B Cell Stimulation through BCR and CD40 Modulates the Response of Chronic Lymphocytic Leukemia Cells to BAFF and APRIL.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1361-1361
Author(s):  
Gerardo Ferrer ◽  
Kate E Hodgson ◽  
Victor Ciria ◽  
Gael Roue ◽  
Dolors Colomer ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Cd40 Ligation ◽  
Stimulation Of

Abstract Abstract 1361 The two TNF family proteins (B-cell activating factor [BAFF] and a proliferation-inducing ligand [APRIL]) and their three receptors (transmembrane activator and CAML interactor [TACI], B-cell maturation antigen [BCMA], and BAFF receptor [BAFF-R]) play a critical role in the process of differentiation, maturation and survival of normal B cells. Additionally, recent studies indicate that activation or inhibitory signals can modulate the sensitivity of normal B cells to BAFF and APRIL through the regulation of their receptors. In chronic lymphocytic leukemia (CLL), BAFF and APRIL have been shown to increase survival of neoplastic B cells in vitro. We investigated whether stimulation of CLL cells through the B cell receptor (BCR) or CD40 ligation could regulate the expression of BAFF-R, TACI and BCMA and enhance BAFF and APRIL sensitivity. Purified B cells were obtained from 23 CLL patients and nine healthy controls. Receptor expression was measured by flow cytometry at baseline and at 48 hours after stimulation with F(ab’)2 antihuman IgM (10 μg/ml) and CD40L (500ng/ml) plus IL-4 (20ng/ml). Cell activation and viability, as assessed by labeling CD69 and Annexin V/TO-PRO-3, were evaluated at 48, and at 72 hours after co-stimulation with either soluble BAFF (100ng/ml) or APRIL (500ng/ml). Baseline analyses showed that BAFF-R was the most highly expressed receptor in CLL cells and normal B cells (Mean fluorescence intensity (MFI) ratios, 213.5 and 185.8, respectively). TACI and BCMA were also expressed in all CLL cells and normal B cells (MFI ratios TACI: 2.5 and 1.9; BCMA: 14.8 and 6.6, respectively), but at a significantly lower level than BAFF-R (p<0.001). Furthermore, BCMA MFI ratio was significantly higher in CLL than in normal B cells (p=0.015). After 48h of culture, an increase of all three receptors was observed in normal B cells in response to either BCR stimulation or CD40 ligation. In contrast, in CLL cells BCR stimulation induced almost no variation in the receptors expression in all cases. This was accompanied by a failure of cell activation and a significant decreased viability of CLL cells (from 36% to 24% p=0.013). By contrast, CD40 ligation in CLL cells induced a significant upregulation of TACI expression (p=0.007) and a significant reduction of BCMA (p=0.007), which correlated with an increase of CLL cell activation and viability (p<0.001). BAFF-R levels did not change. The addition of exogenous soluble BAFF or APRIL showed increase in the viability of normal B cells at 72 hours independently of whether cells were unstimulated or stimulated through the BCR or by CD40 ligation. In CLL cells, however, the viability was significantly increased in CD40-stimulated cells whereas in either unstimulated or BCR-stimulated CLL cells, the addition of BAFF and APRIL had a modest effect on viability (Table). These findings indicate that stimulation of CLL cells through the BCR and CD40 modifies the sensitivity of CLL cells to respond to BAFF and APRIL which reflects the regulation of BCMA, TACI and BAFF-R. In contrast to normal B cells, CD40-ligation in CLL cells upregulated only TACI expression. The fact that the addition of CD40L plus IL-4 and BAFF increased viability in CLL cells while BAFF alone had almost no effect may be related to the ability of CD40 ligation to increase TACI expression. Although BCR stimulation failed to increase the expression of the receptors, co-stimulation by BAFF plus BCR increased viability in CLL cells. Disclosures: No relevant conflicts of interest to declare.

