Bone Marrow Development Research Articles

Pim1 is Critical in T-cell-independent B-cell Response and MAPK Activation in B Cells

The Pim family consists of three members that encode a distinct class of highly conserved serine/threonine kinases. In this study, we generated and examined mice with hematopoiesis-specific deletion of Pim1 and bone marrow (BM) chimeric mice with B-cell-specific targeted deletion of Pim1. Pim1 was expressed at all stages of B-cell development and hematopoietic-specific deletion of Pim1 altered B-cell development in BM, spleen and peritoneal. However, Pim1 deficiency did not affect T-cell development. Studies of BM chimeric mice showed that Pim1 is required in a cell-intrinsic manner to maintain normal B-cell development. Pim1 deficiency led to significant changes in B cell antibody responses. Additionally, Pim1 deficiency resulted in reduced B cell receptor (BCR)-induced cell proliferation and cell cycle progression. Examination of the various BCR-activated signaling pathways in Pim1-deficient B cells reveals defective activation of mitogen-activated protein kinases (MAPKs), which are known to regulate genes involved in cell proliferation and survival. qRT-PCR analysis of BCR-engaged B cells from Pim1-deficient B cells revealed reduced expression of cyclin-dependent kinase (CDK) and cyclin genes, including CDK2, CCNB1 and CCNE1. In conclusion, Pim1 plays a crucial role in B-cell development and B cell activation.

NONO regulates B-cell development and B-cell receptor signaling.

The paraspeckle protein NONO is a multifunctional nuclear protein participating in the regulation of transcriptional regulation, mRNA splicing and DNA repair. However, whether NONO plays a role in lymphopoiesis is not known. In this study, we generated mice with global deletion of NONO and bone marrow (BM) chimeric mice in which NONO is deleted in all of mature B cells. We found that the global deletion of NONO in mice did not affect T-cell development but impaired early B-cell development in BM at pro- to pre-B-cell transition stage and B-cell maturation in the spleen. Studies of BM chimeric mice demonstrated that the impaired B-cell development in NONO-deficient mice is B-cell-intrinsic. NONO-deficient B cells displayed normal BCR-induced cell proliferation but increased BCR-induced cell apoptosis. Moreover, we found that NONO deficiency impaired BCR-induced activation of ERK, AKT, and NF-κB pathways in B cells, and altered BCR-induced gene expression profile. Thus, NONO plays a critical role in B-cell development and BCR-induced B-cell activation.

Exploring the Tumor Clonal Dynamics between Diffuse Large B-Cell Lymphomas and Bone Marrow Involvement By Deep Immunoglobulin Heavy Chain VDJ Sequencing

