scholarly journals Role of mTORC1 and mTORC2 during Chronic Lymphocytic Leukaemia (CLL) Initiation and Progression

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3123-3123
Author(s):  
Natasha Malik ◽  
Karen Dunn ◽  
Owen Sansom ◽  
Alison M Michie
Keyword(s):  
B Cell ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Cell Lineage ◽  
Cell Development ◽  
B Cell Development ◽  
Cell Cycling ◽  
And Migration

Abstract Mechanistic target of rapamycin (mTOR) functions within a complex signalling cascade, through its activity in two unique complexes mTORC1 and mTORC2, to promote a multitude of different cellular functions including autophagy, protein synthesis and survival. The exact role of these complexes during leukaemia initiation/maintenance remains to be elucidated. Here, using transgenic knockout (KO) mouse models, we determine the individual roles of mTORC1 (targeting raptor) and mTORC2 (targeting rictor) in normal haemopoiesis and in CLL initiation/maintenance. Our results demonstrate that mice carrying a targeted KO of raptor at the haemopoietic stem cell (HSC) stage (Vav-cre+Raptorfl/fl ) do not survive post birth. This is due to anaemia resulting from a significant decrease in Ter119+ population, a significant decrease in KLF1 and KLF2 gene expression, and a significant increase in the megakaryocyte-erythroid population (MEP), suggesting a block at the MEP stage in Vav-cre+Raptorfl/fl foetal liver. While mTORC1 plays a fundamental role in RBC development, we show that mTORC2 plays a role in RBC regulation, as Rictor-deficient HSPCs exhibit an increase in RBC colony formation ex vivo. Conditional KO (cKO) of Raptor (Mx1-cre+Raptorfl/fl) in adult mice results in splenomegaly accompanied by increased spleen organ cellularity. There is a significant decrease in the B cell lineage, with a block in B cell development at the Lin-Sca-1+CD117+ (LSK) stage in the BM. mTORC2, on the other hand regulates late B cell maintenance as indicated by a significant decrease in transitional B cells (T1/T2), marginal zone progenitor (MZP), and follicular 1 (Fol1) cells in Vav-cre+Rictorfl/fl mice compared to controls. To address the role of mTORC1 and mTORC2 in CLL initiation/maintenance in vitro, BM-derived haemopoietic progenitors isolated from control (Cre-), Raptor-deficient (Mx1-cre+Raptorfl/fl) or Rictor-deficient (Vav-cre+Rictorfl/fl) mice were retrovirally-transduced with a kinase dead PKCα (PKCα-KR) construct to induce an aggressive CLL-like disease. Raptor-deficient BM progenitors exhibited reduced proliferation and failed to generate a CLL-like disease, due to a block in B cell lineage commitment. However, there was an increase in cell cycling and migration in PKCα-KR CLL-like cells with Rictor- deficiency suggesting a role of mTORC2 in disease maintenance. To determine a role for mTORC1 in disease maintenance in vivo, NSG mice were transplanted with PKCα-KR-transduced BM-isolated from either Mx1-cre-Raptorfl/fl or Mx1-cre+Raptorfl/fl. Once disease was established in vivo, cKO was induced and disease load and progression was monitored. Our data indicate a significant decrease in disease load with Raptor cKO, together with a trend towards increased survival. Ongoing experiments with Mx1-cre+Rictorfl/fl mice will give us an insight into the role of mTORC2 in CLL. Taken together, mTORC1 plays an essential role in haemopoiesis, with Raptor-deficiency causing a block in RBC and B cell development at the MEP and LSK stage respectively. In comparison, Rictor-deficiency regulates later B cell lineages and promotes RBC colony formation, potentially through mTORC1 activation. Importantly, CLL-like cells lacking mTORC2 have increased cell cycling and migration whereas mTORC1 deficiency causes a decrease in disease load. Therefore, mTORC1 and mTORC2 play complementary roles in haemopoietic development and leukaemia initiation/progression. These studies provide a strong foundation for further studies testing clinical mTOR inhibitors for CLL in our models. Disclosures No relevant conflicts of interest to declare.