Concomitant, T-Independent TLR9-Mediated and BCR-Mediated Activation Provides Signals For Optimal Telomerase Induction In Chronic Lymphocytic Leukemia Cells Regardless Of IGHV Mutation Status

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4142-4142
Author(s):  
Rajendra N Damle ◽  
Sonal Temburni ◽  
Ryon M. Andersen ◽  
Jacqueline C. Barrientos ◽  
Jonathan E. Kolitz ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Activation ◽  
Telomerase Activity ◽  
Lymphocytic Leukemia ◽  
Tlr9 Activation ◽  
Cells Cultured ◽  
Stimulation Of

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the clonal amplification of CD5-expressing B cells that appear to develop and evolve based on signals from the microenvironment. In vitro and in vivo evidence suggests that the B-cell antigen receptor (BCR) and Toll-like receptors (TLRs) may be keys to this stimulation. Because clonal turnover can lead to the release of naked nuclear material into the cellular microenvironment, these remnants of dying/dead cells may contribute to disease progression by repeated low level T-independent activation of CLL cells through the combination of the BCR and TLRs. To test this hypothesis, we assessed TLR9-driven or BCR + TLR9-driven CLL B-cell activation, focusing on its impact on telomerase activation in CLL cells, which is known to be important in the disease and which we have shown to be selectively activated by BCR stimulation in Ig V-unmutated (U-CLL) clones but not in Ig V-mutated (M-CLL) clones. B cells, isolated by negative selection from peripheral blood of IgM+ CLL patients and cryopreserved until use, were cultured for 16 hr without/ with TLR9 agonist, ODN 2006, alone and were assayed for apoptosis using Annexin V and flow cytometry. To study the relative contribution of simultaneous TLR9 activation and BCR activation, B cells were exposed to ODN2006 alone or HB57dex (monoclonal anti IgM Ab conjugated onto dextran) alone or a combination of the two reagents. Extracts from cells cultured for a period of 3 days were assayed for functional telomerase activity using TRAP. Parallel cultures of B cells exposed to the same stimuli were harvested at day 3 and assayed for cell activation and proliferation, which was assessed by 3H thymidine incorporation. CLL cells cultured with ODN2006 exhibited significant apoptosis within 16 hours in 6/12 cases. However at day 3, the same stimulus elicited significant increases in percentages of CD69-expressing cells and densities of HLA-DR in all CLL cases studied. As compared to BCR activation, which upregulates telomerase activity in U-CLL only, TLR9-mediated activation of CLL induced telomerase activation in all CLL cases. Furthermore, ODN2006 elicited significantly higher induction of telomerase activity in M-CLL cases compared to U-CLL cases (p=0.01). In addition, in M-CLL cases, simultaneous activation via TLR9 and BCR significantly upregulated the telomerase activity (p=0.05) that was induced by TLR9 activation alone. IRAK-1/4 inhibitor down modulated both TLR9 mediated and TLR9 +BCR mediated telomerase activity to a greater extent in M-CLL cases than in U-CLL cases. TLR9 activation of CLL cells induced a 3.75 + 0.8 fold (range 1.1 to 19.6; n=32) increase in cell proliferation. When segregated by Ig V mutation, U-CLL cells (n=16) responded significantly better (6.0 + 1.6 fold) compared to M-CLL cells (2.1 + 0.3 fold, n=16; p=0.03). However, co-stimulation of cells via their BCR significantly increased TLR-mediated responses only in M-CLL cases (from 2.3 + 0.4 fold to 5.4 + 1.7 fold; p=0.05). IRAK-1/4 inhibitor did not exert a significant effect on TLR9 mediated cell proliferation in either the U-CLL or M-CLL cases. Co-culture of CLL cells with human stromal cells, HS5, further upregulated the concerted TLR9 + BCR induced proliferative responses in 70% of the cases studied. Together, these results indicate that simultaneous stimulation of CLL cells via both their TLR9 and BCR molecules positively impacts on telomerase activity in all patients studied. Since telomerase is crucial in maintaining longevity of repeatedly stimulated cells, this could represent a mechanism for worse clinical outcome in CLL. These studies stress the need for devising therapeutic agents or combinations thereof to effectively target multiple pathways downstream of these signaling receptors and to ultimately eradicate newly evolving CLL cells. Disclosures: No relevant conflicts of interest to declare.

Comprehensive Analysis of BAFF Binding Receptor Profiles and Receptor Occupancy in B Cell Chronic Lymphocytic Leukemia: Identification of Discrete Phenotypic Subgroups.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1135-1135
Author(s):  
Renee C. Tschumper ◽  
Jaime R. Darce ◽  
Xiaosheng Wu ◽  
Stephen A. Mihalcik ◽  
Diane F. Jelinek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Activation ◽  
Receptor Occupancy ◽  
Lymphocytic Leukemia ◽  
B Cell Activation ◽  
Normal Peripheral Blood ◽  
Fold Induction ◽  
Cell Chronic Lymphocytic Leukemia

