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.

scholarly journals Altered Erythrocytes and a Leaky Block in B-Cell Development in CD24/HSA-Deficient Mice

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1058-1067 ◽  
Author(s):  
P.J. Nielsen ◽  
B. Lorenz ◽  
A.M. Müller ◽  
R.H. Wenger ◽  
F. Brombacher ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
B Cell ◽  
Embryonic Stem ◽  
Cell Development ◽  
B Cell Development ◽  
Surface Glycoprotein ◽  
Malarial Parasite ◽  
Heat Stable Antigen ◽  
Deficient Mice

Abstract The heat stable antigen (HSA, or murine CD24) is a glycosyl phosphatidylinositol-linked surface glycoprotein expressed on immature cells of most, if not all, major hematopoietic lineages, as well as in developing neural and epithelial cells. It has been widely used to stage the maturation of B and T lymphocytes because it is strongly induced and then repressed again during their maturation. Terminally differentiated lymphocytes, as well as most myeloid lineages, are negative for HSA. Erythrocytes are an exception in that they maintain high levels of HSA expression. HSA on naive B cells has been shown to mediate cell-cell adhesion, while HSA on antigen-presenting cells has been shown to mediate a costimulatory signal important for activating T lymphocytes during an immune response. Here, we characterize mice that lack a functional HSA gene, constructed by homologous recombination in embryonic stem cells. While T-cell and myeloid development appears normal, these mice show a leaky block in B-cell development with a reduction in late pre-B and immature B-cell populations in the bone marrow. Nevertheless, peripheral B-cell numbers are normal and no impairment of immune function could be detected in these mice in a variety of immunization and infection models. We also observed that erythrocytes are altered in HSA-deficient mice. They show a higher tendency to aggregate and are more susceptible to hypotonic lysis in vitro. In vivo, the mean half-life of HSA-deficient erythrocytes was reduced. When infected with the malarial parasite Plasmodium chabaudi chabaudi, the levels of parasite-bearing erythrocytes in HSA-deficient mice were also significantly elevated, but the mice were able to clear the infection with kinetics similar to wild-type mice and were immune to a second challenge. Thus, apart from alterations in erythrocytes and a mild block in B-cell development, the regulated expression of HSA appears to be dispensable for the maturation and functioning of those cell lineages that normally express it.

In Vitro Analysis of Murine B-Cell Development

1985 ◽  
Vol 3 (1) ◽  
pp. 213-235 ◽  
Author(s):  
C Whitlock ◽  
K Denis ◽  
D Robertson ◽  
O Witte
Keyword(s):  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Vitro Analysis ◽  
In Vitro Analysis

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.

scholarly journals In Vivo Tungsten Exposure Alters B-Cell Development and Increases DNA Damage in Murine Bone Marrow

2012 ◽  
Vol 131 (2) ◽  
pp. 434-446 ◽  
Author(s):  
Alexander D. R. Kelly ◽  
Maryse Lemaire ◽  
Yoon Kow Young ◽  
Jules H. Eustache ◽  
Cynthia Guilbert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Murine Bone Marrow

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

2018 ◽  
Vol 19 (9) ◽  
pp. 2522 ◽  
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.

scholarly journals A Dual Role for Src Homology 2 Domain–Containing Inositol-5-Phosphatase (Ship) in Immunity

2000 ◽  
Vol 191 (5) ◽  
pp. 781-794 ◽  
Author(s):  
Cheryl D. Helgason ◽  
Christian P. Kalberer ◽  
Jacqueline E. Damen ◽  
Suzanne M. Chappel ◽  
Nicolas Pineault ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Lymphoid Development ◽  
Src Homology 2 ◽  
Src Homology ◽  
Src Homology 2 Domain ◽  
B Lymphoid

In this report, we demonstrate that the Src homology 2 domain–containing inositol-5-phosphatase (SHIP) plays a critical role in regulating both B cell development and responsiveness to antigen stimulation. SHIP−/− mice exhibit a transplantable alteration in B lymphoid development that results in reduced numbers of precursor B (fraction C) and immature B cells in the bone marrow. In vitro, purified SHIP−/− B cells exhibit enhanced proliferation in response to B cell receptor stimulation in both the presence and absence of Fcγ receptor IIB coligation. This enhancement is associated with increased phosphorylation of both mitogen-activated protein kinase and Akt, as well as with increased survival and cell cycling. SHIP−/− mice manifest elevated serum immunoglobulin (Ig) levels and an exaggerated IgG response to the T cell–independent type 2 antigen trinitrophenyl Ficoll. However, only altered B cell development was apparent upon transplantation into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice. The in vitro hyperresponsiveness, together with the in vivo findings, suggests that SHIP regulates B lymphoid development and antigen responsiveness by both intrinsic and extrinsic mechanisms.

