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

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 Science 2007) or Notch1 loss (Lee and Maeda, unpublished) rescues the defects in early B cell development seen in LRFF/FMx1-Cre+ mice.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.

Download Full-text

Induction of metabolic quiescence defines the transitional to follicular B cell switch

Science Signaling ◽

10.1126/scisignal.aaw5573 ◽

2019 ◽

Vol 12 (604) ◽

pp. eaaw5573 ◽

Cited By ~ 3

Author(s):

Jocelyn R. Farmer ◽

Hugues Allard-Chamard ◽

Na Sun ◽

Maimuna Ahmad ◽

Alice Bertocchi ◽

...

Keyword(s):

B Cells ◽

Cell Surface ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Ampk Activation ◽

Cell Stage ◽

Extracellular Adenosine ◽

Transitional B Cells ◽

Follicular B Cells

Transitional B cells must actively undergo selection for self-tolerance before maturing into their resting follicular B cell successors. We found that metabolic quiescence was acquired at the follicular B cell stage in both humans and mice. In follicular B cells, the expression of genes involved in ribosome biogenesis, aerobic respiration, and mammalian target of rapamycin complex 1 (mTORC1) signaling was reduced when compared to that in transitional B cells. Functional metabolism studies, profiling of whole-cell metabolites, and analysis of cell surface proteins in human B cells suggested that this transition was also associated with increased extracellular adenosine salvage. Follicular B cells increased the abundance of the cell surface ectonucleotidase CD73, which coincided with adenosine 5′-monophosphate–activated protein kinase (AMPK) activation. Differentiation to the follicular B cell stage in vitro correlated with surface acquisition of CD73 on human transitional B cells and was augmented with the AMPK agonist, AICAR. Last, individuals with gain-of-function PIK3CD (PI3Kδ) mutations and increased pS6 activation exhibited a near absence of circulating follicular B cells. Together, our data suggest that mTORC1 attenuation may be necessary for human follicular B cell development. These data identify a distinct metabolic switch during human B cell development at the transitional to follicular stages, which is characterized by an induction of extracellular adenosine salvage, AMPK activation, and the acquisition of metabolic quiescence.

Download Full-text

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

Blood ◽

10.1182/blood-2007-12-128140 ◽

2008 ◽

Vol 111 (9) ◽

pp. 4627-4636 ◽

Cited By ~ 29

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.

Download Full-text

LRF Critically Regulates Mature B Cell Fate and Germinal Center Response Via Notch-Dependent and -Independent Mechanisms.

Blood ◽

10.1182/blood.v114.22.1961.1961 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1961-1961

Author(s):

