RNAi Screening Identifies A Novel Role for A-Kinase Anchoring Protein 12 (AKAP12) in B Cell Development and Function

Abstract Abstract 855 The cAMP signaling pathway has emerged as a key regulator of hematopoietic cell proliferation, differentiation, and apoptosis. Signal specificity is achieved through local activation of signaling enzymes that are anchored to subcellular organelles and membranes. In particular, A-kinase anchoring proteins (AKAPs) coordinate and control cAMP responsive events. AKAPs were originally classified based on their ability to bind cAMP-dependent protein kinase (protein kinase A; PKA). The activity of PKA is regulated by its two regulatory subunits, which from a dimer that binds to the two catalytic subunits. Binding of cAMP to the regulatory dimer dissociates the catalytic subunits and activates PKA. Anchoring of PKA by AKAPs constrains PKA activity to a relevant subset of potential substrates. Thus, AKAPs contribute to the precision of intracellular signaling events by directing anchored enzyme pools to a subset of their physiological substrates at specific subcellular localizations. Using an in vitro short hairpin RNA (shRNA) screen against potentially druggable targets, we have uncovered a requirement for AKAP12 in the proliferation of a cultured pre-B cell leukemia cell line. In the hematopoietic system of mice and humans, expression of AKAP12 is tightly restricted to the pro/pre/immature stages of B lymphopoiesis, suggesting a potential role in pre-B cell receptor (pre-BCR) or BCR signaling. We find that retroviral knockdown or germline knockout of AKAP12 in mice leads to an increase in pre B and immature B cells in the bone marrow. In contrast, B cell numbers in the spleen are significantly reduced, as are recirculating B cells in the bone marrow. Transplantation of AKAP12 null hematopoietic stem and progenitor cells from fetal liver into wildtype recipients demonstrates an autonomous defect in the development of AKAP12−/− B cells. Competitive bone marrow transplantations confirm that this defect is cell autonomous and not due to a defective bone marrow environment or secretion of a B cell inhibitory factor. To identify AKAP12 interaction partners, we overexpressed FLAG-epitope tagged AKAP12 in a pre-B cell leukemia cell line. Affinity purification of AKAP12 showed a repeated co-immunoprecipitation of poorly characterized RIO kinase 1 (RIOK1). Our current efforts are focused on investigating the interaction between RIOK1 and AKAP12 and their role in the control of B cell development and cell cycle progression. Further, we are focusing on a likely role for AKAP12 in the scaffolding of PKA, PKC and phosphodiesterases by analyzing the activation of signaling cascades in cultured primary wildtype and AKAP12−/− pre B cells. Additionally, we are investigating the role of the BCR in vivo by testing if enforced expression of BCR components rescue B cell development in a AKAP12−/− BCR transgenic mouse model (SWHEL mouse). In summary, we have confirmed a novel role for AKAP12 in B lymphopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Chronic GvHD Is Characterized By Impaired B Cell Development in the Bone Marrow

Blood ◽

10.1182/blood.v126.23.1883.1883 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1883-1883

Author(s):

