IL7 Counteraction with Notch Signaling Followed by EBF1 Expression Marks the B-Cell Commitment in CLP Stage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2452-2452
Author(s):  
Sasan Zandi ◽  
Panagiotis Tsapogas ◽  
Robert Månsson ◽  
Mikael Sigvardsson
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
B Cell ◽  
Single Cell ◽  
Reporter Gene ◽  
B Cell Development ◽  
Expression Level ◽  
Single Cell Pcr ◽  
Cell Commitment

Abstract Development of B-cell lineage from hematopoietic Stem cells in bone marrow is a stepwise process associated with a gradual loss of myeloid and T cell potential. This process involves a complex interaction of transcription factors like EBF1 and E2A, and extrinsic signals including IL7. It has been suggested that IL7 plays an inductive role in B-cell commitment through EBF activation in early B-Cell development. Mice deficient in Il-7 signaling show a dramatic reduction in the number of B-cell progenitors and reduced expression of EBF1 in the common lymphoid progenitor (CLP) compartment and ectopic expression of EBF can partially rescue the B-cell phenotype. However, the rather limited ability of EBF1 to rescue the phenotype as well as the powerful function of Il-7 in the expansion of committed cells creates a complex situation with an inherent difficulty to separate instructive and permissive actions of Il-7. Using transgenic mice carrying a reporter gene under the control of the EBF1 dependent Igll1 promoter, we were able to identify a B220−CD19− committed B-cell progenitor likely to represent the earliest committed population in the mouse bone marrow. This has opened the possibility to investigate lineage commitment in cells not expressing classical surface markers creating increased possibilities to study lineage choices. In order to investigate the inductive role of Il-7 we crossed the Igll1 reporter mice to Il-7 deficient mice. Analysis of reporter gene expression, gene expression by multiplex single cell PCR as well as functional analysis by in vitro differentiation assays, all supported that the committed lineage negative population was dramatically decreased in the absence of Il-7. These data all support the idea that Il-7 is critical not only for expansion of B-lineage progenitors but also for commitment per se. Investigation of the expression of EBF-1 by Real time and single cell PCR suggested that the expression level of EBF was on an average 50% lower in Il-7 deficient progenitors as compared to wild type cells. This expression level was recapitulated in mice heterozygote for a mutation in the EBF1 gene but since the formation of the early committed cells was not as dramatically effected in these mice, we found a need to look for a further function of Il-7 in its instructive role in B-cell development. This prompted us to investigate a potential function of Il-7 in the modulation of Notch signals known to counteract B-cell commitment and EBF function. This revealed that the addition of Il-7 largely inhibits the Notch response in pro-B cells in vitro. Therefore we suggest that Il-7 is directly involved in B-cell commitment and that this function is achieved by modulation of EBF1 both at the transcriptional and functional level.

scholarly journals Mesenchymal Stromal Cells in the Bone Marrow Niche Consist of Multi-Populations With Distinct Transcriptional and Epigenetic Properties

2021 ◽  
Author(s):  
Sanshiro Kanazawa ◽  
Hironori Hojo ◽  
Shinsuke Ohba ◽  
Junichi Iwata ◽  
Makoto Komura ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Single Cell ◽  
Expression Profiles ◽  
Marker Genes ◽  
Mouse Bone Marrow ◽  
Bone Marrow Niche ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Abstract Although multiple studies have investigated the mesenchymal stem and progenitor cells (MSCs) that give rise to mature bone marrow, high heterogeneity in their morphologies and properties causes difficulties in molecular separation of their distinct populations. In this study, by taking advantage of the resolution of the single cell transcriptome, we analyzed Sca-1 and PDGFR-α fraction in the mouse bone marrow tissue. The single cell transcriptome enabled us to further classify the population into seven populations according to their gene expression profiles. We then separately obtained the seven populations based on candidate marker genes, and specified their gene expression properties and epigenetic landscape by ATAC-seq. Our findings will enable to elucidate the stem cell niche signal in the bone marrow microenvironment, reconstitute bone marrow in vitro, and shed light on the potentially important role of identified subpopulation in various clinical applications to the treatment of bone- and bone marrow-related diseases.