The Proto-Oncogene LRF Is Essential for Normal Mature B Cell Development and Germinal Center Formation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1788-1788
Author(s):  
Nagisa Sakurai ◽  
Manami Maeda ◽  
Sung-UK Lee ◽  
Julie Teruya-Feldstein ◽  
Takahiro Maeda
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Cell Stage ◽  
Transitional B Cells ◽  
Notch Pathways ◽  
Secondary Lymphoid Organs

Abstract LRF (Leukemia/Lymphoma Related Factor, also known as Pokemon, FBI-1, OCZF and ZBTB7a) was originally identified as an interaction partner of the oncoprotein BCL6. LRF can act as a proto-oncogene by repressing the tumor suppressor ARF and cooperates with BCL6 in MEF (mouse embryonic fibroblasts) immortalization. It is highly expressed in human Non-Hodgkin Lymphoma (NHL) cases, in the pathogenesis of which BCL6 is known to be involved (Maeda et al. Nature 2005). Inducible inactivation of the LRF gene in mouse Hematopoietic Stem Cells (HSCs) results in complete block of early B cell development at the HSC/progenitor stages and concomitant development of double positive (DP) T cells in the bone marrow (BM) (Maeda et al. Science 2007). While these findings clearly illustrate key roles of LRF in normal and malignant B cell development, it is not fully identified as to which B cell stages LRF is required during normal B cell development. To elucidate the role of LRF in B cells in vivo, we established and characterized B cell-specific LRF conditional knockout (KO) mice. We took advantage of mb-1 Cre knock-in mice, in which Cre expression is restricted to the B cells after the ProB cell stage. B cell compartments in the BM (PreProB, ProB, PreB and immatureB) are grossly normal in LRFF/ Fmb1-Cre mice. The LRF gene was efficiently eliminated in BM CD19+ B cells revealed by quantitative real-time PCR assay. Furthermore, LRF protein was not detected in purified CD19+ B cells, but seen in CD19-non-B cells, confirming the specific inactivation of the LRF gene in B cells. Thus, despite its critical role at the HSC/progenitor stages, LRF was found to be dispensable for the survival of normal BM B cells. These findings are consistent with the fact that GSI treatment (Maeda et al. Science 2007) or Notch1 loss (Lee and Maeda, unpublished) rescues the defects in early B cell development seen in LRFF/FMx1-Cre+ mice. Notch signaling is necessary for the transitional B cells to commit to the marginal zone B cells (MZB). Inactivation of the component of the Notch pathways in mice results in no MZB development. On the contrary, deletion of the MINT/SHARP gene, a suppressor of Notch signaling, leads to increase of MZB cells and concomitant reduction of follicular B (FOB) cells, indicating that Notch induces MZB cell fate at the transitional B cell stage. Given that LRF is a potent Notch suppressor at the HSC/progenitor stages, we hypothesized that LRF opposes Notch pathway in mature B cells as well. To test this hypothesis, we characterized mature B cell development in LRFF/Fmb1-Cre mice. While transitional B cells were largely unaffected in LRFF/Fmb1-Cre mice, we observed a slight but statistically significant reduction of follicular (FO) B cells (B220+CD19+AA4.1-CD1d-CD23+) and concomitant increase of MZB cells (B220+CD19+AA4.1-CD1d+CD23-) as seen in MINT/SHARP knockout mice. Thus, LRF may also oppose Notch pathways at the branching point for the FOB vs. MZB fate decision. Finally, to determine the role of LRF in Germinal Center (GC) formation in vivo, we characterized secondary lymphoid organs of LRFF/Fmb1-Cre mice after antigen stimulation. Both spleen and Peyer’s Patches were analyzed two weeks after immunization with Chicken Gamma Globulin (NP-CGG). While a GC reaction was robustly induced in control mice upon immunization, GC formation was significantly impaired in LRFF/Fmb1-Cre mice as revealed by immuno-histochemical analysis (IHC) and FACS. Only few GC cells (B220+CD19+FAS+CD38-PNA+) were observed in spleens, and the absolute numbers of GC cells were drastically reduced in LRFF/Fmb1-Cre mice. Residual LRF-deficient GC B cells were mostly negative for CXCR4, which is predominantly expressed in proliferating centroblasts within GCs, suggesting that LRF-deficient GC B cells may have defects in cellular proliferation in response to antigen stimuli. Our data indicates that LRF plays key roles in mature B cell development in the secondary lymphoid organs, but dispensable for the maintenance of early BM B cells.