Oleg Kolupaev ◽

Michelle West ◽

Bruce R. Blazar ◽

Stephen Tilley ◽

James Coghill ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

B Cell ◽

Cd4 T Cells ◽

Critical Role ◽

Cell Development ◽

B Cell Development ◽

B Lymphopoiesis

Abstract Background. Chronic-graft-versus-host disease (cGvHD) continues to be a major complication following allogeneic hematopoietic stem cell transplantation (HSCT). Despite significant progress, mechanisms underlying development of the pathology are yet to be fully understood. Recent studies utilizing mouse models and patient samples have demonstrated a critical role for B cells in GvHD pathogenesis. Bone marrow (BM)-derived B cells can produce auto-reactive antibodies causing tissue fibrosis and multiorgan cGvHD. Impaired B cell homeostasis in the periphery, activation due to abnormally high levels of B cell-activating factor (BAFF), increased survival of auto-reactive B cells and aberrant BCR signaling are shown to be important for disease progression in cGvHD patients. Murine models also highlighted the critical role of germinal center reactions, particularly interactions between T follicular helper (Tfh) cells and B cells for generation of auto-antibodies which are responsible for triggering immune responses and cell-mediated toxicity. A growing body of evidence has emerged highlighting the fact that BM itself is a target organ during acute GvHD (aGvHD) with recent work suggesting a role for donor CD4+ T cells in BM specific aGvHD. Our group has shown that patients with higher numbers of BM B cell precursors were less likely to develop cGvHD after allogeneic HSCT (Fedoriw et al., 2012). These observations indicate clinical relevance of impaired BM B lymphopoiesis for cGvHD development. Methods. In order to investigate the effect of cGvHD on BM B cell development, we used the well-characterized major mismatch B6 into B10.BR model of systemic cGvHD. Recipient mice were treated with cyclophosphamide on day -3 and -2, irradiated with 700 cGy on day -1, and injected with 107 T cell depleted (TCD) BM with or without total splenic T cells (0.5-1x105). Mice were monitored for 30 days, and BM and spleen was harvested and analyzed using flow cytometry. Results. Consistent with patient data, we observed a decrease in the frequency and number of donor-derived uncommitted common lymphoid progenitors (CLP) and B cell progenitors in the BM+ allogeneic T cells group (CLP: 0.17±0.03% vs. 0.06±0.01%, p <0.01; pro B: 2.2 ± 0.5% vs. 0.7 ± 0.3%, p<0.05; pre B: 15.3±1.8% vs. 6.3±2.4%, p<0.05; immature B cells: 5.7±0.7% vs. 2.1±0.7%, p<0.01) (Fig.1). As previously reported for this model, we also found a decrease in the frequency of follicular (FO) B cells (Flynn et al., 2014). We hypothesized that during cGvHD the B cell progenitor BM niche is affected by donor CD4+ T cells leading to impaired B lymphopoiesis. Bone marrow from BM+T cell animals had a significantly higher frequency of CD4+ cells compared to the control group (0.45±0.06% vs. 0.2±0.02%). Depletion of CD4+ T cells using anti-CD4 antibody during the first two weeks after transplant improved pathology scores and prevented weight loss in BM+T cells mice. We also observedpartial recovery of B cell progenitors and Lin-CD45-CD31-CD51+ osteoblasts (OB) in animals treated with anti-CD4 antibodies (pre B 3.5±1.1% vs. 20.4±4.5%, p<0.05; immature B: 1.9±0.9% vs. 3.5±0.3%; OB: 0.8±0.1% vs.1.2±0.2%). A recent study showed that activation and proliferation of conventional T cells in aGvHD model can be prevented by in vivo expansion of regulatory T cells (Tregs) using αDR3 antibody (4C12). We adopted this approach to determine whether Tregs can suppress the cytotoxic effect of donor CD4+ T cells in BM in cGvHD model. Animals that received T cells from 4C12-treated donors had an increase in survival and lower cGvHD pathology scores. These mice also had higher frequency of pro B, pre B, and immature B cells compared to the mice infused with T cells from isotype-treated donors. Conclusions. These studies demonstrate that BM development of B lymphocytes is impaired in a mouse model of systemic cGvHD. Our data suggests that donor-derived CD4+ T cells are involved in the destruction of hematopoietic niches in BM, particularly OB, which support B lymphopoiesis. Moreover, depletion of CD4+ T cells and infusion with in vivo expanded Tregs reduced the severity of cGvHD. Thus, Treg therapy in patients with cGvHD may be important for BM B cell development, and improvement of clinical outcomes. Disclosures No relevant conflicts of interest to declare.

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G-CSF-Induced Alterations in the Bone Marrow Microenvironment Suppress B Lymphopoiesis

Blood ◽

10.1182/blood.v120.21.1246.1246 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1246-1246

Author(s):