scholarly journals Developmental Stage Specific B-Progenitor Expansion in Normal Fetal Bone Marrow Is Absent in Down Syndrome: Implications for Infant ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4331-4331
Author(s):  
Anindita Roy ◽  
Georg Bohn ◽  
Katerina Goudevenou ◽  
Gillian Cowan ◽  
Neha Bhatnagar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Fetal Life ◽  
Second Trimester ◽  
Leukemic Transformation ◽  
Developmentally Regulated ◽  
Cd34 Cells

Abstract Although distinct fetal and adult B-cell lineage development has been demonstrated in murine studies, human fetal B-lymphopoiesis remains poorly understood. Previous work from our lab identified a population of PreProB progenitors (CD34+CD19+CD10-) in fetal liver (FL), co-existing with adult-type CD34+CD19+CD10+ ProB progenitors (Roy, A., Cowan G et al. PNAS: 109, 17579-17584; 2012). This supports other work demonstrating a PreProB progenitor population in normal cord blood (Sanz, E., et al. PNAS: 107, 5925-5930; 2010) and suggests that fetal pathways of B-cell development are also likely to be markedly different from adult bone marrow (AdBM) in humans. FL PreProB progenitors give rise solely to B-cells in vitro and display a gene expression pattern of successive activation of B-cell transcription factors as determined by Fluidigm RQ-PCR. In addition, increasing evidence indicates that infant ALL and many cases of childhood ALL arise in fetal life, suggesting that ontogeny-related changes in B-cell development may provide the cellular and microenvironmental context for in utero leukemia initiation. We therefore investigated B-cell development in normal human fetal BM from the onset of BM hematopoiesis in late first trimester. The composition and function of the early lymphoid and committed B-progenitor compartments of fetal BM(12-22 weeks; n=20) were compared with FL (n=25) at the same gestation, paediatric (Paed) BM (n=6) and AdBM (n=7), by multiparameter flow cytometry, differentiation in stromal co-culture assays and clonogenic assays. All stages of B-cell development were demonstrable in human fetal BM. However, there was a significantly higher frequency of B-progenitors in fetal BM (45.8±2.7% of CD34+ cells) compared to FL (10.3±0.97%; p<0.0001), PaedBM (28.2±4.2%; p=0.001) and AdBM (25.8±2.8%; p<0.0001). As in FL, both CD34+CD19+CD10- (PreProB) and CD34+CD19+10+ (ProB) progenitors were identified in fetal BM. PreProB progenitors were significantly higher in fetal BM (21.9±2.3% of CD34+ cells) compared to FL (3.8±0.4%), PaedBM (4.2±0.9%) and AdBM (3.4±0.9%) (p<0.0001 for all). Fetal BM PreProB progenitors gave rise solely to B-cells when co-cultured on MS5 stromal cells with FLT3L, SCF and IL7 in contrast to multilineage output of fetal BM HSC and LMPP cultured under identical conditions. Furthermore, fetal BM Lin-CD10-CD34+ cells cultured in vitro acquire CD19 expression before CD10 expression (n=6) consistent with a unique, fetal-specific B-cell differentiation pathway. The progressive decline in the proportion of PreProB progenitors from 47.8% to 36.8%, 14.8% and 13% of total B-progenitors in fetal BM, FL, PaedBM and AdBM respectively points towards a developmental stage-specific emergence of these progenitors. Finally, the fetal BM B-progenitor: B-cell ratio falls rapidly from ~4:1 at 12 weeks gestation to 1:1 at 18 weeks gestation as mature B-cell production in fetal BM gradually increases. This is consistent with a developmentally-regulated drive to B-progenitor proliferation, at the expense of differentiation, early in the second trimester which might represent a target population vulnerable to leukemic transformation in fetal life. Since children with Down syndrome (DS) do not develop infant ALL, we examined DS fetal BM as they may lack susceptible target cells for leukemic transformation. Consistent with this, PreProB progenitors in DS fetal BM (n=7) were >6-fold lower than normal fetal BM (3.3±0.8% vs. 21.9±2.3% of CD34+ cells; p<0.0001). Furthermore, CD19+ cells with an infant ALL-associated immunophenotype (CD19+CD10-CD20-) are detectable in normal fetal BM mononuclear cells, but are rarely found in DS fetal BM. Conclusion: There is a marked expansion of PreProB progenitors in normal second trimester human fetal BM which is virtually absent in DS fetal BM, in normal PaedBM and in normal AdBM. We suggest that developmentally-regulated, functional and molecular characteristics of these fetal-specific B-progenitors may provide the 'oncogenic' cellular context necessary to co-operate with genetic events, such as MLL rearrangements, to induce ALL in infants without DS. Disclosures No relevant conflicts of interest to declare.

