scholarly journals Phenotypic and functional analysis of B cell lines from patients with multiple myeloma

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 444-446
Author(s):  
M Goldstein ◽  
J Hoxie ◽  
D Zembryki ◽  
D Matthews ◽  
AI Levinson
Keyword(s):  
Multiple Myeloma ◽  
Functional Analysis ◽  
Cell Differentiation ◽  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Plaque Assay ◽  
Functional Markers ◽  
B Cell Differentiation

We characterized phenotypic and functional properties of B cell lines obtained from patients with multiple myeloma to determine how well they conform to particular stages of B cell differentiation. This information is a prerequisite for using such lines as tools for studying B cell growth and the regulation thereof. Two lines, GM1312 and GM1500, expressed B1 and Ia, determinants on early B cells, but expressed little, if any, T10, a determinant expressed on plasma cells. By contrast, B1 and Ia were poorly expressed on two other lines, GM2132 and U266. T10 was expressed on GM2132 but not on U266. Using a reverse hemolytic plaque assay, we also assessed the numbers of cells actively secreting immunoglobulin (IgSCs) in such cultures to provide a functional marker of B cell differentiation. We observed consistently higher numbers of IgSCs in cultures of GM2132 than in GM1500 and GM1312. These phenotypic and functional markers were stable over several months. The data suggest that such cell lines represent early (GM1312, GM1500) and later stages (GM2132, U266) of B cell differentiation, although all lines were derived from patients with multiple myeloma.

scholarly journals Phenotypic and functional analysis of B cell lines from patients with multiple myeloma

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 444-446 ◽  
Author(s):  
M Goldstein ◽  
J Hoxie ◽  
D Zembryki ◽  
D Matthews ◽  
AI Levinson
Keyword(s):  
Multiple Myeloma ◽  
Functional Analysis ◽  
Cell Differentiation ◽  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Plaque Assay ◽  
Functional Markers ◽  
B Cell Differentiation

Abstract We characterized phenotypic and functional properties of B cell lines obtained from patients with multiple myeloma to determine how well they conform to particular stages of B cell differentiation. This information is a prerequisite for using such lines as tools for studying B cell growth and the regulation thereof. Two lines, GM1312 and GM1500, expressed B1 and Ia, determinants on early B cells, but expressed little, if any, T10, a determinant expressed on plasma cells. By contrast, B1 and Ia were poorly expressed on two other lines, GM2132 and U266. T10 was expressed on GM2132 but not on U266. Using a reverse hemolytic plaque assay, we also assessed the numbers of cells actively secreting immunoglobulin (IgSCs) in such cultures to provide a functional marker of B cell differentiation. We observed consistently higher numbers of IgSCs in cultures of GM2132 than in GM1500 and GM1312. These phenotypic and functional markers were stable over several months. The data suggest that such cell lines represent early (GM1312, GM1500) and later stages (GM2132, U266) of B cell differentiation, although all lines were derived from patients with multiple myeloma.

scholarly journals Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells

2021 ◽  
Author(s):  
Casper Marsman ◽  
Dorit Verhoeven
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Formation ◽  
B Cell Differentiation ◽  
Differentiation Potential ◽  
Human B Cell

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19+ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

scholarly journals Modulation of spontaneous B-cell differentiation in macroglobulinemia by retinoic acid

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2206-2210 ◽  
Author(s):  
Y Levy ◽  
S Labaume ◽  
MC Gendron ◽  
JC Brouet
Keyword(s):  
Retinoic Acid ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Culture Supernatants

Abstract We previously showed that clonal blood B cells from patients with macroglobulinemia spontaneously differentiate in vitro to plasma cells. This process is dependent on an interleukin (IL)-6 autocrine pathway. We investigate here whether all-trans-retinoic acid (RA) interferes with B-cell differentiation either in patients with IgM gammapathy of undetermined significance (MGUS) or Waldenstrom's macroglobulinemia (WM). RA at a concentration of 10(-5) to 10(-8) mol/L inhibited by 50% to 80% the in vitro differentiation of purified B cells from four of five patients with MGUS and from one of five patients with WM as assessed by the IgM content of day 7 culture supernatants. We next determined whether this effect could be related to an inhibition of IL- 6 secretion by cultured B cells and/or a downregulation of the IL-6 receptor (IL-6R), which was constitutively expressed on patients' blood B cells. A 50% to 100% (mean, 80%) inhibition of IL-6 production was found in seven of 10 patients (five with MGUS and two with WM). The IL- 6R was no more detectable on cells from patients with MGUS after 2 days of treatment with RA and slightly downregulated in patients with WM. It was of interest that B cells susceptible to the action of RA belonged mostly to patients with IgM MGUS, which reinforces our previous data showing distinct requirements for IL-6-dependent differentiation of blood B cells from patients with VM or IgM MGUS.

