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

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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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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The development of functional B lymphocytes in conditional PU.1 knock-out mice

Blood ◽

10.1182/blood-2005-01-0283 ◽

2005 ◽

Vol 106 (6) ◽

pp. 2083-2090 ◽

Cited By ~ 55

Author(s):

Matthew Polli ◽

Aleksandar Dakic ◽

Amanda Light ◽

Li Wu ◽

David M. Tarlinton ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Fetal Liver ◽

Cell Receptor ◽

Interleukin 4 ◽

Knock Out ◽

B Lineage ◽

And Function

Abstract An abundance of research has entrenched the view that the Ets domain containing transcription factor PU.1 is fundamental to the development and function of B lymphocytes. In this study, we have made use of a conditional PU.1 allele to test this notion. Complete deletion of PU.1 resulted in the loss of B cells and all other lineage-positive cells in the fetal liver and death between E18.5 and birth; however, specific deletion of PU.1 in the B lineage had no effect on B-cell development. Furthermore, deletion of PU.1 in B cells did not compromise their ability to establish and maintain an immune response. An increased level of apoptosis was observed in vitro upon B-cell receptor (BCR) cross-linking; however, this was partially rescued by interleukin-4 (IL-4). These findings suggest that PU.1 is not essential for the development of functional B lymphocytes beyond the pre-B stage. (Blood. 2005;106:2083-2090)

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

Journal of Experimental Medicine ◽

10.1084/jem.20020784 ◽

2002 ◽

Vol 196 (5) ◽

pp. 705-711 ◽

Cited By ~ 141

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.

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

Journal of Experimental Medicine ◽

10.1084/jem.193.4.417 ◽

2001 ◽

Vol 193 (4) ◽

pp. 417-426 ◽

Cited By ~ 147

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.

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

Blood ◽

10.1182/blood.v106.11.1287.1287 ◽

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.

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

Blood ◽

10.1182/blood.v112.11.706.706 ◽

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.

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Single Cell Trajectory Detection Orders Hallmarks of Early Human B Cell Development

Blood ◽

10.1182/blood.v120.21.1044.1044 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1044-1044 ◽

Cited By ~ 3

Author(s):

Kara L. Davis ◽

Sean C Bendall ◽

El-ad D. Amir ◽

Erin F. Simonds ◽

Astraea Jager ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Single Cell ◽

Developmental Process ◽

Cell Receptor ◽

Cell Development ◽

B Cell Development ◽

Hematopoietic Stem ◽

Human B Cell

Abstract Abstract 1044 Background: Normal B cell development in the bone marrow (BM) is a seemingly well-understood, progressive process and thus represents a suitable test system in which to apply an algorithmic approach to modeling cellular differentiation. In humans, hematopoietic stem cells form lymphoid progenitor cells that develop into pro- then pre- B cells and finally those cells that escape negative selection become immature B cells that leave the BM for the peripheral immune organs. Flow cytometry can track these stages using the expression of immunophenotypic cell surface markers, including those for progenitors (CD34, CD38), early B cell populations (CD10), as well as those of more mature B cells (CD20, IgM). Expression of the B cell transcription factor PAX5, and immune diversity conferring enzymes terminal deoxynucleotidyl transferase (TdT) and recombination-activating gene (RAG) can also be tracked at the single cell level. Regulatory signaling by factors in the BM orchestrates critical checkpoints in the B cell developmental program, such as Interleukin (IL)-7-mediated STAT5 phosphorylation and signaling downstream the preB cell receptor/B cell receptor (BCR) (p-BLNK, p-Syk, p-PLCγ2, p-Erk). Successful coordination of these signals with immunoglobulin gene rearrangement results in a burst of proliferative expansion prior to maturation/exit to the periphery. Failure of any one of these processes results in B cell deletion while certain dysregulations driven by oncogenic processes can result in malignancy. While much of this core understanding has been founded in murine models, the rarity of early B cell progenitors and lack of genetic tools has complicated definition of B cell development in humans. Using 42 parameter mass cytometry in combination with a novel single-cell trajectory finding algorithm, we have now laid a human B cell developmental process in primary human BM to an unprecedented level of detail, mapping out the expression pattern of virtually all relevant B cell immunophenotypic markers as well as intracellular enzyme, transcription factor and regulatory modification simultaneously, at the single cell level. Methods: The mononuclear cell fraction of multiple healthy human marrows was characterized by simultaneously analyzing 42 antibody parameters with mass cytometry targeting a multitude of phenotypic markers, intracellular signaling molecules, hallmarks of cell cycle and apoptosis all in the context of in vitro perturbations relevant to B cell development (including IL-7 and BCR crosslinking). The resulting multidimensional data was modeled using a novel, scalable, robust graph-based trajectory algorithm that iteratively refines a solution trajectory using random landmarks to reduce variability. Populations of interest were prospectively isolated and a novel qPCR assay was created to quantitate immunoglobulin heavy chain (IgH) rearrangement in genomic DNA. Results/Conclusion: Modeling of the resulting data was undertaken using this algorithm (termed Wanderlust) that devised and ordered cellular relationships based on the average phenotypic progression from our defined starting point, in this case, CD34+CD38- hematopoietic stem cells, in order to calculate a developmental trajectory. The predicted trajectory was then used to inform a traditional 'gating' analysis of the data and provide a higher resolution view of human B cell development than previously published. It both confirmed established steps in human B cell progression, and importantly, revealed new populations of early B cell progenitors based on expression of CD34, CD38, CD24 and TdT. These populations were corroborated to be of B-lineage and ordered as predicted based on the progressive rearrangement of the IgH locus by qPCR of extracted genomic DNA. We aligned previously unregistered key developmental checkpoints such as STAT5 activation in response to IL-7 and proliferation in response preBCR expression with traditional immunophenotypic cell populations. While predicted in silico, and then molecularly verified and staged in vitro, these regulatory events all lay within discrete cell subsets that can now be demarcated using conventional cytometric methods. Together, this provides a backbone on which to further examine both healthy regulatory events as well as the corruption of this developmental process such as in malignant or immunodeficient states. Disclosures: No relevant conflicts of interest to declare.

