Mimicking the tumour microenvironment: three different co‐culture systems induce a similar phenotype but distinct proliferative signals in primary chronic lymphocytic leukaemia cells

Hamilton, Emma; Pearce, Laurence; Morgan, Laura; Robinson, Sophie; Ware, Victoria Lesley; Brennan, Paul; Thomas, N. Shaun B.; Yallop, Deborah; Devereux, Stephen; Fegan, Christopher; Buggins, Andrea G.S.; Pepper, Christopher John

doi:10.1111/j.1365-2141.2012.09191.x

Cited by 47 publications

(44 citation statements)

References 21 publications

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“…[18][19][20][22][23][24] In keeping with previous findings, 21 CLL cells were protected from apoptosis in the presence of both NTL and TL(CD40L) when compared to liquid culture (LC) alone (Figure 2A; NTL versus LC, P=0.02; TL(CD40L) versus LC, P=0.05). There was no significant difference in percent apoptotic CLL cells between the NTL and TL(CD40L) cultures.…”

Section: Blinatumomab Induces Death Of Chronic Lymphocytic Leukemia Csupporting

confidence: 74%

“…21 For co-cultures, irradiated (80 Gy) NTL or TL(CD40L) cells were seeded into a 48-well plate, and allowed to adhere before addition of CLL PBMC and blinatumomab (10 or 100 ng/mL) for 7 days. CLL PBMC were removed from the surface of the fibroblast monolayer, and analyzed by flow cytometry (annexin V, CD5, CD8 and CD4).…”

Section: Flow Cytometry-based Cytotoxicity Assaymentioning

confidence: 99%

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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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Section: Blinatumomab Induces Death Of Chronic Lymphocytic Leukemia Csupporting

confidence: 74%

Section: Flow Cytometry-based Cytotoxicity Assaymentioning

confidence: 99%

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

et al. 2013

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“…8 In the peripheral circulation, CLL cells transiently interact with endothelial cells, which stimulate survival 9 but not proliferation. 10 These findings suggest a 2-compartment model of disease in which CLL cells traffic between the peripheral vasculature and the lymphoid tissues. In support of this, Herishanu et al compared the gene expression of CLL cells in different compartments and identified the LN as the predominant site of CLL cell activation and proliferation.…”

Section: Introductionmentioning

confidence: 97%

Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration

Pasikowska

Walsby

Apollonio

et al. 2016

Blood

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Key Points• LN-derived CLL cells have increased capacity for T-cell activation and superior immune synapse formation compared with those from PB.• Enhanced CLL cell immunologic function is also linked to PB circulating cells with the propensity to migrate.Several lines of evidence suggest that homing of tumor cells to lymphoid tissue contributes to disease progression in chronic lymphocytic leukemia (CLL). Here, we demonstrate that lymph node (LN)-derived CLL cells possess a distinct phenotype, and exhibit enhanced capacity for T-cell activation and superior immune synapse formation when compared with paired peripheral blood (PB) samples. LN-derived CLL cells manifest a proliferative, CXCR4 dim CD5 bright phenotype compared with those in the PB and higher expression of T-cell activation molecules including CD80, CD86, and HLA-D-related (DR). In addition, LN-CLL cells have higher expression of a4b1 (CD49d) which, as well as being a co-stimulatory molecule, is required for CLL cells to undergo transendothelial migration (TEM) and enter the proliferation centers of the LNs. Using an in vitro system that models circulation and TEM, we showed that the small population of CLL cells that migrate are CXCR4 dim CD5 bright with higher CD49d, CD80, CD86, and HLA-DR compared with those that remain circulating; a phenotype strikingly similar to LN-derived CLL cells. Furthermore, sorted CD49d hi CLL cells showed an enhanced capacity to activate T cells compared with CD49d lo subpopulations from the same patient. Thus, although PB-CLL cells have a reduced capacity to form immune synapses and activate CD4 1 T cells, this was not the case for LN-CLL cells or those with the propensity to undergo TEM. Taken together, our study suggests that CLL cell immunologic function is not only modulated by microenvironmental interactions but is also a feature of a subpopulation of PB-CLL cells that are primed for lymphoid tissue homing and interaction with T cells. (Blood. 2016;128(4):563-573)