Clonal Analysis of IgM Cells in Multiple Myeloma Patients Suggests the Co-Existence of Normal and Malignant Arms of the Myeloma Clone.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1415-1415
Author(s):  
Brian J. Taylor ◽  
Ming Ye ◽  
Erin R. Strachan ◽  
Tara M. Tiffinger ◽  
Andrew R. Belch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Immunomagnetic Separation ◽  
B Cell Differentiation ◽  
Reading Frame ◽  
A Minor ◽  
Pcr Targeting ◽  
Insight Into

Abstract Analysis of immunoglobulin V genes, which undergo stepwise changes during B cell differentiation such as VDJ rearrangement, somatic hypermutation, and class switch recombination, provides insight into the point of transformation of B cell tumors. In Multiple Myeloma (MM), clonotypic VDJ sequences of malignant plasma cells are mutated, homogeneous, and associated with post-switch constant regions (either IgG or IgA, called the clinical isotype), suggesting the malignant arm of the MM clone arises from transformation events in the late stages of the germinal centre reaction. By contrast, the existence of clonotypic VDJ associated with pre-switch IgM is well established, and we have shown persistent clonotypic IgM is associated with advanced disease at diagnosis and poor survival in MM. Whether clonotypic IgM cells represent a malignant progenitor or a non-malignant population that parallels disease severity is unclear. To address these possibilities, we focused our analysis of clonotypic VDJ mutation profiles on IgM+ cells sorted by immunomagnetic separation from MM patient peripheral blood cells (PBMC). IgM clonotypic transcripts were amplified by hemi-nested RT-PCR targeting the CDR2-C mu constant region in IgM+ cells from 4/7 patients. These products were cloned, and 122, 28, 27, and 25 IgM clonotypic colonies were identified by specific CDR2/CDR3 PCR for patients 1–4 respectively. Each of these clones was sequenced, and mutations were identified by comparison with the closest germline V gene and tumor derived plasma cell VDJ sequences. An average mutation frequency of 0.005, significantly greater than the Taq error rate, was obtained for the 250–280 bp fragment downstream of CDR2, including the D-J-C mu region. Typically, MM clones were observed with 1–2 mutations in this region, many localizing to the D-J-C mu region. Small deletions that preserve reading frame were also observed in the D region of single clones of patients 1 and 4 respectively. The detection of intraclonal heterogeneity amongst clonotypic IgM cells may reflect a normal arm of the myeloma clone that co-exists with the post-switch malignant arm. In previous work examining bulk PBMC populations we had detected diversified clonotypic cells in the non-clinical isotype compartment of one patient, but, in accordance with studies performed by several other groups, were unable to detect diversified pre-switch counterparts. In this work we have focused on IgM+ MM B cells, a compartment of the MM clone that may remain driven by antigenic selection and undergo persistent clonal expansion. Our analysis gives insight into the nature of this proposed normal arm of the myeloma clone, revealing two coexisting subsets of pre-switch clonotypic IgM cells: a major set exhibiting homogeneity, identity with post-switch tumor VDJ, and questionable transformation status, and a minor clonally heterogeneous set which may represent the pre-malignant clone from which myeloma arose.

CtBP1 Is a Novel and Essential Corepressor for BCL6 Autoregulation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3581-3581
Author(s):  
Lourdes M. Mendez ◽  
Jose Polo ◽  
Melissa Krupski ◽  
Jessica Yu ◽  
Ari M. Melnick ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Cell Lines ◽  
Cell Biology ◽  
Target Genes ◽  
Point Mutations ◽  
B Cell Lymphoma ◽  
B Cell Differentiation ◽  
Wild Type ◽  
Negative Autoregulation

