyme B expressionPBMC were cultured (3 days) with 10 ng/mL blinatumomab, before treatment with Golgi plug and Golgi stop (BD biosciences), and incubation with an anti-CD107 antibody (5 h at 37°C). Intracellular expression of granzyme B or interferon (IFN)-g and tumor necrosis factor (TNF)-α in CD4 or CD8 T cells was measured by flow cytometry. Absolute counts of T cells and chronic lymphocytic leukemia cellsPBMC samples that had or had not been treated with blinatumomab (7 days) were surface-stained with antibodies against annexin V, CD5, CD8 and CD4. Absolute counts were determined by flow cytometry using Cytocount beads (Dako, Stockport, UK). Flow cytometry-based cytotoxicity assayMouse fibroblast cells transfected with CD40L [TL(CD40L)] and non-transfected cells (NTL) were cultured as previously described. 21 For co-cultures, irradiated (80 Gy) NTL or TL(CD40L) cells were seeded into a 48-well plate, and allow...

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“…21 The increased level of CD38 expression, which here typified B-CLL SP cells from CD38 þ patients, is usually observed for highly proliferating B-CLL cells 29 and correlates with a poor clinical outcome, 30 supporting the view that B-CLL SP cells are important for progression of the disease. The fact that whether in vitro SP cells exposed to pertinent stimuli and microenvironmental cells 31 actually undergo more mitosis than the bulk of quiescent B-CLL cells remains elusive, although likely. There is now a large amount of scientific evidence suggesting that the proliferating pool of B-CLL cells is localized into specialized structures in the lymph nodes called the proliferation centers.…”

Section: Discussionmentioning

confidence: 99%

B-chronic lymphocytic leukemia chemoresistance involves innate and acquired leukemic side population cells

Gross

Fe²,

Ysebaert

et al. 2010

Leukemia

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Different proliferative and survival capacity of CLL-cells in a newly established in vitro model for pseudofollicles

Cited by 58 publications

References 48 publications

The functional in vitro response to CD40 ligation reflects a different clinical outcome in patients with chronic lymphocytic leukemia

The functional in vitro response to CD40 ligation reflects a different clinical outcome in patients with chronic lymphocytic leukemia

Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

B-chronic lymphocytic leukemia chemoresistance involves innate and acquired leukemic side population cells

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