Involvement of Anti-Apoptotic Signaling in Flt3-Dependent B Lymphopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2177-2177
Author(s):  
Christina T. Jensen ◽  
Yutaka Sasaki ◽  
Ewa Sitnicka ◽  
Sten Eirik W. Jacobsen
Keyword(s):  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Cytokine Signaling ◽  
Tyrosine Kinase Receptor ◽  
B Lymphopoiesis ◽  
Wild Type ◽  
Apoptotic Signaling ◽  
Apoptotic Pathways ◽  
Cell Commitment

Abstract Cytokine signaling through the tyrosine kinase receptor flt3 and the hematopoietin Interleukin-7-receptor a chain (IL7Ra) is critically involved in regulation of B lymphopoiesis. Specifically, we have recently demonstrated that mice double deficient in flt3 and IL7Ra signaling fail to support B cell commitment and development during embryogenesis as well as in adult hematopietic tissues (Sitnicka et al, J. Exp. Med; 198:1495, 2003). Previous studies have through generation of IL7Ra knockout mouse on a Bcl-2 transgenic background suggested that IL-7 induced triggering of anti-apoptotic pathways is critically involved in T cell but not B cell development (Akashi et al, Cell; 89:1033, 1997, Kondo et al, Immunity; 7:155, 1997). Thus, we here investigated whether anti-apoptotic signaling might rather be involved in flt3-dependent B cell development, through generation of a H2k driven human Bcl-2 transgenic/Flt3 ligand (FL)−/− (Bcl-2/FL−/−) mice. Strikingly, numbers of Common Lymphoid Progenitors (CLP; lin−c-kitloSca-1loIL7Ra+), pro-B (B220+CD19+AA4.1+CD43+), pre-B (B220+CD43−IgM−) and mature B cells (B220+CD43−IgM+) in the BM of 7–8 weeks old Bcl-2/FL−/− mice were all comparable to those observed in wild type mice. More importantly, the pronounced reductions in CLP, pro-B and pre-B progenitors seen in FL−/− mice on a wild type background were compensated by approximately 50% when FL-deficiency was rather induced on a bcl-2 transgenic background (Bcl-2/FL−/−). Thus, in contrast to IL7Ra, the critical role of flt3 in B cell commitment and development might involve triggering of anti-apoptotic pathways.

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

Blood ◽  
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.

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.

scholarly journals In vitro requirement for periostin in B lymphopoiesis

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

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

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

Blood ◽  
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.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 855-855 ◽  
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.

The Bcl6 RD2 Domain Is Essential For Pre-Germinal Center B Cell Development

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 783-783
Author(s):  
Chuanxin Huang ◽  
Ann Haberman ◽  
Ari M. Melnick
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Transcriptional Repression ◽  
Cell Development ◽  
B Cell Development ◽  
Wild Type ◽  
Btb Domain ◽  
Repression Domain

Abstract The transcriptional repressor Bcl6 is a master regulator of the germinal center (GC) reaction through directing naïve B cells and CD4+ T cells to differentiate into GC B cells and follicular T helper (TFH) cells respectively. Bcl6 mediates its action largely by recruitment of co-repressors through its N-terminal BTB domain and its middle second repression domain (RD2). The BTB domain repression function is critical for GC B cell survival and proliferation, but not important for TFH cell differentiation. However, the in vivobiological function of RD2 remains unknown. To explore the specific role of RD2 transcriptional repression in the GC reaction, we generated a knockin mouse model in which the endogenous Bcl6 locus encodes a mutant form of the protein that specifically disrupts RD2 mediated transcriptional repression. RD2 mutant mice were developmentally indistinguishable from wild-type mice and displayed normal B cell development prior to the GC phase. However, these mice failed to accumulate GCs after immunization with sheep blood cells and exhibited remarkably impaired production of high-affinity antibodies 21 days after T-cell dependent antigen immunization, indicative of severe deficiency of the GC reaction. Mixed bone marrow transplantation experiments showed that RD2 loss of function led to complete loss of GC B cells and partial impairment of TFH cell differentiation in cell-intrinsic manner. Intravital imaging analysis indicated that RD2-deficent antigen-engaged B cells migrate normally to the inter-follicular zone of lymph nodes and interacted normally with cognate T helper cells. To further understand the nature of the functional defect of RD2 mutant B-cells, hen egg lysosome (HEL)-specific RD2-deficient GFP B cells and wild type RFP B cells (with the ratio 1:1) were transferred together with non-fluorescent ovalbumin (OVA)-specific T cells into SMARTA hosts, which were then immunized at the footpad with HEL-OVA two days later. On day 5 after immunization, draining popliteal lymph nodes were harvested and subjected for immunofluorescence histology analysis. At this time point, wild-type RFP B cells have started to cluster into tiny GC, whereas RD2-deficient GFP B cells did not form GCs. Moreover, wild-type B cells in the follicular interior were predominantly Bcl6hi, a characteristic of pre-GC B cells, suggesting that they could serve as a source of GC B cells. By contrast, RD2-deficient GFP B cells were primarily extra-follicular, and infrequently observed in the follicle interior. Most importantly, these cells were typically Bcl6lo, demonstrating that RD2 repression function is essential for pre-GC B cell differentiation. BCL6 knockout mice display a lethal inflammatory phenotype due to aberrant T-cell and macrophage activation. In striking contrast, RD2-deficient mice experienced normal healthy lives with no inflammation, and had nearly normal inflammation cytokine production in B cells and macrophages as well as differentiation of Th1,Th2 and Th17 subtypes. Hence the RD2 repression domain is specifically involved in humoral immunity but has minimal participation in the anti-inflammatory functions of BCL6. Instead we observed that the BCL6 zing finger domain plays the key role in anti-inflammatory functions in macrophages, and through ChIP-competition assays show that this is mediated by directly competing with STATs for binding to chemokine genes. These results highlight an essential role of RD2-mediated transcriptional repression in pre-GC B cell development specifically at the early B-cell activation phase. This is different than mice with BCL6 BTB mutations where early activation is normal and the defect occurs later on in the proliferative phase of GCs. The data suggest a surprising development and cellular context-specific biochemical functions of Bcl6 governing each distinct phase of the humoral immune response and inflammation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Essential role of EBF1 in the generation and function of distinct mature B cell types

