Detailed Analysis of Apoptotic Responses to Cytokine Stimulation in B-Cell Chronic Lymphocytic Leukaemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4773-4773
Author(s):  
Andrew P. Jewell ◽  
Wendy Smith ◽  
Rob van Schie ◽  
Nina Porakishvili ◽  
Peter M. Lydyard
Keyword(s):  
B Cell ◽  
Interferon Gamma ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Live Cells ◽  
Tnf Alpha ◽  
Gm Csf ◽  
Cytokine Stimulation

Abstract Introduction B cell Chronic lymphocytic leukemia (BCLL) is characterized by the accumulation of monoclonal malignant CD5+ B cells that show resistance to apoptosis in vivo, but are susceptible to apoptosis when incubated in vitro. A number of cytokines have been reported to protect against apoptosis in vitro, but it is not clear whether the effects of these cytokines are mediated in similar ways. We have therefore studied the regulation of apoptosis in B-CLL cells in vitro, in response to various cytokines. Methods B-CLL cells were purified and incubated with the following agents; Interferon-gamma, IL-4, IL-10, IL-13, TNF-alpha and GM-CSF, each at 3 different concentrations. Apoptosis was measured after 70 hours by Annexin/PI staining and analysed for early apoptosis (Annexin V positive), late apoptotic (Annexin V and PI positive), and necrotic (PI positive). Results The percentage of live cells in unstimulated B-CLL cells was 42.4±2.3%. Co-incubation of B-CLL cells with IL-4 at 100ng/ml increased the percentage of live cells to 63.2±8.4%, while in the presence of interferon-gamma the percentage of live cells was 48.7±5.9%. In contrast, co-incubation with TNF-alpha reduced the percentage of live cells to 30.8±6.8%. IL-10, IL-13 and GM-CSF had no effect on B-CLL cells survival at any of the concentrations used.. No significant differences in the pattern of early/late apoptosis or necrosis were seen between the different cytokines used. Figure Figure

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 Chimeric CLL-1 Antibody Fusion Proteins Containing Granulocyte-Macrophage Colony-Stimulating Factor or Interleukin-2 With Specificity for B-Cell Malignancies Exhibit Enhanced Effector Functions While Retaining Tumor Targeting Properties

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4437-4447 ◽  
Author(s):  
Jason L. Hornick ◽  
Leslie A. Khawli ◽  
Peisheng Hu ◽  
Maureen Lynch ◽  
Peter M. Anderson ◽  
...  
Keyword(s):  
B Cell ◽  
Fusion Proteins ◽  
Tumor Targeting ◽  
B Cell Malignancies ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Although monoclonal antibody (MoAb) therapy of the human malignant lymphomas has shown success in clinical trials, its full potential for the treatment of hematologic malignancies has yet to be realized. To expand the clinical potential of a promising human-mouse chimeric antihuman B-cell MoAb (chCLL-1) constructed using the variable domains cloned from the murine Lym-2 (muLym-2) hybridoma, fusion proteins containing granulocyte-macrophage colony-stimulating factor (GM-CSF) (chCLL-1/GM–CSF) or interleukin (IL)-2 (chCLL-1/IL–2) were generated and evaluated for in vitro cytotoxicity and in vivo tumor targeting. The glutamine synthetase gene amplification system was employed for high level expression of the recombinant fusion proteins. Antigenic specificity was confirmed by a competition radioimmunoassay against ARH-77 human myeloma cells. The activity of chCLL-1/GM–CSF was established by a colony formation assay, and the bioactivity of chCLL-1/IL–2 was confirmed by supporting the growth of an IL-2–dependent T-cell line. Antibody-dependent cellular cytotoxicity against ARH-77 target cells demonstrated that both fusion proteins mediate enhanced tumor cell lysis by human mononuclear cells. Finally, biodistribution and imaging studies in nude mice bearing ARH-77 xenografts indicated that the fusion proteins specifically target the tumors. These in vitro and in vivo data suggest that chCLL-1/GM–CSF and chCLL-1/IL–2 have potential as immunotherapeutic reagents for the treatment of B-cell malignancies.

scholarly journals Receptors for tumor necrosis factor on neoplastic B cells from chronic lymphocytic leukemia are expressed in vitro but not in vivo

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1607-1613 ◽  
Author(s):  
W Digel ◽  
W Schoniger ◽  
M Stefanic ◽  
H Janssen ◽  
C Buck ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Necrosis ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Tnf Alpha ◽  
Tnf Receptor ◽  
Tnf Receptors ◽  
Necrosis Factor