B Cell Development in the Absence of PU.1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 226-226 ◽  
Author(s):  
Min Ye ◽  
Olga Ermaermakova-Cirilli ◽  
Thomas Graf
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Target Genes ◽  
Fetal Liver ◽  
Cell Formation ◽  
Cell Development ◽  
B Cell Development ◽  
B Lineage

Abstract Mice deficient of the ETS-family transcription factor PU.1 lack B cells as well as macrophages. While most macrophage specific genes are known to be regulated by high levels of PU.1, the reason for the defect in B cell formation is not known. Here we analyzed a mouse strain in which a floxed version of the PU.1 gene, surrounding exon 4 and 5, which encode the DNA, binding and PEST domains (developed by C. Somoza and D. Tenen), was excised by Cre mediated recombination. As expected, this strain lacks both B cells and macrophages and die at birth. Surprisingly, however, we were able to establish lymphoid cell lines from fetal livers of these mice (day 14 to day 18), which proliferated on S17 stromal cells supplemented with IL-7 and stem cell factor. These cells expressed the B lineage cell surface markers CD19, CD43, BP-1 and CD24, but not B220. They also expressed B cell transcription factors, EBF, E47, Pax5, and their target genes, Rag1, IL7R, λ5 and v-preB, as detected by RT-PCR, exhibited DJ and VDJ immunoglobulin heavy chain rearrangements, and expressed IgM after IL-7 withdrawal. We then tested the effect of PU.1 deletion in B cells in adult animals by crossing the floxed PU.1 strain with a CD19 Cre mouse line. The spleen and peripheral blood (but not bone marrow) of these mice contained B cells that were CD19+ IgMlow, IgDhigh but B220 negative and instead expressed CD43. Thus PU.1 is not essential for immunoglobulin production and late B cell development. Although PU.1−/− fetal liver cells can give rise to cells, resembling Pre-B in vitro, the process of B cell formation was delayed by almost 12 days, compared with wt fetal liver, and the efficiency was reduced approximately 25-fold. In addition, PU.1 deficient B cells demonstrated an impaired ability to engraft into the bone marrow, when injected into irradiated SCID mice. We have found that PU.1 deficient B progenitors showed reduced or undetectable levels of the SDF1 receptor CXCR4, a receptor that has been implicated in B cell homing. Taken together, our observations suggest that PU.1 plays two different roles during B cell development: for early B cell formation and for proper migration and engraftment, which might be mediated through regulation of CXCR4 expression.

C-Kit Signaling Blocks B Cell Differentiation of Common Lymphoid Progenitor Cells by Negatively Regulating Stat5-Dependent Activation of Ebf Expressionc-Kit Signaling Blocks B Cell Differentiation of Common Lymphoid Progenitor Cells by Negatively Regulating Stat5-Dependent Activation of Ebf1 Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1209-1209
Author(s):  
Oleg Kolupaev ◽  
Melissa Thal ◽  
Christopher Klug
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
B Cell ◽  
Progenitor Cells ◽  
Signaling Pathway ◽  
B Cell Development ◽  
B Cell Differentiation ◽  
Flt3 Ligand ◽  
Cell Commitment

