Induction of a CTL-Like Phenotype In B-CLL Cells Stimulated with Interleukin 21 and CpG Oligodeoxynucleotides

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3906-3906
Author(s):  
Bernd Jahrsdoerfer ◽  
Sue E Blackwell ◽  
Magdalena Hagn ◽  
Verena Ebel ◽  
Thamara Beyer ◽  
...  
Keyword(s):  
B Cells ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Cpg Odn ◽  
Apoptotic Response ◽  
Hematological Diseases ◽  
Cpg Oligodeoxynucleotides ◽  
Interleukin 21 ◽  
Dependent Pathway ◽  
Stimulation Of

Abstract Abstract 3906 Interleukin 21 (IL-21) and CpG oligodeoxynucleotides (CpG ODN) are two novel and highly promising agents for the treatment of hematological diseases. Recently, we reported that IL-21 and CpG ODN induce granzyme B (GrB) and GrB-dependent apoptosis in malignant B cells from patients with B chronic lymphocytic leukemia (B-CLL), but not in healthy peripheral B cells. Here we further characterized the factors accountable for the different apoptotic response of B-CLL cells versus normal B cells. GrB induction in B-CLL cells after stimulation with IL-21 and CpG ODN was associated with upregulation of molecules, which are normally involved in the differentiation of cytotoxic T lymphocytes (CTL) including T-bet, perforin, monokine-induced by interferon-gamma (MIG) and IP-10, a finding not observed in normal healthy B cells. Furthermore, the induction of GrB in B-CLL cells by IL-21 and CpG ODN required signaling via a JAK/STAT-dependent pathway, as suggested by upregulation of phosphorylated STAT1/3. Finally, stimulation of B-CLL cells with IL-21 and CpG ODN upregulated molecules involved in cell adhesion (CD54), antigen presentation (MHC class I), co-stimulation (CD40, CD86) and GrB uptake (CD222, mannose-6-phosphate receptor), suggesting B-CLL cells activated with IL-21 and CpG ODN are able to contact other immune cells and may be able to reabsorb secreted GrB. In summary, B-CLL cells can express factors involved in cytotoxic differentiation of CTL as well as GrB in response to stimulation with IL-21 and CpG ODN. Our data provide novel insights into the aberrant signaling state of B-CLL cells and may pave the way for the development of novel immunotherapeutic treatment approaches for B-CLL. Disclosures: No relevant conflicts of interest to declare.

Stimulation of B-CLL Cells with Interleukin 21 and Toll Like Receptor Agonists Induces a CTL-Like Transcriptional Profile.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3685-3685
Author(s):  
Magdalena Hagn ◽  
Verena Ebel ◽  
Kai Sontheimer ◽  
Thamara Beyer ◽  
Sue E. Blackwell ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cells ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Cpg Odn ◽  
Toll Like Receptor ◽  
Activated T Cells ◽  
Cpg Oligodeoxynucleotides ◽  
Interleukin 21 ◽  
Stimulation Of

Abstract Abstract 3685 Poster Board III-621 Interleukin 21 (IL-21) and CpG oligodeoxynucleotides (CpG ODN) are two novel and highly promising agents for the treatment of hematological diseases. Recently, we reported that IL-21 and CpG ODN induce granzyme B (GrB) and GrB-dependent apoptosis in malignant B cells from patients with B chronic lymphocytic leukemia (B-CLL), but not in healthy peripheral B cells. Using various techniques including FACS analysis, Western immunoblotting and RT-PCR we further characterized the factors accountable for the different apoptotic response of B-CLL cells versus normal B cells. GrB induction in B-CLL cells after stimulation with IL-21 and CpG ODN was associated with upregulation of transcription factors, which are normally involved in the differentiation of cytotoxic T lymphocytes (CTL) including T-bet, Eomesodermin (EOMES) and nuclear factor of activated T cells (NFAT), a finding not observed in normal healthy B cells. Furthermore, the induction of GrB in B-CLL cells by IL-21 and CpG ODN required signaling via a JAK/STAT-dependent pathway, as suggested by simultaneous upregulation of phosphorylated STAT3 and complete abrogation of GrB expression by the pan-JAK inhibitor pyridone 6. Stimulation of B-CLL cells with IL-21 and CpG ODN upregulated molecules involved in cell adhesion (CD54), antigen presentation (MHC class I), co-stimulation (CD40, CD86), and GrB uptake (CD222), suggesting B-CLL cells activated with IL-21 and CpG ODN are able to contact other immune cells and may be able to reabsorb secreted GrB. Similar findings resulted when the toll-like receptor (TLR)7 agonist imiquimod was used instead of the TLR9 agonist CpG ODN, suggesting that comparable differentiation programs are initiated by TLR7 and TLR9. In summary, B-CLL cells can express transcription factors involved in cytotoxic differentiation of CTL as well as GrB in response to IL-21 and TLR stimulation. The B-CLL cell differentiation program described in this study could explain our recent findings of GrB-dependent apoptosis in B-CLL cells but not in benign B cells. Moreover, our data provide novel insights into the aberrant signaling state of B-CLL cells. Disclosures: No relevant conflicts of interest to declare.

