Purified B-cell Chronic Lymphocytic Leukemia Cells Research Articles

Mechanistic Insights into CpG DNA and IL-15 Synergy in Promoting B Cell Chronic Lymphocytic Leukemia Clonal Expansion.

Malignant cell growth within patients with B cell chronic lymphocytic leukemia (B-CLL) is largely restricted to lymphoid tissues, particularly lymph nodes. The recent in vitro finding that TLR-9 ligand (oligodeoxynucleotide [ODN]) and IL-15 exhibit strong synergy in promoting B-CLL growth may be particularly relevant to growth in these sites. This study shows IL-15-producing cells are prevalent within B-CLL-infiltrated lymph nodes and, using purified B-CLL cells from blood, investigates the mechanism for ODN and IL-15 synergy in driving B-CLL growth. ODN boosts baseline levels of phospho-RelA(S529) in B-CLL and promotes NF-κB-driven increases in IL15RA and IL2RB mRNA, followed by elevated IL-15Rα and IL-2/IL-15Rβ (CD122) protein. IL-15→CD122 signaling during a critical interval, 20 to 36-48 h following initial ODN exposure, is required for optimal induction of the cycling process. Furthermore, experiments with neutralizing anti-IL-15 and anti-CD122 mAbs indicate that clonal expansion requires continued IL-15/CD122 signaling during cycling. The latter is consistent with evidence of heightened IL2RB mRNA in the fraction of recently proliferated B-CLL cells within patient peripheral blood. Compromised ODN+IL-15 growth with limited cell density is consistent with a role for upregulated IL-15Rα in facilitating homotypic trans IL-15 signaling, although there may be other explanations. Together, the findings show that ODN and IL-15 elicit temporally distinct signals that function in a coordinated manner to drive B-CLL clonal expansion.

Correlation of clinical activity of lumiliximab in combination with FCR and ZAP 70 expression on B-cell chronic lymphocytic leukemia cells

7073 Background: Lumiliximab is an anti-CD23 monoclonal antibody under investigation for the treatment of patients with relapsed B-cell chronic lymphocytic leukemia (BCLL). Lumiliximab binds specifically to human CD23, a glycoprotein expressed on the majority of BCLL cells. As ZAP70 is a poor prognostic factor of CLL, expression of the ZAP70 antigen by malignant B cells was analyzed on samples taken pre-treatment and day 2 post-treatment in a subset of patients with relapsed BCLL enrolled in a clinical study evaluating lumiliximab as monotherapy. The ability of lumiliximab to induce ex-vivo apoptosis was also evaluated in BCLL samples collected in this subset. Subsequently, pre-treatment ZAP70 expression was analyzed in patients treated lumiliximab in combination with fludarabine, cyclophosphamide, and rituximab (L + FCR). Methods: In the lumiliximab monotherapy study, ZAP70 expression was quantified by Western blot assay using lysates from purified BCLL cells and apoptosis was evaluated by flow cytometric assessment of Caspase-3 activation on pre- treatment and day 2 treated samples (ex-vivo). In the L + FCR study, frequencies of BCLL cells and ZAP70 positive cells were estimated by four color flow cytometry using standard commercially available antibodies. BCLL cells were identified by first gating on viable CD45+ cells and subselecting with antibodies to CD5 and CD19 receptors (CD5+CD19+ phenotype). ZAP70 expression was reported as % BCLL cells. Results: Analysis of data from the monotherapy study suggested induction of apoptosis and decrease in CD5+CD19+ cells in patients expressing high levels of ZAP70 on purified B cells. Moreover, apoptosis was also observed in a patient that expressed activated ZAP70. Results from the L + FCR study demonstrated that clinical activity in this combination trial is observed in patients having high or low levels of ZAP70 expression. Conclusions: Lumiliximab potentially provides clinical benefit in BCLL patients with both low and high levels of ZAP70, whose expression is associated with aggressive disease and poor prognosis. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Biogen Idec Biogen Idec Biogen Idec

Attacking Oncogene Addiction in B-CLL: Seliciclib First Engages the Mcl-1/Noxa Axis, after Which Gradual Exhaustion of Bcl-2 Protection Leads to Bax Activation.

