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

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.

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B Cell Development in the Absence of PU.1.

Blood ◽

10.1182/blood.v104.11.226.226 ◽

2004 ◽

Vol 104 (11) ◽

pp. 226-226 ◽

Cited By ~ 1

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.

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B Cell Lineage-Specific Deletion of Icsbp/IRF8 Reveals Roles for IRF8 in the Regulation of Marginal Zone and Follicular B Cell Development.

Blood ◽

10.1182/blood.v110.11.1334.1334 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1334-1334

Author(s):

Hongsheng Wang ◽

Jianxun Feng ◽

Chang Hoon Lee ◽

Herbert Morse

Keyword(s):

B Cells ◽

B Cell ◽

Marginal Zone ◽

Cell Development ◽

B Cell Development ◽

Conditional Knockout ◽

Myelogenous Leukemia ◽

Exon 2 ◽

B Lineage ◽

B Lineage Cells

Abstract Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a transcription factor that expresses in T cells, B cells and macrophages and plays a role in myeloid development. Targeted deletion of IRF8 in mice (IRF8−/−) induced progressive increase in the numbers of granulocytes in various lymphoid organs and development of a syndrome similar to human chronic myelogenous leukemia. In addition to defective development of macrophages and dendritic cells, B cell development was also impaired in IRF8−/− mice. This includes decreased numbers of early B cells, expanded marginal zone (MZ) B cells and diminished follicular (OF) B2 cells. Because abnormal myeloid cells could alter microenvironment required for normal B cell development, we have generated IRF8 conditional knockout mice to specifically investigate the function of IRF8 in B lineage cells. Mice were engineered to have exon 2, encoding the DNA binding domain of IRF8, flanked by loxP sites (designated IRF8f/+). These mice were then crossed with the CD19Cre strain in which the expression of Cre-recombinase is controlled by the endogenous CD19 locus. Homozygous mice (designated (IRF8f/f x Cre)F1) underwent germline excision of IRF8 in CD19+ B lineage cells. As a result, there was no detectable mRNA and protein of IRF8 in their splenic B cells. Flow cytometry analysis revealed expanded MZ B cells and reduced OF B2 cells in the spleen of (IRF8f/f x Cre)F1 mice. Interestingly, the expression level of CD23 on OF B cells was significantly decreased in (IRF8f/f x Cre)F1 mice, indicating that IRF8 is required for maintaining a normal OF phenotype. In the peritoneum of (IRF8f/f x Cre)F1 mice, while the numbers of B1a and B2 cells were slightly decreased, the number of B1b cells was slightly increased. Furthermore, BXH2 mice carrying a mutation (C915T) in the Icsbp1 gene exhibited similar expansion of MZ B cells and low expression of CD23 in OF B cells. Taken together, these analyses indicate that IRF8 is required for development of normal MZ and B2 cells.

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Role of Microbes in B-Cell Lymphopoiesis and Early Ig Repertoire Development

Blood ◽

10.1182/blood.v124.21.sci-47.sci-47 ◽

2014 ◽

Vol 124 (21) ◽

pp. SCI-47-SCI-47

Author(s):

Duane R. Wesemann ◽

Akritee Shrestha ◽

Jennifer Magee ◽

Yuezhou Chen ◽

Jared Silver ◽

...

Keyword(s):