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“…CLL cells that are ZAP-70 Pos experience dynamic expression of CD38 (22)(23)(24), a plasma-membrane ectoenzyme that can interact with a ligand on endothelial cells (CD31) to initiate signaling that leads to enhanced leukemia-cell proliferation and/or survival (25). Also, expression of CD38 and ZAP-70 identifies CLL cells with enhanced migration to the chemokine CXCL12 (26), suggesting a functional link between these two proteins.…”

Section: Discussionmentioning

confidence: 99%

Targeting chronic lymphocytic leukemia cells with a humanized monoclonal antibody specific for CD44

Zhang

Fecteau

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Chronic lymphocytic leukemia (CLL) cells express high levels of CD44, a cell-surface glycoprotein receptor for hyaluronic acid. We found that a humanized mAb specific for CD44 (RG7356) was directly cytotoxic for leukemia B cells, but had little effect on normal B cells. Moreover, RG7356 could induce CLL cells that expressed the zeta-associated protein of 70 kDa (ZAP-70) to undergo caspasedependent apoptosis, independent of complement or cytotoxic effector cells. The cytotoxic effect of this mAb was not mitigated when the CLL cells were cocultured with mesenchymal stromal cells (MSCs) or hyaluronic acid or when they were stimulated via ligation of the B-cell receptor with anti-μ. RG7356 induced rapid internalization of CD44 on CLL cells at 37°C, resulting in reduced expression of ZAP-70, which we found was complexed with CD44. Administration of this mAb at a concentration of 1 mg/kg to immune-deficient mice engrafted with human CLL cells resulted in complete clearance of engrafted leukemia cells. These studies indicate that this mAb might have therapeutic activity, particularly in patients with CLL that express ZAP-70.cell survival | preclinical studies | animal model | antibody therapy B -cell chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of mature, antigen-stimulated CD5+/ CD23+ B cells in blood, secondary lymphoid tissues, and marrow (1). Most of the circulating CLL cells in patients are arrested in the G0/G1 phase of the cell cycle and express high levels of antiapoptotic proteins (2). CLL therefore has been characterized as a process of defective apoptosis, rather than increased proliferation. However, despite their apparent longevity in vivo, CLL cells undergo spontaneous and drug-induced apoptosis in vitro, unless rescued by monocyte-derived Nurse-like cells (NLCs), follicular dendritic cells, or mesenchymal stromal cells (MSCs) (3-6). Thus, it has been postulated that CLL cells receive survival signals from these accessory cells, which constitute part of the CLL B-cell microenvironment in secondary lymphoid tissues and marrow (6). These survival signals can inhibit spontaneous or drug-induced apoptosis, particularly for CLL cells that express unmutated Ig heavy-chain variable genes (IGHVs) and/or the zeta-associated protein of 70 kDa (ZAP-70), which typically is not expressed by normal B cells (7). Patients with leukemia cells that possess such characteristics typically have a relatively short interval from diagnosis to initial therapy compared with patients with CLL cells that express mutated IGHVs or that lack expression of .One of the survival signals received by leukemia cells may be mediated via CD44, a surface glycoprotein receptor for the nonsulfated glycosaminoglycan hyaluronic acid (HA), which typically is found in the microenvironment of lymphoid tissues (13). CLL cells express high levels of CD44, particularly those that express unmutated IGHVs and/or . Upon binding HA in the extracellular matrix, CD44 activates the phosphoinositol 3-kinase (PI3K)/AKT and MAPK/ERK ...

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Mimicking the tumour microenvironment: three different co‐culture systems induce a similar phenotype but distinct proliferative signals in primary chronic lymphocytic leukaemia cells

Cited by 47 publications

References 21 publications

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

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

Phenotype and immune function of lymph node and peripheral blood CLL cells are linked to transendothelial migration

Targeting chronic lymphocytic leukemia cells with a humanized monoclonal antibody specific for CD44

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