Ryan B. Day ◽

Adam Greenbaum ◽

Daniel C. Link

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Stromal Cells ◽

Cell Development ◽

B Cell Development ◽

Bone Marrow Microenvironment ◽

B Lymphopoiesis ◽

Treatment Results ◽

Cell Suppression

Abstract Abstract 1246 Infectious stress is associated with a shift in the bone marrow from lymphopoiesis to granulopoiesis. Expression of granulocyte colony-stimulating factor (G-CSF), the principal cytokine regulating granulopoiesis, is often induced during infection. We previously reported that G-CSF treatment is associated with marked suppression of B lymphopoiesis in murine bone marrow. After 5 days of G-CSF treatment (250 μg/kg), total B cells in the bone marrow were reduced 8.1 ± 0.9-fold. Pre-pro-B cells were reduced 1.6 ± 0.3-fold, pro-B cells 12.4 ± 1.9-fold, pre-B cells 5.6 ± 0.8-fold, immature B cells 7.5 ± 1.2-fold, and mature naïve B cells 83 ± 7.6-fold. B-committed lymphoid progenitors (BLP) were modestly but significantly decreased (1.4 ± 0.2-fold), while common lymphoid progenitors (CLP) were not affected by G-CSF treatment. Increased apoptosis of mature naïve B cells in the bone marrow was observed. Studies of G-CSF receptor deficient (Csf3r−/−) bone marrow chimeras show that G-CSF acts in a non-cell intrinsic fashion to suppress B lymphopoiesis. Consistent with this observation, we show that G-CSF treatment results in decreased expression in the bone marrow microenvironment of multiple B-supportive factors including CXCL12, interleukin-6, interleukin-7, and B cell activating factor (BAFF). Prior studies have established that CXCL12-abundant reticular (CAR) cells in the bone marrow play a key role in B cell development. CAR cells are perivascular stromal cells that express very high levels of CXCL12 and are in direct contact with pre-pro-B cells. G-CSF treatment did not affect CAR cell number. However, RNA expression profiling of sorted CAR cells showed that expression of several genes associated with B cell development are significantly decreased by G-CSF, including CXCL12 (4.2 ± 1.5-fold). In addition to CAR cells, other stromal cells in the bone marrow express CXCL12, including osteoblasts and endothelial cells. To assess the role of CXCL12 production by each of these cell types to B lymphopoiesis, we generated Cxcl12flox mice and crossed them with mice expressing the following tissue-specific Cre-recombinase transgenes: Osteocalcin-Cre (Oc-Cre) targeting mature mineralizing osteoblasts; Osterix-Cre (Osx-Cre) targeting CAR cells and all osteolineage cells; or Prx1-Cre targeting mesenchymal progenitors and their progeny. Deletion of Cxcl12 using Oc-Cre or Osx-Cre had a similar effect on B cell development, with an isolated loss of mature naïve B cells in the bone marrow (2.7 ± 0.5 and 4.1 ± 1.7-fold, respectively). In contrast, deletion of Cxcl12 using Prx1-Cre resulted in severe suppression of B lymphopoiesis that included a loss of CLP (3.3 ± 2.0-fold), BLP (5.6 ± 4.3-fold), and pre-pro-B cells (12.4 ± 5.1-fold). Interestingly, treatment of Prx1-Cre Cxcl12flox/- mice with G-CSF resulted in additional B cell loss, indicating that deletion of Cxcl12 in mesenchymal stromal cells is not sufficient to fully recapitulate G-CSF-induced B cell suppression. In summary, G-CSF treatment results in marked changes in the bone marrow microenvironment that lead to a suppression of B lymphopoiesis. While G-CSF-induced inhibition of CXCL12 expression from stromal cells contributes to B cell suppression, additional alterations in the microenvironment also contribute to this phenotype. Disclosures: No relevant conflicts of interest to declare.

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G-CSF Induces Alterations in the Bone Marrow Microenvironment That Suppress B Lymphopoiesis,

Blood ◽

10.1182/blood.v118.21.3243.3243 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3243-3243

Author(s):

Ryan B Day ◽

Adam Greenbaum ◽

Mahil Rao ◽

Daniel Link

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Bone Marrow Microenvironment ◽