Bone Marrow ◽

B Cell ◽

Intestinal Mucosa ◽

Cell Development ◽

B Cell Development ◽

Recombination Reaction ◽

Small Intestinal Mucosa ◽

Receptor Editing ◽

B Lineage ◽

B Lineage Cells

Abstract Progenitor (pro-) and precursor (pre-) B cells express the recombination-activating gene (RAG1/RAG2) endonuclease, which initiates the V(D)J recombination reaction that assembles Immunoglobulin heavy (IgH) and light (IgL) chain variable region exons from germline gene segments. Expression of productively assembled IgH μ chains and IgL (Igκ or Igλ) chains generates IgM molecules that form the B-cell antigen binding receptor (BCR) with a diverse primary repertoire of binding specificities. As the newly assembled and expressed IgM interacts with local antigens, RAG expression can continue, allowing continued Igκ V(D)J recombination that can replace the previously assembled VκJκ exon with one that generates a new specificity. This "receptor editing" process provides an antigen-mediated regulatory checkpoint that helps shape the pre-immune BCR repertoire. As the principal location of early B-cell development, the bone marrow antigenic environment is the main force affecting receptor editing. However, the extent to which self-antigens outside of the bone marrow — as well as environmental antigens — may shape the primary Ig repertoire is not known. Here we show that early B-cell development takes place in peripheral sites, including the small intestinal mucosa, in young mice. Cells in these sites co-express RAG with cytoplasmic Igκ and Igμ, indicating active receptor editing; and, correspondingly, they have significant differences in Vκ usage compared to RAG2-expressing bone marrow B-lineage cells and other RAG2-expressing B-lineage cells from other peripheral sites. In addition, the luminal microbial environment appears to also affect Igλ/Igκ ratios. We conclude that early B-cell development can occur in the periphery, including the intestinal mucosa, where commensal microbes may influence BCR diversification and gut pre-immune Ig repertoires. Disclosures No relevant conflicts of interest to declare.

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IKKα-mediated signaling circuitry regulates early B lymphopoiesis during hematopoiesis

Blood ◽

10.1182/blood-2012-01-401547 ◽

2012 ◽

Vol 119 (23) ◽

pp. 5467-5477 ◽

Cited By ~ 16

Author(s):

Mumtaz Yaseen Balkhi ◽

Jami Willette-Brown ◽

Feng Zhu ◽

Zhisong Chen ◽

Shuang Liu ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Fetal Liver ◽

Cell Development ◽

B Cell Development ◽

Erythroid Cell ◽

Early Event ◽

Lineage Differentiation ◽

Iκb Kinase ◽

Cell Commitment

Abstract Multiple transcription factors regulate B-cell commitment, which is coordinated with myeloid-erythroid lineage differentiation. NF-κB has long been speculated to regulate early B-cell development; however, this issue remains controversial. IκB kinase-α (IKKα) is required for splenic B-cell maturation but not for BM B-cell development. In the present study, we unexpectedly found defective BM B-cell development and increased myeloid-erythroid lineages in kinase-dead IKKα (KA/KA) knock-in mice. Markedly increased cytosolic p100, an NF-κB2–inhibitory form, and reduced nuclear NF-κB p65, RelB, p50, and p52, and IKKα were observed in KA/KA splenic and BM B cells. Several B- and myeloid-erythroid–cell regulators, including Pax5, were deregulated in KA/KA BM B cells. Using fetal liver and BM congenic transplantations and deleting IKKα from early hematopoietic cells in mice, this defect was identified as being B cell–intrinsic and an early event during hematopoiesis. Reintroducing IKKα, Pax5, or combined NF-κB molecules promoted B-cell development but repressed myeloid-erythroid cell differentiation in KA/KA BM B cells. The results of the present study demonstrate that IKKα regulates B-lineage commitment via combined canonical and noncanonical NF-κB transcriptional activities to target Pax5 expression during hematopoiesis.

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

Journal of Experimental Medicine ◽

10.1084/jem.20160852 ◽

2017 ◽

Vol 214 (7) ◽

pp. 2041-2058 ◽

Cited By ~ 9

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.

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Fates of human B-cell precursors

Blood ◽

10.1182/blood.v96.1.9 ◽

2000 ◽

Vol 96 (1) ◽

pp. 9-23 ◽

Cited By ~ 160

Author(s):

Tucker W. LeBien

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

Cell Development ◽

B Cell Development ◽

B Lineage ◽

Human B Cell ◽

B Lineage Cells

Abstract Development of mammalian B-lineage cells is characterized by progression through a series of checkpoints defined primarily by rearrangement and expression of immunoglobulin genes. Progression through these checkpoints is also influenced by stromal cells in the microenvironment of the primary tissues wherein B-cell development occurs, ie, fetal liver and bone marrow and adult bone marrow. This review focuses on the developmental biology of human bone marrow B-lineage cells, including perturbations that contribute to the origin and evolution of B-lineage acute lymphoblastic leukemia and primary immunodeficiency diseases characterized by agammaglobulinemia. Recently described in vitro and in vivo models that support development and expansion of human B-lineage cells through multiple checkpoints provide new tools for identifying the bone marrow stromal cell–derived molecules necessary for survival and proliferation. Mutations in genes encoding subunits of the pre-B cell receptor and molecules involved in pre-B cell receptor signaling culminate in X-linked and non–X-linked agammaglobulinemia. A cardinal feature of these immunodeficiencies is an apparent apoptotic sensitivity of B-lineage cells at the pro-B to pre-B transition. On the other end of the spectrum is the apoptotic resistance that accompanies the development of B-lineage acute lymphoblastic leukemia, potentially a reflection of genetic abnormalities that subvert normal apoptotic programs. The triad of laboratory models that mimic the bone marrow microenvironment, immunodeficiency diseases with specific defects in B-cell development, and B-lineage acute lymphoblastic leukemia can now be integrated to deepen our understanding of human B-cell development.