scholarly journals PON2 Exemplifies a Unique Dependency of B Cell Lineage ALL Cells on Detoxifying Lactonases

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 882-882
Author(s):  
Gang Xiao ◽  
Chao Hong ◽  
Huimin Geng ◽  
Markus Muschen
Keyword(s):  
B Cell ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Cell Lineage ◽  
Specific Treatment ◽  
Cell Development ◽  
B Cell Development ◽  
Mrna Levels ◽  
Expression Levels ◽  
B Lineage

Abstract Background and significance : Paraoxonase 2 (PON2) is a member of mammalian detoxifying enzymes that are located to the mitochondrial membrane, hydrolyze lactone metabolites and interact with coenzyme Q10 to diminish oxidative stress. While PON2 is highly expressed in the CNS and multiple fetal tissues, expression levels in normal hematopoietic cells are low. We began to study the function of PON2 in B cell lineage ALL, because microarray analyses suggested high mRNA levels of PON2 in B cell lineage ALL cells. In addition, PON2 is used as a diagnostic marker on a 15 gene diagnostic LDA panel (e.g. in AALL1131; NCT02883049) that is used for the identification of Ph-like ALL, a subgroup with particularly poor outcome and specific treatment requirements. Results: Analyzing data from pediatric and adult clinical trials, we found that greater than median PON2 mRNA levels at the time of diagnosis predicted poor clinical outcomes for both children (COG P9906; n=207; P=1.09e-05) and adults (ECOG; n=215; P=0.003) with B-lineage ALL. Studying expression levels of PON2 by quantitative RT-PCR and Western blot in normal bone marrow B cell precursors from healthy donors and patient-derived pre-B ALL cells including Ph+ and Ph -like ALL, we found 3-10-fold increased mRNA and protein levels of PON2 throughout multiple pre-B ALL samples. To elucidate potential functions of PON2 in normal B cells and B-lineage ALL, we studied normal B cell development and ALL-models in Pon2-/- mice. While B cell development was unperturbed in Pon2 -deficient mice, deletion of Pon2 had profound effects on both BCR-ABL1- and N RASG12D-driven leukemogenesis. Compared to wildtype, Pon2-/- pre-B ALL cells failed to form colonies in semisolid agar. Pon2-/-ALL cells were arrested in G0/G1 phase and expressed 2-5-fold increased levels of Arf and p21, compared to Pon2+/+ ALL cells. Strikingly, >50% of Pon2-/-ALL cells spontaneously underwent cellular senescence, as shown by b-galactosidase staining in conjunction with increased Arf expression levels. These in vitro findings suggest an important role of PON2 in B-lymphoid leukemogenesis, which was confirmed in transplant experiments based on BCR-ABL1 and N RASG12D ALL models. While Pon2 -deficiency substantially prolonged survival of recipient mice of BCR-ABL1 ALL cells (P=0.0001), mice transplanted with Pon2 -deficient N RASG12D ALL cells survived for indefinite periods of time. Targeting PON2 expression in relapse ALL: Given that PON2 is expressed at very high levels in relapse ALL samples, we tested the concept of targeting PON2 lactonase activity in a prodrug-approach. While PON2 activity typically results in detoxification of lactone-metabolites, lactone-hydrolysis of the N-(3-oxododecanoyl)-homoserine lactone (3OC12) prodrug results in cytotoxic byproducts (Guoping et al., 2016). We therefore tested the therapeutic potential of 3OC12. For genetic validation, we treated wildtype and Pon2-/- ALL cells with 3OC12 and found strong cytotoxic effects in wildtype but not Pon2-/- ALL cells. Likewise, inducible overexpression of Pon2 in patient-derived pre-B ALL cells exacerbated toxicity of 3OC12 compared to empty vector controls. Likewise, CRISPR-Cas9 mediated ablation of PON2 in human ALL PDX reversed sensitivity of the pre-B ALL cells to 3OC12. Conclusion: Here we describe the previously unknown function of the detoxifying PON2 lactonase as an essential prerequisite for pre-B cell transformation and leukemogenesis. PON2 expression is specific for leukemia cells, an outcome predictor for patients with pre-B ALL and a biomarker of Ph-like ALL. While PON2 protects ALL cells and enables malignant growth, we demonstrate that its lactonase-activity can be leveraged for pharmacological targeting as exemplified by the lactone-prodrug 3OC12. Disclosures No relevant conflicts of interest to declare.