Abstract Abstract 1209 B-lymphocyte commitment from common lymphoid progenitor cells (CLP) is orchestrated by a number of transcription factors and external cues such as IL-7, stem cell factor (SCF) and Flt3 ligand. IL-7 signaling plays a critical role in promoting B-cell differentiation by inducing the expression of transcription factor Ebf1 via the Jak-Stat5 signaling pathway (Kikuchi et al., 2007; Tsapogas et al., 2011). Previously, it has been shown that Flt3 ligand acts synergistically with IL-7 during the earliest stages of B-cell commitment (CLP and pre-pro-B fraction)(Åhsberg et al., 2010), but is downregulated by the pre-B stage to ensure normal development of B lymphocytes (Holmes et al., 2006). The role of SCF signaling in this early cell fate decision, however, remains unclear. Here, we demonstrate that SCF dominantly inhibits IL-7-induced B-cell differentiation from CLP by negatively regulating the Jak-Stat5 signaling pathway. Co-culture of bone marrow Lin-Kit+Sca-1+(LSK) cells with OP9 feeder cells in the presence of IL-7 (10 ng/ml) and SCF (10 ng/ml) resulted in 1.5–2-fold reduction in the frequency and absolute numbers of B220+CD19+ cells compared to cultures stimulated with IL-7 alone. To test whether c-Kit signaling inhibits B cell commitment in vivo, BM progenitor cells overexpressing a constitutively active form of KIT (MSCV-Kit-V814D-IRES-VEX) were transplanted into lethally irradiated mice. VEX+ cells in reconstituted mice had a very limited potential for B-lymphoid lineage differentiation, as judged by severe reduction in the frequency of B220+ cells in BM at 7 weeks post-transplant [B220+CD19-IgM-: 1.5%±0.3 (control) vs 0.6%±0.2 (V814D), p<0.05, n=6); B220+CD19+: 19.4%± 2.7 vs 2.1%±0.6, p<0.001; CD19+IgM+: 6.7%±1.3 vs 0.9%±0.3, p<0.01)]. Analysis of CLP cells isolated from IL-7−/− mice revealed that co-culture with IL-7 and SCF inhibited IL-7-induced upregulation of Ebf1 as well as other Stat5 target genes (Socs3, Cish, Osm). Furthermore, short-term treatment of BM cells isolated from IL-7−/− mice with a combination of SCF and IL-7 resulted in a significant reduction of phospho-Stat5 in CLP cells compared to cells treated with IL-7 alone. Class III receptor tyrosine kinases such as Flt3 and c-Kit are able to activate multiple signaling pathways via a juxtamembrane SH2-docking site. Mutation of the SH2 domain in the context of the Kit-V814D mutation (Kit-V814D-Y567/569F) did not rescue B-cell development when this mutant protein was expressed in primary murine bone marrow cells in vivo. In contrast, expression of a mutant form of Kit-V814D that inhibited binding of the SH2B adapter protein 2 (APS) (Kit-V814D-I570A/L937A) rescued B-lymphopoiesis, indicating that a signaling pathway coupled to APS dominantly suppresses B cell commitment when CLP are co-stimulated with SCF and IL-7. Based on the observations that APS is a known adapter for the E2-ubiquitin ligase, c-Cbl, which has been shown to negatively regulate Stat5 in other systems, we speculate that c-Kit signaling inhibits IL-7-induced Stat5 activation via an APS-Cbl pathway during early B-cell development. Further characterization of this inhibitory developmental pathway will be discussed. Disclosures: No relevant conflicts of interest to declare.