B Lymphopoiesis ◽

Wild Type ◽

Treatment Results ◽

A Cell

Abstract Abstract 3243 During infectious stress, there is a marked shift in the bone marrow from lymphopoiesis to granulopoiesis. Granulocyte colony-stimulating factor (G-CSF) is the principal cytokine regulating granulopoiesis, and its expression is induced during infection. In this study, we show that G-CSF treatment in mice is associated with a marked suppression of lymphopoiesis in the bone marrow. Specifically, after 5 days of G-CSF treatment (250 μg/kg), the number of B cells in the bone marrow was reduced 8.6 ± 1.3-fold, the number of T cells reduced 14.8 ± 3.8-fold, and the number of NK cells reduced 7.5 ± 1.6-fold. Though modest increases in splenic and blood lymphocytes were observed following G-CSF treatment, this did not account for the loss in the bone marrow. To assess B cell development, modified Hardy fractions were analyzed. All stages of B cell development were significantly reduced by G-CSF, but to different degrees. Fraction A (pre-pro B cells) declined 2.1 ± 0.5-fold; fraction B/C (mostly pro-B cells): 9.4 ± 1.7-fold; fraction D cells (pre-B cells): 5.9 ±1.1-fold; fraction E (immature B cells): 8.1 ± 1.6-fold; and fraction F (mature B cells): 87 ±13-fold. In addition, mature plasma cells declined 1.3 ± 0.07-fold while immature plasmablasts decreased 7.7 ± 1.7-fold. Interestingly, preliminary analysis suggests that there is no significant change in the number of common lymphoid progenitors in the bone marrow. Since there are reports of G-CSF receptor (G-CSFR) expression on certain B cell subsets, we next asked whether G-CSFR signals act in a cell-intrinsic fashion to suppress B lymphopoiesis. Mixed bone marrow chimeras were generated that contain both wild type and G-CSFR−/− bone marrow cells. G-CSF treatment of these mixed chimeras demonstrated equal suppression of wild type and G-CSFR−/− B cells. Thus, G-CSF works in a cell-extrinsic fashion to suppress B lymphopoiesis. Certain bone marrow stromal cell populations are known to regulate B lymphopoiesis, including osteoblasts and CXCL12-abundant reticular (CAR) cells. We previously showed that G-CSF treatment results in a loss of mature osteoblasts. To examine CAR cells, we analyzed mice in which green fluorescent protein (GFP) has been knocked-in to the Cxcl12 locus, allowing for CAR cell identification (Tokoyoda et al. 2004). Whereas G-CSF treatment did not alter the number of CAR cells, a significant decrease in GFP expression per CAR cell was observed. Consistent with this observation, we observed a significant decrease in CXCL12 mRNA expression in the bone marrow following G-CSF treatment. Interestingly, we also noted significant decreases in RNA and/or protein expression of a number of B-supportive cytokines, including interleukin-6, interleukin-7, and B cell activating factor (BAFF) protein. In summary, G-CSF treatment results in marked changes in the bone marrow microenvironment that lead to a suppression of B lymphopoiesis. The ability of G-CSF to disrupt homeostatic signals required for B cell maintenance at multiple stages of development suggest that upfront G-CSF treatment may be a novel strategy to sensitize certain B cell malignancies to chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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Gas7 Induces The Proliferation Of Ph+ ALL Cells and Prevents The Differentiation Of Early B Cell Progenitors Into CD25high Small Pre-B Cells

Blood ◽

10.1182/blood.v122.21.2506.2506 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2506-2506 ◽

Cited By ~ 1

Author(s):

Jaewoong Lee ◽

Maike Buchner ◽

Huimin Geng ◽

Srividya Swaminathan ◽

Eugene Park ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Cell Surface ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Cell Development ◽

