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

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

Blood ◽

10.1182/blood.v120.21.855.855 ◽

2012 ◽

Vol 120 (21) ◽

pp. 855-855 ◽

Cited By ~ 1

Author(s):

Mutlu Kartal-Kaess ◽

Luisa Cimmino ◽

Simona Infantino ◽

Mehmet Yabas ◽

Jian-Guo Zhang ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Leukemia Cell ◽

Cell Development ◽

B Cell Development ◽

Leukemia Cell Line ◽

B Lymphopoiesis ◽

Catalytic Subunits ◽

B Cell Leukemia

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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A Defect in the Bone Marrow Microenvironment of ERdj4GT/GT Mice Impacts B Cell Development

Blood ◽

10.1182/blood.v120.21.3463.3463 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3463-3463

Author(s):

Jill M Fritz ◽

Timothy E Weaver

Keyword(s):

Bone Marrow ◽

B Cell ◽

Bone Cells ◽

Conflicts Of Interest ◽

Cell Development ◽

Gene Trap ◽

B Cell Development ◽

Bone Marrow Microenvironment ◽

B Lymphopoiesis ◽

The Unfolded Protein Response

Abstract Abstract 3463 ERdj4 is a critical component of the unfolded protein response (UPR) that facilitates the removal of unfolded/misfolded substrates from the ER lumen for proteasomal degradation. Since previous studies have linked the UPR to B lymphopoiesis [Reimold, A.M. et al., Nature, 2001; Zhang, K. et al., JCI, 2005], we hypothesized that a deficiency in ERdj4 would impact B cell development. The ERdj4 allele was disrupted by a gene trap leading to hypomorphic expression of ERdj4 in mice homozygous for the gene trap allele (ERdj4GT/GT). Pro-B, pre-B, immature, and mature B cell populations were significantly reduced in the bone marrow of ERdj4GT/GT mice, which was associated with an increase in pro-B cell death. ERdj4GT/GT donor cells transplanted into ERdj4+/+ recipients rescued all stages of B cell development, implicating a defect in the bone marrow microenvironment. Osteoblasts support B cell development through VCAM-1, SDF-1 and IL-7 signaling [Visnjic, D. et al., Blood, 2004; Zhu, J. et al., Blood, 2007]; coincidently, the UPR is essential for osteoblast differentiation [Tohmonda, T. et al., EMBO, 2011; Wei, J. et al., J Cell Physiol, 2008]. ERdj4 deficiency in primary bone cells was associated with increased ER stress. Osteoblast-specific markers and the B cell growth factor SDF-1 was also significantly reduced in primary ERdj4GT/GT bone cells. Importantly, osteoblasts were reduced in femur tissue sections of ERdj4GT/GT mice. Collectively, these data suggest that the loss of ERdj4 in osteoblasts is associated with a defect in B lymphopoiesis. This research was supported by the NHLBI (HL103923 and HL086492). Disclosures: No relevant conflicts of interest to declare.

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Sclerostin Deficiency Alters Peripheral B Lymphocyte Responses in Mice

10.1101/357772 ◽

2018 ◽

Cited By ~ 2

Author(s):

Arthur Chow ◽

Jourdan Mason ◽

Larrisha Coney ◽

Jamila Bajwa ◽

Cameron Carlisle ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Response ◽

Cell Development ◽

B Cell Development ◽

Bone Marrow Microenvironment ◽

Protein Antigens ◽

Hematopoietic Stem ◽

B Cell Response

AbstractUnderstanding how changes in bone physiology and homeostasis affect immune responses will inform how to retain strong immunity in patients with bone disease and in aged individuals. We previously identified sclerostin (Sost) as a mediator of cell communication between the skeletal and the immune system. Elevated bone mineral density in Sost-knockout (Sost-/-) mice contributes to an altered bone marrow microenvironment and adversely affects B cell development. B cells originate from hematopoietic stem cells within the bone marrow and mature in peripheral lymphoid organs to produce antibodies in response to infection and/or vaccination. In this study, we investigated whether the aberrant B cell development observed in the bone marrow of Sost-/- mice extends to peripheral B cells in the spleen during immune challenge, and if these changes were age-dependent. Concomitant with more severe changes in bone architecture, B cell development in the bone marrow and in the spleen worsened with age in Sost-/- mice. B cell responses to T-independent antigens were enhanced in young Sost-/- mice, whereas responses to T-dependent antigens were impaired. Our results support the hypothesis that the adverse effects of B cell development in the Sost-deficient bone marrow microenvironment extends to the peripheral B cell immune response to protein antigens, and suggest that the B cell response to routine vaccinations should be monitored regularly in patients being treated with sclerostin antibody therapy. In addition, our results open the possibility that Sost regulates the T-independent B cell response, which might be applicable to the improvement of vaccines towards non-protein antigens.

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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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IL-7 Effects on Thymocyte Progenitors.

Blood ◽

10.1182/blood.v106.11.3318.3318 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3318-3318

Author(s):