Abstract BCL6 is POZ/BTB transcription repressor that is required for the germinal center (GC)- stage of B cell development and its deregulated expression underlies the development of many GC-derived B cell lymphomas such as diffuse large B cell lymphoma (DLBCL). BCL6 carries out its biological function by repressing target genes involved in various aspects of B cell biology such as DNA damage response, cell-cycle regulation and plasma cell differentiation. Recent publications indicate that BCL6 differentially utilizes its corepressor partners to silence target genes involved in different biological processes. Negative autoregulation of BCL6 is likely to play an important role in B-cell differentiation, and is frequently disrupted in DLBCL due to translocation or point mutation of the BCL6 promoter. However, from a mechanistic standpoint, it is not known how BCL6 mediates negative autoregulation. BCL6 is reported to repress its target genes through binding of the SMRT, NCoR and BCoR corepressors to its N-terminal POZ domain and through binding of the MTA3 and HDAC2 corepressors to its second repression domain. However, a BCL6 mutant unable to bind these corepressors retained near wild-type repression activity on the BCL6 promoter. The expression of endogenous BCL6 was unchanged in DLBCL cell lines treated with BCL6 Peptide Inhibitor, which selectively disrupts the association between BCL6 and its POZ domain corepressors, or with MTA3 siRNA. This led us to consider the possibility that BCL6 autoregulation proceeds through a novel corepressor. Several POZ transcription factors can interact with CtBP as their corepressor. We found BCL6 and CtBP can interact in both the ectopically expressed and endogenous settings in DLBCL cells. Moreover, our ChIP experiments demonstrate that CtBP is present in the 5′UTR of BCL6 at sites that were previously shown by us and others to mediate BCL6 negative autoregulation. Nearly half of DLBCL patients are estimated to carry translocations and “activating” point mutations in the 5′UTR of BCL6 which allow negative autoregulation to be bypassed. In DLBCL cell lines carrying BCL6 promoter mutations or translocations, CtBP was preferentially bound to the wild-type BCL6 allele. Moreover, CtBP siRNA specifically derepressed the wild-type allele sparing the translocated BCL6 allele driven by heterologous promoters. This allelic analysis of BCL6 is consistent with a model in which BCL6 recruits CtBP to carry out negative autoregulation. Tiling ChIP-on-chip of BCL6 target genes showed colocalization of CtBP in a BCL6 repression complex at only a subset of target genes, including BCL6. However, the BCL6 locus was the only target dependent exclusively on CtBP for repression. In an effort to address the corepressor requirements of BCL6 autoregulation, we have uncovered a novel BCL6 corepressor, CtBP. Our results substantiate the growing body of evidence that BCL6’s mechanism of repression is dynamic, selectively calling upon corepressors to silence different cohorts of target genes perhaps reflecting segregation of biological functions. Our study provides new insight into normal BCL6-driven biology and also informs BCL6-targeted lymphoma therapies.

Quantitative Proteomic Analysis Implicates An Altered B-Cell Differentiation State as a Mechanism Underlying GC-Resistance in An Acute Lymphoblastic Leukaemia (ALL) Cell Line Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1736-1736
Author(s):  
Lindsay Nicholson ◽  
Caroline Evans ◽  
Elizabeth Matheson ◽  
Lynne Minto ◽  
Christopher Keilty ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Acute Lymphoblastic Leukaemia ◽  
B Cell ◽  
Cell Line ◽  
Mismatch Repair ◽  
Cell Lines ◽  
Lymphoblastic Leukaemia ◽  
B Cell Differentiation ◽  
Protein Levels ◽  
Resistant Phenotype

