Preclinical Development Of ROR1 Peptide Based Vaccine With Activity Against Chronic Lymphocytic Leukemia In ROR1 Transgenic Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4174-4174 ◽  
Author(s):  
Jian Yu ◽  
Bing Cui ◽  
George F. Widhopf ◽  
Liguang Chen ◽  
Laura Rassenti ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
High Titer ◽  
Lymphocytic Leukemia ◽  
Orphan Receptor ◽  
Lymphoid Tissues ◽  
Mouse Tissues ◽  
Freund’S Adjuvant ◽  
Freund's Adjuvant

Abstract Receptor tyrosine kinase-like orphan receptor (ROR1) is expressed on chronic lymphocytic leukemia (CLL) and other cancers, but not on normal post-partum tissues, except for a small subset of precursor B cells known as hematogones. Because of its restricted expression on cancer cells, ROR1 is an attractive target for developing novel anti-cancer therapies. In this study, we designed several different peptides, corresponding to distinct epitopes in the extracellular domain of ROR1. Each peptides was conjugated with keyhole-limpet hemocyanin (KLH) to generate a peptide-KLH vaccine, which we used to immunize C57BL/6 mice, first in complete Freund’s adjuvant (CFA) and then again 2 weeks later in incomplete Freund’s adjuvant (IFA). Two weeks following the second injection the sera from immunized mice were tested for binding activity for recombinant ROR1 protein via immunoblot analyses or ELISA or for cell-surface ROR1 by flow cytometry. We identified one peptide-KLH vaccine (R22-KLH) that induced high-titer anti-ROR1 antisera specific for ROR1-expressing cells that did not react with normal human or mouse tissues, which did not express this protein. The antisera generated by mice immunized with R22-KLH were capable of directing specific complement-dependent cytotoxicity (CDC) against neoplastic cells that expressed ROR1 (e.g. human CLL cells, EW36, or JeKo-1), but not against cells that did not express ROR1 (e.g. Jurkat). To study the capacity of R22-KLH to break self-tolerance, we immunized C57BL/6 mice transgenic for human ROR1 controlled by the immunoglobulin μ heavy-chain promoter/enhancer, designated as ROR1-Tg mice. Despite having B cells that express surface ROR1, such animals still were able to generate high-titer anti-ROR1 antisera comparable in binding and CDC activity and specificity as antisera generated in C57BL/6 mice. Production of such antisera was associated with reduced expression of ROR1 by the B cells of such ROR1-Tg mice. Cohorts of C57BL/6 mice or ROR1-Tg mice were immunized with R22-KLH or KLH and then challenged with intravenous injections of 3 × 105 or 2 × 104 ROR1-expressing primary CLL cells of double-transgenic ROR1xTCL1 C57BL/6 mice. Immunization with R22-KLH, but not KLH, significantly inhibited leukemia engraftment, as demonstrated by evaluation of the spleen and lymphoid tissues 4 weeks after adoptive transfer. To generate vaccines that might be suitable for clinical studies, we examined the relative immunogenicity of R22-KLH mixed with newly-developed agonists of toll-like receptor 4 (TLR-4; 1Z105) and TLR-7 (1V270). For this, we compared the immune responses of C57BL/6 mice or ROR1-Tg mice to R22-KLH administered s.c. in CFA/IFA to that of R22-KLH administered s.c. with 1Z105/1V270 in saline. Either C57BL/6 or ROR1-Tg mice generated higher titers of anti-ROR1 antibodies following immunization with R22-KLH in 1Z105/1V270 than did animals immunized with R22-KLH in CFA/IFA. Moreover, injection of R22-KLH in 1Z105/1V270 appeared better tolerated than injection of R22-KLH in CFA/IFA. These data indicate that immunization with R22-KLH can break tolerance to ROR1 and induce high-titer anti-ROR1 antisera. The immunogenicity of R22-KLH can be enhanced by injection with agonists of TLR4/7, obviating use of CFA/IFA. Pharmacology/toxicology studies are ongoing to determine the suitability of R22-KLH in 1Z105/1V270 for clinical trials involving patients with ROR1-expressing cancers. 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.

Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3316-3325 ◽  
Author(s):  
Andrea Bürkle ◽  
Matthias Niedermeier ◽  
Annette Schmitt-Gräff ◽  
William G. Wierda ◽  
Michael J. Keating ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Actin Polymerization ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Accessory Cells ◽  
Mitogen Activated Protein ◽  
B1 Cells

Abstract CXCL13 is a homeostatic chemokine for lymphocyte homing and positioning within follicles of secondary lymphoid tissues, acting through its cognate receptor, CXCR5. Moreover, the CXCR5-CXCL13 axis plays a unique role in trafficking and homing of B1 cells. Here, we report that chronic lymphocytic leukemia (CLL) B cells express high levels of functional CXCR5. CXCR5 expression levels were similar on CLL B cells and normal CD5+ B cells, and higher compared with normal CD5− B cells, follicular B-helper T cells (TFH cells), or neoplastic B cells from other B-cell neoplasias. Stimulation of CLL cells with CXCL13 induces actin polymerization, CXCR5 endocytosis, chemotaxis, and prolonged activation of p44/42 mitogen-activated protein kinases. Anti-CXCR5 antibodies, pertussis toxin, and wortmannin inhibited chemotaxis to CXCL13, demonstrating the importance of Gi proteins and PI3 kinases for CXCR5 signaling. Moreover, CLL patients had significantly higher CXCL13 serum levels than volunteers, and CXCL13 levels correlated with β2 microglobulin. We detected CXCL13 mRNA expression by nurselike cells, and high levels of CXCL13 protein in supernatants of CLL nurselike cell cultures. By immunohistochemistry, we detected CXCL13+ expression by CD68+ macrophages in situ within CLL lymph nodes. These data suggest that CXCR5 plays a role in CLL cell positioning and cognate interactions between CLL and CXCL13-secreting CD68+ accessory cells in lymphoid tissues.

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.

CD1d Expression Is Higher In Chronic Lymphocytic Leukemia Patients With Unfavorable Prognosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5278-5278
Author(s):  
Agnieszka Bojarska-Junak ◽  
Iwona Hus ◽  
Anna Dmoszynska ◽  
Jacek Rolinski
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Disease Progression ◽  
Conflicts Of Interest ◽  
Staining Intensity ◽  
Lymphocytic Leukemia ◽  
Disease Stage ◽  
Inkt Cells ◽  
Cd38 Expression

Abstract Chronic lymphocytic leukemia (CLL) is characterized by a very heterogeneous clinical course, which is slow and indolent in most of the patients, however some patient experience rapid disease progression and anticancer therapy is required shortly after the diagnosis. Many issues in CLL development and progression are still unclear. The functional consequences of CD1d expression on tumour cells are not well understood. However, increasing evidence suggests that they may affect iNKT cells.The role of CD1d expression in CLL immunopathogenesis remains undefined. In this study, we investigated the potential role of CD1d in CLL by analyzing the level of CD1d expression on leukemic B cells in peripheral blood of120 patients and assessed its correlation with prognostic markers such as ZAP-70 and CD38 expression, Rai stages and unfavourable cytogenetic changes.Measuring CD1d expression by flow cytometry and qRT-PCR, we showed lower CD1d molecule and CD1d mRNA expression in B cells of CLL patients than of healthy controls. The frequency of CD1d+/CD19+ cells, CD1d staining intensity and CD1d transcript levels increased with the disease stage. CD1d expression was positively associated with ZAP-70 and CD38 expressions as well as with unfavourable cytogenetic changes (17p deletion, 11q deletio),. We established the relationship between high CD1d expression and shorter time to treatment and overall survival. The percentage of CD1d+/CD19+cells inversely correlated with the percentage of iNKT cells. iNKT cells ζ-chain expression was downregulated in the high-CD1d group.These results suggest that high CD1d expression is associated with poor prognosis of CLL and might be involved in disease progression. Disclosures: No relevant conflicts of interest to declare.