Abstract B cell-activating factor (BAFF) is known to regulate normal B cell development and homeostasis primarily by signaling through the high affinity receptor, BAFF-R, one of three BAFF binding receptors (BBRs). BAFF also binds two other receptors, BCMA and TACI with lesser affinity. We have recently shown that normal peripheral blood (PB) B cells express high levels of prebound soluble BAFF, which is lost upon B cell activation. Because of BAFF’s activity on normal B cells, we have been interested in the roles of BAFF and BBRs in B cell chronic lymphocytic leukemia (B-CLL). We and others have demonstrated that BAFF promotes primary CLL B cell survival and that serum BAFF levels are elevated in some patients. Although CLL B cells are known to express BBRs, a comprehensive and quantitative analysis of BBR levels and CLL B cell capacity to bind BAFF has not yet been done. We began this study by characterizing the level of soluble BAFF bound to freshly isolated CLL B cells, measured by both western blot analysis and flow cytometry. To assess receptor occupancy, cells were incubated with or without exogenous BAFF before assessing anti-BAFF reactivity and changes in median fluorescence intensity (ΔMFI; defined by dividing the MFI of the anti-BAFF antibody by the MFI of the isotype matched control antibody) were calculated. Normal B cells have higher detectable levels of bound BAFF with a ΔMFI ranging from 16 to 35 (mean=22.2). Upon addition of exogenous BAFF, the ΔMFI range increased to 27–96.6 (mean=49.1; n=8). Thus, despite evidence of prebound BAFF, clearly not all BBRs were occupied on normal PB B cells. By contrast, the levels of prebound BAFF on CLL B cells were significantly lower with a ΔMFI ranging from 1 to 13.1 (mean=2.7; n=36). Of note, 10/36 patients did not exhibit increased anti-BAFF reactivity upon incubation with exogenous BAFF (mean fold induction=0.8) whereas 26/36 patients displayed a mean fold induction of anti-BAFF reactivity of 3.5. These observations prompted us to next quantitate CLL B cell BBR expression. All patient CLL B cells expressed BAFF-R but at significantly lower levels than observed in normal B cells (p=0.0009). When CLL patients were categorized into IGHV mutated (M; n=22) and unmutated (UM; n=24), UM patients were observed to express higher levels of BAFF-R (ΔMFI =8.9) than M patients (ΔMFI =5.24). Regarding TACI, we previously demonstrated that normal memory B cells uniformly express TACI (ΔMFI =12.7; n=10) and there is a small population of activated naïve B cells that express TACI at lower levels (ΔMFI =8.3; n=10). In our CLL cohort, 14/22 M patients were TACI+ (ΔMFI =7.0) and 19/24 UM patients were TACI+ (ΔMFI =4.7). Finally, whereas normal PB B cells completely lack BCMA expression, 7/22 M and 4/22 UM patients expressed BCMA. Thus, using the BBR profile and analysis of expression levels relative to normal PB B cells, the following subgroups of B-CLL can be defined: BAFF-R+; BAFF-R/TACI+; BAFF-R/BCMA+; BAFF-R/TACI/BCMA+. It remains to be determined if these BBR profiles correlate with aspects of clinical disease. In addition, given the putative importance of BAFF in this disease, it is interesting to note that in general, CLL B cells display overall lower levels of prebound BAFF. Current studies are focused on determining whether this reflects CLL B cell activation status, increased competition for BAFF, and/or reduced levels of BBR expression.

Dysregulation of TNFα-Induced Necroptotic Signaling In Chronic Lymphocytic Leukemia: Suppression of CYLD Gene by LEF1

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 377-377 ◽  
Author(s):  
Peng Liu ◽  
Bei Xu ◽  
Jianyong Li
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Death Receptors ◽  
Lymphocytic Leukemia ◽  
Ros Production ◽  
A Genome ◽  
Stimulation Of