INTRODUCTION : The BM of patients with diffuse large B-cell lymphomas (DLBCL) can be involved as DLBCL (morphologically concordant), small B-cell lymphoma (SBCL) (morphologically discordant) or a monotypic B-lymphocyte population (MBCL) detected by flow cytometry (FC). The tumor clonal dynamics in the bone marrow (BM) of patients with DLBCL has not been studied and is poorly understood. We examined DLBCL and their paired BM, and comparatively analyzed their clonal evolution patterns and dynamics based on V(D)J usage and IGH somatic hypermutation profiles by deep sequencing.METHODS : DLBCL and paired BM from 39 patients with DLBCL were included. In 14 of 39 cases, there was morphologic, immunophenotypic and/or cytogenetic (CG) evidence of BM involvement: 7 with DLBCL, 3 with SBCL, 3 with MBCL by FC only, and 1 with abnormal CG only. The IGH VDJ regions were PCR amplified using framework 1 (FR1) and/or FR2 primers and the amplicons generated from diagnostic DLBCL and BM were subjected to paired-end sequencing in separate flow cells, resulting in an average of ~0.75 million reads per sample. The V(D)J region with the highest count of read-pairs was defined as the major rearrangement combination. V(D)J usage and somatic hypermutations of all identifiable clones were analyzed. Clonal and subclonal comparisons were visualized by phylogenetic trees of the matched tumor and BM samples.RESULTS : In all 14 cases in which there was prior morphologic, immunophenotypic and/or CG evidence of BM involvement by B-cell lymphoma, VDJ reads of various abundance related to the dominant clone in the original extra-medullary tumors (DoT) could be identified in the BM. They represented either tumor clones identical (DoBM) or divergent/ancestral (DiBM) to the DoT. All of these 14 cases showed tumor clones of the DiBM type in the BM. In 8 of these 14 cases, DoBM-type clones could also be detected, but DiBM-type clones dominated over the DoBM-type in 5 of these 8 cases. In 4 of the 10 cases which were involved by DLBCL or SBCL, the most abundant VDJ clones in the BM had distinctly different VDJ rearrangements compared to the DoT, implying that tumor clones unrelated to DoT represented the most abundant clones in the BM (UnrBM). Overall, DiBM- or UnrBM-type clones were the dominant clones in 90% (9/10) of cases with morphologic evidence of lymphoma involvement in BM. In 5 of 7 (71%) cases in which the BM showed involvement by DLBCL, i.e. morphologically concordant, only UnrBM- and/or DiBM-type clones were identified; in the remaining 2 cases, both DiBM and DoBM-type clones were present, with DiBM-type clone as dominant in 1 of the 2 cases. Minor clones similar to the DiBM- or UnrBM-type clones could be identified in a subset of the diagnostic extramedullary tumors.Among the negative BM cases, deep sequencing detected lymphoma clones at a relatively low abundance in 16 of 25 cases (64%). In 14 of these 16 cases, DoBM-type clones were identified; 6 of these 14 also harbored DiBM-type clones. Two cases (2/16) had DiBM-type clones only. Notably, in two cases, the negative staging BM harbored minute DiBM- or UnrBM-type clones which persisted post-chemotherapy and resembled the dominant clone of the relapsed DLBCL, suggesting that this minute clone was chemo-resistant and may have served as a potential precursor for relapse.CONCLUSIONS : Our studies indicate a complex tumor clonal dynamics in the BM of patients with DLBCL. Lymphoma involvement of the BM in patients with DLBCL, in most cases, represents expansion of tumor cells clonally divergent or unrelated from the dominant tumor clone in the primary tumor. In some instances, such tumor clones in the marrow can be traced back to the original diagnostic tumor. These findings underscore the extent of tumor heterogeneity between the primary DLBCL and the lymphoma in the BM that has not been previously recognized by routine pathologic examination. The frequent detection of low-abundance DoBM or DiBM-type clones in pathologically negative BM suggests that expansion of these clones depends on the appropriate microenvironment, which may be provided by the neoplastic lymphoid tissue but not the BM. In addition, minute clones of the DiBM- or UnrBM-type detected in the BM by deep VDJ sequencing but not by conventional diagnostic methods may be a harbinger of frank lymphoma development in BM or extramedullary relapse; thus, their detection may be of clinical significance. DisclosuresNo relevant conflicts of interest to declare.