Enforced Expression of Pax-5 Results in Developmental Arrest, Immortalisation and Aberrant Expression of B Lineage Genes in Committed Myeloid Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2784-2784
Author(s):  
Kristina Anderson ◽  
Corinne Rusterholz ◽  
Robert Mansson ◽  
Christina Jensen ◽  
Karl Bacos ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Gene Expression Pattern ◽  
Cell Development ◽  
B Cell Development ◽  
Myeloid Differentiation ◽  
Aberrant Expression ◽  
Murine Bone Marrow ◽  
Myeloid Progenitors

Abstract The paired domain transcription factor Pax-5, has been demonstrated to play a crucial role in development and final commitment of the B cell lineage. In fact, otherwise committed B cell progenitors acquire multipotentiality (myelo-lymphoid) potential upon targeted deletion of Pax-5 expression (Nutt et al. Nature.1999; 401:556–562). Thus, in addition, to promoting B cell development through acting as an activator of transcription of B cell specific genes such as CD19, Pax-5 is also thought to act as a suppressor of transcription of genes involved in determination of other blood cell lineages. However, it remains unclear how Pax-5 might repress myeloid development. Thus, we investigated the effect of Pax-5 expression on lympho-myeloid differentiation by overexpressing human (h)Pax-5 (through retroviral transduction) in adult murine bone marrow Linlo/−Sca-1+c-kit+ (LSK) cells. When compared to cells transduced with a control vector, LSK cells ectopically expressing hPax-5 very efficiently developed into hPax-5+B220+CD19+ pro-B cells in response to flt3 ligand and interleukin-7 in vitro. In contrast, when hPax-5+ LSK cells were cultured under myeloid conditions, we consistently observed development of a highly proliferative and immortalised bi-phenotypic (B-myeloid) hPax-5+B220+Gr-1+Mac-1+ population that predominantly consisted of immature myeloblasts but also maturated granulocytes and monocytes. Global gene expression analysis by micro-array, and confirmation by RT-PCR, demonstrated that hPax-5+B220+Gr-1+Mac-1+ cells also possessed a bi-phenotypic gene expression pattern, characteristic for B-cell as well as myeloid lineages including the Pax-5 target B cell genes mb-1 and BLNK as well as GM-CSFRα and low levels of C/EBPα. Similar findings were observed when targeting committed myeloid progenitors. These findings suggest that in addition to promoting B cell development, Pax-5 is capable of inhibiting/blocking myeloid differentiation and inducing immortalization as well as expression of B cell specific genes in otherwise myeloid committed progenitors. These findings motivate a careful investigation of the potential involvement of Pax-5 also in myeloid leukemias.

Molecular Basis of Proliferation/Survival and Migration of CLL in Peripheral Blood, Bone Marrow and Lymph Nodes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 546-546
Author(s):  
Amit K Mittal ◽  
Javeed Iqbal ◽  
Tara Marie Nordgren ◽  
Margaret Moragues ◽  
R. Gregory Bociek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
B Cell ◽  
Chemokine Receptors ◽  
Expression Patterns ◽  
Gene Expression Patterns ◽  
Bcr Signaling ◽  
And Migration