Nagisa Sakurai ◽

Manami Maeda ◽

Sung-UK Lee ◽

Toshiki Saito ◽

Shigeru Chiba ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Fate ◽

Knockout Mice ◽

Critical Role ◽

Notch Pathway ◽

Cell Development ◽

B Cell Development ◽

Secondary Lymphoid Organs

Abstract Abstract 1961 Poster Board I-984 LRF (Leukemia/Lymphoma Related Factor) is a transcriptional repressor originally identified as an interaction partner of the oncoprotein BCL6 (B cell Lymphoma 6). We previously found that LRF acts as a proto-oncogene by repressing tumor suppressor ARF (Alternative Reading Frame, also known as p19 in mice and p14 in humans) and is highly expressed in 60-80% of human Non-Hodgkin Lymphoma (NHL) cases (Maeda et al., Nature 2005). LRF was also found to be indispensable for hematopoietic stem cells (HSCs) to commit to the B cell lineage by opposing Notch function (Maeda et al., Science 2007). Considering that: 1) LRF is normally expressed in Germinal Center B cells (GCB) and overexpressed in NHL tissues and 2) LRF opposes Notch function to maintain normal B cell fate at HSC/progenitor levels, we explored the role of LRF in B cell development and its functional interaction with the Notch pathway in vivo. Upon T cell dependent (TD) immunization, GC formation was severely impaired in secondary lymphoid organs of B cell specific LRF conditional knockout mice (LRFflox/flox mb1-Cre+). While a GC reaction was robustly induced in control mice upon immunization, only few GCB cells were noted in secondary lymphoid organs of LRFflox/flox mb1-Cre+ mice. To assess functional significance of LRF loss in antigen response in vivo, titers of class-switched immunoglobulin (Ig) were measured in the serum; baseline serum titers of IgG1, IgG2b and IgG3 were perturbed, and the primary and secondary antibody response against the TD antigen was impaired in LRFflox/flox mb1-Cre+ mice. Absolute numbers of memory B cells and long-lived BM plasma cells were reduced in LRFflox/flox mb1-Cre+ mice 20 wk after immunization. To determine the cause of defective GC formation, apoptosis and proliferation of GCB cells were examined by FACS. While proportions of apoptotic (AnnexinV positive) GCB cells were similar, regardless of genotypes, LRF deficient GCB cells failed to proliferate upon antigen stimuli. Short-term kinetic analysis demonstrated 5-ethynyl-2'-deoxyuridine (EdU) incorporation was markedly decreased in LRF deficient GCB cells and that the proportion of GCB cells in S phase was reduced in LRFflox/flox mb1-Cre+ mice. In agreement with these findings, quantitative RT-PCR analysis in FACS-sorted GCB cells demonstrated up-regulation of p19Arf and p21, but not p53, mRNA levels in LRF deficient GCB cells. Up-regulation of p19Arf protein levels was also observed in Western Blots. Furthermore, microarray analysis and subsequent Gene Set Enrichment Analysis in FACS-sorted GCB cells showed signatures of defective proliferation, further implicating a critical role for LRF in GCB cell proliferation. Signals mediated by Notch2 are necessary for transitional B cells to commit to the marginal zone B cells (MZB). Inactivation of a component of the Notch pathway in mice resulted in no MZB development and increased follicular B cells (FOB). On the contrary, deletion of the MINT/SHARP gene, a suppressor of Notch signaling, lead to increase of MZB cells and concomitant reduction of FOB cells, indicating that Notch induces MZB cell fate at the transitional B cell stage. While B cell development in the BM was grossly normal, a reduction of FOB cells and a concomitant increase of MZB cells were observed in LRFflox/flox mb1-Cre+ mice. Since the phenotype was reminiscent of that seen in MINT/SHARP knockout mice and opposite to that observed in Notch2 knockout mice, we hypothesized that LRF antagonizes Notch2 mediated signal during the FOB vs. MZB fate determination process. To test this, LRF/Notch2 double knockout mice (LRFflox/flox Notch2flox/flox mb1-Cre+) were established and their mature B cell compartments analyzed. As expected, loss of the Notch2 gene led to an increase of FOB cells and decrease of MZB in LRFflox/flox mb1-Cre+ mice, suggesting that LRF regulates FOB vs. MZB fate in a Notch2 dependent manner. However, Notch2 deficiency did not restore GC formation in LRFflox/flox mb1-Cre+ mice. In summary, our genetic studies strongly indicate that the proto-oncogene LRF is required for normal mature B cell development and function via distinct mechanisms. We propose that LRF is necessary for mature B cell fate by blocking Notch2-mediated signals and plays a critical role in GCB cell proliferation via suppressing p19Arf mediated cell cycle arrests. Our findings provide a further rational for targeting LRF for the treatment of B cell malignancies as well as autoimmune diseases. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

A novel Rac-dependent checkpoint in B cell development controls entry into the splenic white pulp and cell survival

Journal of Experimental Medicine ◽

10.1084/jem.20091489 ◽

2010 ◽

Vol 207 (4) ◽

pp. 837-853 ◽

Cited By ~ 37

Author(s):

Robert B. Henderson ◽

Katarzyna Grys ◽

Anne Vehlow ◽

Carine de Bettignies ◽

Agnieszka Zachacz ◽

...

Keyword(s):

B Cells ◽

Positive Selection ◽

B Cell ◽

Cell Survival ◽

Cell Development ◽

B Cell Development ◽

White Pulp ◽

Cell Stage ◽

Transitional B Cells ◽

Splenic White Pulp

Rac1 and Rac2 GTPases transduce signals from multiple receptors leading to cell migration, adhesion, proliferation, and survival. In the absence of Rac1 and Rac2, B cell development is arrested at an IgD− transitional B cell stage that we term transitional type 0 (T0). We show that T0 cells cannot enter the white pulp of the spleen until they mature into the T1 and T2 stages, and that this entry into the white pulp requires integrin and chemokine receptor signaling and is required for cell survival. In the absence of Rac1 and Rac2, transitional B cells are unable to migrate in response to chemokines and cannot enter the splenic white pulp. We propose that loss of Rac1 and Rac2 causes arrest at the T0 stage at least in part because transitional B cells need to migrate into the white pulp to receive survival signals. Finally, we show that in the absence of Syk, a kinase that transduces B cell antigen receptor signals required for positive selection, development is arrested at the same T0 stage, with transitional B cells excluded from the white pulp. Thus, these studies identify a novel developmental checkpoint that coincides with B cell positive selection.