Abstract Abstract 377 Impaired cell death program has been noted as one of the hallmarks of Chronic lymphocytic leukemia (CLL) and contributes to its accumulation of malignant monoclonal B cells as well as to chemotherapy resistance. A cell can die through apoptosis or necrosis pathway. While apoptosis is known as a regulated cellular program, necrosis is known as an accidental event caused by overwhelming stress. However, accumulating evidence suggests that necrosis can also be executed by regulated mechanisms, especially in apoptotic-deficient conditions. Recently, the term necroptosis has been used to designate one particular form of programmed necrosis induced by stimulating death receptors with agonists such as TNFα, FasL, and TRAIL. Apoptosis suppression by caspase inhibitors such as zVAD may switch apoptotic response to necroptosis or enhance necroptosis. In contrast to well-characterized apoptotic pathway, the detailed molecular mechanisms underlying necroptosis are still not fully understood. A genome wide siRNA screen revealed two members of the receptor interacting protein (RIP) kinase family, RIP1 and RIP3P, to be essential for necroptosis. Upon stimulation of death receptors, RIP3 is recruited to RIP1 to form a necroptosis-inducing complex which is essential for cell death execution. The deubiquitinase cylindromatosis (CYLD) is recruited to TNFα receptor upon its activation and directly regulates RIP1 ubiquitination. In addition, by activating key enzymes of metabolic pathways, RIP3 regulates TNFα-inducing mitochondrial reactive oxygen species (ROS) production, which partly accounts for its ability to potentiate necroptosis. Until now, much less is known about the significance of necroptosis in malignant disease. Here we demonstrate that primary CLL cells failed to undergo necroptosis upon stimulation of TNFα combined with pan-caspase inhibitor zVAD. Upon TNFα+zVAD stimulation, normal CD19+ B cells increased ROS production > 8 fold, while same treatment only resulted in ∼ 2 fold induction in ROS generation in CLL samples. Two core components of necroptotic machine, RIP3 and CYLD, are markedly down-regulated in CLL compared with normal B cells, at both protein and transcription levels. Moreover, we identified LEF1, a downstream effector of Wnt/β-catenin pathway, as a transcription repressor of CYLD in CLL. LEF1 is highly expressed in CLL cells, whereas normal B cells have very low levels of LEF1 expression. Attenuation of LEF1 expression through RNAi technology resulted in a dramatic increase in CYLD levels in CLL cells, as determined by western blot and real time RT-PCR analysis. Dual-luciferase assays showed that forced expression of LEF1 markedly decreased CYLD promoter activity compared with controls. Mutation of LEF1 responsive elements (LERs) on CYLD promoter significantly abolished transcriptional repression of CYLD by LEF1. Chromatin immunoprecipitation assays showed that LEF1 is recruited to LER region within the CYLD promoter in CLL cells. Additionally, Knocking down LEF1 sensitizes CLL cells to TNFα-induced necroptosis. The present investigation provides the first evidence that CLL cells have defects not only in apoptotic program but also in necroptotic signaling. Targeting the key regulators of necroptotic machine such as LEF1 to restore this pathway may represent a novel approach for CLL treatment. Disclosures: No relevant conflicts of interest to declare.

B-Cell Chronic Lymphocytic Leukemia (B-CLL) Cells Unresponsive to CD180 Ligation Fail to Respond to Anti-IgM Stimulation as Well

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3582-3582
Author(s):  
Nino Porakishvili ◽  
Peter Lydyard ◽  
Anna Bremser ◽  
Ketki Vispute ◽  
Azka Memon ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Protein Kinases ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Intracellular Signalling ◽  
Orphan Receptor ◽  
B Cell Activation ◽  
Ki 67