B Cell Development ◽

Specific Gene

Abstract Background Growth arrest-specific gene 7 (Gas7) first discovered in growth-arrested NIH3T3 cells possesses WW, Fes/CIP4 homology (FCH), and coiled-coil domains, which can act as an adaptor for SH2 or 3-containing proteins. Gas7 is abundantly expressed in the brain and is involved in neuronal differentiation. Recently, Gas7-deficient mouse transiently expressing truncated form of Gas7 mutant protein shows motor activity defects due to reduced motor neuron number (Huang BT et al., PLoS One 2012). Interestingly, MLL-GAS7 resulting from t(11;17)(q23;p13) has been reported in a treat-related acute myeloid leukemia (AML) and in a pediatric acute lymphoblastic leukemia (ALL). However the specific role of Gas7 in an area of hematology has not been determined yet. Results We found that Gas7-deficient bone marrow (BM)-derived progenitor B cells, grown in vitro in the presence of interleukin 7 (IL-7), display two distinct populations of CD43highCD25- and CD43lowCD25high representing the small pre-B cells compared to their normal counterparts which show only CD43highCD25- population. As expected, CD43lowCD25high population showed reduced IL7R expression on the cell surface and increased expression of intracellular µHC, Igα and surface Igβ compared to CD43highCD25- population. Consistent with the high expression of CD25, Gas7 deficient B cell progenitors showed significantly increased expression of κ light chains on cell surface with decreased levels of P-AktS473 as well as increased levels of P-Erk T202/Y20, p53 and p21. Moreover, Gas7 mRNA expression was specifically upregulated by >13-fold in pre pro-B (Hardy fraction A) and small pre-B (Hardy fraction D) subsets compared to other subsets of B cell progenitors, suggesting that Gas7 may be involved in the regulation of early B-cell development. To elucidate the function of Gas7 in Ph+ B cell lineage leukemia, we transformed bone marrow B cell progenitors from Gas7-deficient mice with BCR-ABL1. Gas7 deficient Ph+ ALL cells showed decreased proliferation with reduced S phase and increased apoptosis through the inhibition of Stat5Y694 phosphorylation as well as increased levels of p21. We found that Imatinib-mediated suppression of Stat5Y694 phosphorylation dramatically upregulates the expression of Gas7. In agreement with effects of Stat5 on the sensitivity of Ph+ ALL cells against tyrosine kinase inhibitors (TKIs), Gas7 deficient Ph+ ALL cells showed high susceptibility to Imatinib-induced apoptosis. In addition, absence of Gas7 leads to loss of self-renewal capacity and failure to form colonies in methylcellulose assay. Conclusions Here we show that Gas7 may play critical roles in early B-cell development and BCR-ABL1-driven leukemia cell survival. Pathways affected by Gas7 include Stat5, AKT and Erk signaling which is known as a downstream of BCR-ABL1 and as major regulators of B-cell development. Disclosures: No relevant conflicts of interest to declare.

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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.

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Precursor B Cell Acute Lymphoblastic Leukemia Induces Pro-Leukemic Changes in the Bone Marrow Microenvironment That Contribute to Therapeutic Resistance

Blood ◽

10.1182/blood.v120.21.1466.1466 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1466-1466

Author(s):

