Differentiation-Stage-Specific Expression of MicroRNAs in B-Lymphocytes and Diffuse Large B-Cell Lymphomas (DLBCL)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 805-805 ◽  
Author(s):  
Raquel Malumbres ◽  
Robert Tibshirani ◽  
Elena Cubedo ◽  
Kristopher A Sarosiek ◽  
Xiaoyu Jiang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Memory B Cells ◽  
Microrna Expression ◽  
B Cell Differentiation ◽  
Cell Of Origin ◽  
Specific Expression ◽  
Differentiation Stage

Abstract B-cell development and differentiation are complex processes controlled by distinct programs of transcriptional control. A large set of transcriptional factors together or in succession control this process and their deregulation may result in block of differentiation or malignant transformation. MicroRNAs are small RNAs that orchestrate cellular functions by modulating the level of their targeted proteins by either translational arrest or transcript degradation, and play a key role in cell differentiation, apoptosis, proliferation and cancer development. An increasing number of transcription factors are being found targeted by microRNAs. Emerging evidence suggests that differentiation stage-specific expression of microRNAs occurs in the hematopoietic system and during T cell differentiation. Only limited information exists on microRNA expression in normal B cell differentiation and its malignant counterparts. Herein we analyzed microRNA expression profiles in distinct peripheral B cell differentiation stages-naïve, germinal center (GC) centroblasts and memory cells as well as tonsilar T cells. Furthermore, microRNA profiling was performed in germinal center-like (GCB-like) and activated B-cell-like (ABC-like) DLBCL cell lines originating from distinct B-cell differentiation stages. RNA, extracted with mirVana kit (AMBION) from B cell subsets and T cells enriched from normal tonsils was hybridized on LC Sciences (Houston, TX) microarrays harboring 470 human microRNAs probes (Sanger miRBase Release 9.1). Expression of selected microRNAs was confirmed by ABI RT-PCR methodology. Unsupervised clustering of microRNAs with values present in at least 50% of the samples (122 probes) resulted in perfect differentiation-stage clustering of samples. Application of Statistical Analysis of Microarrays (SAM) and Prediction Analysis of Microarrays (PAM) methods (FDR= 10%) identified a 47 microRNA cell of origin classifier for B-cells differentiation stage; 27 of these microRNAs were upregulated and 20 downregulated in centroblasts compared to memory B-cells. MicroRNAs belonging to paralog microRNA clusters (e.g. miR17-92-1, miR363-106a and miR25-106b) demonstrated similar patterns of expression in specific differentiation stages. To identify specific microRNA targets, miRanda, TargetScan and PicTar programs were used. To experimentally confirm the targets, we assessed the effects of specific microRNAs on the expression levels of targeted proteins and on the luciferase reporter under the control of the wild type and mutated 3′ UTR regions of putative target genes. Using this experimental approach we identified lymphocyte-stage-specific microRNAs which expression inversely correlated and might regulate the expression of LMO2, BLIMP1 and IRF4 proteins distinctively expressed at different differentiation stages of B lymphocytes. For example, miR223, which expression is low in GC cells but is high in naïve and memory B cells, downregulates the expression of LMO2. We next analyzed microRNA expression in DLBCL cell lines. Clustering analysis, using the 47 microRNA cell of origin classifier perfectly classified GCB-like and ABC-like cell lines. Interestingly, the expression of microRNAs in both GCB-like and ABC-like DLBCL cell lines was more similar to normal centroblasts than to memory B cells, suggesting that both may originate from distinct subpopulations of GC lymphocytes. The similarity of microRNA expression in cell lines to centroblasts was striking, with only 16 microRNAs (1 upregulated and 15 downregulated in cell lines) showing noticeable differences in levels of expression compared to normal cells. These microRNAs might be involved in the process of lymphoma transformation. SAM analysis aimed to differentiate GCB-like and ABC-like cell lines identified 11 microRNAs, only 3 of which were present in the cell of origin classifier. This observation suggests that there is also a difference in expression of microRNAs not directly related to the distinct cell of origin between the DLBCL subtypes. In summary, our results demonstrate that the microRNA profile changes during the GC reaction as well as during malignant transformation. Specific microRNAs can regulate key transcription factors controlling the processes of lymphocyte differentiation and transformation.