Serum IgM Activates BCR Signaling Pathways and Increases Survival of Chronic Lymphocytic Leukemia B Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4131-4131
Author(s):  
Stefania Gobessi ◽  
Binu K Sasi ◽  
Luca Laurenti ◽  
Dimitar G Efremov
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Signaling Pathways ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Direct Interaction ◽  
Leukemic Cell ◽  
Fold Increase ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling

Abstract Serum IgM would be expected to bind chronic lymphocytic leukemia B cells through two different mechanisms. The first mechanism is via interactions between the immunoglobulin heavy chain CDR3 of the leukemic B cell receptors (BCRs) and internal epitopes located in the FR2 and FR3 regions of serum IgM molecules, analogous to the recently identified cell-autonomous BCR-BCR interaction. The latter interaction represents a general feature of human CLL BCRs and was recently shown to be positively selected during leukemia development in the Eμ-TCL1 transgenic murine model. The second mechanism is by binding of serum IgM to the recently identified Fc receptor for IgM (FcμR), which is overexpressed on CLL B cells. In the present study we investigated the consequences of the interaction between serum IgM and CLL cells. Incubation of CLL cells with Alexa488-conjugated human IgM resulted in strong cell surface labeling, confirming that IgM binds to CLL cells. Binding was substantially inhibited by preculture of CLL cells with Fcμ, suggesting that IgM interacts with CLL B cells primarily through the FcμR. To investigate whether IgM also binds to the leukemic BCRs, we analyzed activation of downstream BCR signaling pathways and expression of a well-defined set of BCR-target genes (Herishanu Y et al, Blood. 2011;117:563-74) in CLL cells cultured in the presence or absence of purified IgM. After three hours in culture with polyclonal or monoclonal human IgM, 5 of the 7 investigated BCR target genes (OAS3, RGS1, GFI1, CCND2 and KLF4) showed a 2- to 9-fold increase with respect to unstimulated CLL cells, whereas the remaining two genes (EGR1 and EGR2) were not induced. The induced BCR target genes were also upregulated to an equal or even greater extent by Fcμ, suggesting that these effects are primarily or exclusively caused by binding of IgM to the FcμR. Analysis of downstream signaling events, such as SYK and ERK phosphorylation, also showed similar induction by IgM and Fcμ. However, intracellular Ca2+ flux was induced to a substantially greater extent with IgM, suggesting that certain effects are mediated by a direct interaction between serum IgM and the leukemic cell BCRs. Since co-ligation of the FcμR was recently shown to enhance the survival of anti-IgM-stimulated murine B lymphocytes (Ouchida R et al, J Immunol. 2015;194:3096-101), we investigated the consequences of IgM binding on CLL cell survival. CLL cells from 18 patients were cultured with or without purified human IgM for 72 hours and then analyzed by Annexin V/PI staining. A modest but significant increase in the percentage of viable CLL cells was observed in the presence of IgM (percentage of viable CLL cells without IgM: 40.5±17.8; with IgM: 43.8±18.4; P =0.016), which was replicated in a smaller series of samples cultured with Fcμ (n=12, percentage of viable CLL cells without Fcμ: 41.1±17.8; with Fcμ: 49.5±15.6; P =0.019). Altogether, these data suggest that binding of serum IgM results in activation of prosurvival pathways in CLL cells and that this effect is most likely mediated by co-triggering the FcμR and BCR. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comprehensive Profiling of the Epitranscriptomic N6-Methyladenosine RNA Methylation in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Ya Zhang ◽  
Xinting Hu ◽  
Hongzhi Xu ◽  
Ying Li ◽  
Lingyan Zhang ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
De Novo ◽  
Conflicts Of Interest ◽  
R Package ◽  
Methylation Pattern ◽  
Lymphocytic Leukemia ◽  
Functional Enrichment ◽  
Read Length ◽  
Rna Methylation