Background : Seliciclib (R-roscovitine) is a cyclin-dependent kinase inhibitor in clinical development. It triggers apoptosis by inhibiting de novo transcription of the short-lived anti-apoptotic Mcl-1 protein, but it is unclear if and how this leads to Bax or Bak activation that is required for most forms of cell death.Aim: To study the effects of seliciclib on apoptosis gene expression and Mcl-1 and Bcl-2 protein interactions in B cell chronic lymphocytic leukemia (B-CLL), a malignancy with known aberrant apoptosis regulation.Methods: Purified B-CLL cells (PBMC consisting of >90% B-CLL cells; n=20) and Ramos cell lines overexpressing different apoptosis regulators were used in this study. The effect of seliciclib on viability, apoptosis gene expression pattern, and protein associations was investigated via RT-Multiplex-Ligation-dependent Probe Amplification (RT-MLPA), Western blotting and co-immunoprecipitation assays. Ramos cells were transduced with retroviral vectors expressing either Noxa siRNA, Bim siRNA or control-GFP virus, and tested for different apoptosis stimuli.Results: We found that although seliciclib resulted in proteasome-dependent Mcl-1 degradation within 4 hrs in B-CLL cells, Bax and Bak activation and apoptosis occurred with a considerable delay, i.e. at 16 hrs. During this period, there was no evidence of transcriptional changes in p53-responsive or apoptosis-related genes. In freshly isolated, viable B-CLL cells, pro-survival Mcl-1 was engaged by the pro-apoptotic proteins Noxa and Bim but not by Bak. The contribution of Noxa [Figure1] and Bim (liberated from McL-1 within 4 hours) as specific mediators of seliciclib-induced apoptosis was demonstrated via RNAi in two model systems. Interestingly, 16 hrs after seliciclib treatment, there was a clear accumulation of Bcl-2, Bim, and Bax in the detergent insoluble (mitochondria containing) fraction of B-CLL cells. This suggests that after Mcl-1 degradation, the remaining apoptosis neutralizing capacity of Bcl-2 is gradually overwhelmed, probably resulting in Bax multimerisation and pore formation in the mitochondria.Conclusions: These data support the 'oncogene-addiction' model in which malignant cells depend on increased Bcl-2 levels, and extend it to include Mcl-1. Furthermore, since Noxa is elevated in B-CLL, its involvement in p53-independent apoptosis suggests this BH3-only protein may be a therapeutic target. [Display omitted]

Gene and surface-antigen expression profilings concordantly identify alpha4-integrin/CD49d as a marker for unmutated (UM) bad prognosis B-cell chronic lymphocytic leukemia (B-CLL)

10076 Background: The highly heterogeneous clinical courses of B-CLL can be foreseen by investigating IgVH gene mutations or expression of specific prognosticators. Gene and surface-antigen expression profilings (GEP and SEP) have been both employed for identifying molecules of prognostic relevance in B-CLL. Methods: i) GEP - Purified B-CLL cells from 19 UM and 38 mutated (M) cases were investigated for differential GEP by using a two-color Operon Human Genome Oligo Set 2.1 platform with normal B cells as common reference and by applying, after data pre-processing, the LIMMA (Linear Model for MicroArray) package with two combined cutoffs (Bayesian log-odds>1; adjusted p value for false discovery rate <10e−4); ii) SEP - B-CLL cells from 60 UM and 101 M cases were investigated for the expression of 36 surface markers by flow cytometry. Results: i) GEP - 77 probes (32 duplicates) were overexpressed in M B-CLLs (UM/M log-ratio range −0.79/−3.62; p range 6.72e−5/3.6e−10) and 81 probes (46 duplicates) in UM B-CLLs (UM/M log-ratio range 0.87/4.57; p range 9.49e−5/3.01e−12); the CD49d gene was overexpressed in UM cases with UM/M log-ratio of 3.21 (p=8.3e-10). ii) SEP - Six markers significantly discriminated UM to M B-CLLs (t-test, p<10ed-3) and separated most UM (49/60) from M B-CLLs by hierarchical clustering; among them, CD49d was significantly overexpressed in UM cases (median % positive cells 71.2±36 vs. 10.0±31; p=2.1e−10). By applying standardized log-rank statistics in 95 pts, all with CD49d expression and survival data, 30% of positive cells was judged optimal cutoff for identifying two groups with different survivals, 41 CD49dhigh pts showing worse prognosis than 54 CD49dlow cases (p=7.4×10e−5). Similarly, 46 UM B-CLLs had shorter survival than 77 M cases (p=1.4×10e−5). By combining IgVH mutations and CD49d expression, 42 concordant M/CD49dlow pts had better prognosis than 25 concordant UM/CD49dhigh cases (p=1.8×10e−5); noteworthy, among 28 discordant cases, 16 with a M/CD49dhigh phenotype had survival similar to bad prognosis cases. Conclusions: CD49d is a novel prognosticator for B-CLL. Given its high expression level in bad prognosis subsets, CD49d may be a promising therapeutic target. No significant financial relationships to disclose.