Regulation of CCR6 chemokine receptor expression and responsiveness to macrophage inflammatory protein-3α/CCL20 in human B cells

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2338-2345 ◽  
Author(s):  
Roman Krzysiek ◽  
Eric A. Lefevre ◽  
Jérôme Bernard ◽  
Arnaud Foussat ◽  
Pierre Galanaud ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Chemokine Receptor ◽  
Memory B Cells ◽  
Receptor Expression ◽  
B Cell Differentiation ◽  
Hematopoietic Stem ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein

Abstract The regulation of CCR6 (chemokine receptor 6) expression during B-cell ontogeny and antigen-driven B-cell differentiation was analyzed. None of the CD34+Lin− hematopoietic stem cell progenitors or the CD34+CD19+ (pro-B) or the CD19+CD10+ (pre-B/immature B cells) B-cell progenitors expressed CCR6. CCR6 is acquired when CD10 is lost and B-cell progeny matures, entering into the surface immunoglobulin D+ (sIgD+) mature B-cell pool. CCR6 is expressed by all bone marrow–, umbilical cord blood–, and peripheral blood–derived naive and/or memory B cells but is absent from germinal center (GC) B cells of secondary lymphoid organs. CCR6 is down-regulated after B-cell antigen receptor triggering and remains absent during differentiation into immunoglobulin-secreting plasma cells, whereas it is reacquired at the stage of post-GC memory B cells. Thus, within the B-cell compartment, CCR6 expression is restricted to functionally mature cells capable of responding to antigen challenge. In transmigration chemotactic assays, macrophage inflammatory protein (MIP)-3α/CC chemokine ligand 20 (CCL20) induced vigorous migration of B cells with differential chemotactic preference toward sIgD− memory B cells. These data suggest that restricted patterns of CCR6 expression and MIP-3α/CCL20 responsiveness are integral parts of the process of B-lineage maturation and antigen-driven B-cell differentiation.

scholarly journals Favorable efficacy of rituximab in ANCA-associated vasculitis patients with excessive B cell differentiation

2020 ◽  
Author(s):  
Yusuke Miyazaki ◽  
Shingo Nakayamada ◽  
Satoshi Kubo ◽  
Yuichi Ishikawa ◽  
Maiko Yoshikawa ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Selection Criteria ◽  
Memory B Cells ◽  
Phenotypic Characterization ◽  
B Cell Differentiation ◽  
Color Flow ◽  
Anca Associated Vasculitis ◽  
Cell Phenotypes

Abstract Objectives: B-cell depletion by rituximab (RTX) is an effective treatment for anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). However, peripheral B cell phenotypes and the selection criteria for RTX therapy in AAV remain unclear.Methods: Phenotypic characterization of circulating B cells was performed by 8-color flow cytometric analysis in 54 newly diagnosed AAV patients (20 granulomatosis with polyangiitis and 34 microscopic polyangiitis). Patients were considered eligible to receive intravenous cyclophosphamide pulse (IV-CY) or RTX. All patients also received high-dose glucocorticoids (GC). We assessed circulating B cell phenotypes and evaluated the efficacy after 6 months of treatment. Results: There were no significant differences in the rate of clinical improvement, relapses, or serious adverse events between patients receiving RTX and IV-CY. The rate of Birmingham Vasculitis Activity Score (BVAS)-improvement at 6 months tended to be higher in the RTX group than in the IV-CY group. The proportion of effector or class-switched memory B cells increased in 24 out of 54 patients (44%). The proportions of peripheral T and B cell phenotypes did not correlate with BVAS at baseline. However, among peripheral B cells, the proportion of class-switched memory B cells negatively correlated with the rate of improvement in BVAS at 6 months after treatment initiation (r = -0.28, p = 0.04). Patients with excessive B cell differentiation were defined as those in whom the proportion of class-switched memory B cells or IgD-CD27- B cells among all B cells was >2 SDs higher than the mean in the HCs. The rate of BVAS-remission in patients with excessive B cell differentiation was significantly lower than that in patients without. In patients with excessive B cell differentiation, the survival rate, the rate of BVAS-remission, and dose reduction of GC were significantly improved in the RTX group compared to those in the IV-CY group after 6 months of treatment. Conclusions: The presence of excessive B cell differentiation was associated with treatment resistance. However, in patients with circulating B cell abnormality, RTX was effective and increased survival compared to IV-CY. The results suggest that multi-color flow cytometry may be useful to determine the selection criteria for RTX therapy in AAV patients. (349/350 words)