Role of Sclerostin in Multiple Myeloma Progression

Multiple Myeloma (MM) is a tumor of plasma cells that originates and expands within bone marrow (BM). The BM microenvironment, including its constituent stromal cells and cytokines contribute to disease progression. The BM stromal cells include osteolineage cells, osteoclasts, adipocytes, endothelial cells etc. Among these, cells of the osteoblast lineage have been identified as the most versatile regulators of hematopoietic cells. The osteolineage cells include preosteoblasts, committed osteoblasts, mature osteoblasts, and the terminally differentiated osteocytes, each with its own characteristic biological activity. The osteolineage cells have been shown to regulate commitment and differentiation of hematopoietic lineage cells including hematopoietic stem cells, B-cells, and myeloid lineage cells.Previously published data have shown that loss of parathyroid hormone (PTH) signaling in osteolineage cells adversely affects B-cell development (Panaroni et al., 2016). PTH is also a strong inhibitor of sclerostin, a Wnt-signaling inhibitor secreted by osteocytes. Moreover, mice lacking osteocytes show decreased lymphopoiesis, which may in part be regulated by sclerostin. A sclerostin-neutralizing antibody has shown promise for treatment for age-induced osteoporosis. We have recently demonstrated that MM patients have increased levels of sclerostin and that treatment with a SOST neutralizing antibody resulted in increased osteoblastic differentiation (Eda et al., 2015) and decreased tumor burden. Based upon these findings, we hypothesized that the osteolineage cells contribute to MM progression, and that this may in part occur via sclerostin. To test this hypothesis, we have used genetic mouse models of osteolineage or sclerostin deficiency. Mouse MM cell line 5TGM1 was used to examine MM progression in these mouse models.Global sclerostin deficient (SOST KO) mice were generated as described previously. Given that sclerostin acts as a negative regulator of bone formation, SOST KO mice displayed robust increase in trabecular and cortical bone density as analyzed by micro-computed tomography. Co-culture with 5TGM1 MM cells showed that the BM stromal cells from SOST KO mice significantly decreased proliferation of MM cells raising the possibility that sclerostin deficiency may decrease MM progression in vivo. The SOST KO mice showed significantly reduced BM cellularity compared to the controls. However, relevant to MM progression, overall B-lymphopoiesis, proliferation or apoptosis was unchanged during any stages of B-cell development in BM of 3-month old SOST KO mice.To determine if sclerostin deficiency alters MM engraftment in vivo, 5TGM1 cells were injected into the tibia of 3-month old control or SOST KO mice and engraftment was followed for 2-months. Complete blood counts (CBC) showed no significant changes between the control and SOST KO mice injected with the MM cells. The 5TGM1 cells were fluorescently tagged before in vivo injections allowing us to track the cells by whole body bioluminescence imaging and by contralateral tibia BM flow cytometry analysis. Both the analyses showed no changes in the distant MM engraftment between the control and the SOST KO mice. Ongoing work is examining local MM engraftment in the tibia with injected MM cells.These data suggest that the sclerostin deficiency does not alter systemic MM engraftment after 2 months of follow-up observations. It is possible, however, that the local progression of MM cells may have altered. We are investigating this possibility. We are also examining MM engraftment in mice lacking mature osteoblasts and osteocytes to identify sclerostin-dependent and independent factors controlling MM progression. DisclosuresRaje:Millenium: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Onyx: Consultancy.

Tumor cells in light-chain amyloidosis and myeloma show distinct transcriptional rewiring of normal plasma cell development

AbstractAlthough light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation–related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies.

Endothelium-derived stromal cells contribute to hematopoietic bone marrow niche formation

Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR+ cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration.

Delineating Human B Cell Precursor Development With Genetically Identified PID Cases as a Model.

B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with surrogate light chains. Appropriate signaling via this pre-BCR complex is followed by rearrangement of the Ig light chain genes, resulting in the formation, and selection of functional BCR molecules. Consecutive production, expression, and functional selection of the pre-BCR and BCR complexes guide the BCP differentiation process that coincides with corresponding immunophenotypic changes. We studied BCP differentiation in human BM samples from healthy controls and patients with a known genetic defect in V(D)J recombination or pre-BCR signaling to unravel normal immunophenotypic changes and to determine the effect of differentiation blocks caused by the specific genetic defects. Accordingly, we designed a 10-color antibody panel to study human BCP development in BM by flow cytometry, which allows identification of classical preB-I, preB-II, and mature B-cells as defined via BCR-related markers with further characterization by additional markers. We observed heterogeneous phenotypes associated with more than one B-cell maturation pathway, particularly for the preB-I and preB-II stages in which V(D)J recombination takes place, with asynchronous marker expression patterns. Next Generation Sequencing of complete IGH gene rearrangements in sorted BCP subsets unraveled their rearrangement status, indicating that BCP differentiation does not follow a single linear pathway. In conclusion, B-cell development in human BM is not a linear process, but a rather complex network of parallel pathways dictated by V(D)J-recombination-driven checkpoints and pre-BCR/BCR mediated-signaling occurring during B-cell production and selection. It can also be described as asynchronous, because precursor B-cells do not differentiate as full population between the different stages, but rather transit as a continuum, which seems influenced (in part) by V-D-J recombination-driven checkpoints.