scholarly journals A critical role of Rap1b in B-cell trafficking and marginal zone B-cell development

Blood ◽  
2008 ◽  
Vol 111 (9) ◽  
pp. 4627-4636 ◽  
Author(s):  
Yuhong Chen ◽  
Mei Yu ◽  
Andrew Podd ◽  
Renren Wen ◽  
Magdalena Chrzanowska-Wodnicka ◽  
...  
Keyword(s):  
B Cells ◽  
Lymph Nodes ◽  
B Cell ◽  
Marginal Zone ◽  
Cell Development ◽  
B Cell Development ◽  
Cell Trafficking

Abstract B-cell development is orchestrated by complex signaling networks. Rap1 is a member of the Ras superfamily of small GTP-binding proteins and has 2 isoforms, Rap1a and Rap1b. Although Rap1 has been suggested to have an important role in a variety of cellular processes, no direct evidence demonstrates a role for Rap1 in B-cell biology. In this study, we found that Rap1b was the dominant isoform of Rap1 in B cells. We discovered that Rap1b deficiency in mice barely affected early development of B cells but markedly reduced marginal zone (MZ) B cells in the spleen and mature B cells in peripheral and mucosal lymph nodes. Rap1b-deficient B cells displayed normal survival and proliferation in vivo and in vitro. However, Rap1b-deficient B cells had impaired adhesion and reduced chemotaxis in vitro, and lessened homing to lymph nodes in vivo. Furthermore, we found that Rap1b deficiency had no marked effect on LPS-, BCR-, or SDF-1–induced activation of mitogen-activated protein kinases and AKT but clearly impaired SDF-1–mediated activation of Pyk-2, a key regulator of SDF-1–mediated B-cell migration. Thus, we have discovered a critical and distinct role of Rap1b in mature B-cell trafficking and development of MZ B cells.

scholarly journals Divergent Lineage-Specific Functions of PP2A in Chronic Phase CML and Lymphoid Blast Crisis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3128-3128
Author(s):  
Gang Xiao ◽  
Huimin Geng ◽  
Lai N Chan ◽  
Zhengshan Chen ◽  
Markus Muschen
Keyword(s):  
B Cell ◽  
Blast Crisis ◽  
Cell Lineage ◽  
Chronic Phase ◽  
Cell Development ◽  
B Cell Development ◽  
Erk Signaling ◽  
Glycolytic Flux ◽  
Myeloid Lineage