Frequent Loss of the SLP-65 Adapter Protein in Childhood Acute B-Precursor Lymphoblastic Leukemia (ALL) with TEL/AML1 Rearrangement.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 741-741 ◽  
Author(s):  
Arndt Borkhardt ◽  
Christine Damm-Welk ◽  
Thomas Wossning ◽  
Bettina Storch ◽  
Uta Fuchs ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
Tumor Suppressor ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Sh2 Domain ◽  
B Cell Differentiation ◽  
Cell Leukemia ◽  
B Cell Leukemia

Abstract The adaptor protein SLP-65 plays an essential role during B cell differentiation. A crucial consequence of SLP-65 deficiency in mice is a high incidence of pre-B-cell leukemia, suggesting a tumor suppressor role for SLP-65 in pre-B-cells. While the link between SLP-65 deficiency and leukemia development is established in mice, experiments mainly using microarrays for gene expression profiling suggested normal expression of SLP-65 in human precursor B-cell ALL. This analysis however does not discriminate between normal and aberrant SLP-65 transcripts with the latter being unable to generate functional protein. To examine the correlation between SLP-65 deficiency and childhood precursor B-cell ALL, we determined SLP-65 expression in 119 precursor B-cell ALL samples by both RNA and protein methods. The expression of SLP-65 was compared to clinical and laboratory findings, cytogenetics as well as to the outcome data within this uniformly treated cohort of patients. Loss of slp-65 protein was significantly associated with the occurrence of the TEL/AML1 rearrangement (p=0.026) but not with any other clinical or cytogenetic feature. We found a profound disconnection between slp-65 mRNA and protein expression in 38 out of the 119 leukemic samples pointing to a posttranscriptional regulation of slp-65 (Table). To confirm that SLP-65 transcript expression does not automatically correlate with its protein expression, we analyzed a panel of human cell lines derived from precursor B-cell ALL patients. The cell lines HPB-NULL and BV-173 showed a deficiency in SLP-65 protein expression, although SLP-65 transcripts can easily be detected in both lines. Together, the data suggest that SLP-65 expression might be regulated at the posttranscriptional level and that the presence of SLP-65 transcripts does not necessarily lead to SLP-65 protein and function. In one particular patient, we found a truncated slp-65 transcript and the predicted slp-65 protein lacks its SH2 domain. We tested whether this SLP-65 protein lacking the SH2 domain is functional in pre-B cells. To this end, we transfected murine SLP-65 −/− pre-B cells with retroviral constructs for either wild-type (wt SLP-65) or truncated SLP-65 (SLP-65delSH2) and analysed pre-BCR downregulation, Ca2+ release and pre-B cell differentiation. The results showed that, in contrast to wt SLP-65, SLP-65delSH2 failed to induce any effects in the performed experiments. Together with previous findings showing that SLP-65-deficient mice develop pre-B cell leukemia, the data suggest that SLP-65 acts as a tumor suppressor that limits pre-B cell proliferation by inducing differentiation. Disconnection between slp-65 transcripts and protein expression total slp-65 protein+ (51 patients) slp-65 protein weak (19 patients) slp-65 protein- (49 patients) PCR+ 108 51(9 TEL/AML+, 42 TEL/AML-) 19 (9 TEL/AML+, 10 TEL/AML-) 38 (15 TEL/AML+, 23 TEL/AML-) PCR- 11 0 0 11 (T-ALL)

