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.

scholarly journals The transcription factor Gli3 promotes B cell development in fetal liver through repression of Shh

2017 ◽  
Vol 214 (7) ◽  
pp. 2041-2058 ◽  
Author(s):  
Anisha Solanki ◽  
Ching-In Lau ◽  
José Ignacio Saldaña ◽  
Susan Ross ◽  
Tessa Crompton
Keyword(s):  
B Cells ◽  
B Cell ◽  
Fetal Liver ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
Pathway Activation ◽  
Genes Expression ◽  
B Lineage

Before birth, B cells develop in the fetal liver (FL). In this study, we show that Gli3 activity in the FL stroma is required for B cell development. In the Gli3-deficient FL, B cell development was reduced at multiple stages, whereas the Sonic hedgehog (Hh [Shh])–deficient FL showed increased B cell development, and Gli3 functioned to repress Shh transcription. Use of a transgenic Hh-reporter mouse showed that Shh signals directly to developing B cells and that Hh pathway activation was increased in developing B cells from Gli3-deficient FLs. RNA sequencing confirmed that Hh-mediated transcription is increased in B-lineage cells from Gli3-deficient FL and showed that these cells expressed reduced levels of B-lineage transcription factors and B cell receptor (BCR)/pre-BCR–signaling genes. Expression of the master regulators of B cell development Ebf1 and Pax5 was reduced in developing B cells from Gli3-deficient FL but increased in Shh-deficient FL, and in vitro Shh treatment or neutralization reduced or increased their expression, respectively.

scholarly journals The Earliest Step in B Lineage Differentiation from Common Lymphoid Progenitors Is Critically Dependent upon Interleukin 7

2002 ◽  
Vol 196 (5) ◽  
pp. 705-711 ◽  
Author(s):  
Juli P. Miller ◽  
David Izon ◽  
William DeMuth ◽  
Rachel Gerstein ◽  
Avinash Bhandoola ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Gamma Chain ◽  
Lineage Differentiation ◽  
Lymphoid Progenitors ◽  
B Lineage

Little is known about the signals that promote early B lineage differentiation from common lymphoid progenitors (CLPs). Using a stromal-free culture system, we show that interleukin (IL)-7 is sufficient to promote the in vitro differentiation of CLPs into B220+ CD19+ B lineage progenitors. Consistent with current models of early B cell development, surface expression of B220 was initiated before CD19 and was accompanied by the loss of T lineage potential. To address whether IL-7 receptor (R) activity is essential for early B lineage development in vivo, we examined the frequencies of CLPs and downstream pre–pro- and pro-B cells in adult mice lacking either the α chain or the common gamma chain (γc) of the IL-7R. The data indicate that although γc−/− mice have normal frequencies of CLPs, both γc−/− and IL-7Rα−/− mice lack detectable numbers of all downstream early B lineage precursors, including pre–pro-B cells. These findings challenge previous notions regarding the point in B cell development affected by the loss of IL-7R signaling and suggest that IL-7 plays a key and requisite role during the earliest phases of B cell development.

scholarly journals Iκb Kinase α Is Essential for Mature B Cell Development and Function

2001 ◽  
Vol 193 (4) ◽  
pp. 417-426 ◽  
Author(s):  
Tsuneyasu Kaisho ◽  
Kiyoshi Takeda ◽  
Tohru Tsujimura ◽  
Taro Kawai ◽  
Fumiko Nomura ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Fetal Liver ◽  
Cell Development ◽  
B Cell Development ◽  
Cytokine Signaling ◽  
Iκb Kinase ◽  
And Function

IκB kinase (IKK) α and β phosphorylate IκB proteins and activate the transcription factor, nuclear factor (NF)-κB. Although both are highly homologous kinases, gene targeting experiments revealed their differential roles in vivo. IKKα is involved in skin and limb morphogenesis, whereas IKKβ is essential for cytokine signaling. To elucidate in vivo roles of IKKα in hematopoietic cells, we have generated bone marrow chimeras by transferring control and IKKα-deficient fetal liver cells. The mature B cell population was decreased in IKKα−/− chimeras. IKKα−/− chimeras also exhibited a decrease of serum immunoglobulin basal level and impaired antigen-specific immune responses. Histologically, they also manifested marked disruption of germinal center formation and splenic microarchitectures that depend on mature B cells. IKKα−/− B cells not only showed impairment of survival and mitogenic responses in vitro, accompanied by decreased, although inducible, NF-κB activity, but also increased turnover rate in vivo. In addition, transgene expression of bcl-2 could only partially rescue impaired B cell development in IKKα−/− chimeras. Taken together, these results demonstrate that IKKα is critically involved in the prevention of cell death and functional development of mature B 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.

scholarly journals Interactions Between c-kit and Stem Cell Factor Are Not Required for B-Cell Development In Vivo