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 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.

Differential Effects Of Triggering Via Toll Like Receptors In Stable Or Aggressive Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5296-5296
Author(s):  
Min Chen ◽  
Claude Capron ◽  
Gilbert Faure ◽  
Marc Maynadie ◽  
Pierre Feugier ◽  
...  
Keyword(s):  
Immune System ◽  
B Cells ◽  
Lymphocytic Leukemia ◽  
Cpg Odn ◽  
Spontaneous Apoptosis ◽  
Aggressive Disease ◽  
Mutational Status ◽  
Stimulation Of

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the peripheral blood, secondary lymphoid tissues and bone marrow of functionally defective clonal B lymphocytes with prolonged survival in vivo. Despite therapeutic achievements have been accomplished in the management of this disease, however CLL remains incurable partially because of the resistance to apoptosis of CLL B-cells and of the altered immune system of CLL patients. CLL is hetereogenous, but its evolution is mostly slow, probably linked to some level of immune control of the leukemic cells. Indeed, clinical observations have been reported of spontaneous remissions associated with the intense immunological activity following a viral infection. Clinical responses have also been observed after treatment by immunomodulating cytokines and long-term survival is described, without disease, after allogeneic stem cells transplantation. All these data suggest that immunotherapy could be useful in the treatment of CLL, possibly as an adjuvant therapy after classical immunochemotherapy schedules. Toll like receptors (TLR) are proteins of the innate immune system belonging to the family of Pattern Recognition receptors (PRRs). Recognition of their ligands by the TLR present on neutrophils, macrophages, dendritic cells and B-cells are important in the initiation of adaptive immune responses. TLR-7 and 9 are expressed by CLL B-cells (Grandjenette, Hematologica, 2007). Previous studies have shown that stimulation of TLR-9 by CpG ODN (oligodinucleotides) induces an activation of CLL B-cells while triggering TLR-7 increases in vitro apoptosis of these cells. Here we show that these effects of TLR-9 and TLR-7 stimulation differ depending on the clinical form (stable or aggressive) of the disease and on the mutational status of CLL B-cells. In vitro stimulation with three doses of Imiquimod R837 or ODN CpG M362 was carried out for three days on cells from 40 patients (22 stable, 18 aggressive, mutational status known for 35, 18 IgVH mutated, 17 unmutated). Flow cytometry was used to measure apoptosis, proliferation after carboxyfluorescein succinimidyl ester (CFSE) labeling and modulation of surface differentiation antigens. Signaling pathways after incubation were further studied by antibody arrays and western blot. Spontaneous apoptosis occurring in vitro was demonstrated to involve the mitochondrial pathway. CLL B-cells were also confirmed to proliferate strongly and produce large amounts of IL-6 and IL-8 upon triggering by phorbol myrsistate (positive control), this compound almost completely aborting in vitro apoptosis. Cells from patients with a stable disease were significantly more prone to rapid apoptosis after TLR-7 triggering with Imiquimod, compared to cells from patients with an aggressive disease which displayed only spontaneous apoptosis. This rapid apoptosis in stable patients involved the p38 MAP-Kinase pathway. It was concomitant to an important production of IL-8 and IL-6. Conversely, CpG ODN conferred a protection against apoptosis to CLL B-cells from patients with an aggressive disease. This was accompanied by the activation of numerous anti-apoptotic proteins in the cells. CpG ODN also significantly increased CLL B-cells proliferation, concomitantly to the phosphorylation of Erk and Akt proteins. ODN finally increased the expression of CD20 and CD19 on the cells’surface. The same differences in reactivity were observed comparing mutated (∼stable) and unmutated (∼aggressive) cases. These data indicate that CLL B-cells from patients with a stable or aggressive (mutated/unmutated) disease answer differently when triggered through their surface TLRs. This might have an incidence on the behavior of these cells in vivo, in answer to stimulations by microbial compounds naturally binding these structures. These properties could also be used to develop adjuvant immunotherapies by loading CpG ODN-activated CLL B-cells with autologous apoptotic fragments issued from stimulation of part of the same cells with Imiquimod. Disclosures: No relevant conflicts of interest to declare.