CD38 is a signaling molecule in B-cell chronic lymphocytic leukemia cells

AbstractThe prognosis for patients with B-cell chronic lymphocytic leukemia (B-CLL) is generally less favorable for those expressing CD38. Our working hypothesis is that CD38 is not merely a marker in B-CLL, but that it plays a receptor role with pathogenetic potential ruling the proliferation of the malignant clone. CD38 levels were generally low in the patients examined and monoclonal antibody (mAb) ligation was inefficient in signaling. Other cellular models indicated that molecular density and surface organization are critical for CD38 functionality. Interleukin 2 (IL-2) induced a marked up-modulation and surface rearrangement of CD38 in all the patients studied. On reaching a specific expression threshold, CD38 becomes an efficient receptor in purified B-CLL cells. Indeed, mAb ligation is followed by Ca2+ fluxes and by a markedly increased proliferation. The unsuitability of CD38 to perform as a receptor is obviated through close interaction with the B-cell–receptor (BCR) complex and CD19. On mAb binding, CD38 translocates to the membrane lipid microdomains, as shown by a colocalization with the GM1 ganglioside and with CD81, a raft-resident protein. Finally, CD38 signaling in IL-2–treated B-CLL cells prolonged survival and induced the appearance of plasmablasts, providing a pathogenetic hypothesis for the occurrence of Richter syndrome.

Chronic lymphocytic leukemia B cells inhibit spontaneous Ig production by autologous bone marrow cells: role of CD95-CD95L interaction

A variable degree of humoral immunodeficiency is a common feature in patients with B-cell chronic lymphocytic leukemia (B-CLL). The aim of this study was to explore the possibility that B-CLL cells play a direct role in this phenomenon. To this end, patients' bone marrow (BM) immunoglobulin (Ig)-secreting cells were cocultured with autologous purified B-CLL cells. The results show that tumoral cells inhibited the spontaneous IgG secretion by BM plasma cells, and this effect increased after PMA-induction of B-CLL cells. This inhibitory process was proportional to the number of B-CLL cells added and depended on cellular contact. Adhesion molecules did not appear to be involved in the cellular interaction, because the inclusion of blocking antibody to a variety of these proteins did not reverse the inhibitory phenomenon. However, the addition of monoclonal antibody that blocked the function of either CD95 or CD95L clearly reversed B-CLL cell inhibition on autologous BM plasma cells. These latter cells were shown to express CD95, and B-CLL cells contained detectable quantities of CD95L at the level of messenger RNA and protein. Annexin V–binding experiments revealed increased apoptosis of BM Ig-secreting cells when cocultured with autologous B-CLL cells. Finally, this inhibitory phenomenon might be operative in vivo because (a) there was a good correlation between the intensity of the inhibitory effect in vitro and the serum IgG level exhibited by every patient and (b) B-CLL cells also inhibited in vivo antigen-induced IgG-tetanus toxoid–secreting cells obtained from normal immunized subjects. Collectively, these data suggest that B-CLL cells inhibit autologous CD95-bearing Ig-secreting cells by the interaction with CD95L present on B-CLL cells and, hence, contribute to the state of humoral immunodeficiency that occurs in these patients.

Chronic lymphocytic leukemia B cells inhibit spontaneous Ig production by autologous bone marrow cells: role of CD95-CD95L interaction