Abstract B-cell chronic lymphocytic leukemia (CLL) is a heterogenous and incurable B-cell malignancy. CLL cells migrate and accumulate in different sites including the peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) in vivo, but undergo apoptosis in vitro. Therefore, we hypothesized that CLL cells at these sites are different and receive different microenvironmental signals that regulate their proliferation/survival and migration. Most reports on the microenvironmental influence on CLL cells have used in vitro models consisting of stromal and CLL cells. However, in this study, to better understand the influence of site-specific microenvironments in vivo, gene expression patterns of CLL cells obtained from PB, BM and LN were investigated. CLL cells were isolated from patients’ PB (PB-CLL, n= 20), BM (BM-CLL, n=14) and LN (LN-CLL, n=15) and used to determine the gene expression patterns by microarray analysis. In addition, we also included PB-CLL cases from our previous study (n=40) to further validate the findings of this study. Significant Analyses of Microarray (SAM) revealed differential expression of more than 500 genes among these three sites. To understand the potential roles of these differentially-expressed genes and their association with relevant functional pathways in CLL, Gene Set Enrichment Analysis (GSEA) was performed. The validation of pathway specific genes was further confirmed by quantitative real time PCR. Among the pathways identified, the most active pathways associated with the migration and proliferation/survival of CLL cells, namely chemokine-signaling, BCR signaling, BAFF/APRIL-signaling, and NFκB-signaling pathways, were selected for further analyses. We hypothesized that chemokines and their receptors mediate the migration of CLL cells between PB and LN or BM, and that molecules of the BCR, BAFF/APRIL and NFκB pathways regulate proliferation/survival. To determine the role of chemokines and their receptors in CLL cell migration, we studied the expression of 52 chemokine/chemokine receptors and found that PB-CLL cells significantly (p&lt;0.005) overexpressed CXCR4 and CCR7 compared to BM-CLL and LN-CLL cells. The ligands CCL21 and CXCL13 were significantly overexpressed (p&lt;0.005 and p&lt;0.01 respectively) in LN-CLL. These results indicate that PB-CLL cells express distinct chemokine receptors which may lead them to home to BM or LN and receive stimuli to form proliferation centers. Based on GSEA analysis, the stimuli for proliferation/survival for CLL cells in the LN and BM are provided by Syk and Btk (BCR signaling), BAFF and TRAF2 (BAFF/APRIL signaling), and several targets of the NFκB pathways. Syk and Btk were significantly overexpressed in LN-CLL (p&lt;0.05) and PB-CLL (p&lt;0.005) compared to BM-CLL, with the highest expression in LN-CLL, suggesting chronic activation of CLL cells in lymph node. Similarly, BAFF and TRAF2 were significantly overexpressed (p&lt;0.03) in LN-CLL compared to PB-CLL and BM-CLL. Furthermore, the NFκB pathway, which is important for the proliferation and survival, also showed distinct association in different CLL-cell compartments. The RELA, NFκB1, NFκB2, TNFAIP3 and NFκB regulators such as NFκBIA, NFκBIE were also significantly (p&lt;0.01) overexpressed in PB-CLL and BM-CLL compared to LN-CLL with highest expression in BM-CLL. Whereas few NFκB associated genes such as NFκB1L1 and RelB were significantly (p&lt;0.02) expressed in LN-CLL cells. Thus, differentially-expressed NFkB genes among PB-CLL, BM-CLL and LN-CLL cells indicate that these different CLL cells utilize different NFκB molecules for proliferation/survival. Together, our results show that CLL cells from different in vivo microenvironments such as PB, BM and LN exhibit differential gene expression patterns, and many of the genes are involved in regulation of migration and proliferation/survival. Furthermore, LN-CLL cells expressing chemokine ligands, BCR, BAFF and NFκB signaling molecules attract other cells including more CLL cells to form an optimal microenvironment which provide prosurvival and proliferative signals to CLL cells.

The MiR-23a MicroRNA Cluster Inhibits B Cell Development.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1465-1465
Author(s):  
Jason Mullenix ◽  
Kimi Y Kong ◽  
Kristin Severns Owens ◽  
Jason Rogers ◽  
Shannon FitzPatrick ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Mature Mirnas ◽  
Cell Development ◽  
B Cell Development

Abstract Abstract 1465 Poster Board I-488 The miR-23a microRNA (miRNAs) cluster inhibits both [ITALIC]in vitro[/ITALIC] and [ITALIC]in vivo[/ITALIC] B cell development. When murine hematopoietic progenitor cells expressing the 23a cluster miRNAs were cultured in B cell promoting conditions we observed over a five-fold decrease in the generation of CD19+ B cells compared to control cultures. Conversely, we observed over a five-fold increase in CD11b+ myeloid cells. When irradiated mice were transplanted with bone marrow expressing the miR-23a cluster we observed a two-fold decrease in bone marrow and splenic B cells, 8 weeks post-transplant compared to control mice. The miR-23a cluster codes for a single pri-transcript, which when processed yields three mature miRNAs: miR-23a, miR-27a, and miR-24-2. All three mature miRNAs are more abundant in myeloid cells compared to other hematopoietic cells. In vitro miR-24 alone is necessary and sufficient to inhibit B cell development. The promoter for the cluster contains conserved binding sites for the essential myeloid transcription factors PU.1 and C/EBP alpha. Chromatin immunoprecipitations demonstrated that PU.1 and C/EBP alpha are associated with the promoter in myeloid cells. In addition, C/EBP alpha is bound to several highly conserved regions upstream of the promoter. Both PU.1 and C/EBP alpha promote myeloid development at the expense of lymphopoiesis. Our work suggests that the miR-23a cluster may be a critical downstream target of PU.1 and C/EBP alpha in the specification of myeloid cell fate. Although miRNAs have been identified downstream of PU.1 and C/EBP alpha in mediating the development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in the regulating lymphoid cell fate acquisition. We are currently identifying targets of miR-24 that may mediate the inhibitory effect on B lymphopoiesis. Disclosures No relevant conflicts of interest to declare.