Introduction N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNA. Accumulating evidence suggests that RNA m6A methylation exerts crucial roles in oncogenesis. However, the mRNA m6A methylation pattern in chronic lymphocytic leukemia (CLL) has not been investigated. Hence, the aim of this study is to perform a comprehensive profiling to identify distinct m6A methylation signatures in CLL patients. Methods Peripheral blood samples from de novo CLL patients were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong First Medical University. CD19+ B cells were isolated with informed consents from healthy donors. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was conduct to profile mRNA m6A methylation of CLL-B cells and normal CD19+ B cells at Novogene (Beijing, China). The library preparations were sequenced on an Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols. The sequencing was carried out with 3 independent biological replicates. After mapping reads to the reference genome, exomePeak R package was used for the m6A peak identification in each anti-m6A immunoprecipitation group with the corresponding input samples serving as a control, and q-value threshold of enrichment of 0.05 was used for all data sets. The m6A-enriched motifs of each group were identified by HOMER. Differential peak calling was performed using exomePeak R package with parameters of p-value &lt; 0.05 and fold_change &gt; 1. Functional enrichment analyses of gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) of differentiated peaks associated genes were performed. All investigators comply with the guiding principles for experimental procedures found in the Declaration of Helsinki of the World Medical Association. Results By MeRIP sequencing, significant distribution of methylation peaks were detected in 5'UTR, 3'UTR and CDS regions of CLL primary cells (Figure 1A) and normal B cells (Figure 1B). Figure 1C-D illustrated the percentage of methylation peaks in the five regions, suggesting distinct m6A patterns in CLL cells. Moreover, Figure 1E-F revealed the positions of m6A methylation peaks in chromosomes of CLL and normal B cells. Furthermore, the compared distributions of m6A methylation peaks in CLL and normal B cells were presented in Figure 2A. Besides, the bean plot visibly displayed the obvious differentiation of methylation peaks in CLL group and normal B cells in read density (Figure 2B). Importantly, a total of 1836 significantly changed peaks, of which 1519 were significantly up-regulated and 317 peaks were significantly down-regulated (p&lt;0.05, |log2Foldchange|&gt;1; Figure 2C). These m6A peaks were located across 1850 genes. Functional enrichment analyses identified that differentiated peaks associated genes were potentially regulate RNA metabolic process via oncogenic pathways in CLL pathogenesis (Figure 3A-B). In addition, HOMER analysis identified 38 significant de novo m6A peak motifs, top 10 most significant peak motifs of which were presented (Figure 3C), illuminating potential detailed mechanism of m6A RNA methylation in the tumorigenesis and progression of CLL. Conclusion Taken together, our investigations explored for the first time the m6A methylation pattern of mRNA in CLL. m6A modifications play crucial roles in the progression and survival of CLL patients,highlighting m6A modifications-targeted intervention formulating a novel treatment paradigm in progressed CLL that warrants clinical investigation. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Piers E. M. Patten ◽  
Gerardo Ferrer ◽  
Shih-Shih Chen ◽  
Jonathan E. Kolitz ◽  
Kanti R. Rai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Intraperitoneal Administration ◽  
Human Condition ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Quiescent State

Patient-derived xenograft models of chronic lymphocytic leukemia (CLL) can be created using highly immunodeficient animals, allowing analysis of primary tumor cells in an in vivo setting. However, unlike many other tumors, CLL B lymphocytes do not reproducibly grow in xenografts without manipulation, proliferating only when there is concomitant expansion of T cells. Here we show that in vitro pre-activation of CLL-derived T lymphocytes allows for a reliable and robust system for primary CLL cell growth within a fully autologous system that uses small numbers of cells and does not require pre-conditioning. In this system, growth of normal T and leukemic B cells follows four distinct temporal phases, each with characteristic blood and tissue findings. Phase 1 constitutes a period during which resting CLL B cells predominate, with cells aggregating at perivascular areas most often in the spleen. In Phase 2, T cells expand and provide T-cell help to promote B-cell division and expansion. Growth of CLL B and T cells persists in Phase 3, although some leukemic B cells undergo differentiation to more mature B-lineage cells (plasmablasts and plasma cells). By Phase 4, CLL B cells are for the most part lost with only T cells remaining. The required B-T cell interactions are not dependent on other human hematopoietic cells nor on murine macrophages or follicular dendritic cells, which appear to be relatively excluded from the perivascular lymphoid aggregates. Notably, the growth kinetics and degree of anatomic localization of CLL B and T cells is significantly influenced by intravenous versus intraperitoneal administration. Importantly, B cells delivered intraperitoneally either remain within the peritoneal cavity in a quiescent state, despite the presence of dividing T cells, or migrate to lymphoid tissues where they actively divide; this dichotomy mimics the human condition in that cells in primary lymphoid tissues and the blood are predominately resting, whereas those in secondary lymphoid tissues proliferate. Finally, the utility of this approach is illustrated by documenting the effects of a bispecific antibody reactive with B and T cells. Collectively, this model represents a powerful tool to evaluate CLL biology and novel therapeutics in vivo.

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.

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