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

Developmental Immunology ◽

10.1080/1044667031000088057 ◽

2002 ◽

Vol 9 (2) ◽

pp. 86-95 ◽

Cited By ~ 8

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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Identification of an Essential Cytoplasmic Region of Interleukin-7 Receptor α Subunit in B-Cell Development

International Journal of Molecular Sciences ◽

10.3390/ijms19092522 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2522 ◽

Cited By ~ 2

Author(s):

Hirotake Kasai ◽

Taku Kuwabara ◽

Yukihide Matsui ◽

Koichi Nakajima ◽

Motonari Kondo

Keyword(s):

B Cells ◽

B Cell ◽

Myeloid Cell ◽

Cell Development ◽

B Cell Development ◽

Α Subunit ◽

Interleukin 7 ◽

In Cells

Interleukin-7 (IL-7) is essential for lymphocyte development. To identify the functional subdomains in the cytoplasmic tail of the IL-7 receptor (IL-7R) α chain, here, we constructed a series of IL-7Rα deletion mutants. We found that IL-7Rα-deficient hematopoietic progenitor cells (HPCs) gave rise to B cells both in vitro and in vivo when a wild-type (WT) IL-7Rα chain was introduced; however, no B cells were observed under the same conditions from IL-7Rα-deficient HPCs with introduction of the exogenous IL-7Rα subunit, which lacked the amino acid region at positions 414–441 (d414–441 mutant). Signal transducer and activator of transcription 5 (STAT5) was phosphorylated in cells with the d414–441 mutant, similar to that in WT cells, in response to IL-7 stimulation. In contrast, more truncated STAT5 (tSTAT5) was generated in cells with the d414–441 mutant than in WT cells. Additionally, the introduction of exogenous tSTAT5 blocked B lymphopoiesis but not myeloid cell development from WT HPCs in vivo. These results suggested that amino acids 414–441 in the IL-7Rα chain formed a critical subdomain necessary for the supportive roles of IL-7 in B-cell development.

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Essential Immunoregulatory Role for BCAP in B Cell Development and Function

Journal of Experimental Medicine ◽

10.1084/jem.20011751 ◽

2002 ◽

Vol 195 (5) ◽

pp. 535-545 ◽

Cited By ~ 89

Author(s):

Tetsuo Yamazaki ◽

Kiyoshi Takeda ◽

Kumiko Gotoh ◽

Hiroshi Takeshima ◽

Shizuo Akira ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Receptor ◽

Cell Development ◽

B Cell Development ◽

Humoral Immune ◽

Humoral Immune Responses ◽

Phosphoinositide 3 Kinase ◽

And Function ◽

B Cell Deficiency

BCAP was recently cloned as a binding molecule to phosphoinositide 3-kinase (PI3K). To investigate the role of BCAP, mutant mice deficient in BCAP were generated. While BCAP-deficient mice are viable, they have decreased numbers of mature B cells and B1 B cell deficiency. The mice produce lower titers of serum immunoglobulin (Ig)M and IgG3, and mount attenuated responses to T cell–independent type II antigen. Upon B cell receptor cross-linking, BCAP-deficient B cells exhibit reduced Ca2+ mobilization and poor proliferative responses. These findings demonstrate that BCAP plays a pivotal immunoregulatory role in B cell development and humoral immune responses.

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