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B-cell development fails in the absence of the Pbx1 proto-oncogene

Blood ◽

10.1182/blood-2006-10-054213 ◽

2007 ◽

Vol 109 (10) ◽

pp. 4191-4199 ◽

Cited By ~ 38

Author(s):

Mrinmoy Sanyal ◽

James W. Tung ◽

Holger Karsunky ◽

Hong Zeng ◽

Licia Selleri ◽

...

Keyword(s):

B Cell ◽

De Novo ◽

Fetal Liver ◽

Es Cells ◽

Critical Role ◽

Embryonic Stem ◽

Cell Development ◽

B Cell Development ◽

Hematopoietic Stem ◽

Cell Commitment

AbstractPbx1, a homeodomain transcription factor that was originally identified as the product of a proto-oncogene in acute pre-B–cell leukemia, is a global regulator of embryonic development. However, embryonic lethality in its absence has prevented an assessment of its role in B-cell development. Here, using Rag1-deficient blastocyst complementation assays, we demonstrate that Pbx1 null embryonic stem (ES) cells fail to generate common lymphoid progenitors (CLPs) resulting in a complete lack of B and NK cells, and a partial impairment of T-cell development in chimeric mice. A critical role for Pbx1 was confirmed by rescue of B-cell development from CLPs following restoration of its expression in Pbx1-deficient ES cells. In adoptive transfer experiments, B-cell development from Pbx1-deficient fetal liver cells was also severely compromised, but not erased, since transient B lymphopoiesis was detected in Rag-deficient recipients. Conditional inactivation of Pbx1 in pro-B (CD19+) cells and thereafter revealed that Pbx1 is not necessary for B-cell development to proceed from the pro-B–cell stage. Thus, Pbx1 critically functions at a stage between hematopoietic stem cell development and B-cell commitment and, therefore, is one of the earliest-acting transcription factors that regulate de novo B-lineage lymphopoiesis.

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Gradient of E2A Activity in B-Cell Development

Molecular and Cellular Biology ◽

10.1128/mcb.22.3.886-900.2002 ◽

2002 ◽

Vol 22 (3) ◽

pp. 886-900 ◽

Cited By ~ 61

Author(s):

Sabine Herblot ◽

Peter D. Aplan ◽

Trang Hoang

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Gene Dosage ◽

Lymphoblastic Leukemia ◽

Control Cell ◽

Cell Development ◽

B Cell Development ◽

Immunoglobulin M ◽

B Lineage

ABSTRACT The E2A locus is a frequent target of chromosomal translocations in B-cell acute lymphoblastic leukemia (B-ALL). E2A encodes two products, E12 and E47, that are part of the basic helix-loop-helix (bHLH) family of transcription factors and are central in B lineage differentiation. E2A haplo-insufficiency hinders progression through three major checkpoints in B-cell development: commitment into the B lineage, at the pro-B to pre-B transition, and in the induction of immunoglobulin M (IgM) expression required for a functional BCR. These observations underscore the importance of E2A gene dosage in B-cell development. Here we show that a higher proportion of pro-B cells in E2A+/− mice is in the cell cycle compared to that in wild-type littermates. This increase correlates with lower p21waf/cip1 levels, indicating that E2A has an antiproliferative function in B-cell progenitors. Ectopic expression in the B lineage of SCL/Tal1, a tissue-specific bHLH factor that inhibits E2A function, blocks commitment into the B lineage without affecting progression through later stages of differentiation. Furthermore, ectopic SCL expression exacerbates E2A haplo-insufficiency in B-cell differentiation, indicating that SCL genetically interacts with E2A. Taken together, these observations provide evidence for a gradient of E2A activity that increases from the pre-pro-B to the pre-B stage and suggest a model in which low levels of E2A (as in pro-B cells) are sufficient to control cell growth, while high levels (in pre-B cells) are required for cell differentiation. The antiproliferative function of E2A further suggests that in B-ALL associated with t(1;19) and t(17;19), the disruption of one E2A allele contributes to leukemogenesis, in addition to other anomalies induced by E2A fusion proteins.