scholarly journals In vitro requirement for periostin in B lymphopoiesis

Blood ◽  
2011 ◽  
Vol 117 (14) ◽  
pp. 3770-3779 ◽  
Author(s):  
Basile T. Siewe ◽  
Susan L. Kalis ◽  
Phong T. Le ◽  
Pamela L. Witte ◽  
Sangdun Choi ◽  
...  
Keyword(s):  
B Cell ◽  
Small Interfering Rna ◽  
Matrix Protein ◽  
Cell Development ◽  
B Cell Development ◽  
Extracellular Matrix Protein ◽  
Adult Rabbit ◽  
B Lymphopoiesis ◽  
Interfering Rna

Abstract B lymphopoiesis arrests in rabbits by 4 months of age. To identify molecules that contribute to this arrest, cDNA–representational difference analysis on BM stromal cells from young and adult rabbits showed that expression of Postn that encodes for the extracellular matrix protein periostin dramatically reduced with age. Postn–small interfering RNA OP9 cells lost their capacity to support B-cell development from rabbit or murine BM cells, and reexpression of periostin restored this potential, indicating an in vitro requirement for periostin in B lymphopoiesis. In our system, we determined that periostin deficiency leads to increased cell death and decreased proliferation of B-lineage progenitors. Further, RGD peptide inhibition of periostin/αvβ3 interaction resulted in a marked decrease in B lymphopoiesis in vitro. Microarray analysis of the Postn–small interfering RNA OP9 cells showed decreased expression of key B-lymphopoietic factors, including IL-7 and CXCL12. In vivo, unidentified molecule(s) probably compensate periostin loss because Postn−/− mice had normal numbers of B-cell progenitors in BM. We conclude that the decline in periostin expression in adult rabbit BM does not solely explain the arrest of B lymphopoiesis. However, the interaction of periostin with αvβ3 on lymphoid progenitors probably provides both proliferative and survival signals for cells in the B-cell development pathway.

ABCG2 Is Expressed in a Small Population of Circulating CLL B-Cells Characterized by Expression of Self-Renewal and Early B-Cell Development Genes and Resistance to Therapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3156-3156 ◽  
Author(s):  
Grzegorz S. Nowakowski ◽  
Xiaosheng Wu ◽  
Jennifer L. Abrahamzon ◽  
Renee Tschumper ◽  
Neil E. Kay ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
B Cells ◽  
B Cell ◽  
Residual Disease ◽  
Cell Development ◽  
Small Population ◽  
B Cell Development ◽  
Self Renewal ◽  
Resistance To Therapy