A Putative Role for VEGFR-1 (FLT-1) in B Cell Commitment and Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1152-1152
Author(s):  
Rita Fragoso ◽  
Catia Igreja ◽  
Claudia Appleton ◽  
Alexandra Henriques ◽  
Nuno Clode ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Myeloid Differentiation ◽  
B Cell Differentiation ◽  
Cd34 Cells ◽  
Cell Commitment

Abstract VEGF and its receptors are expressed in the hematopoietic system. A role for FLT-1 in particular was described in monocyte-macrophage migration and lineage differentiation (Sawano A et al, 2001), megakaryocytes maturation (Casella I et al, 2003) and dendritic cell differentiation (Dikov M et al, 2005). Given that the expression of this receptor in the lymphoid lineage is not known, we to studied FLT-1 expression and a putative function in normal lymphoid progenitors. To address this question we induced in vitro CD34+ cells differentiation into the B cell lineage using a well established assay (on S17 stromal cells). With this approach, we observed that FLT-1 is expressed throughout B cell differentiation increasing along the differentiation process, and reaching its highest at the “immature B cell” stage. We also neutralized FLT-1 during B cell differentiation in vitro. Surprisingly, in the presence of the FLT-1 neutralizing antibody (6.12 monoclonal Ab, from ImClone systems), at the end of the assays (4 different experiments) a significantly higher number of CD19+ cells (mainly immature B cells) were detected. Analyzing some of the transcription factors known to be involved in the commitment and differentiation of lymphoid B cells, we observed that the expression of PU.1, Pax5 and E47 was up-regulated by FLT-1 neutralization. Next, given that FLT-1 function was mainly associated with cell migration, and since it is expressed in B cells that are ready to exit the bone marrow into secondary lymphoid organs, we reasoned that FLT-1 might have a role in B cells exit from the bone marrow. For this purpose, we treated mice with the FLT-1 neutralizing Ab for 3 days and analyzed B cells levels in bone marrow and peripheral blood. FLT-1 neutralization led to a significant decrease (p&lt;0.05) in B cells in the bone marrow and peripheral blood. Taken together, our data supports a clear role for FLT-1 in B cell commitment. To understand if VEGF/PlGF signalling through FLT-1 promotes myeloid differentiation, suppresses B cell differentiation or simply regulates the quiescent state of hematopoietic stem cells, we differentiated in vitro CD34+/FLT-1− cells and CD34+/FLT-1+ cells (10% of CD34+ cells) using the assay described above. Interestingly, CD34+/FLT-1− differentiation in vitro largely promoted B cell differentiation, while CD34+/FLT-1+ cells originated mostly myeloid cell differentiation. We are currently exploiting the molecular basis whereby FLT-1 signalling may impair B cells commitment and possibly promotes myeloid differentiation.

scholarly journals Nonpermissive bone marrow environment impairs early B-cell development in common variable immunodeficiency

Blood ◽  
2020 ◽  
Vol 135 (17) ◽  
pp. 1452-1457 ◽  
Author(s):  
Arianna Troilo ◽  
Claudia Wehr ◽  
Iga Janowska ◽  
Nils Venhoff ◽  
Jens Thiel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Immature B Cells ◽  
Cell Repopulation