Abstract Background & Hypothesis: Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase and functions as negative regulator of Akt- and Erk-signaling. PP2A attenuates downstream signaling of BCR-ABL1 and thereby functions as important tumor suppressor in chronic myeloid leukemia (CML). PP2A-activating drugs have been proposed for the treatment of CML and eradication of tyrosine kinase inhibitor resistant leukemia stem cells in CML (Neviani et al., 2013). Studying Cre-mediated ablation of PP2A in mouse models for BCR-ABL1-driven leukemia including myeloid CML in chronic phase (CML-CP), B lymphoid blast crisis (LBC) and B cell lineage Ph+ ALL, we were able to provide genetic evidence for this treatment concept in CML. Seemingly paradoxically, however, inducible ablation of PP2A in B cell lineage LBC and Ph+ ALL cells has the opposite effect and induced rapid cell death. These findings highlight fundamental differences in the regulation of Akt- and Erk-signaling in myeloid lineage and B cell lineage cells and have implications for specific therapy concepts that distinguish between chronic phase CML and lymphoid blast crisis/ Ph+ ALL. Results: Interestingly, PP2A protein levels are significantly higher in B cell lineage Ph+ ALL and CML lymphoid blast crisis (LBC) compared to chronic phase CML. To explore the function of PP2A during early B cell development, we deleted the α isoform of the PP2A subunit A in pro-B cells by introducing Mb1-Cre into Ppp2r1afl/fl mice. Analysis of bone marrow from Ppp2r1afl/flMb1-Cre (PP2A-cko) mice showed obvious B cell development block at the pre-B cell receptor checkpoint, although V(D) recombination and RAG1/2 activity were intact in PP2A-cko pre-B cells. Since high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL, we studied the function of PP2A in mouse models for B cell lineage ALL including Ph+ ALL and CML lymphoid blast crisis (LBC). After Cre-induced deletion of Ppp2r1a, we observed reduced protein expression of both PP2A subunit A and subunit C accompanied with decreased PP2A phosphatase. The PP2A-cKO ALL cells showed normal proliferation but significantly impaired colony formation capability and increased apoptosis, both of which could be rescued by overexpression of wildtype PP2A in PP2A-cKO ALL cells. We next transplanted luciferase expressing PP2A-KO ALL cells into recipient mice and monitored cell growth and leukemia progression. Cre-mediated deletion significantly prolonged overall survival of recipient mice that were transplanted with Ppp2r1afl/fl ALL cells. Although all of those mice died eventually from leukemia, the ALL cells isolated from the mice retained Ppp2r1a floxed alleles suggesting that these mice died from clones that had escaped Cre-mediated deletion of PP2A. Cre-mediated deletion increased phosphorylation levels of Stat5 and Erk, resulted in accumulation of p53 and increased output of the PI3K-AKT signaling pathway as measured by increased phosphorylation of FoxO factors, p70S6K and S6 ribosomal protein. Based on the involvement of PI3K-AKT in glucose metabolism, we measured the effects of inducible PP2A-deletion on glycolytic function in CML and Ph+ ALL/LBC cells. Interestingly, inducible deletion of PP2A induced profound imbalances of glucose metabolism in B cell lineage Ph+ ALL/LBC but not myeloid lineage CML-like cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-KO ALL cells indicated impaired balance of glycolytic flux and may account for increased apoptosis of those cells. This notion was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-cKO cells. In agreement with reported tumor suppressor role of PP2A in CML cells (Neviani et al., 2005), we observed that CEBPα-driven lineage conversion of B cell lineage Ph+ ALL/LBC into not myeloid lineage. Conclusion: Here we reported the requirement of PP2A in normal B cell development and lineage-specific oncogenic role of PP2A in BCR-ABL1-driven leukemia. Divergent roles of PP2A in Ph+ ALL/LBC and myeloid lineage CML may lead to future insights into mechanisms of LBC-transformation of CML and lineage-specific requirement of BCR-ABL1-transformation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Lgr5 Functions As a Critical Negative Regulator of Wnt/β-Catenin Signaling and Is Essential for B-Lymphopoiesis and Malignant B-Cell Transformation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 748-748
Author(s):  
Kadriye Nehir Cosgun ◽  
Gauri Deb ◽  
Xin Yang ◽  
Gang Xiao ◽  
Teresa Sadras ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Survival ◽  
Cell Lines ◽  
Cell Lineage ◽  
Cell Development ◽  
B Cell Development ◽  
Negative Regulator ◽  
B Lymphopoiesis ◽  
B Cell Survival