MicroRNAs Regulate Mature B Cell Differentiation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 705-705
Author(s):  
Jenny Zhang ◽  
Dereje D. Jima ◽  
Cassandra L. Jacobs ◽  
Eva Gottwein ◽  
Grace Huang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Target Genes ◽  
Expression Patterns ◽  
Microrna Expression ◽  
Seed Sequence ◽  
B Cell Differentiation ◽  
Cell Stage

Abstract Background: Mature B cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells are common and constitute the majority of leukemias and lymphomas. MicroRNAs are known to play a role in oncogenesis, lineage-selection, and immune cell function, including early B cell differentiation. However, the full extent and function of microRNA expression during mature B cell differentiation and in B cell malignancies are not known. Methods: From normal young patients undergoing tonsillectomies, we sorted the mature B cell subsets (naive, germinal center, memory and plasma) using FACS, based on their expression of CD19, CD38, IgD and CD27. These sorted B cells were profiled for microRNA expression using a highly sensitive multiplexed real-time PCR assay, as well as for gene expression at the whole genome level using Affymetrix U133plus microarrays. miRNA targets can be predicted based on seed sequence matching of their 2–8 nt to the 3′UTR of gene transcripts. For each B cell stage, we experimentally validated microRNA regulation of predicted target genes of interest, LMO2, MYBL1 and PRDM1, by microRNA over-expression experiments and luciferase assays. Results: We found that microRNAs have a characteristic expression pattern that defines each mature B cell stage. Examination of both microRNA and mRNA expression showed that in each B cell population, the target genes predicted based on seed matching were expressed at lower levels, results that were highly significant (P<1E-10). We found that differential microRNA expression is important at every B cell stage transition, and differentially expressed microRNAs frequently target differentially expressed transcription factors. In the naive to germinal center B cell and germinal center B cell to memory cell transitions, we found that miR-223 had an inverse relationship with its predicted target genes LMO2 and MYBL1. To test this relationship predicted based on seed pairing, in Germinal Center-derived BJAB cells, we over-expressed miR-223 by introducing its precursor, and saw a subsequent knockdown of LMO2 and MYBL1 at both the mRNA and protein level. We confirmed seed sequence specificity by comparing miR-223 knockdown of luciferase reporter activity on the LMO2 3′UTR compared to its seed sequence mutant. We further found that miR-9 and miR-30 family members directly regulate PRDM1 (blimp1), a master regulator of the GC to PC transition. In U266 cells (PC-derived), introduction of miR-9 and miR-30 family precursor resulted in decreased PRDM1 protein expression, although transcript levels were not changed, consistent with previous evidence that miRNA can regulate at the post-transcriptional steps. We further profiled over 50 tumors derived from various B cell malignancies (small lymphocytic lymphoma, Burkitt lymphoma, and the molecular subsets of diffuse large B cell lymphoma) and found that these malignancies maintain the expression patterns of their respective lineage; microRNA expression profiles of normal B cells could correctly classify the lineage of these tumors in over 80% of the cases. In contrast to other malignancies, common lymphomas do not down-regulate microRNAs, but rather maintain the microRNA-expression patterns of their normal B-cell counterparts. Conclusion: Through concomitant microRNA and mRNA-profiling, we demonstrate a regulatory role for microRNAs at every stage in mature B-cell differentiation. Further, we have experimentally identified a direct role for the microRNA-regulation of key transcription factors in B-cell differentiation: LMO2, MYBL1 and PRDM1 (Blimp1). Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B-cells.

scholarly journals IL-21 regulates germinal center B cell differentiation and proliferation through a B cell–intrinsic mechanism

2010 ◽  
Vol 207 (2) ◽  
pp. 365-378 ◽  
Author(s):  
Dimitra Zotos ◽  
Jonathan M. Coquet ◽  
Yang Zhang ◽  
Amanda Light ◽  
Kathy D'Costa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Affinity Maturation ◽  
Memory B Cells ◽  
Target Cells ◽  
B Cell Differentiation ◽  
Follicular Helper