Nahed El Kassar ◽

Baishakhi Choudhury ◽

Francis Flomerfelt ◽

Philip J. Lucas ◽

Veena Kapoor ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cell ◽

Notch Signaling ◽

Stromal Cells ◽

T Cell Development ◽

Fold Increase ◽

Cell Development ◽

B Cell Development ◽

Over Expression

Abstract IL-7 is a non-redundant cytokine in T cell development. We studied the role of IL-7 in early T-cell development using a model of transgenic (Tg) mice with the murine IL-7 gene under control of the lck proximal promoter. At high IL-7 over-expression (x39 fold increase at day 1 in total thymic tissue), we observed a disruption of TCRαβ development along with increased B cell development in the thymus (7- to 13-fold increase) (El Kassar, Blood, 2004). In order to further explore abnormal T and B cell thymic development in these mice, we first confirmed that they both arise in parallel and were non-cell autonomous, by in vivo injection of neutralizing anti-IL-7 MAb and mixed bone marrow chimera experiments. Using a six color flow cytometry analysis, we found a dramatic decrease of the early thymocyte progenitors (ETPs, lin−CD44+CD25−c-kithiIL-7R−/lo) in the adult Tg mice (x4.7 fold decrease). Lin−CD44+CD25−c-kit+ thymocytes were sorted and cultured on OP9 and OP9 delta-like1 (OP9-DL1) stromal cells (kindly provided by Pr Zuniga Pflucker). At day 14, we observed an important decrease of T cell development (54% vs. 1% of DP cells) and an increase of NK cells (x5 fold increase) in the Tg-derived DN1 cell culture. DN2 (Lin−CD44+CD25−c-kit+) Tg thymocytes showed the same, but less dramatic abnormalities. While DN1 progenitors developed effectively into B220+CD19+ cells on OP9 stromal cells, no B cell development was observed on OP-DL stromal cells from DN1-Tg derived progenitors or by addition of increasingly high doses of IL-7 (x10, x40, x160) to normal B6-derived DN1 progenitors. Instead, a block of T-cell development was observed with increased IL-7. We hypothesized a down regulation of Notch signaling by IL-7 over-expression and analyzed by FACS Notch expression in the DN thymocytes. By staining the intra-cellular part of Notch cleaved after Notch 1/Notch ligand activation, Tg-derived DN2 cells showed decreased Notch signaling. More importantly, HES expression was decreased in the DN2, DN3 and DN4 fractions by semi-quantitative PCR. Sorted Pro/Pre B cells from Tg thymi showed TCR Dβ1-Jβ1 rearrangement indicating their T specific origin, in opposition to Pro/Pre B cells sorted from the bone marrow of the same mice. We suggest that more than one immature progenitor seeds the thymus from the bone marrow. While ETPs had T and NK proliferative capacity, another thymic progenitor with B potential may be responsible for thymic B cell development in normal and IL-7 Tg mice. Finally, IL-7 over-expression may induce a decreased Notch signaling in thymic progenitors, inducing a switch of T vs. B lineage development.

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Dlk-1 Regulates B Cell Development Through Altered Bone Marrow Stromal Microenvironment, Not by Affecting Hematopoitic Stem/Progenitor Cells

Blood ◽

10.1182/blood.v120.21.3465.3465 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3465-3465

Author(s):

Edyta Pawelczyk ◽

Heba A Degheidy ◽

Allison L Branchaw ◽

Kenn Holmbeck ◽

Steven R Bauer

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cell ◽

Methyl Cellulose ◽

Cell Development ◽

B Cell Development ◽

Bone Marrow Microenvironment ◽

Wild Type ◽

Mineral Density ◽

Hematopoietic Stem

Abstract Abstract 3465 Introduction: DLK-1(delta-like 1) is a member of the EGF-like homeotic protein family whose expression is known to influence cell fate decisions through cell-cell interactions. It is also known to influence the differentiation of bone marrow stromal cells (BMSC) and hematopoietic stem cells (HSC) in bone marrow. Recently, we reported the essential role of DLK-1 in B cell development, which showed that the absence of DLK-1 led to accumulation of the earliest B cell progenitors (pre-pro B cells or Fraction A (Fr A)) in bone marrow, an altered pattern of B cell development in the spleen, and an altered humoral immune response. The objective of this study was to determine whether alterations in the HSC compartment or the BMSC microenvironment contributed to Fr A accumulation in mdlk1−/− mice. Methods: The mdlk1−/− and wild type bone marrow osteoblast and HSC compartments were analyzed by multicolor flow cytometry and in vitro methyl-cellulose colony forming cell assays. Bone marrow harvested from mdlk1−/− and wild type mice was assessed for BMSCs colony forming efficiency (CFU-F) and cultured. Supernatants from cultured BMSCs were analyzed by protein arrays. Since osteoblasts are an important component of the bone marrow microenvironment, OPN+CD45-TER119-ALP+ osteoblasts were identified in the bone marrow and quantified by flow cytometry. Finally, the femurs of mdlk1−/− and wild type mice were analyzed by micro-computed tomography (uCT) scanning. Results: Using flow cytometry, we observed no statistically significant changes in the HSC and progenitor populations in the absence of DLK-1 in mice at 4 and 16 weeks of age. The results of methyl-cellulose assay confirmed the findings of flow cytometry experiments and showed no statistically significant differences in the number of CFU-G, CFU-GM, and CFU-M of 4 and 16 week old mdlk1−/− mice as compared to wild-type control mice. However, significant alterations in the microenvironment of the mdlkl −/− were observed. CFU-F efficiency of mdlk1−/− bone marrow BMSC isolated from 4 week old mice was significantly decreased when compared to age-matched controls. Furthermore, the uCT scans showed the mineral density of the femoral bone significantly decreased in 4 week old mdlk1−/− mice and the number of osteoblast cells analyzed by flow cytometry was decreased by 10%. The analysis of BMSC supernatants revealed a striking down regulation of factors associated with osteoblast function and differentiation such as osteoactivin, PF-4, Follstatin-like 1, Frizzled-6, IGF-1, M-CSF, DKK-1 and others. Conclusions: Our results indicate that accumulation of the earliest B cell progenitors with DLK-1 ablation is the result of multiple defects in the bone marrow microenvironment including decreased CFU-F, decreased number of osteoblasts, decreased bone mineral density or alterations in factors important for osteoblast function but not from increase in numbers of hematopoietic stem or progenitors cells. Our laboratory is investigating this further. Disclosures: Pawelczyk: Baxter Inc.: currently employed by Baxter Inc. Other.

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