Abstract Abstract 1736 Poster Board I-762 Introduction Glucocorticoids (GC) are pivotal agents used in the treatment of childhood acute lymphoblastic leukaemia (ALL). GC-resistance is a significant prognostic indicator of a poor treatment outcome in childhood ALL, but the underlying molecular basis remains unclear. Previous studies using cell lines have identified mutation/deletion of the glucocorticoid receptor (GR) as a mechanism of GC-resistance. However, genetic aberration of the GR is rare in clinical samples1. This disparity may be due to the mismatch repair deficient status of many ALL cell lines which consequently have a greater likelihood of acquiring mutations under GC-selection. We have used a discovery proteomics approach for hypothesis generation on potential mechanisms for resistance. To achieve this, we compared a well-characterized mismatch repair proficient GC-sensitive cell line, PreB 697, and a GC-resistant sub-clone (R3F9) both bearing wildtype GR, in a comparative proteomics experiment using 4-channel isobaric tagging for relative and absolute quantitation (the iTRAQ approach). Methods Cells were treated with either vehicle control or 0.1μM dexamethasone for 24 hours and subjected to subcellular fractionation to prepare a nuclear fraction. Each sample was labelled with a distinct isobaric tag for relative quantification and analysed by 2-dimensional liquid chromatography/ tandem mass spectrometry. The proteins were identified and relatively quantified using Protein Pilot software (Applied Biosystems). Ratios were calculated for dexamethasone-treated ‘versus’ control vehicle for each cell line and an ITRAQ ratio of greater than or equal to ± 1.2 or less than 0.8 fold change were considered to be differentially expressed. Results The comparative dataset highlighted two transcription factors which are involved in B-cell differentiation, PAX5 and IRF4, to be differentially expressed in the PreB 697 compared to the R3F9 cell line. The GC-resistant R3F9 cell line had reduced PAX5 and IRF4 protein expression compared to the parental cell line and this was further validated in other GC-resistant sub-clones derived from the PreB 697 cell line by western blot analysis. The reduced PAX5 level in the GC-resistant cell lines was not due to monoallelic loss, as measured by a QRT-PCR method or mutation as determined by DHPLC analysis of ‘hot-spot’ exons. In addition, PAX5 mRNA levels were not significantly altered, thus suggestive of a post-transcriptional mechanism for PAX5 protein reduction. To test the direct role of PAX5 in GC-resistance, we reduced PAX5 mRNA and protein levels using RNA interference in the parental GC-sensitive, PreB 697 cell line. PAX5 protein levels were reduced by at least 80% and were maintained for 48 hours post-transfection. The PreB 697 cell line was transfected with siRNA directed to PAX5 using electroporation, the cells were allowed to recover for 24 hours and the levels of cell kill were assessed in response to a 48 hour incubation with 1 μM dexamethasone by Annexin V staining and the MTS assay. Paradoxically, PAX5 knockdown increased GC-sensitivity (mean 60.4% apoptosis, S.D. 16.8, N=3) in comparison to a non-specific siRNA (mean 31.0% apoptosis, S.D. 5.2, N=3) but did not influence sensitivity to either vincristine or daunorubicin. Thus, this response was specific to glucocorticoids. Conclusion Using a proteomic approach we have shown alterations in PAX5 protein levels are associated with a GC-resistant phenotype which an mRNA-based technology would fail to detect. Modulation of PAX5 in ALL cells may influence the response to GC-therapy. It is known that GC-sensitivity alters during B-cell development, with early lymphoid precursors being highly sensitive and more mature B cells being highly resistant to GC-induced apoptosis. We propose that reduced PAX5 protein levels may reflect an altered differentiation state of the sub-clones of PreB 697 which are associated with a GC resistant phenotype. 1Irving et al, Cancer Res, 2005 2Schmidt et al, FASEB, 2006 Disclosures No relevant conflicts of interest to declare.

Microrna Expression Profile Identifies Distinct Clinically Relevant Sub-Groups in Multiple Myeloma: Novel Prognostic Markers and Potential Targets for Therapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 96-96 ◽  
Author(s):  
Sophia Adamia ◽  
Herve AvetLoiseau ◽  
Samirkumar B Amin ◽  
Yu-Tzu Tai ◽  
Steven P. Treon ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Differentiation ◽  
Clinical Outcome ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Plasma Cells ◽  
Normal Plasma ◽  
Group A ◽  
Group B