Germinal centers (GCs) are sites of B cell proliferation, somatic hypermutation, and selection of variants with improved affinity for antigen. Long-lived memory B cells and plasma cells are also generated in GCs, although how B cell differentiation in GCs is regulated is unclear. IL-21, secreted by T follicular helper cells, is important for adaptive immune responses, although there are conflicting reports on its target cells and mode of action in vivo. We show that the absence of IL-21 signaling profoundly affects the B cell response to protein antigen, reducing splenic and bone marrow plasma cell formation and GC persistence and function, influencing their proliferation, transition into memory B cells, and affinity maturation. Using bone marrow chimeras, we show that these activities are primarily a result of CD3-expressing cells producing IL-21 that acts directly on B cells. Molecularly, IL-21 maintains expression of Bcl-6 in GC B cells. The absence of IL-21 or IL-21 receptor does not abrogate the appearance of T cells in GCs or the appearance of CD4 T cells with a follicular helper phenotype. IL-21 thus controls fate choices of GC B cells directly.

scholarly journals Patterns of microRNA expression characterize stages of human B-cell differentiation

Blood ◽  
2009 ◽  
Vol 113 (19) ◽  
pp. 4586-4594 ◽  
Author(s):  
Jenny Zhang ◽  
Dereje D. Jima ◽  
Cassandra Jacobs ◽  
Randy Fischer ◽  
Eva Gottwein ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Microrna Expression ◽  
Lymphocytic Leukemia ◽  
Cell Phenotype ◽  
Tumor Type ◽  
B Cell Differentiation ◽  
B Cell Malignancies

Abstract Mature B-cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells constitute the majority of leukemias and lymphomas. These malignancies often maintain the characteristics of the normal B cells that they are derived from, a feature that is frequently used in their diagnosis. The role of microRNAs in mature B cells is largely unknown. Through concomitant microRNA and mRNA profiling, we demonstrate a potential regulatory role for microRNAs at every stage of the mature B-cell differentiation process. In addition, we have experimentally identified a direct role for the microRNA regulation of key transcription factors in B-cell differentiation: LMO2 and PRDM1 (Blimp1). We also profiled the microRNA of B-cell tumors derived from diffuse large B-cell lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia. We found that, in contrast to many other malignancies, common B-cell malignancies do not down-regulate microRNA expression. Although these tumors could be distinguished from each other with use of microRNA expression, each tumor type maintained the expression of the lineage-specific microRNAs. Expression of these lineage-specific microRNAs could correctly predict the lineage of B-cell malignancies in more than 95% of the cases. Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B cells.

scholarly journals Favorable efficacy of rituximab in ANCA-associated vasculitis patients with excessive B cell differentiation

2020 ◽  
Author(s):  
Yusuke Miyazaki ◽  
Shingo Nakayamada ◽  
Satoshi Kubo ◽  
Yuichi Ishikawa ◽  
Maiko Yoshikawa ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Selection Criteria ◽  
Memory B Cells ◽  
Phenotypic Characterization ◽  
B Cell Differentiation ◽  
Color Flow ◽  
Anca Associated Vasculitis ◽  
Cell Phenotypes