Abstract Abstract 3582 Introduction: We have demonstrated that CD180, an orphan receptor of the Toll-like receptor family, is expressed heterogeneously on B-CLL cells, mainly on those with mutated IGVH genes. We further showed that specific ligation of CD180 with mAbs induced activation and cycling of only ~50% CD180+ B-CLL clones (“R”: responders), while CD180+ B-CLL cells unresponsive to CD180 ligation (“NR”: non-responders) or CD180− B-CLL cells could not be activated through either CD40 or IL-4 suggesting anergy. Because CD180 has a short intracellular domain, it presumably, signals through pathways associated with other receptors, such as smIgM. Indeed, engagement of smIgM or CD180 induces Lyn and Syk phosphorylation. Here we compare activation, cycling and phosphorylation of intracellular protein kinases in R and NR and CD180− B-CLL clones and B lymphocytes from healthy subjects upon ligation of smIgM. Methods: B-CLL cells were analyzed for smCD180 and smIgM, and sm CD180+IgM+ B-CLL clones were categorized as R and NR by responsiveness to CD180 ligation. Leukemic clones from 15 smCD180+IgM+R, 14 smCD180+IgM+NR, 12 smCD180−IgM+ untreated B-CLL patients and 14 healthy age-matched individuals were stimulated with goat F(ab’)2 anti-human IgM pAbs for 72h, and stained with PE~anti-CD86 mAbs, or fixed, permeabilized and stained with PE~anti-Ki-67 to assess B-cell activation and cycling, respectively. In order to study early intracellular signalling events, cells were stimulated with the same antibodies for 20 min, fixed, permeabilized and stained with Alexa Fluor~rabbit/mouse antibodies to phospho-Akt, phospho-ERK, phospho-p38MAPK, and phospho-ZAP70/Syk. Unstimulated cells in medium were used as controls. Results were assessed by flow cytometry and analyzed with the Mann-Whitney U test and paired t-test where appropriate. Results: ligation of sIgM on smCD180+IgM+R B-CLL cells resulted in a significant increase in CD86+ cells (66.3±21.7% vs 18.7±12.0%, p=0.00004) and Ki-67+ cells (38.9±10.5% vs 11.1±5.9%, p=0.0001) compared to medium controls; this was not different from the increase in activation and cycling of normal B cells (not shown). In contrast, smCD180+IgM+NR B-CLL cells failed to significantly upregulate CD86 in response to anti-IgM pAbs (20.6±13.8% vs 17.6±13.7%, p=0.334) and Ki-67 (8.4±4.6% vs 5.3±1.4%, p=0.063). Interestingly, smCD180−IgM+ B-CLL cells demonstrated diminished CD86 upregulation following sIgM ligation: 36.9±21.7% vs 11.0±4.7% in medium, p=0.058 (difference with smCD180+IgM+R B-CLL, p=0.0069). Cell cycling was also decreased: 9.7±4.1% vs 5.4±3.6% in medium, p=0.015 (difference with smCD180+IgM+R, p=0.0022). The proximal stages of anti-smIgM responses were further studied by intracellular signalling of protein kinases associated with the IgM-signalling pathway. While ligation of sIgM on control B cells and smCD180+IgM+R B-CLL cells resulted in phosphorylation of all four enzymes studied, smCD180+IgM+NR cells failed to signal downstream from ZAP70/Syk following sIgM ligation (Table 1), although there was a greater heterogeneity in smCD180+IgM+R B-CLL responses, compared to normal B cells. Importantly, smIgM ligation of smCD180−IgM+ B-CLL cells did not increase phosphorylation of Erk or p38MAPK, although some such clones responded to smIgM ligation by phosphorylation of ZAP70/Syk and Akt (data not shown). Conclusions: B-CLL clones that are smCD180+IgM+ but unresponsive to CD180 ligation (~30% of all B-CLL cases) are also unresponsive (anergic) to smIgM ligation measured by intracellular signalling, cell activation and cycling. Meanwhile, smCD180−IgM+ B-CLL clones respond heterogeneously to IgM crosslinking. Disclosures: No relevant conflicts of interest to declare.

CCR4:CCL17 Interaction Influences TLR-9 Mediated Cell Survival and Proliferation In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3593-3593
Author(s):  
Sonal C. Temburni ◽  
Ryon M. Andersen ◽  
Luke Janson ◽  
Xiao-Jie Yan ◽  
Barbara Sherry ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Dose Range ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
Novel Therapy

Abstract Abstract 3593 Unlike other hematologic disorders, chronic lymphocytic leukemia(CLL) exhibits remarkable heterogeneity in the rates of disease progression among cases. CLL cells survive by receiving signals from the microenvironment via various receptors: B-cell antigen receptor (BCR), Toll-like receptors (TLRs) and cytokine and chemokine receptors. We previously reported that CLL clones with somatically mutated IGHVs and high (≥30%) percentage of CD38 expressing cells have the highest percentage of CCR4-expressing cells. To further explore the functional contribution of the CCR4:CCL17 axis in CLL, we studied CCL17-induced chemotactic behavior in 16 CLL cases. In transwell cultures we observed a bimodal migratory response to CCL17 at 2 doses in a dose range of 0.78– 25ng/ml, in ~60% of cases; the remaining cases showed maximal migration at a single dose (1.56 or 3.12ng/ml). A comparison of phenotypes of the migrated and non-migrated cell populations was undertaken in 10 cases, analyzing CXCR3, CXCR4, CCR4 and CCR7 that are involved in homing of cells to sites favoring growth, and CD31, CD38 and CD69, activation related molecules. The migrated cells consistently showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 in the CCR4-mediated downstream pathway. CCR4 ligand, CCL17, is constitutively expressed in the thymus and is produced by dendritic cells, endothelial cells, keratinocytes and fibroblasts, whereas CCL22 is produced by tumor cells and the tumor microenvironment. Serum levels of both these ligands in untreated patients were quantified by ELISA. CCL17 levels ranged between 45-1, 229 pg/ml in U-CLL cases (n=23) and between 43-1, 418 pg/ml in M-CLL cases (n=30). CCL22 levels ranged between 121-5, 497 pg/ml in U-CLL cases (n=23) and 409-5, 502 pg/ml in M-CLL cases (n=30). The percentages of CCR4- expressing B cells directly correlated with percentages of T cells expressing CCR4 in individual cases, whereas they inversely correlated with both, serum levels of CCL17 (p< 0.01) and CCL22 (p< 0.05). CCL17 produced by DCs in peripheral organs may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells. We therefore tested if CCL17 supported BCR- and TLR-mediated proliferative responses in a cohort of 31 (16 U-CLL and 15M-CLL) CLL cases. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, but only in 3/15 (20%) M-CLL cases. On the other hand, CCL17 showed an additive effect in promoting TLR-9-mediated cell proliferation in 13/15 (87%) M-CLL cases at a dose of 2ng/nl (approximating that detected in serum); it also augmented TLR-9 mediated B cell proliferation in 6/16 U-CLL cases but at a 5-fold or higher dose (10-25 ng/ml). In a subset of this cohort (8 cases) CCL17-induced modulation of molecules involved in the apoptotic process was studied. We found upregulation of anti-apoptotic proteins Mcl-1 and Bcl2 and down-regulation of pro-apoptotic molecules Bim, PUMA, and Bid in 5 of these cases. The pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating TLR-9-mediated signaling and migration in CLL. Therefore, inhibition of CCR4:CCL17 interaction in vivo represents a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival. Disclosures: No relevant conflicts of interest to declare.