Blood ◽  
1997 ◽  
Vol 89 (2) ◽  
pp. 518-525 ◽  
Author(s):  
Shunichi Takeda ◽  
Takeyuki Shimizu ◽  
Hans-Reimer Rodewald
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Fetal Liver ◽  
Critical Role ◽  
Cell Development ◽  
B Cell Development ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract The receptor-type tyrosine kinase, c-kit is expressed in hematopoietic stem cells (HSC), myeloid, and lymphoid precursors. In c-kit ligand-deficient mice, absolute numbers of HSC are mildly reduced suggesting that c-kit is not essential for HSC development. However, c-kit− HSC cannot form spleen colonies or reconstitute hematopoietic functions in lethally irradiated recipient mice. Based on in in vitro experiments, a critical role of c-kit in B-cell development was suggested. Here we have investigated the B-cell development of c-kitnull mutant (W/W ) mice in vivo. Furthermore, day 13 fetal liver cells from wild type or W/W mice were transferred into immunodeficient RAG-2−/− mice. Surprisingly, transferred c-kit− cells gave rise to all stages of immature B cells in the bone marrow and subsequently to mature conventional B2, as well as B1, type B cells in the recipients to the same extent as transferred wild type cells. Hence, in contrast to important roles of c-kit in the expansion of HSC and the generation of erythroid and myeloid lineages and T-cell precursors, c-kit− HSC can colonize the recipient bone marrow and differentiate into B cells in the absence of c-kit.

Rescue of B Cells Development in an Animal Model of X-Linked Agammaglobulinemia(XLA) Via B Lineage-Specific Lentiviral Gene Therapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1287-1287
Author(s):  
S. Humblet-Baron ◽  
W. Zhang ◽  
K. Kipp ◽  
S. Khim ◽  
J. Jarjour ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Lentiviral Vector ◽  
Cell Development ◽  
B Cell Development ◽  
Gfp Expression ◽  
B Lineage

Abstract X-linked agammaglobulinemia (XLA) is a human immunodeficiency caused by mutations in Bruton’s tyrosine kinase (Btk) and characterized by an arrest in early B-cell development, absence of serum immunoglobulin, and recurrent bacterial infections. Using Btk and Tec double deficient (Btk/Tec−/ −) mice as a model for XLA, we recently showed that onco-retroviral-mediated Btk gene transfer into hematopoietic stem cells (HSC) reconstituted in vivo Btk-dependent B-cell development and function (Yu et al. Blood 104(5):1281–90). In order to increase the safety of this approach, we developed a SIN-lentiviral vector with a B cell specific enhancer/promoter element, Eμ B29. Using SIN-lentiviral vectors expressing GFP, we observed that Eμ B29 consistently promoted 3–5 fold higher GFP expression in human B lineage cells derived from transduced HSC in vitro and in vivo (ASGT 2002 abstract #1302). We also evaluated this vector, CSOM-Eμ B29-GFP-WPRE, in lentiviral transgenic mice where it exhibited the highest GFP expression in peripheral B cells compared with all other hematopoietic lineages. Specifically, in more than 8 independent founder strains the MFI for GFP expression in B cells was > 3 fold higher than that in T cells (p=0.0002). Based upon these findings we developed Eμ B29-huBtk SIN-lentiviral vectors with or without the insulator element derived from the chicken β-globulin insulator (HS4). Using both vectors to transduce Btk −/ − DT40 B cells, followed by cloning by limiting dilution, we demonstrate Btk protein expression by intracellular staining and western blotting and full rescue of Btk-dependent, B cell receptor (BCR)-mediated Ca2+ signaling in all clones evaluated including those exhibiting a single viral integration. Next we tested the capacity of these vectors to reconstitute Btk-dependent B-cell development and function in a cohort of Btk/Tec−/ − mice. Marrow from 5-FU treated Btk/Tec −/ − mice was harvested, cultured on fibronectin coated plates with growth factors (mIL-3,mIL-6, mSCF, mTPO and mFLT3ligand) and concentrated lentivirus (2.3x107pg/106 cells measured by p24 level). After 48h of in vitro culture, cells were transplanted into lethally irradiated animals and transplanted animals were serially evaluated for presence of B cells in the peripheral blood. B-cell numbers progressively increased with a significant difference as early as within 6 weeks in mice receiving transduced (16–18% B220+ cells) vs. control marrow (8–9%; mock transduced). Further, mature B cells (B220+IgMlowIgDhi) represented 14–20% of total B cells in treated compared to <5% in control mice. Finally, mice receiving transduced cells exhibited a rescue of total serum IgM and IgG3 levels and responses to TI-II dependent immunization. Results of two additional animal cohorts will be presented. In summary, our data demonstrate that Eμ B29-Btk SIN-lentiviral vector specifically promotes Btk expression in B lineage cells, and correction of the Btk-deficient phenotype in vitro and in vivo. Peripheral blood B cells were analyzed for relative IgM and IgD expression at 6 weeks post reconstitution. Representative data from animals receiving mock-vs/ EμB29-Btk transduced marrow are shown. Upper left quadrant shows percentage of circulating mature B cells. Peripheral blood B cells were analyzed for relative IgM and IgD expression at 6 weeks post reconstitution. Representative data from animals receiving mock-vs/ EμB29-Btk transduced marrow are shown. Upper left quadrant shows percentage of circulating mature B cells.