Abstract Objectives: B-cell depletion by rituximab (RTX) is an effective treatment for anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). However, peripheral B cell phenotypes and the selection criteria for RTX therapy in AAV remain unclear.Methods: Phenotypic characterization of circulating B cells was performed by 8-color flow cytometric analysis in 54 newly diagnosed AAV patients (20 granulomatosis with polyangiitis and 34 microscopic polyangiitis). Patients were considered eligible to receive intravenous cyclophosphamide pulse (IV-CY) or RTX. All patients also received high-dose glucocorticoids (GC). We assessed circulating B cell phenotypes and evaluated the efficacy after 6 months of treatment. Results: There were no significant differences in the rate of clinical improvement, relapses, or serious adverse events between patients receiving RTX and IV-CY. The proportion of effector or class-switched memory B cells increased in 24 out of 54 patients (44%). The proportions of peripheral T and B cell phenotypes did not correlate with BVAS at baseline. However, among peripheral B cells, the proportion of class-switched memory B cells negatively correlated with the rate of improvement in BVAS at 6 months after treatment initiation (r = -0.28, p = 0.04). Patients with excessive B cell differentiation were defined as those in whom the proportion of class-switched memory B cells or IgD-CD- B cells among all B cells was >2 SDs higher than the mean in the HCs. The rate of Birmingham Vasculitis Activity Score (BVAS) remission in patients with excessive B cell differentiation was significantly lower than that in patients without. In patients with excessive B cell differentiation, the survival rate, the rate of BVAS remission, and dose reduction of GC were significantly improved in the RTX group compared to those in the IV-CY group after 6 months of treatment. Conclusions: The presence of excessive B cell differentiation was associated with treatment resistance. However, in patients with circulating B cell abnormality, RTX was effective and increased survival compared to IV-CY. The results suggest that multi-color flow cytometry may be useful to determine the selection criteria for RTX therapy in AAV patients.

EBV MicroRNA Expression in Virus Driven B-Cell Differentiation and Lymphomagenesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 93-93
Author(s):  
Jamie P Nourse ◽  
Pauline Crooks ◽  
Do Nguyen Van ◽  
Kimberley Jones ◽  
Nathan Ross ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Phase 1 ◽  
Differentiation Process ◽  
B Cell Differentiation ◽  
B Cell Lymphomas

Abstract Abstract 93 Lymphomagenesis is a complex process, in part reflecting the nature of the transforming event, as well as the developmental stage of the cell. In the B-cell differentiation represents a continuum that is initiated when a naïve B-cell encounters antigen, undergoes a germinal centre (GC) reaction and ends with terminal differentiation into either a memory or plasma B-cell. Interruption of this process by a transforming event may result in a clonal proliferation where differentiation of the cell is blocked at this stage. The majority of B-cell lymphomas are derived from GC or post-GC B-cells. As physiologically relevant human models that emulate the various stages of B-cell differentiation are lacking we rationalized that in-vitro utilization of the B-cell lymphotrophic Epstein-Barr virus (EBV) would provide insights into this process. In one scenario, EBV infects naïve B-cells and drives a differentiation process paralleling the GC reaction through a well-characterized series of latency gene expression programs. EBV is also implicated in a range of GC and post-GC derived B-cell lymphomas (including Burkitt's, Hodgkin's, PTLD and DLBCL). Using high efficiency EBV infection of isolated naïve B-cells from EBV seronegative subjects, we have demonstrated that EBV infection provides a highly relevant in-vitro model that accurately reflects three distinct phases in the GC differentiation process. Alterations in the expression of a broad range of genes associated with the differentiation of the naïve B-cell were observed within 24 hours of infection and within four days of infection a process exhibiting many similarities to the GC reaction had taken place. These included BCL6, the levels of which were rapidly down-regulated within 24 hours indicating activation of the naïve B-cell. Levels of the memory cell marker CD27 steadily increased over 24 to 96 hours, while BLIMP1 expression increased, peaking at 48 hours. An increase in AID expression over 8 to 48 hours was consistent with somatic hypermutation and isotype switching. Finally a dramatic elevation in expression of the GC associated oncogene LMO2 was observed after two days followed by an equally dramatic downregulation after two weeks. Within two weeks of infection (phase 1), B-cells progressed through a GC-like phase followed by a one week transition state (phase 2) after which continued culture resulted in further differentiation to cells with the phenotypic hallmarks of post-GC cells (phase 3). MicroRNAs (miRNAs) are small non-coding RNAs, which act as negative regulators of gene expression. miRNA expression reflects the developmental lineage and differentiation state of several human cancers and over-expression is implicated in lymphomagenesis. They are also associated with the development of the GC reaction. EBV expresses at least 39 unique miRNAs from the BART and BHRF1 clusters within the viral genome. These EBV miRNAs are differentially expressed in tumour cell lines, suggesting roles during EBV-driven B-cell differentiation and lymphomagenesis. The relationship between EBV miRNAs and the kinetics of EBV driven B-cell differentiation has not been characterized. In our model we find distinct miRNA expression kinetics, coincidental with gene expression changes during B-cell differentiation, suggesting that these regulatory molecules may be involved in the GC process. Although a small number of EBV miRNAs were expressed at low levels early in the GC-like phase 1, the majority were up-regulated during the transition phase 2, exhibiting a subsequent partial down-regulation in the post-GC-like phase 3. The three phases were coincident with differential BART and BHRF1 promoter usage and alternate splicing. Strikingly, application of the infection model to primary patient samples and lymphoma cell-lines revealed that lymphomas clustered within distinct phases, reflecting the full continuum of the B-cell differentiation process. Interestingly, the majority of PTLD samples clustered within the transition phase, whereas Burkitt's and Hodgkin's lymphoma sample segregated with the GC stage. Application of our gene expression and miRNA data to cell-lines and a range of GC and post-GC EBV-positive lymphomas of various histological types indicate that our B-cell differentiation model can be used to accurately classify B-cell lymphomas in a physiologically relevant manner according to the stage of arrested B-cell differentiation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of A-myb, but not c-myb and B-myb, is restricted to Burkitt's lymphoma, sIg+ B-acute lymphoblastic leukemia, and a subset of chronic lymphocytic leukemias