Introduction: When early B-cell precursors have successfully undergone V(D)J rearrangement of immunoglobulin genes, they receive a strong positive selection signal, which initiates clonal expansion and the first wave of B-lymphopoiesis. Interestingly, this expansion following successful V(D)J recombination is marked by massive up-regulation of Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5). Results: To test the function of Lgr5 at this particular stage and later in B-cell development, we crossed Lgr5fl/fl mice with Mb1cre and CD21cre mice respectively. While Lgr5 deletion at the time of V(D)J recombination resulted in near complete ablation of the B cell lineage in the bone marrow and periphery, deletion at later stages of B-cell development had no significant impact on B-cell survival and proliferation. Here, we identified Lgr5 as a B-cell specific negative regulator of Wnt/β-catenin signaling, which is essential for normal B-lymphopoiesis but also for initiation of B-cell lineage acute lymphoblastic leukemia (B-ALL). Indeed, in children with B-ALL, higher than median mRNA levels of LGR5 at the time of diagnosis was identified as a predictor of poor clinical outcome (COG P9906, n=207, p=0.008). LGR5 over-expression conferred growth advantage to B-ALL cells, enhanced serial plating capacity and the ability to initiate leukemia in NSG mice. Limiting dilution transplant experiments showed that LGR5 overexpression increased LIC-frequencies by 4-fold. Inducible deletion of Lgr5 in BCR-ABL1- or NRASG12D-transformed B-ALL cell lines led to growth arrest, abolished colony forming capacity and compromised the ability of leukemia cells to initiate fatal disease in NSG recipients. Lgr5 is thought to function as a potentiator of Wnt/β-catenin signaling in epithelial cells. Upon deletion of Lgr5 in B-ALL cells, however, we observed massive accumulation of nuclear β-catenin and increased expression of β-catenin target genes, which suggests that Lgr5 acts as an essential negative regulator of β-catenin in the B-cell context. Phosphoproteomic approaches identified β-catenin to be differentially phosphorylated on S675 upon Lgr5-deletion, which enhances nuclear translocation and transcriptional activity of β-catenin. Deletion of LGR5 in human colon cancer cell lines did not result in β-catenin accumulation, which indicates a lineage specific role of Lgr5 in B-lymphocytes. Studying activating CTNNB1 mutations in 89,325 samples from 23 types of cancer, we found 7,777 activating mutations (8.7%). However, among 2,375 B-cell tumor samples, we did not observe a single activating mutation. Indeed, compared to solid tumors, B-ALL and B-cell lymphomas have very low amounts of nuclear β-catenin. B-ALL cells are extremely sensitive to β-catenin activation, resulting in cell cycle arrest and loss of colony forming ability, which parallels the phenotype observed in the absence of Lgr5. CRISPR-Cas9 mediated deletion of Ctnnb1 in Lgr5 knock-out B-ALL cells strikingly rescued proliferation and colony formation capacity, further corroborating the role of Lgr5 in promoting B-cell leukemogenesis by opposing aberrant activation of β-catenin. Therapeutic implication: Given that Lgr5 is required for leukemia-initiation and marks LICs, we tested an Lgr5-MMAE antibody-drug conjugate (ADC) in the presence and absence of dexamethasone. Combination treatment with dexamethasone not only enforced persistently high surface expression of LGR5, but also potentiated efficacy of by Lgr5-MMAE in B-ALL eradication. Conclusion: Lgr5 is critical for normal B cell development and tumor initiation in B-ALL. Lgr5, restricts the levels of nuclear β-catenin and enables B cell survival through negative regulation of Wnt/β-catenin signaling. Moreover, LGR5 represents a promising target for eradicating leukemia initiating cells. Disclosures No relevant conflicts of interest to declare.

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 Ligand-independent Signaling Functions for the B Lymphocyte Antigen Receptor and Their Role in Positive Selection during B Lymphopoiesis

2001 ◽  
Vol 194 (11) ◽  
pp. 1583-1596 ◽  
Author(s):  
Gregory Bannish ◽  
Ezequiel M. Fuentes-Pananá ◽  
John C. Cambier ◽  
Warren S. Pear ◽  
John G. Monroe
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocyte ◽  
Antigen Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
B Lymphopoiesis ◽  
Biologically Relevant ◽  
Developmental Progression

Signal transduction through the B cell antigen receptor (BCR) is determined by a balance of positive and negative regulators. This balance is shifted by aggregation that results from binding to extracellular ligand. Aggregation of the BCR is necessary for eliciting negative selection or activation by BCR-expressing B cells. However, ligand-independent signaling through intermediate and mature forms of the BCR has been postulated to regulate B cell development and peripheral homeostasis. To address the importance of ligand-independent BCR signaling functions and their regulation during B cell development, we have designed a model that allows us to isolate the basal signaling functions of immunoglobulin (Ig)α/Igβ-containing BCR complexes from those that are dependent upon ligand-mediated aggregation. In vivo, we find that basal signaling is sufficient to facilitate pro-B → pre-B cell transition and to generate immature/mature peripheral B cells. The ability to generate basal signals and to drive developmental progression were both dependent on plasma membrane association of Igα/Igβ complexes and intact immunoregulatory tyrosine activation motifs (ITAM), thereby establishing a correlation between these processes. We believe that these studies are the first to directly demonstrate biologically relevant basal signaling through the BCR where the ability to interact with both conventional as well as nonconventional extracellular ligands is eliminated.

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