Christopher D Chien ◽

Elizabeth D Hicks ◽

Paul P Su ◽

Haiying Qin ◽

Terry J Fry

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Cell Development ◽

B Cell Development ◽

Bone Marrow Microenvironment ◽

Therapeutic Resistance

Abstract Abstract 1466 Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although cure rates for this disease are approximately 90%, ALL remains one of the leading causes cancer-related deaths in children. Thus, new treatments are needed for those patients that do not respond to or recur following standard chemotherapy. Understanding the mechanisms underlying resistance of pediatric ALL to therapy offers one approach to improving outcomes. Recent studies have demonstrated the importance of communication between cancer cells and their microenvironment and how this contributes to the progression and therapeutic resistance but this has not been well studied in the context of ALL. Since the bone marrow is presumed to be the site of initiation of B precursor ALL we set out in our study to determine how ALL cells utilize the bone marrow milieu in a syngeneic transplantable model of preB cell ALL in immunocompetent mice. In this model, intravenously injected preB ALL develops first in the bone marrow, followed by infiltration into the spleen, lymph node, and liver. Using flow cytometry to detect the CD45.2 isoform following injection into B6CD45.1+ congenic recipients, leukemic cells can be identified in the bone marrow as early as 5 days after IV injection with a sensitivity of 0.01%-0.1%. The pre-B ALL line is B220+/CD19+/CD43+/BP1+/IL-7Ralpha (CD127)+/CD25-/Surface IgM-/cytoplasmic IgM+ consistent with a pre-pro B cell phenotype. We find that increasing amounts of leukemic infiltration in the bone marrow leads to an accumulation of non-malignant developing B cells at stages immediately prior to the pre-pro B cell (CD43+BP1-CD25-) and a reduction in non-malignant developing pre B cells at the developmental stage just after to the pre-pro B cell stage (CD43+BP1+CD25+). These data potentially suggest occupancy of normal B cell developmental niches by leukemia resulting in block in normal B cell development. Further supporting this hypothesis, we find significant reduction in early progression of ALL in aged (10–12 month old) mice known to have a deficiency in B cell developmental niches. We next explored whether specific factors that support normal B cell development can contribute to progression of precursor B cell leukemia. The normal B cell niche has only recently been characterized and the specific contribution of this niche to early ALL progression has not been extensively studied. Using a candidate approach, we examined the role of specific cytokines such as Interleukin-7 (IL-7) and thymic stromal lymphopoietin (TSLP) in early ALL progression. Our preB ALL line expresses high levels of IL-7Ralpha and low but detectable levels of TLSPR. In the presence of IL-7 (0.1 ng/ml) and TSLP (50 ng/ml) phosphSTAT5 is detectable indicating that these receptors are functional but that supraphysiologic levels of TSLP are required. Consistent with the importance of IL-7 in leukemia progression, preliminary data demonstrates reduced lethality of pr-B cell ALL in IL-7 deficient mice. Overexpression of TSLP receptor (TSLPR) has been associated with high rates of relapse and poor overall survival in precursor B cell ALL. We are currently generating a TSLPR overepressing preBALL line to determine the effect on early ALL progression and are using GFP-expressing preB ALL cells to identify the initial location of preB ALL occupancy in the bone marrow. In conclusion, or model of early ALL progression provides insight into the role of the bone marrow microenvironment in early ALL progression and provides an opportunity to examine how these microenvironmental factors contribute to therapeutic resistance. Given recent advances in immunotherapy for hematologic malignancies, the ability to study this in an immunocompetent host will be critical. Disclosures: No relevant conflicts of interest to declare.

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B Cell Development in the Bone Marrow Is Regulated by Homeostatic Feedback Exerted by Mature B Cells

Frontiers in Immunology ◽

10.3389/fimmu.2016.00077 ◽

2016 ◽

Vol 7 ◽

Cited By ~ 9

Author(s):

Gitit Shahaf ◽

Simona Zisman-Rozen ◽

David Benhamou ◽

Doron Melamed ◽

Ramit Mehr

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Development ◽

B Cell Development

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Regulated Expression of Human Histocompatibility Leukocyte Antigen (HLA)-DO During Antigen-dependent and Antigen-independent Phases of B Cell Development

Journal of Experimental Medicine ◽

10.1084/jem.20012066 ◽

2002 ◽

Vol 195 (8) ◽

pp. 1053-1062 ◽

Cited By ~ 47

Author(s):

Xinjian Chen ◽

Oskar Laur ◽

Taku Kambayashi ◽

Shiyong Li ◽

Robert A. Bray ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Class Ii ◽

Cell Development ◽

B Cell Development ◽

Negative Regulator ◽

Leukocyte Antigen ◽

Peptide Loading

Human histocompatibility leukocyte antigen (HLA)-DO, a lysosomal resident major histocompatibility complex class II molecule expressed in B cells, has previously been shown to be a negative regulator of HLA-DM peptide loading function. We analyze the expression of DO in human peripheral blood, lymph node, tonsil, and bone marrow to determine if DO expression is modulated in the physiological setting. B cells, but not monocytes or monocyte-derived dendritic cells, are observed to express this protein. Preclearing experiments demonstrate that ∼50% of HLA-DM is bound to DO in peripheral blood B cells. HLA-DM and HLA-DR expression is demonstrated early in B cell development, beginning at the pro-B stage in adult human bone marrow. In contrast, DO expression is initiated only after B cell development is complete. In all situations, there is a striking correlation between intracellular DO expression and cell surface class II–associated invariant chain peptide expression, which suggests that DO substantially inhibits DM function in primary human B cells. We report that the expression of DO is markedly downmodulated in human germinal center B cells. Modulation of DO expression may provide a mechanism to regulate peptide loading activity and antigen presentation by B cells during the development of humoral immune responses.