Abstract Recombinant tumor necrosis factor-alpha (TNF-alpha) is a cytokine that induces proliferation of neoplastic B cells from patients with chronic lymphocytic leukemia (CLL). To gain insight into the mechanisms involved in regulating TNF responsiveness, we have examined TNF receptor expression on neoplastic B-CLL cells. We have demonstrated that freshly isolated neoplastic B cells from patients with CLL did not express TNF receptors. After 1 day of incubation in culture medium, TNF receptors were detectable in the range of 540 to 1,500/cell. Kinetic experiments revealed that receptor expression was half-maximal after 3 hours of culturing and required de novo protein synthesis. The Scatchard plots of TNF-alpha binding indicated a single set of high- affinity TNF receptors with a dissociation constant of 70 pmol/L. TNF receptor expression in vitro was found in all examined cases. All cytokines tested, with the exception of IL-2, did not influence the expression of TNF receptors. The TNF receptor expression is enhanced in B-CLL cells cultured in the presence of interleukin-2 when compared with the receptor expression of cells cultured in medium alone. Our data suggest that neoplastic B-CLL cells in patients with stable disease do not express TNF receptors in vivo and that an unknown mechanism suppressing TNF receptor expression in vivo may play a role in growth regulation of neoplastic B cells.

Nurture versus Nature: The Microenvironment in Chronic Lymphocytic Leukemia

Hematology ◽  
2011 ◽  
Vol 2011 (1) ◽  
pp. 96-103 ◽  
Author(s):  
Jan A. Burger
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Disease Progression ◽  
Drug Testing ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
In Vivo Models

Abstract Intrinsic factors such as genetic lesions, anti-apoptotic proteins, and aberrant signaling networks within leukemia cells have long been the main focus of chronic lymphocytic leukemia (CLL) research. However, over the past decade, it became increasingly clear that external signals from the leukemia microenvironment make pivotal contributions to disease progression in CLL and other B-cell malignancies. Consequently, increasing emphasis is now placed on exploring and targeting the CLL microenvironment. This review highlights critical cellular and molecular pathways of CLL-microenvironment cross-talk. In vitro and in vivo models for studying the CLL microenvironment are discussed, along with their use in searching for therapeutic targets and in drug testing. Clinically, CXCR4 antagonists and small-molecule antagonists of B cell receptor (BCR)-associated kinases (spleen tyrosine kinase [Syk], Bruton's tyrosine kinase [Btk], and PI3Kδ) are the most advanced drugs for targeting specific interactions between CLL cells and the miocroenvironment. Preclinical and first clinical evidence suggests that high-risk CLL patients can particularly benefit from these alternative agents. These findings indicate that interplay between leukemia-inherent and environmental factors, nature and nurture determines disease progression in CLL.

scholarly journals Effect of a thymic factor on T lymphocytes in B cell chronic lymphocytic leukemia: in vitro and in vivo studies

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 667-671 ◽  
Author(s):  
F Lauria ◽  
D Raspadori ◽  
S Tura
Keyword(s):  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
B Cell ◽  
Proliferative Response ◽  
Lymphocytic Leukemia ◽  
Before And After ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Abnormalities of T lymphocytes in B cell chronic lymphocytic leukemia (B-CLL) have been extensively documented by several immunologic investigations. Following recent studies pointing to the favorable effect of TP-1, a partially purified extract of calf thymus, on the T cell-mediated immunity of several diseases, including Hodgkin's disease, we have used monoclonal antibodies and the enriched T lymphocytes of 16 untreated B-CLL patients to evaluate the proportion of T cell subsets before and after the administration of TP-1. In addition, the proliferative response to phytohemagglutinin (PHA) and the helper function in a pokeweed mitogen (PWM) system were assessed. In ten cases, the effect of TP-1 was also studied in vitro by evaluating the same parameters before and after incubation of B-CLL T cells with the drug. The study demonstrated that in vivo administration of TP-1 increases significantly (P less than .001) the proportion of the defective helper/inducer T cell population (OKT4-positive cells) in B-CLL, leading to a near normal OKT4/OKT8 ratio. Furthermore, the improved phenotypic profile was accompanied by an increased proliferative response to PHA and, in particular, by a significant increase (P less than .01) of T helper capacity; this increase was, however, insufficient to enable the normalization of the serum immunoglobulin levels. The in vitro incubation of B-CLL T lymphocytes did not succeed in producing significant modifications in distribution and function.

Phase I trial of recombinant interferon gamma in cancer patients.