Download Full-text

B Cell Development Is Arrested at the Immature B Cell Stage in Mice Carrying a Mutation in the Cytoplasmic Domain of Immunoglobulin β

Journal of Experimental Medicine ◽

10.1084/jem.193.1.13 ◽

2000 ◽

Vol 193 (1) ◽

pp. 13-24 ◽

Cited By ~ 83

Author(s):

Amy Reichlin ◽

Yun Hu ◽

Eric Meffre ◽

Hitoshi Nagaoka ◽

Shiaoching Gong ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Receptor ◽

Cytoplasmic Domain ◽

Cell Development ◽

Immature Stage ◽

B Cell Development ◽

Cell Stage ◽

And Function

The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)α and Igβ, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Igβ, we produced mice that carry a deletion of the cytoplasmic domain of Igβ (IgβΔC mice) and compared them to mice that carry a similar mutation in Igα (MB1ΔC, herein referred to as IgαΔC mice). IgβΔC mice differ from IgαΔC mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca2+ flux. However, IgβΔC B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Igβ is required for B cell development beyond the immature B cell stage and that Igα and Igβ have distinct biologic activities in vivo.

Download Full-text

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.

Download Full-text

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

Journal of Experimental Medicine ◽

10.1084/jem.194.11.1583 ◽

2001 ◽

Vol 194 (11) ◽

pp. 1583-1596 ◽

Cited By ~ 112

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.

Download Full-text

Altered BCR and TLR signals promote enhanced positive selection of autoreactive transitional B cells in Wiskott-Aldrich syndrome

Journal of Experimental Medicine ◽

10.1084/jem.20150585 ◽

2015 ◽

Vol 212 (10) ◽

pp. 1663-1677 ◽

Cited By ~ 41

Author(s):

Nikita S. Kolhatkar ◽

Archana Brahmandam ◽

Christopher D. Thouvenel ◽

Shirly Becker-Herman ◽

Holly M. Jacobs ◽

...

Keyword(s):

B Cells ◽

Positive Selection ◽

B Cell ◽

Heavy Chain ◽

High Throughput Sequencing ◽

Cell Stage ◽

Wiskott Aldrich Syndrome ◽

Transitional B Cells ◽

Selection Of

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder frequently associated with systemic autoimmunity, including autoantibody-mediated cytopenias. WAS protein (WASp)–deficient B cells have increased B cell receptor (BCR) and Toll-like receptor (TLR) signaling, suggesting that these pathways might impact establishment of the mature, naive BCR repertoire. To directly investigate this possibility, we evaluated naive B cell specificity and composition in WASp-deficient mice and WAS subjects (n = 12). High-throughput sequencing and single-cell cloning analysis of the BCR repertoire revealed altered heavy chain usage and enrichment for low-affinity self-reactive specificities in murine marginal zone and human naive B cells. Although negative selection mechanisms including deletion, anergy, and receptor editing were relatively unperturbed, WASp-deficient transitional B cells showed enhanced proliferation in vivo mediated by antigen- and Myd88-dependent signals. Finally, using both BCR sequencing and cell surface analysis with a monoclonal antibody recognizing an intrinsically autoreactive heavy chain, we show enrichment in self-reactive cells specifically at the transitional to naive mature B cell stage in WAS subjects. Our combined data support a model wherein modest alterations in B cell–intrinsic, BCR, and TLR signals in WAS, and likely other autoimmune disorders, are sufficient to alter B cell tolerance via positive selection of self-reactive transitional B cells.

Download Full-text

IL-7 receptor signaling is necessary for stage transition in adult B cell development through up-regulation of EBF

Journal of Experimental Medicine ◽

10.1084/jem.20050158 ◽

2005 ◽

Vol 201 (8) ◽

pp. 1197-1203 ◽

Cited By ~ 154

Author(s):

Kazu Kikuchi ◽

Anne Y. Lai ◽

Chia-Lin Hsu ◽

Motonari Kondo

Keyword(s):

Transcription Factor ◽

B Cells ◽

B Cell ◽

Target Genes ◽

Cell Development ◽

B Cell Development ◽

Receptor Signaling ◽

Cell Stage ◽

Transcription Factor Network ◽

Stage Transition

Cytokine receptor signals have been suggested to stimulate cell differentiation during hemato/lymphopoiesis. Such action, however, has not been clearly demonstrated. Here, we show that adult B cell development in IL-7−/− and IL-7Rα2/− mice is arrested at the pre–pro-B cell stage due to insufficient expression of the B cell–specific transcription factor EBF and its target genes, which form a transcription factor network in determining B lineage specification. EBF expression is restored in IL-7−/− pre–pro-B cells upon IL-7 stimulation or in IL-7Rα−/− pre–pro-B cells by activation of STAT5, a major signaling molecule downstream of the IL-7R signaling pathway. Furthermore, enforced EBF expression partially rescues B cell development in IL-7Rα−/− mice. Thus, IL-7 receptor signaling is a participant in the formation of the transcription factor network during B lymphopoiesis by up-regulating EBF, allowing stage transition from the pre–pro-B to further maturational stages.

Download Full-text