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1900-1911 ◽  
Author(s):  
J Golay ◽  
M Luppi ◽  
S Songia ◽  
C Palvarini ◽  
L Lombardi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Differentiation ◽  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Burkitt’S Lymphoma ◽  
Burkitt's Lymphoma ◽  
B Cell Differentiation

Abstract The A-myb gene encodes a transcription factor that is related both functionally and structurally to the v-myb oncogene. Following our observations that A-myb is expressed in a restricted subset of normal mature human B lymphocytes, with the phenotype CD38+, CD39-, slgM-, we have now investigated the pattern of A-myb expression in neoplastic B cells representating the whole spectrum of B-cell differentiation and compared it to that of c-myb and B-myb. In a panel of 32 B-cell lines, A-myb was very strongly expressed in most Burkitt's lymphoma (BL) cell lines, but weak or negative in 2 pre-B acute lymphoblastic leukemia (ALL), 4 non-Hodgkin's lymphoma (NHL), 6 Epstein-Barr virus- immortalized lymphoblastoid cell lines, and 6 myeloma lines. Protein expression paralleled that of the RNA. We have also investigated A-myb expression in 49 fresh cases of B leukemias. Among 24 ALL, 6 were of the null and 11 of the common type and all these were negative for A- myb expression; on the other hand, all 7 B-ALL cases (slg+), as well as one fresh BL case with bone marrow infiltration, expressed A-myb. A-myb was undetectable in 4 prolymphocytic leukemias (PLL) but was strongly expressed in 5/20 (25%) of chronic lymphocytic leukemia (CLL) samples. In the latter A-myb did not correlate with phenotype or clinical stage. Finally, we have studied the progression of one case of CLL into Richter's syndrome and have found that the Richter's cells expressed about 25-fold less A-myb RNA than the CLL cells from the same patient. The pattern of c-myb and B-myb was clearly distinct from that of A-myb. C-myb and B-myb were expressed in all neoplastic groups, except in CLL cells. Thus, A-myb expression, unlike that of c-myb and B-myb, is restricted to a subset of B-cell neoplasias (in particular BL and slg+B- ALL) representative of a specific stage of B-cell differentiation. This expression may in part reflect expression of A-myb by the normal germinal center B cells that are the normal counterpart of these transformed B cells. The data presented strongly support a role for this transcription factor in B-cell differentiation and perhaps in B- cell transformation in some neoplasias.

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