AbstractA variable degree of humoral immunodeficiency is a common feature in patients with B-cell chronic lymphocytic leukemia (B-CLL). The aim of this study was to explore the possibility that B-CLL cells play a direct role in this phenomenon. To this end, patients' bone marrow (BM) immunoglobulin (Ig)-secreting cells were cocultured with autologous purified B-CLL cells. The results show that tumoral cells inhibited the spontaneous IgG secretion by BM plasma cells, and this effect increased after PMA-induction of B-CLL cells. This inhibitory process was proportional to the number of B-CLL cells added and depended on cellular contact. Adhesion molecules did not appear to be involved in the cellular interaction, because the inclusion of blocking antibody to a variety of these proteins did not reverse the inhibitory phenomenon. However, the addition of monoclonal antibody that blocked the function of either CD95 or CD95L clearly reversed B-CLL cell inhibition on autologous BM plasma cells. These latter cells were shown to express CD95, and B-CLL cells contained detectable quantities of CD95L at the level of messenger RNA and protein. Annexin V–binding experiments revealed increased apoptosis of BM Ig-secreting cells when cocultured with autologous B-CLL cells. Finally, this inhibitory phenomenon might be operative in vivo because (a) there was a good correlation between the intensity of the inhibitory effect in vitro and the serum IgG level exhibited by every patient and (b) B-CLL cells also inhibited in vivo antigen-induced IgG-tetanus toxoid–secreting cells obtained from normal immunized subjects. Collectively, these data suggest that B-CLL cells inhibit autologous CD95-bearing Ig-secreting cells by the interaction with CD95L present on B-CLL cells and, hence, contribute to the state of humoral immunodeficiency that occurs in these patients.

IFN-alpha stimulates proliferation and cytokine secretion of CD40-stimulated B cell chronic lymphocytic leukemia cells in vitro.

Interferon (IFN)-alpha has a therapeutic effect in several B cell malignancies, including low-grade non-Hodgkin's lymphoma (NHL), multiple myeloma, and hairy cell leukemia, whereas its efficacy in the treatment of B cell chronic lymphocytic leukemia (B-CLL) is rather limited. In the present study, we investigated the effect of IFN-alpha on the biologic functions of B-CLL cells, which were stimulated by cross-linking of the CD40 antigen. In cell samples from 16 B-CLL patients, the addition of IFN-alpha to CD40-stimulated purified B-CLL cells caused a significant increase in [3H]thymidine uptake (p < 0.003). In B-CLL cells maximally activated by CD40 cross-linking and interleukin-2 (IL-2)/IL-10, proliferation was not further enhanced or inhibited by IFN-alpha. In contrast, proliferation of normal tonsillar B cells stimulated by the combination CD40/IL-2/IL-10 was significantly inhibited by IFN-alpha. Because B-CLL activation might be enhanced by induction of the autocrine growth factor tumor necrosis factor-alpha (TNF-alpha), we investigated the effect of IFN-alpha on the secretion of B cell tropic cytokines. In fact, IFN-alpha significantly stimulated the secretion of IL-6 and TNF-alpha in CD40-activated B-CLL cells (p < 0.01). Although exogenous addition of TNF-alpha did not influence activation of CD40-stimulated B-CLL cells, neutralization of TNF-alpha by polyclonal antibodies led to a complete inhibition of CD40-mediated proliferation of B-CLL cells, suggesting that enhanced proliferation and increased cytokine production by CD40-activated B-CLL cells are independent IFN-alpha-mediated events. The studies presented here provide evidence that IFN-alpha mediates costimulatory signals in the context of T cell-mediated B-CLL cell activation.

Cytokine gene expression in B-cell chronic lymphocytic leukemia: evidence of constitutive interleukin-8 (IL-8) mRNA expression and secretion of biologically active IL-8 protein

To extent our knowledge on the cytokines possibly involved in the pathophysiology of B-cell chronic lymphocytic leukemia (B-CLL), the mRNA expression of a panel of 10 cytokines was investigated on purified B-CLL cells using a reverse-transcriptase polymerase chain reaction method. Whereas negative RT-PCR signals were recorded for interleukin-1 alpha (IL-1 alpha), IL-2, IL-3, IL-4, IL-5, IL-7, tumor necrosis factor beta (TNF beta), and granulocyte-macrophage colony-stimulating factor, we detected the expression of IL-1 beta, IL-6 and TNF alpha. Furthermore, the constitutive expression of IL-8 mRNA was observed in all 17 B-CLL samples analyzed. mRNA expression was associated with the capacity of the leukemic cells to release IL-8 both constitutively (4.6 +/- 8.1 SD ng/mL) and, to a further extent, after stimulation (14.5 +/- 19.4 ng/mL). The circulating levels of IL-8 were also evaluated in 12 untreated B-CLL sera samples and the overall mean level was significantly higher (P &lt;.01) than in normal sera. In addition, supernatants of purified B-CLL cells cultured in the presence of 12-O- tetradecanoylphorbol-13-acetate showed chemotactic activity towards neutrophils; this activity was neutralized in the presence of an anti- IL-8 antiserum. The mRNA for IL-8 was absent in five B-cell preparations from hairy cell leukemia cases and in four B-cell lines. Normal tonsil CD5+ B cells showed a low expression of IL-8 mRNA only in two of the nine preparations tested and the overall quantity of IL-8 released by these cells after 3 days' incubation was significantly lower compared with that released by B-CLL cells (0.4 +/- 0.3 and 1.6 +/- 0.9 ng/mL under basal and stimulated conditions, respectively). These findings point to an involvement of a member of the proinflammatory chemokine supergene family in human CD5+ B lymphocytes. The different IL-8 behavior observed between B-CLL cells and their normal counterpart is likely to reflect an activation state of the leukemic population.