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Differential Regulation of B Cell Development, Activation, and Death by the Src Homology 2 Domain–Containing 5′ Inositol Phosphatase (Ship)

Journal of Experimental Medicine ◽

10.1084/jem.191.9.1545 ◽

2000 ◽

Vol 191 (9) ◽

pp. 1545-1554 ◽

Cited By ~ 96

Author(s):

Anne Brauweiler ◽

Idan Tamir ◽

Joseph Dal Porto ◽

Robert J. Benschop ◽

Cheryl D. Helgason ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Inositol Phosphatase ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain ◽

Deficient Mice

Although the Src homology 2 domain–containing 5′ inositol phosphatase (SHIP) is a well-known mediator of inhibitory signals after B cell antigen receptor (BCR) coaggregation with the low affinity Fc receptor, it is not known whether SHIP functions to inhibit signals after stimulation through the BCR alone. Here, we show using gene-ablated mice that SHIP is a crucial regulator of BCR-mediated signaling, B cell activation, and B cell development. We demonstrate a critical role for SHIP in termination of phosphatidylinositol 3,4,5-triphosphate (PI[3,4,5]P3) signals that follow BCR aggregation. Consistent with enhanced PI(3,4,5)P3 signaling, we find that splenic B cells from SHIP-deficient mice display enhanced sensitivity to BCR-mediated induction of the activation markers CD86 and CD69. We further demonstrate that SHIP regulates the rate of B cell development in the bone marrow and spleen, as B cell precursors from SHIP-deficient mice progress more rapidly through the immature and transitional developmental stages. Finally, we observe that SHIP-deficient B cells have increased resistance to BCR-mediated cell death. These results demonstrate a central role for SHIP in regulation of BCR signaling and B cell biology, from signal driven development in the bone marrow and spleen, to activation and death in the periphery.

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The Tetraspanin CD53 Regulates Early B Cell Development By Promoting IL-7R Signaling

Blood ◽

10.1182/blood-2019-131641 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 79-79

Author(s):

Zev J. Greenberg ◽

Darlene A. Monlish ◽

Rachel L. Bartnett ◽

Jeffrey J. Bednarski ◽

Laura G. Schuettpelz

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Development ◽

B Cell Development ◽

Cellular Migration ◽

Physical Interaction ◽

Hematopoietic Stem ◽

Human Phenotype

The tetraspanin CD53 has been implicated in B cell development and function. Tetraspanins are a family of transmembrane proteins important for organization of the plasma membrane and regulation of cellular migration, adhesion, and activation. CD53 has been shown to be a transcriptional target of EBF1, a critical transcription factor for early B cell development. Additional signaling for early B cell development occurs through the IL-7 receptor (IL-7R), where ligation promotes continued B cell differentiation and pro-survival/anti-apoptotic gene expression. Human deficiency of CD53 results in recurrent infections and reduced serum immunoglobulins. While prior studies have implicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. Herein, we show that CD53 expression rapidly increases throughout B cell development, beginning at the pre-pro-B cell stage. With a CRISPR-generated knockout mouse, we show that Cd53-/- mice have significantly reduced bone marrow (25% fewer, p<0.005), splenic (35% fewer, p<0.05), lymphatic (65% fewer, p<0.0001), and peripheral (30% fewer, p<0.005) B cells compared to wild-type (WT) littermate controls. Mirroring the human phenotype, Cd53-/- mice have significantly reduced serum IgG and IgM (40% reduced, p<0.01). In addition, hematopoietic stem cells isolated from Cd53-/- mice give rise to 30% fewer B cells compared to controls in vitro (p=0.005). Analysis of bone marrow B cell development demonstrates that this loss of B cells originates with early B cell progenitors, which express nearly 50% less IL-7Ra than WT and reduced IL-7 signaling. Using mass cytometry, we identified differential signaling pathways downstream of IL-7R in B cell progenitors. Specifically, we observe impaired PI3K and STAT5 activation in pre-pro- and pro-B cells in the absence of CD53, with a consequent increase in apoptosis in these populations (p<0.01). Decreased STAT5 phosphorylation was confirmed by western blot. Finally, co-immunoprecipitation studies demonstrate a physical interaction between CD53 and IL-7Ra, suggesting that these proteins associate at the cell surface. Together, these data suggest a novel role for CD53 during IL-7 signaling to promote early B cell development. Ongoing studies are focused on determining the CD53 residues required for interaction with IL-7R. Disclosures No relevant conflicts of interest to declare.

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