Abstract MicroRNA, an abundant class of small endogenous RNAs, regulate target genes through inducing translational inhibition and cleavage of targeted transcripts. To date, microRNAs have been implicated in normal biological processes, including development, cell differentiation, apoptosis and proliferation as well as in malignant transformation. However, their role in multiple myeloma (MM) remains unknown. Here we investigated role of microRNAs in myelomagenesis, and their influence on prognosis and clinical outcome. We evaluated profiles of 384 microRNAs in bone marrow derived CD138+ plasma cells (PC) from 79 uniformly treated MM patients, 11 MM cell lines and 9 healthy donors using qRT-PCR based microRNA array. The relative expression was calculated using comparative Ct method, and data was normalized using endogenous controls and analyzed using SDS, RQ manager, R and dChip softwares. MicroRNA expression profiles detected in MM patients were correlated with clinical outcome measures. We observed significant modulate expression of 61 microRNAs in myeloma cells compared to normal plasma cells. When more stringent criteria were used, we identified 24 differentially expressed microRNAs in patient samples. Further, unsupervised hierarchical clustering of filtered microRNAs, based on their DCt values, identified two major groups within the MM population (groups A and group B). Samples of Group A clusters with MM cell lines, indicating more proliferative nature of MM patient cells. Within B group, a second degree node group B2, clusters with normal plasma cells indicating more indolent course, while patients in an additional node B1 represented an assorted pattern. The unsupervised clustering of all MM samples showed consistent changes in miR-30b, -30c, -30d, -142-5p, -24, -191, -181d, -374, -146b, -140, -145, -125a, -151, -223, -155, let7b, indicative of a role of these microRNA in myelomagenesis; while supervised analysis of samples within groups A and B identified modulated expression of different sets of miRNAs. In group A miR-585 and let-7f were upregulated 8–12 fold, while miRs -125a, -126, -155, -223, -146a, -374 -19a, -20a, -26a, -30a -5p, -30b, and -30d were significantly downregulated; in group B, all differentially expressed microRNAs were downregulated (p<0.001) compared to normal plasma cells. These modulated miRNAs target critical signaling pathways including apoptosis, hematopoietic cell differentiation and proliferation, survival and angiogenesis by upregulating function of HOX9, c-myc, VCAM-1, Bcl-2, E2F1, SHP1, SHP2, VEGF, and DUSp6 molecules. We further analyzed the effect of microRNA on clinical outcome. We have observed significantly superior event free and overall survival of patients in group B2 compared to patients in group A (2 yr estimated EFS 79% versus 54% respectively; p=0.05; and 2 yr estimated OS 94% versus 70% respectively; p =0.017). Taken together this data identifies critical microRNAs as modulators of gene expression and signaling pathways and provides potential novel microRNA and gene targets in MM to both understand biological behavior and for therapeutic application.

Acadesine Inhibits Proliferation and Induces Apoptosis In Multiple Myeloma Cells, and Synergizes with Standard of Care Antimyeloma Agents

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5023-5023
Author(s):  
Susana Hernández-García ◽  
Mercè de Frias ◽  
Clara Campàs ◽  
Bruno Paiva ◽  
Enrique M. Ocio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Lymphoma ◽  
Hematologic Malignancy ◽  
B Cell Lymphoma ◽  
In Vivo Studies ◽  
Mutational Status

Abstract Abstract 5023 Multiple myeloma (MM) is a malignancy characterized by the accumulation of plasma cells. The disease represents the second most common hematologic malignancy and remains incurable, despite recent advances in its treatment. Therefore, studies to develop new therapies are still necessary, particularly in patients with bad prognostic factors, such as 17p deleted/p53 mutated patients. In this study we describe the preclinical activity of 5-Aminoimidazole-4-carboxamide-1–4-ribofuranoside (AICAR or acadesine) in multiple myeloma. Acadesine is an analog of AMP that is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy changes. Acadesine induces apoptosis in different cell types including CLL, mantle cell lymphoma (MCL) and splenic marginal zone B-cell lymphoma (SMZL) cells and tumor cell lines, without affecting primary T lymphocytes. Thus, acadesine is a promising drug for the treatment of B-cell neoplasms. A clinical phase I/II study of acadesine is currently being performed in CLL patients. We studied the effects of acadesine on the MTT metabolization of several multiple myeloma cell lines (MM1S, MM1R, RPMI-8266, RPMI-LR5, U266, U266-LR7, U266 Dox4, MM144, MGG, SJR, OPM-2, NCIH-929). Acadesine inhibited MM cell growth and induced apoptosis, with IC50 values in the micromolar range, and independently of the p53 mutational status. Cancer treatment, including myeloma, is generally based on combinations of drugs with different mechanisms of action. Thus, we studied the effect of acadesine in double combinations with drugs used in myeloma therapy, such as dexamethasone, melphalan, doxorubicin, bortezomib, and lenalidomide. Analyses of these data using the Chou and Talalay method indicated that acadesine was synergistic with dexamethasone (CI values of 0.60), and particularly with lenalidomide (CI values of 0.42). These promising results with double combinations promoted the investigation of triple combinations in the MM1S cell line. Triple combination of acadesine plus dexamethasone plus lenalidomide or bortezomib notably improved the efficacy of the respective double combinations, being the combination of acadesine plus lenalidomide plus dexamethasone especially efficient. Further studies to determinate the mechanism of action, and in vivo studies in MM1S xenograph are ongoing. Disclosures: de Frias: Advancell: Employment. Campàs:Advancell: Employment.