1986 ◽  
Vol 4 (2) ◽  
pp. 137-146 ◽  
Author(s):  
S Vadhan-Raj ◽  
A Al-Katib ◽  
R Bhalla ◽  
L Pelus ◽  
C F Nathan ◽  
...  
Keyword(s):  
Interferon Gamma ◽  
Phase 1 ◽  
Rest Period ◽  
Lymphocytic Leukemia ◽  
Fixed Dose ◽  
Rapid Decline ◽  
Recombinant Interferon ◽  
Advanced Malignancy

Interferon gamma (IFN-gamma) is a lymphokine with potent in vitro effects on cell growth and immune function. We have investigated the effects of rIFN-gamma (sp act approximately 2 X 10(7) U/mg, purity greater than 99%) in 16 evaluable patients with advanced malignancy in a phase 1 trial. Patients were treated with six-hour intravenous (IV) infusions daily, five days a week for 2 weeks. After a 2-week rest period, the IV treatment cycle was repeated. Responders were maintained on repeated IV treatment cycles or daily intramuscular (IM) injections. Patients were entered at fixed dose levels of 0.1, 0.5, or 1.0 mg/m2/d. The maximum safely tolerated dose was 0.5 mg/m2. The most common side effects were constitutional symptoms, including fever, chills, fatigue, and myalgias. Reversible and transient increases in hepatic transaminase and decrease in granulocyte counts were seen. Treatment was associated with a dose-dependent increase in serum levels of beta 2 microglobulin. Partial responses (PRs) were observed in one patient with Hodgkin's disease and one patient with chronic lymphocytic leukemia. Fairly constant levels of serum IFN were found at four and six hours during infusion, followed by a rapid decline within one to two hours. We conclude that rIFN-gamma can be safely administered by a six-hour IV infusion and that it can induce in vivo some of the biologic effects reported in in vitro studies.

scholarly journals Growth- and differentiation-associated expression of bcl-2 in B-chronic lymphocytic leukemia cells

Blood ◽  
1992 ◽  
Vol 79 (11) ◽  
pp. 2981-2989 ◽  
Author(s):  
M Schena ◽  
LG Larsson ◽  
D Gottardi ◽  
G Gaidano ◽  
M Carlsson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Messenger Rna ◽  
Phorbol Esters ◽  
Lymphocytic Leukemia ◽  
Mantle Zone ◽  
Cell Stage ◽  
High Level

Abstract The bcl-2 gene is translocated into the Ig loci in about 80% of human follicular lymphomas and in 10% of B-type chronic lymphocytic leukemias (B-CLL), resulting in a high level of expression. We have compared the expression of bcl-2 transcripts and protein in B-CLL cells in their normal equivalent CD5+ B cells and in normal B-cell populations representative of different in vivo and in vitro stages of activation and proliferation. We report here that bcl-2 was expressed in 11 of 11 cases of CD5+ B-CLL clones, contrasting with the absent expression in normal CD5+ B cells. Activation of 173 and 183 B-CLL cells by phorbol esters (12-O-tetradecanoylphorbol-13-acetate [TPA]) to IgM secretion without concomitant DNA synthesis resulted in a rapid but transient downregulation of bcl-2 expression. In contrast, the reduction of bcl-2 at both the messenger RNA and protein levels was sustained after mitogenic stimulation, suggesting that bcl-2 expression and proliferation are inversely related in these cells. This notion was further supported by immunocytochemical analysis showing that bcl-2 was primarily expressed in small resting lymphocytes and in cells differentiating to the plasma cell stage, but less expressed in Ki67- positive proliferating B blasts. Moreover, it was also supported by the low level of bcl-2 in exponentially growing Epstein-Barr virus-carrying lymphoblastoid and B-CLL cell lines. The regulation of bcl-2 expression in B-CLL resembled that of normal tonsillar follicular B cells, in which a high level of expression was found in resting mantle zone B cells but not in the proliferating germinal center B cells. Based on these findings and the role of bcl-2 in maintaining B-cell memory, we propose that the phenotype of B-CLL cells corresponds to a mantle zone memory-type B cell.

scholarly journals Interferon gamma inhibits apoptotic cell death in B cell chronic lymphocytic leukemia.

1993 ◽  
Vol 177 (1) ◽  
pp. 213-218 ◽  
Author(s):  
M Buschle ◽  
D Campana ◽  
S R Carding ◽  
C Richard ◽  
A V Hoffbrand ◽  
...  
Keyword(s):  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Programmed Cell Death ◽  
B Cell ◽  
Interferon Gamma ◽  
Lymphocytic Leukemia ◽  
Malignant Cells ◽  
Ifn Gamma ◽  
Cell Chronic Lymphocytic Leukemia