Abstract Background: Normal and tumor stem cells are characterized by high activity of multidrug resistance (MDR) transporters. One of these, ABCG2 (ATP-binding cassette, sub-family G member 2 protein), is an ATP dependent transporter and putative stem cell marker responsible for verapamil sensitive Hoechst efflux. While ABCG2 is known to be expressed in normal and leukemic stem cells, as well as a small population of normal lymphocytes and some B-cell malignancies, its expression in chronic lymphocytic leukemia (CLL) is unknown. It has been postulated that leukemic stem cells due to their quiescent nature and expression of MDR transporters represent a population resistant to therapy and that this residual population is critical for tumor persistence and recurrence. Hypothesis: We hypothesized that ABCG2 is expressed in a small percentage of primary CLL B cells; gene expression profiles of ABCG2 positive versus ABCG2 negative CLL B cells differ in respect to expression of self renewal and lymphoid development genes; the frequency of ABCG2+ CLL B cells increases after treatment in patients responding to therapy. Methods: We analyzed ABCG2 expression by primary CD5+, CD19+ CLL-B cells from untreated CLL patients of all Rai stages by flow cytometry. In a subset of patients we used fluorescence activated cell sorting (FACS) to sort CD19+, CD5+ ABCG2+ and CD19+, CD5+ ABCG2- cells. Gene expression profiling was then performed using the U133 plus 2.0 Affymetrix microarray platform. In a separate cohort of patients treated in a clinical trial of pentostatin, cyclophosphamide and rituximab (PCR), the percentage of ABCG2+, CD19+, CD5+, CD79b dim cells at baseline and then two months after completion of 6 cycles of PCR therapy where patients had minimal residual disease (MRD) was assessed and correlated with clinical response. Results: ABCG2+ CD19+, CD5+ detectable populations were seen in all 20 CLL assessed patients (median percentage 0.6%; range 0.08%–3.8%). There was no difference in percentage of ABCG2+ cells based on Rai stage, IGVH mutational status, Zap70 or CD38 expression. Preliminary analysis of the gene expression profiling of ABCG2 positive versus negative CLL B cells from four randomly selected patients revealed significantly higher expression of genes associated with self-renewal, cell cycle and early B-cell development including: cyclin-dependent kinase inhibitor 1C (CDKN1C, p=0.034), transcription factor 7-like 2 (TCF7L2, involved in WNT pathway regulation, p=0.016), beta-catenin (p=0.034) and pre-B-cell colony enhancing factor 1 (PBEF-1, p=0.037). Flow based assessment of the levels of ABCG2 positive populations at baseline and after therapy with PCR in patients with minimal residual disease showed a dramatic increase in frequency of ABCG2 positive CLL B cells. The percentage of ABCG2+ cells went from a median level of 0.19% (range 0.04%–0.19%) prior to therapy to a median level of 10.93% (range 0.15%–25.12%), p<0.001. In contrast two patients who did not reach MRD (partial responses by NCI-WG criteria) had no significant increase in percentage of ABCG2 positive cells (0.14%; 0.23% and 0.16%; 0.21% prior and after therapy, respectively, p=0.68). Conclusion: Our data indicate that ABCG2 positive CLL B-cells constitute 0.1–3.8% of circulating CLL B-cells in untreated patients. The frequency of ABCG2+ CLL B-cells appears to dramatically increase after therapy in the MRD state; this could be related to their relative resistance to therapy and/or a shift from extravascular compartments post therapy. Since ABCG2 positive CLL B-cells demonstrate expression of early B-cell development and self-renewal genes we believe that that this population could represent a putative self renewing CLL B-cell compartment. Further studies to characterize features of ABCG2 CLL-B –cells in relation to their capacity to be self renewing and resistance to therapy are warranted.

Acute Lymphoblastic Leukemia-Associated PAX5 Mutations Induce Aberrant B Cell Development

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 10-10
Author(s):  
Joy Nakitandwe ◽  
Shann-Ching Chen ◽  
Noel T. Lenny ◽  
Christopher B. Miller ◽  
Xiaoping Su ◽  
...  
Keyword(s):  
Dna Binding ◽  
B Cell ◽  
Fusion Proteins ◽  
Transcriptional Activity ◽  
Lymphoblastic Leukemia ◽  
Cell Development ◽  
B Cell Development ◽  
Dna Binding Domain ◽  
B Cell Differentiation