Role Of Lyn Kinase In The Pathogenesis Of Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 668-668
Author(s):  
Phuong-Hien Nguyen ◽  
Nina Reinart ◽  
Michael Hallek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Lymphoid Tissues ◽  
Malignant Cells ◽  
Deficient Mice

Abstract The Src family kinase Lyn is predominantly expressed in B cells and plays a central role in initiating B cell receptor (BCR) signaling. Lyn is associated with BCR complexes and is renowned for its role in B cell activation and proliferation. Active Lyn contributes to positive regulation of signalling through tyrosine phosphorylation of components of the BCR. Intriguingly, Lyn was also shown as a negative regulator of BCR signal transduction. Lyn plays an essential role in negative regulation of signalling through its unique ability to phosphorylate immunoreceptor tyrosine based inhibition motifs (ITIM) in inhibitory cell surface receptors. ITIM phosphorylation induces the recruitment of inhibitory phosphatases such as SHP-1/2 and SHIP-1, which attenuate BCR signalling. Lyn-deficient mice have reduced number of B cells and increased numbers of myeloid progenitors. It was reported that expression and activity of Lyn in human chronic lymphocytic leukemia (CLL) is elevated compared to healthy B cells. Besides, higher levels of Lyn are associated with a shorter treatment-free survival of CLL patients. This rises up a hypothesis about Lyn’s significant role in B cell tumorigenesis, malignant transformation of B cells, and the balance between myeloid cells and B lymphocytes. We generated Eµ-TCL1 transgenic LYN-deficient mice (TCL1+/wtLYN-/-) and monitored them in order to identify the population of malignant B cells and to characterize the development of malignant cells in these mice in comparison with Eµ-TCL1 transgenic mice (TCL1+/wtLYNwt/wt). In comparison to TCL1+/wtLYNwt/wt mice, TCL1+/wtLYN-/- mice show a significantly reduced number of malignant B cells in the peripheral blood, as well as a reduced leukocyte count. Besides, TCL1+/wtLYN-/- mice have significantly decreased infiltration of malignant B cells in lymphoid tissues such as spleen, liver, lymph node and bone marrow. This result is also resembled in a hepato-splenomegaly in the TCL1+/wtLYNwt/wt mice. These mice develop severe splenomegaly and hepatomegaly due to infiltration of malignant cells, while TCL1+/wtLYN-/- mice do not develop hepatomegaly. The non-transgenic LYN-/- control mice develop splenomegaly due to infiltration of myeloid cells. Although TCL1+/wtLYN-/- mice have hindered development of TCL1-induced CLL, preliminary data suggest it is not only due to LYN-deficiency in B cell compartment of these mice. Indeed, B cell of TCL1+/wtLYN-/- mice show enhanced proliferation and better survival ex vivo compared to TCL1+/wtLYNwt/wt mice. Notably, TCL1+/wtLYN-/- mice developed a skewed microenvironment which might contribute to CLL down regulation. LYN-/- microenvironment, particularly in aged mice, does not support engraftment of TCL1-induced leukemic B cell as well as LYNwt/wt mice in our transplantation model. These results point to a complex regulation of Lyn signalling in CLL involving not only leukemic cells but also cells of the micromillieu, that needs further investigation. Disclosures: No relevant conflicts of interest to declare.