The malignant, CD5+ B lymphocytes of B cell chronic lymphocytic leukemia (B-CLL) die by apoptosis in vitro. This is in contrast to the prolonged life span of the leukemic cells in vivo and likely reflects the lack of essential growth factors in the tissue culture medium. We found that interferon gamma (IFN-gamma) inhibits programmed cell death and promotes survival of B-CLL cells in culture. This effect may also be important in vivo: increased serum levels of IFN-gamma, ranging from 60 to > 2,200 pg/ml, were found in 7 of 10 B-CLL samples tested, whereas the sera of 10 healthy individuals did not contain detectable levels of this cytokine (< 20 pg/ml). High levels of IFN-gamma message were detected in RNA from T cell-depleted B-CLL peripheral blood samples by Northern blot analysis. Synthesis of IFN-gamma by B-CLL lymphocytes was confirmed by in situ hybridization and flow cytometry. The majority of B-CLL cells (74-82%) expressed detectable levels of IFN-gamma mRNA, and CD19+ B-CLL cells were labeled with anti-IFN-gamma monoclonal antibodies. These results show that IFN-gamma inhibits programmed cell death in B-CLL cells and suggest that the malignant cells are able to synthesize this cytokine. By delaying apoptosis, IFN-gamma may extend the life span of the malignant cells and thereby contribute to their clonal accumulation.

scholarly journals Pathological RANK signaling in B cells drives autoimmunity and chronic lymphocytic leukemia

2020 ◽  
Vol 218 (2) ◽  
Author(s):  
Begüm Alankus ◽  
Veronika Ecker ◽  
Nathalie Vahl ◽  
Martina Braun ◽  
Wilko Weichert ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Lupus Erythematosus ◽  
Lymphocytic Leukemia ◽  
B Cell Tolerance ◽  
B Cell Malignancy ◽  
Human Lymphoma ◽  
Rank Signaling

Clinical evidence suggests alterations in receptor activator of NF-κB (RANK) signaling are key contributors to B cell autoimmunity and malignancy, but the pathophysiological consequences of aberrant B cell–intrinsic RANK signaling remain unknown. We generated mice that express a human lymphoma–derived, hyperactive RANKK240E variant in B lymphocytes in vivo. Forced RANK signaling disrupted B cell tolerance and induced a fully penetrant systemic lupus erythematosus–like disease in addition to the development of chronic lymphocytic leukemia (CLL). Importantly, RANKK240E transgenic CLL cells as well as CLL cells of independent murine and of human origin depend on microenvironmental RANK ligand (RANKL) for tumor cell survival. Consequently, inhibition of the RANKL–RANK axis with anti-RANKL antibodies killed murine and human CLL cells in vitro and in vivo. These results establish pathological B cell–intrinsic RANK signaling as a potential driver of autoimmunity and B cell malignancy, and they suggest the exploitation of clinically available anti-RANKL compounds for CLL treatment.

scholarly journals Expression and regulation of tumor necrosis factor, interleukin-2, and hematopoietic growth factor receptors in B-cell chronic lymphocytic leukemia

Blood ◽  
1994 ◽  
Vol 84 (12) ◽  
pp. 4249-4256 ◽  
Author(s):  
L Trentin ◽  
R Zambello ◽  
C Agostini ◽  
C Enthammer ◽  
A Cerutti ◽  
...  
Keyword(s):  
B Cells ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Tnf Alpha ◽  
Gm Csf ◽  
Hematopoietic Factors ◽  
Stimulating Factor ◽  
Necrosis Factor

Leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) express tumor necrosis factor (TNF) and interleukin-2 (IL-2) receptors, but only a low proliferative response can be elicited in vitro by TNF alpha and IL-2. To investigate the functional properties of IL-2 and TNF alpha on leukemic B cells, we evaluated (1) the regulation of expression of TNF receptors (TNF-R) and IL-2 receptors on leukemic B cells after culture with TNF alpha and IL-2; (2) the effect of the combination of TNF alpha and IL-2 in a proliferative in vitro assay; and (3) the expression and regulation by these cytokines of receptors for hematopoietic factors, including IL-3, granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage colony- stimulating factor (GM-CSF). Flow cytometry analysis showed that freshly isolated leukemic cells from B-CLL patients bear the 75-kD TNF- R and the 55-kD IL-2R; TNF alpha was able to upregulate the 55-kD IL-2R but not the 75-kD TNF-R. On the other hand, IL-2 was not able to modify the expression of the above-mentioned receptors. Although each cytokine alone was unable to induce a relevant proliferation of leukemic cells, a synergistic proliferative effect was detected when these cytokines were used in combination. Leukemic B cells from B-CLL patients bear receptors for hematopoietic factors (IL-3, G-CSF, and GM-CSF) that were upregulated in vitro by IL-2 via the 55-kD IL-2R. On the contrary, TNF alpha was unable to affect the expression of the above-mentioned receptors. These results indicate (1) that IL-2 and TNF receptors are related to each other on leukemic cells in B-CLL and (2) that the IL-2R is involved in the regulation of other structures, ie, CSF receptors, thus pointing to another functional role of this receptor complex and the related cytokine in leukemic cells.

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