Abstract Abstract 10 Over 60% of pediatric B progenitor acute lymphoblastic leukemia (ALL) cases contain somatic mutations in genes regulating B cell development, with PAX5 being the most common target of mutation (>32% of cases). The transcription factor PAX5 is required for commitment and maintenance of the B lymphoid lineage. A variety of PAX5 mutations has been identified including, mono-allelic deletions, sequence mutations, internal deletions, frame-shift mutations and translocations. We have previously shown that these PAX5 mutations result in reduced transcriptional activity either as a result of haploinsufficiency or the generation of altered PAX5 isoforms with reduced DNA-binding and/or transcriptional activity. However, the direct effect of the mutations on normal B cell development remains unknown. To address this question, we assessed the ability of a series of PAX5 mutations to rescue normal B cell development in Pax5-/- bone marrow (BM) cells using a murine in vitro culture system. Whole BM or transduced cells were grown in IL-7 producing stromal-supported cultures for two weeks and then assessed for their extent of B cell differentiation using flow cytometry. Under these in vitro conditions, both Pax5+/+ and Pax5+/− BM cells differentiated to a Hardy fraction D pre-B cell stage of differentiation (CD43−/B220+/CD19+/BP1+), with only a slight decrease in the level of expression of BP1 detected in the Pax5+/− cells. By contrast, Pax5-/- cells failed to undergo significant differentiation under these in vitro growth conditions and were arrested at an early pro-B stage of development (CD43+/−/B220+/CD19−/BP1−). To assess the biological activity of the identified PAX5 mutants, we then transduced lineage-depleted BM cells from Pax5+/+, Pax5+/− and Pax5-/- mice with MSCV-based retroviral vectors expressing either wild type (WT) or mutant PAX5 followed by in vitro culture. Three classes of PAX5 mutations were assessed: DNA binding domain mutations (P80R, P34Q, and V26G), an internal deletion mutation (Δe6-8), and translocation-induced PAX5 chimeric genes (PAX5-ETV6, PAX5-FOXP1 and PAX5-ZNF521). As expected, expression of WTPAX5 resulted in full rescue of Pax5-/- cells and induced no significant effects on the ability of Pax5+/+ and +/− cells to differentiate. By contrast, PAX5 DNA-binding domain mutants resulted in only partial rescue of Pax5-/- cells, with P80R inducing B220+/CD19−/BP1−, P34Q producing B220+/CD19+ cells with weak BP1 expression, and V26G yielding CD19+/BP1+ cells with minimally reduced levels of BP1. Similarly, expression of Δe6-8 resulted in partial rescue with the expansion of B220+/CD19+/−/BP1− cells. In stark contrast, expression of the translocation encoded PAX5 fusion proteins failed to induce any evidence of rescue. Moreover, these fusion proteins induced only minimal perturbations in the ability of Pax5+/+ and +/− cells to differentiate, suggesting that these fusion proteins were weak competitive inhibitors of normal Pax5 transcriptional activity under intra-cellular conditions. To further characterize the effects of these PAX5 mutations on B cell differentiation, we next analyzed the gene expression patterns of the resultant cell populations using the Mouse Genome 430 2.0 Arrays (Affymetrix) and compared the profiles to those obtained from purified Hardy fractions from normal murine BM. The expression signatures of the Pax5-/- cells were identical to those for normal Hardy fraction A and shifted to the signature of Hardy fraction C following rescue with WTPAX5. Transduction of Pax5-/- cells with either V26G or P34Q resulted in a near complete rescue with expression signatures similar to those obtained for Hardy fractions B/C. By contrast, transduction with P80R or Δe6-8 yielded a more incomplete rescue with expression profiles that were between Hardy fractions A and B. Interestingly, a number of genes within the B cell receptor signaling pathway were altered in cells rescued by P80R and Δe6-8, including the down regulation of CD19, Btk and Blnk. In summary, our data demonstrate that leukemia-associated PAX5 mutations have a graded effect on the transcriptional network that controls normal B cell development and differentiation. Defining the differential target gene specificity of the various PAX5 mutants should provide valuable insights into the molecular mechanisms through which these genetic lesions contribute to leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IRF-4 functions as a tumor suppressor in early B-cell development

Blood ◽  
2008 ◽  
Vol 112 (9) ◽  
pp. 3798-3806 ◽  
Author(s):  
Jaime Acquaviva ◽  
Xiaoren Chen ◽  
Ruibao Ren
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Cell Development ◽  
B Cell Development ◽  
Lymphoid Progenitors ◽  
B Lymphoid

Interferon regulatory factor-4 (IRF-4) is a hematopoietic cell–restricted transcription factor important for hematopoietic development and immune response regulation. It was also originally identified as the product of a proto-oncogene involved in chromosomal translocations in multiple myeloma. In contrast to its oncogenic function in late stages of B lymphopoiesis, expression of IRF-4 is down-regulated in certain myeloid and early B-lymphoid malignancies. In this study, we found that the IRF-4 protein levels are increased in lymphoblastic cells transformed by the BCR/ABL oncogene in response to BCR/ABL tyrosine kinase inhibitor imatinib. We further found that IRF-4 deficiency enhances BCR/ABL transformation of B-lymphoid progenitors in vitro and accelerates disease progression of BCR/ABL-induced acute B-lymphoblastic leukemia (B-ALL) in mice, whereas forced expression of IRF-4 potently suppresses BCR/ABL transformation of B-lymphoid progenitors in vitro and BCR/ABL-induced B-ALL in vivo. Further analysis showed that IRF-4 inhibits growth of BCR/ABL+ B lymphoblasts primarily through negative regulation of cell-cycle progression. These results demonstrate that IRF-4 functions as tumor suppressor in early B-cell development and may allow elucidation of new molecular pathways significant to the lymphoid leukemogenesis by BCR/ABL. The context dependent roles of IRF-4 in oncogenesis should be an important consideration in developing cancer therapies targeting IRF-4.

Sign in / Sign up

Export Citation Format

Share Document