An Alternatively Spliced Form of CD79b Gene May Account for Altered B-Cell Receptor Expression in B-Chronic Lymphocytic Leukemia

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2327-2335 ◽  
Author(s):  
A. Alfarano ◽  
S. Indraccolo ◽  
P. Circosta ◽  
S. Minuzzo ◽  
A. Vallario ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Rt Pcr ◽  
Alternatively Spliced

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

Humanized Anti CD-40 Antibody SGN-40 Effectively Induces Cytotoxicity Against Chronic Lymphocytic Leukemia (CLL) Cells through Antibody Mediated Cytotoxicity and Demonstrates Modest Biologic Evidence of CD40 Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2966-2966 ◽  
Author(s):  
Aruna C. Gowda ◽  
Xiaobin B. Zhao ◽  
Carolyn Cheney ◽  
Najma Mehter ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Activation ◽  
Cd40 Ligand ◽  
Lymphocytic Leukemia ◽  
Extrinsic Pathway ◽  
Concurrent Administration ◽  
Cd40 Ligation ◽  
Cd40 Activation ◽  
Induced Apoptosis

Abstract The CD40 antigen is involved in cell survival and differentiation of B-cells and is uniformly expressed on chronic lymphocytic leukemia (CLL) cells. The CD40/CD40L interaction stimulates B-cells, dendritic cells and monocytes to proliferate, differentiate, up regulate co-stimulatory molecules and increase antigen presentation. While activation of CD40 can protect CLL cells against early fludarabine-induced apoptosis, these cells become sensitive to delayed death by extrinsic pathway apoptosis. (Blood, 105: 3193–8, 2005). SGN-40 is a humanized anti-CD40 antibody entering clinical trials and has been reported to have weak agonistic properties following CD40 ligation. To pursue rational clinical development of SGN-40, we studied the effects of this antibody in fresh, non-cryopreserved primary CLL cells. These studies included classic antibody mediated killing mechanisms and evidence of both CLL cell activation and protection against early fludarabine-mediated apoptosis. CLL cells treated with SGN-40 (10 mcg/ml) for 2 hours (hrs) in the presence of human serum promoted no complement mediated cytoxicity (CDC) in 8 pts tested. Direct SGN-40 induced apoptosis of human CLL cells with or without anti-Fc IgG cross-linking at 24, 48 and 72 hrs was not increased over that observed with the isotype control antibody trastuzumab in 8 pts studied. In contrast, SGN-40 induced antibody dependent cellular cytotoxicity (ADCC) against CLL cells an average of 12% (±11.39 SD, range 2–32%) killing at 4 hrs (effector to target cell ratio 25:1) in 6 pts tested. The SGN-40 induced ADCC against CLL cells were similar to that observed with alemtuzumab (average 19%, SD 6.9, range10–30%) or rituximab (average 18%, SD 12.48, range 8–42.5%). SGN-40 also mediated death in Raji and 697 lymphoblastic lymphoma cell lines via ADCC. Similar to reports by others with CD40 ligand, SGN-40 mediated activation was noted with modest up-regulation of CD80 and HLA-DR at 48hrs. When administered prior to fludarabine, SGN-40 also protected against death in 5 consecutive samples, although this was less than observed with CD40 ligand transfected HeLa cells, consistent with incomplete CD40 activation. Concurrent administration of SGN-40 and fludarabine did not protect from drug-mediated apoptosis. In conclusion, these findings suggest that SGN-40 has dual property of mediating cytotoxic effect by ADCC and partial CD40 activation. Development of SGN-40 as a therapeutic agent in CLL is justified and future studies by our group are focusing on enhancing SGN-40 mediated ADCC against CLL cells and potentially designing combination studies with SGN-40 to exploit this agent’s ability to engage the CD40/CD40L network.