Cytokine Gene Expression in B-Cell Chronic Lymphocytic Leukemia: Evidence of Constitutive Interleukin-8 (IL-8) mRNA Expression and Secretion of Biologically Active IL-8 Protein

To extent our knowledge on the cytokines possibly involved in the pathophysiology of B-cell chronic lymphocytic leukemia (B-CLL), the mRNA expression of a panel of 10 cytokines was investigated on purified B-CLL cells using a reverse-transcriptase polymerase chain reaction method. Whereas negative RT-PCR signals were recorded for interleukin-1 alpha (IL-1 alpha), IL-2, IL-3, IL-4, IL-5, IL-7, tumor necrosis factor beta (TNF beta), and granulocyte-macrophage colony-stimulating factor, we detected the expression of IL-1 beta, IL-6 and TNF alpha. Furthermore, the constitutive expression of IL-8 mRNA was observed in all 17 B-CLL samples analyzed. mRNA expression was associated with the capacity of the leukemic cells to release IL-8 both constitutively (4.6 +/- 8.1 SD ng/mL) and, to a further extent, after stimulation (14.5 +/- 19.4 ng/mL). The circulating levels of IL-8 were also evaluated in 12 untreated B-CLL sera samples and the overall mean level was significantly higher (P < .01) than in normal sera. In addition, supernatants of purified B-CLL cells cultured in the presence of 12-O-tetradecanoylphorbol-13-acetate showed chemotactic activity towards neutrophils; this activity was neutralized in the presence of an anti-IL-8 antiserum. The mRNA for IL-8 was absent in five B-cell preparations from hairy cell leukemia cases and in four B-cell lines. Normal tonsil CD5+ B cells showed a low expression of IL-8 mRNA only in two of the nine preparations tested and the overall quantity of IL-8 released by these cells after 3 days' incubation was significantly lower compared with that released by B-CLL cells (0.4 +/- 0.3 and 1.6 +/- 0.9 ng/mL under basal and stimulated conditions, respectively). These findings point to an involvement of a member of the proinflammatory chemokine supergene family in human CD5+ B lymphocytes. The different IL-8 behavior observed between B-CLL cells and their normal counterpart is likely to reflect an activation state of the leukemic population.

Demonstration of high-affinity interleukin-2 receptors on B-chronic lymphocytic leukemia cells: Functional and structural characterization

Functional and structural characteristics of interleukin 2 (IL-2) receptors on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed by a proliferation assay, IL-2 binding assay and cross-linking study. In the 3H-thymidine incorporation assay, purified B-CLL cells from four out of sixteen cases, in which the percentage of Tac antigen (Tac Ag) positive cells in peripheral blood lymphocytes ranged from 0 to 48.8%, responded to IL-2 (100 U/ml) after both 3- and 6-day incubation. No relationship was found between the responsiveness to IL-2 and the percentage of Tac Ag positive cells. In the radiolabeled IL-2 binding assay, however, B-CLL cells from all seven cases examined, including three cases with mitogenic response to IL-2 and four cases without mitogenic response, were shown to have both high- and low-affinity receptors. The number of high- and low-affinity receptors per cell ranged from 29-186 and from 420 to 1,800, respectively. Furthermore, with the affinity cross-linking method p55 (Tac Ag) and p70/75 were found even in cases without mitogenic response in their B-CLL cells. In conclusion, the B-CLL cells so far examined possessed high-affinity IL-2 receptors consisting of p55 and p70/75; nevertheless, this was not sufficient to respond to the mitogenic signal of IL-2.