2012 ◽  
Vol 209 (4) ◽  
pp. 775-792 ◽  
Author(s):  
Bojan Vilagos ◽  
Mareike Hoffmann ◽  
Abdallah Souabni ◽  
Qiong Sun ◽  
Barbara Werner ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Intracellular Signaling ◽  
Ectopic Expression ◽  
Cell Types ◽  
Cell Development ◽  
B Cell Development ◽  
Cell Stage ◽  
Cell Immunity ◽  
Cell Commitment

The transcription factor EBF1 is essential for lineage specification in early B cell development. In this study, we demonstrate by conditional mutagenesis that EBF1 is required for B cell commitment, pro–B cell development, and subsequent transition to the pre–B cell stage. Later in B cell development, EBF1 was essential for the generation and maintenance of several mature B cell types. Marginal zone and B-1 B cells were lost, whereas follicular (FO) and germinal center (GC) B cells were reduced in the absence of EBF1. Activation of the B cell receptor resulted in impaired intracellular signaling, proliferation and survival of EBF1-deficient FO B cells. Immune responses were severely reduced upon Ebf1 inactivation, as GCs were formed but not maintained. ChIP- and RNA-sequencing of FO B cells identified EBF1-activated genes that encode receptors, signal transducers, and transcriptional regulators implicated in B cell signaling. Notably, ectopic expression of EBF1 efficiently induced the development of B-1 cells at the expense of conventional B cells. These gain- and loss-of-function analyses uncovered novel important functions of EBF1 in controlling B cell immunity.

scholarly journals Essential Role of Phospholipase Cγ2 in Early B-Cell Development and Myc-Mediated Lymphomagenesis

2006 ◽  
Vol 26 (24) ◽  
pp. 9364-9376 ◽  
Author(s):  
Renren Wen ◽  
Yuhong Chen ◽  
Li Bai ◽  
Guoping Fu ◽  
James Schuman ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Wild Type ◽  
Interleukin 7

ABSTRACT Phospholipase Cγ2 (PLCγ2) is a critical signaling effector of the B-cell receptor (BCR). Here we show that PLCγ2 deficiency impedes early B-cell development, resulting in an increase of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B cells. PLCγ2 deficiency impairs pre-BCR-mediated functions, leading to enhanced interleukin-7 (IL-7) signaling and elevated levels of RAGs in the selected large pre-B cells. Consequently, PLCγ2 deficiency renders large pre-B cells susceptible to transformation, resulting in dramatic acceleration of Myc-induced lymphomagenesis. PLCγ2 −/− Eμ-Myc transgenic mice mainly develop lymphomas of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B-cell origin, which are uncommon in wild-type Eμ-Myc transgenics. Furthermore, lymphomas from PLCγ2 −/− Eμ-Myc transgenic mice exhibited a loss of p27Kip1 and often displayed alterations in Arf or p53. Thus, PLCγ2 plays an important role in pre-BCR-mediated early B-cell development, and its deficiency leads to markedly increased pools of the most at-risk large pre-B cells, which display hyperresponsiveness to IL-7 and express high levels of RAGs, making them prone to secondary mutations and Myc-induced malignancy.

scholarly journals Iκb Kinase α Is Essential for Mature B Cell Development and Function

2001 ◽  
Vol 193 (4) ◽  
pp. 417-426 ◽  
Author(s):  
Tsuneyasu Kaisho ◽  
Kiyoshi Takeda ◽  
Tohru Tsujimura ◽  
Taro Kawai ◽  
Fumiko Nomura ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Fetal Liver ◽  
Cell Development ◽  
B Cell Development ◽  
Cytokine Signaling ◽  
Iκb Kinase ◽  
And Function

IκB kinase (IKK) α and β phosphorylate IκB proteins and activate the transcription factor, nuclear factor (NF)-κB. Although both are highly homologous kinases, gene targeting experiments revealed their differential roles in vivo. IKKα is involved in skin and limb morphogenesis, whereas IKKβ is essential for cytokine signaling. To elucidate in vivo roles of IKKα in hematopoietic cells, we have generated bone marrow chimeras by transferring control and IKKα-deficient fetal liver cells. The mature B cell population was decreased in IKKα−/− chimeras. IKKα−/− chimeras also exhibited a decrease of serum immunoglobulin basal level and impaired antigen-specific immune responses. Histologically, they also manifested marked disruption of germinal center formation and splenic microarchitectures that depend on mature B cells. IKKα−/− B cells not only showed impairment of survival and mitogenic responses in vitro, accompanied by decreased, although inducible, NF-κB activity, but also increased turnover rate in vivo. In addition, transgene expression of bcl-2 could only partially rescue impaired B cell development in IKKα−/− chimeras. Taken together, these results demonstrate that IKKα is critically involved in the prevention of cell death and functional development of mature B cells.

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

Blood ◽  
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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