Expression of ZAP-70 in Chronic Lymphocytic Leukemia Cells Enhances CD79b Phosphorylation Following Surface IgM Ligation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2799-2799
Author(s):  
Liguang Chen ◽  
John Apgar ◽  
Li Tang ◽  
Thomas J. Kipps
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Intracellular Signaling ◽  
Critical Role ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Surface Molecule ◽  
Bcr Signaling ◽  
Accessory Molecules

Abstract CD79b is B-cell surface molecule that non-covalently associates with CD79a and surface immunoglobulin (sIg), which together serve as the B-cell receptor complex (BCR). Both CD79a and CD79b have cytosolic immunoreceptor tyrosine-based activation motifs (ITAMs) that can become phosphorylated following sIg ligation, thereby allowing for recruitment to the BCR complex of cytosolic kinases, such as p72Syk , which then can initiate downstream intracellular signaling events. Compared to normal B cells, chronic lymphocytic leukemia (CLL) B cells typically expresses low levels of CD79b, which is speculated to contribute to the relatively poor capacity of CLL cells to initiate intracellular signaling following BCR ligation despite having apparently adequate levels of p72Syk. BCR signaling in CLL cells can be enhanced by expression of the zeta-associated protein of 70 kD (ZAP-70), a tyrosine kinase that initially was identified in T cells, where it plays a critical role in the phosphorylation of ITAMs of the accessory molecules of the T-cell receptor (TCR) complex for antigen following TCR ligation. We investigated for phosphorylation of CD79b following BCR ligation with F(ab)2 anti- μ antibody in CLL cell samples that did or did not express ZAP-70. All CLL cell samples expressed similar amounts of surface IgM and p72Syk, as assessed via flow cytometry and immunoblot analysis. Within 10 minutes after treatment with anti-μ the CLL cell samples that expressed ZAP-70 (n = 28) experienced a mean increase in phosphorylation of CD79b of 21.5% (± 14.0% S.D.), which was significantly greater than the 7.5% increase (± 7.9% S.D.) experienced by similarly treated CLL cell samples that did not express ZAP-70 (n = 19) (P< 0.01). Immune precipitation studies demonstrated association of CD79b with p72Syk in CLL B cells. CLL cell samples (n = 5) lacking expression of ZAP-70 were transfected with a control vector or an expression vector encoding ZAP-70, allowing us to examine the effect that engineered-expression of ZAP-70 has on CD79 phosphorylation following treatment with anti-μ. Anti-μ treatment induced significantly higher mean levels of CD79b phosphorylation in CLL samples made to express ZAP-70 (33% ± 16%) than in control mock-transfected CLL cells (4% ± 2%). This also was associated with enhanced anti-μ induced phosphorylation of p72Syk. We conclude that expression of ZAP-70 in CLL B cells enhances phosphorylation of the accessory molecules in the BCR complex following sIg ligation, potentially allowing for improved recruitment of cytosolic kinases and adapter proteins to these accessory molecules for enhanced BCR signaling.

scholarly journals Nurselike cells express BAFF and APRIL, which can promote survival of chronic lymphocytic leukemia cells via a paracrine pathway distinct from that of SDF-1α

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 1012-1020 ◽  
Author(s):  
Mitsufumi Nishio ◽  
Tomoyuki Endo ◽  
Nobuhiro Tsukada ◽  
Junko Ohata ◽  
Shinichi Kitada ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Survival ◽  
Cell Activation ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Stromal Cell Derived Factor

AbstractWe examined expression of B cell–activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) on chronic lymphocytic leukemia (CLL) B cells and nurselike cells (NLCs), which differentiate from CD14+ cells when cultured with CLL B cells. NLCs expressed significantly higher levels of APRIL than monocytes and significantly higher levels of BAFF and APRIL than CLL B cells. Also, the viability of CLL B cells cultured with NLCs was significantly reduced when CLL B cells were cultured with decoy receptor of B-cell maturation antigen (BCMA), which can bind both BAFF and APRIL, but not with BAFF receptor:Fc (BAFF-R:Fc), which binds only to BAFF. The effect(s) of BAFF or APRIL on leukemia cell survival appeared additive and distinct from that of stromal cell–derived factor-1α (SDF-1α), which in contrast to BAFF or APRIL induced leukemia cell phosphorylation of p44/42 mitogen-activated protein kinase (extracellular signal-regulated kinase-1/2 [ERK1/2]) and AKT. Conversely, BAFF and APRIL, but not SDF-1α, induced CLL-cell activation of the nuclear factor–κB1 (NF-κB1) and enhanced CLL-cell expression of the antiapoptotic protein Mcl-1. However, BAFF, but not APRIL, also induced CLL-cell activation of NF-κB2. We conclude that BAFF and APRIL from NLCs can function in a paracrine manner to support leukemia cell survival via mechanisms that are distinct from those of SDF-1α, indicating that NLCs use multiple distinct pathways to support CLL-cell survival.

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