Induction Of Proliferation and Survival Of Multiple Myeloma Cells By CD137 Ligand Reverse Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2537-2537
Author(s):  
Chengcheng Fu ◽  
Hui Liu ◽  
Juan Wang ◽  
Ling Ma ◽  
Songguang Ju ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Counting ◽  
Myeloma Cells ◽  
Reverse Signaling

Abstract CD137 and its ligand are members of the Tumor Necrosis Factor (TNF) receptor and TNF superfamilies, respectively, regulate cell activation and proliferation of immune system. CD137L, in addition to its ability to costimulate T cells by triggering CD137 receptor, also signals back into antigen presenting cells inducing proliferation, prolonging survival and enhancing secretion of proinflammatory cytokines. The expression of CD137L and its function on multiple myeloma cells is unknown. We identified the constitutive expression of CD137L by flow cytometry on U266, RPMI 8226, LP1, MY5 and KMS-11 of Multiple myeloma (MM) cell lines as high as 96%, 97.5%, 89%, 93% and 94%.But, CD137 expressed on the cell surface was low as 4%, 5%, 1%, 2%, 5% respectively. Now that, CD137L was expressed very strongly on MM cell lines, next, we investigated CD137L expression of MM cells from 85 BM samples of patients seen in the hematological Dept of the First Affiliated Hosp. of Soochow University between January 2012 and June 2013 and diagnosed of active multiple MM, including the patients of newly-diagnosed (n=35), relapsed (n=5) and after 2- 4 prior therapies (n=45). The BM samples were examined using antibodies against CD45RO PE-Cy7, CD138 APC-H7, CD38 FITC and CD137L PE, according to standard protocols for surface staining. Indeed, CD137L protein was expressed by a select group of CD45-CD38++CD138+cells as higher than 95%, the same, CD38 and CD138 are expressed more than 90% of the cells of CD45-CD137L+.There were 22 samples from 11 cases collected before and after treatment and this was further evidence that CD137L molecule was consistently expressed on the MM cell surface. However, CD137L expression was not or hardly detectable on normal plasma cells confirmed by CD45+CD38++CD138+ CD56- CD19+, indicating that CD137L was ectopically expressed by MM cells and probably a specific marker of MM cells. The ectopic CD137L expression was not a mere epiphenomenon but was selected for, what function of it? We hypothesized that it would also act as a growth stimulus for B cell cancers. Then we selected U266-a MM cell line to explore the biological effect of CD137L reverse signaling and its underlying mechanism. As a result, in vitro study, U266 cells(2X105/ml))were cultured plate pre-coated with mAb 1F1 or irrelevant mouse IgG at l ug/ml in PBS and at 400 ul per well of 24-well plate or 80 ul per well of 96-well plate and washed twice after overnight incubation at 4°C. The proliferation and survival of U266 was enhanced by stimulating- CD137L mAb (1F1) than those induced by control mouse IgG by cell counting (4.2 X105/ml VS 3.3 X105/ml), WST-8(1.15 VS 0.81) and CFSE assay (930 VS 991) at incubation for 48h. In addition, the cell cycle analysis showed that CD137L induces proliferation and increases the number of cells in the S phase from 36.1% to 42.5% after 72h incubation. The percentage of apoptosis cells (Annexin V+ and PI+) was 19.6% VS 21.2% with no statistical significance. In order to explore the mechanism of the function of CD137L on MM cells, we surveyed the cytokine profiles during the incubation of U266 cells cultured for 2 days with different stimuli with mAb 1F1 compared with the control group. Intracellular cytokine staining showed that treatment of cells with 1F1 increased the production of IL-6 from 3.8% to 63.9% by Flow cytometry. When neutralizing anti-IL-6 mAb (5 ug/ml) was added to the culture medium, the cells(2X105/ml))were cultured for 48 h in pure medium or plus 10 ng/ml Fc or CD137–Fc protein and the cell proliferation measured by WST-8 was 0.79 VS 0.80 VS 0.72.1F1-induced cell proliferation was effectively inhibited. IL-6 can promote cell proliferation and survival of MM. An increase of these cytokines might explain why CD137L expression could stimulate the proliferation of U266. Finally, the U266 cells were treated with bortezomib and the growth of cells was analyzed by WST-8 assay. It demonstrated that bortezomib could inhibit the function of 1F1 and the inhibition ratio of bortezomib was 22%, 51% and 58% at 24h, 48h and 72h. MM is a B-cell malignancy characterized by the clonal expansion and accumulation of malignant plasma cells in the bone marrow. In our study, CD137L is not only a novel ectopic constitutive marker of MM, but also a promoting proliferation factor. This suggests the possibility that its expression on MM cells may be directly target for immunomodulatory therapy for MM. Disclosures: No relevant conflicts of interest to declare.

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