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Fates of human B-cell precursors

Blood ◽

10.1182/blood.v96.1.9.013k27_9_23 ◽

2000 ◽

Vol 96 (1) ◽

pp. 9-23 ◽

Cited By ~ 8

Author(s):

Tucker W. LeBien

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

Cell Development ◽

B Cell Development ◽

B Lineage ◽

Human B Cell ◽

B Lineage Cells

Development of mammalian B-lineage cells is characterized by progression through a series of checkpoints defined primarily by rearrangement and expression of immunoglobulin genes. Progression through these checkpoints is also influenced by stromal cells in the microenvironment of the primary tissues wherein B-cell development occurs, ie, fetal liver and bone marrow and adult bone marrow. This review focuses on the developmental biology of human bone marrow B-lineage cells, including perturbations that contribute to the origin and evolution of B-lineage acute lymphoblastic leukemia and primary immunodeficiency diseases characterized by agammaglobulinemia. Recently described in vitro and in vivo models that support development and expansion of human B-lineage cells through multiple checkpoints provide new tools for identifying the bone marrow stromal cell–derived molecules necessary for survival and proliferation. Mutations in genes encoding subunits of the pre-B cell receptor and molecules involved in pre-B cell receptor signaling culminate in X-linked and non–X-linked agammaglobulinemia. A cardinal feature of these immunodeficiencies is an apparent apoptotic sensitivity of B-lineage cells at the pro-B to pre-B transition. On the other end of the spectrum is the apoptotic resistance that accompanies the development of B-lineage acute lymphoblastic leukemia, potentially a reflection of genetic abnormalities that subvert normal apoptotic programs. The triad of laboratory models that mimic the bone marrow microenvironment, immunodeficiency diseases with specific defects in B-cell development, and B-lineage acute lymphoblastic leukemia can now be integrated to deepen our understanding of human B-cell development.

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One synchronous wave of B cell development in mouse fetal liver changes at day 16 of gestation from dependence to independence of a stromal cell environment.

Journal of Experimental Medicine ◽

10.1084/jem.170.6.1973 ◽

1989 ◽

Vol 170 (6) ◽

pp. 1973-1986 ◽

Cited By ~ 48

Author(s):

A Strasser ◽

A Rolink ◽

F Melchers

Keyword(s):

B Cell ◽

Stromal Cells ◽

Stromal Cell ◽

Fetal Liver ◽

Cell Development ◽

B Cell Development ◽

Specific Gene ◽

Sudden Increase ◽

B Lineage ◽

Liver Changes

Precursor cells of the B lineage can be enriched from mouse fetal liver by FACS with the aid of the pre-B cell-specific mAb G-5-2. The cells are concomitantly enriched for cells expressing the pre-B cell-specific gene lambda 5, and for cells developing to LPS-reactive mature B cells. The enriched purified precursors are not influenced by rIL-2 through -7, alone or in combination, to develop to mitogen-reactive, sIg+ cells. Marginal proliferation of the precursors is observed in response to IL-3 plus -4, and IL-6 plus -7, and this does not change in the presence of stromal cells. Development to mitogen-reactive, sIg+ cells is dependent on interactions with embryonic stromal cells from fetal liver. Two mAbs raised against the stromal cells inhibit this development. Two phases of precursor cell development can be distinguished in fetal liver. Between days 13 and 15 of gestation, it is dependent on stromal cell interactions, thereafter, from days 16 to 19, it is independent. A sudden increase in the number of mitogen-reactive, sIg+ B lineage cells occurs within 24 h between days 16 and 17. All these results indicate that B cell development occurs in one wave with synchronous steps of changes from a mitogen-insensitive, sIg-, stromal cell dependent to a mitogen-reactive, sIg+, stromal cell-independent B lineage line.

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