Ott1 (Rbm15) Is Required for the Large to Small Pre B-Cell Transition

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 706-706
Author(s):  
Michael Kharas ◽  
John Manis ◽  
D. Gary Gilliland ◽  
Glen D. Raffel
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Fetal Liver ◽  
Cell Development ◽  
B Cell Development ◽  
Transformed Cell Lines ◽  
Cell Transition

Abstract The OTT1 gene is fused with the MAL gene in t(1;22) infant-associated acute megakaryocytic leukemia generating a chimeric protein, OTT1-MAL. OTT1 is a transcriptional activator/repressor related to the spen/SHARP/Mint family. Mint was shown to regulate follicular versus marginal zone B-cell development in the spleen; however, Ott1’s physiologic role in B cell development is not fully understood. Recent data utilizing conditional deletion of a targeted Ott1 allele in mice delineated multiple regulatory roles for Ott1 in myeloid and lymphoid differentiation. Previous work by our laboratory showed that loss of Ott1 in addition to causing a myeloid and megakaryocytic expansion, results in a block in B development prior to the B220+CD43-IgM+ stage. We have characterized Ott1-deficient B-cell progenitors to identify stage- and process-specific requirements for Ott1 in pre B development. Ott1-deleted bone marrow and fetal liver could not generate pre B colonies in methylcellulose, however, we were able to establish IL-7-dependent pro B cell lines in vitro and observed no significant differences in proliferation, apoptosis or the ability to form v-Abl-transformed cell lines. Activated Ras or overexpression of Bcl2 failed to rescue pre B colony formation. In vivo, Ott1 null fetal liver pre B-cells expressed Ig heavy chain but failed to express the B-cell receptor (BCR) on their surface even though kappa rearrangement was detectable in vitro. In comparison to wildtype cells, B220+CD43-IgH+ Ott1 null cells were larger in size, had lower levels of IL-7R, but proliferated at higher levels and with an associated increase in apoptosis. Moreover, these cells had normal pre-BCR proximal signaling as judged by phospho-Blnk and phospho-Erk, but increased phosphorylation of S6 after IL7 and Ig stimulation. Ott1 null large pre B-cells had normal expression of Myc, but higher levels of expression of Cyclin D1. Taken together, these data indicate that loss of Ott1 results in enhanced proliferation and apoptosis in the pre B-cell compartment causing a developmental block at the large to small pre B-cell transition. Differentiation blocks at the pro and pre B stage through mutations in B cell regulatory genes, such as PAX5, BTK and BLNK, have been recently demonstrated in acute lymphocytic leukemias. It is plausible that mutations in OTT1, given its position in the tightly regulated process of B cell development, may likewise contribute to pre B-leukemogenesis.

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.

scholarly journals PU.1 is not strictly required for B cell development and its absence induces a B-2 to B-1 cell switch

2005 ◽  
Vol 202 (10) ◽  
pp. 1411-1422 ◽  
Author(s):  
Min Ye ◽  
Olga Ermakova ◽  
Thomas Graf
Keyword(s):  
B Cell ◽  
Target Genes ◽  
Fetal Liver ◽  
Cell Development ◽  
B Cell Development ◽  
Immunoglobulin M ◽  
Cell Phenotype ◽  
B Lineage ◽  
Cell Commitment ◽  
B Lineage Cells

In this paper, we describe the unexpected outgrowth of B lineage cells from PU.1−/− fetal liver cultures. The cells express all early B cell genes tested, including the putative PU.1 target genes IL-7R and EBF but not B220, and can produce immunoglobulin M. However, we observed a delay in the PU.1−/− B cell outgrowth and reduced precursor frequencies, indicating that although PU.1 is not strictly required for B cell commitment, it facilitates B cell development. We also ablated PU.1 in CD19-expressing B lineage cells in vivo, using a Cre-lox approach that allows them to be tracked. PU.1 excision resulted in a shift from B-2 cells to B-1–like cells, which dramatically increased with the age of the mice. Our data indicate that this shift is predominantly caused by a B-2 to B-1 cell reprogramming. Furthermore, we found that B-2 cells express substantially more PU.1 than B-1 cells, which is consistent with the idea that maintenance of the B-2 cell phenotype requires relatively high levels of PU.1, but B-1 cells require little.

scholarly journals Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

2002 ◽  
Vol 9 (2) ◽  
pp. 86-95 ◽  
Author(s):  
Denise A. Kaminski ◽  
John J. Letterio ◽  
Peter D. Burrows
Keyword(s):  
B Cells ◽  
Growth Factor ◽  
B Cell ◽  
Transforming Growth Factor ◽  
Plasma Cells ◽  
Population Analysis ◽  
Cell Development ◽  
B Cell Development

Transforming growth factor β (TGFβ) can inhibit thein vitroproliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1-/-mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1-pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1+pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell developmentin vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.

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