Growth factor requirements for the stimulation of germinal center B cells: evidence for an IL-2-dependent pathway of development

1991 ◽  
Vol 3 (12) ◽  
pp. 1243-1251 ◽  
Author(s):  
Michelle J. Holder ◽  
Yong-Jun Liu ◽  
Thierry Defrance ◽  
Leopoldo Flores-Romo ◽  
lan C. M. MacLennan ◽  
...  
Keyword(s):  
B Cells ◽  
Growth Factor ◽  
Germinal Center ◽  
Dependent Pathway ◽  
Stimulation Of ◽  
Germinal Center B Cells

scholarly journals Lenalidomide Induces Interleukin-21 Production by T Cells and Enhances IL21-Mediated Cytotoxicity in Chronic Lymphocytic Leukemia B Cells

2016 ◽  
Vol 4 (8) ◽  
pp. 698-707 ◽  
Author(s):  
Rebekah L. Browning ◽  
William H. Byrd ◽  
Nikhil Gupta ◽  
Jeffrey Jones ◽  
Xiaokui Mo ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Interleukin 21

Interleukin-21 receptor (IL-21R) is up-regulated by CD40 triggering and mediates proapoptotic signals in chronic lymphocytic leukemia B cells

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3708-3715 ◽  
Author(s):  
Daniela de Totero ◽  
Raffaella Meazza ◽  
Simona Zupo ◽  
Giovanna Cutrona ◽  
Serena Matis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Caspase 3 ◽  
Active Form ◽  
Growth Arrest ◽  
Lymphocytic Leukemia ◽  
Signaling Cascade ◽  
Caspase 8 ◽  
Interleukin 21

Interleukin-21 (IL-21) is a member of the IL-2 cytokine family, which mediates proliferation or growth arrest and apoptosis of normal B cells, depending on their activation state. Here we demonstrate that surface IL-21 receptor (R) is expressed at variable levels by chronic lymphocytic leukemia (CLL) B cells freshly isolated from 33 different patients. IL-21R expression was up-regulated following cell stimulation via surface CD40. Therefore, IL-21 effects were more evident in CD40-activated CLL B cells. IL-21 induced an early signaling cascade in CLL B cells, which included JAK-1 and JAK-3 autophosphorylation and tyrosine phosphorylation of STAT-1, STAT-3, and STAT-5. IL-21 signaling failed to stimulate CLL B-cell proliferation, but induced their apoptosis. In addition, IL-21 counteracted the proliferative and antiapoptotic signals delivered by IL-15 to CLL B cells. IL-21-mediated apoptosis involved activation of caspase-8 and caspase-3, cleavage of Bid to its active form t-Bid, and cleavage of PARP and of p27Kip-1. Recent data indicate that CLL B cells require interaction with the microenvironment for their survival and expansion. The present findings thus provide a set of new mechanisms involved in the balance between cell-survival and apoptotic signals in CLL B cells.

IL-21 Plus CpG ODN Induces Granzyme B-Dependent Induction of Apoptosis in CD5-Positive B Cells Including B-CLL Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2823-2823
Author(s):  
Sue E. Blackwell ◽  
Bernd Jahrsdoerfer ◽  
James E. Wooldridge ◽  
Jian Huang ◽  
Melinda W. Andreski ◽  
...  
Keyword(s):  
B Cells ◽  
Cord Blood ◽  
Granzyme B ◽  
Lymphocytic Leukemia ◽  
Cpg Odn ◽  
T And B Cells ◽  
B1 Cells ◽  
Lymphocyte Populations

Abstract Interleukin 21 (IL-21), a recently discovered cytokine with structural homology to IL-2, IL-4 and IL-15, has pleiotropic effects on lymphocyte populations including NK, T and B cells and is currently undergoing early clinical evaluation. We explored the effect of the combination of IL-21 and immunostimulatory CpG ODN on B chronic lymphocytic leukemia (B-CLL), and other CD5-positive B cells. IL-21 plus CpG ODN were synergistic in their ability to induce apoptosis of the B-CLL cells, and also induced production and secretion of granzyme B from the B-CLL cells. B-CLL cells treated with IL-21 and CpG ODN were capable of inducing apoptosis of untreated autologous B-CLL cells. This bystander killing was inhibited by anti-granzyme B antibodies. The effect was observed in all cases of CD5-positive B-CLL, but not in CD5-negative B-CLL samples. IL-21 plus CpG ODN also induced granzyme B production and apoptosis of benign CD5-positive B1 cells obtained from umbilical cord blood. In contrast, the number of CD5-negative B2 cells increased in the same samples during in vitro culture, resulting in a decreased ratio of CD5-positive to CD5-negative cord blood B cells (Fig. 1). Our results indicate the combination of IL-21 and CpG ODN is able to induce apoptosis of both benign and malignant CD5-positive B cells. Given the suspected role of B1 cells in autoimmune diseases, our findings could have important implications for the understanding of their pathogenetic mechanisms. These results might also open new avenues for the development of novel therapies for both autoimmune dieseases and CD5-positive B-CLL. Figure 1. IL- 21 and CpG ODN therapy selectively eliminates CD5 positive B cells in cord blood. Figure 1. IL- 21 and CpG ODN therapy selectively eliminates CD5 positive B cells in cord blood.

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.

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