Abstract Common variable immunodeficiency (CVID) is a disease characterized by increased susceptibility to infections, hypogammaglobulinemia, and immune dysregulation. Although CVID is thought to be a disorder of the peripheral B-cell compartment, in 25% of patients, early B-cell development in the bone marrow is impaired. Because poor B-cell reconstitution after hematopoietic stem cell transplantation has been observed, we hypothesized that in some patients the bone marrow environment is not permissive to B-cell development. Studying the differentiation dynamics of bone marrow-derived CD34+ cells into immature B cells in vitro allowed us to distinguish patients with B-cell intrinsic defects and patients with a nonpermissive bone marrow environment. In the former, immature B cells did not develop and in the latter CD34+ cells differentiated into immature cells in vitro, but less efficiently in vivo. In a further group of patients, the uncommitted precursors were unable to support the constant development of B cells in vitro, indicating a possible low frequency or exhaustion of the precursor population. Hematopoietic stem cell transplantation would result in normal B-cell repopulation in case of intrinsic B-cell defect, but in defective B-cell repopulation in a nonpermissive environment. Our study points to the importance of the bone marrow niche in the pathogenesis of CVID.

Change of the Notch1 Expression Level in Lymphoid Malignancies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4425-4425
Author(s):  
Haitao Zheng ◽  
Lijian Yang ◽  
Shao-Hua Chen ◽  
Liye Zhong ◽  
Yangqiu Li
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
B Cell ◽  
Natural Science ◽  
Research Funding ◽  
Gene Expression Level ◽  
Expression Level ◽  
Lymphoid Malignancies ◽  
Healthy Control ◽  
Cell Commitment

Abstract Abstract 4425 Notch gene family encodes single pass transmembrane receptors able to transducer intercellular signals involved in cell-fate determination. Notch signaling is the leading causes to the development of malignant tumors. Notch pathway plays a role in haematopoiesis affecting both stem cells and committed progenitors. The expression of Notch1 in thymic stromal cells and in developing thymocytes is consistent with Notch function. Notch1 is turn on before T-cell commitment along the lymphoid development; a variety of model systems indicates that Notch1 signaling is required for the earliest stages of T-cell commitment in the thymus and that, in its absence, cells develop toward the B-lineage. Overexpression of Notch1 gene was found in T-ALL, however, little is known the expression pattern of Notch1 gene in the different lymphoid malignancies. In the present study, the expression level of Notch1 gene was analyzed in patients with lymphoid malignancies. Real-time PCR with SYBR Green I technique was used for detecting Notch1 gene expression level in peripheral blood mononuclear cells of 43 patients with lymphoid malignancies [including 7 cases with T-ALL, 6 cases with T-NHL, 9 cases with B-ALL, 5 cases with B-CLL, 16 cases with B-NHL] and 20 healthy individuals. β2-microglobulin gene (β2M) was used as an endogenous reference. Relative changes in Notch1 gene expression level were used by the 2-ΔCt×100% ×100% method. The expression level of Notch1 in both T-cell lymphoid malignancies group (Median: 0.897%) and B-cell lymphoid malignancies group (Median: 0.726%) was significant higher than those in the healthy control (Median: 0.288%) (P<0.01, P<0.01). Moreover, the expression level of Notch1 was different in the five types of lymphoid malignancies. That is, the highest in the B-CLL group, secondary in the T-NHL group, the lowest in the B-NHL group. And, it is not significant different between the B-NHL and healthy control groups. In conclusions, Overexpression of Notch1 gene was found not only in T-ALL, but also in T-NHL and B-cell leukemia and suggests that the abnormal overexpression pattern of Notch1 might play an important role in the pathogenesis of lymphoid malignancies. Down-regulation of Notch1 might be considered as target therapeutic strategy for lymphocytic malignancies. Disclosures: Zheng: The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Yang:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Chen:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Zhong:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Li:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding.

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