Hoxa9 silencing of p16 ink4a is essential for murine lymphopoiesis

Abstract The homeo-domain transcription factor Hoxa9 is required for normal B and T lymphocyte development in bone marrow (BM) and thymus, respectively. Here we show that Hoxa9-deficiency is accompanied by increased expression of the cdkn2a tumor suppressor locus that encodes the cell cycle inhibitor p16Ink4a. Furthermore, we found that somatic deletion of p16Ink4a is sufficient to rescue the B and T cell progenitor deficiencies, as well as the BM lymphoid progenitor pools, in hoxa9−/− mice. In contrast, the hematopoietic stem cell (HSC) and multipotential progenitor (MPP) abnormalities characteristic of Hoxa9-deficient mice were not corrected by loss of p16Ink4a. These new data reveal that Hoxa9 repression of p16ink4a is essential for lymphoid lineage development in BM and thymus. Importantly, they highlight distinct functions for Hoxa9 in regulation of primitive hematopoietic progenitors versus those committed to the B or T cell fates.

Concise Review: Conceptualizing Paralogous Stem-Cell Niches and Unfolding Bone Marrow Progenitor Cell Identities.

Lineage commitment and differentiation of skeletal stem cells/bone marrow stromal cells (SSCs/BMSCs, often called bone marrow-derived "mesenchymal stem/stromal" cells) offer an important opportunity to study skeletal and hematopoietic diseases, and for tissue engineering and regenerative medicine. Currently, many studies in this field have relied on cell lineage tracing methods in mouse models, which have provided a significant advancement in our knowledge of skeletal and hematopoietic stem-cell niches in bone marrow (BM). However, there is a lack of agreement in numerous fundamental areas, including origins of various BM stem-cell niches, cell identities, and their physiological roles in the BM. In order to resolve these issues, we propose a new hypothesis of "paralogous" stem-cell niches (PSNs); that is, progressively altered parallel niches within an individual species throughout the life span of the organism. A putative PSN code seems to be plausible based on analysis of transcriptional signatures in two representative genes that encode Nes-GFP and leptin receptors, which are frequently used to monitor SSC lineage development in BM. Furthermore, we suggest a dynamic paralogous BM niche (PBMN) model that elucidates the coupling and uncoupling mechanisms between BM stem-cell niches and their zones of active regeneration during different developmental stages. Elucidation of these PBMNs would enable us to resolve the existing controversies, thus paving the way to achieving precision regenerative medicine and pharmaceutical applications based on these BM cell resources. Stem Cells 2018;36:11-21.

VLA-4 Affinity Assay for Murine Bone Marrow-derived Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are defined by their functional ability to self-renew and to differentiate into all blood cell lineages. The majority of HSC reside in specific anatomical locations in the bone marrow (BM) microenvironment, in a quiescent non motile mode. Adhesion interactions between HSCs and their supporting BM microenvironment cells are critical for maintaining stem cell quiescence and protection from DNA damaging agents to prevent hematology failure and death. Multiple signaling proteins play a role in controlling retention and migration of bone marrow HSCs. Adhesion molecules are involved in both processes regulating hematopoiesis and stem- and progenitor-cell BM retention, migration and development. The mechanisms underlying the movement of stem cells from and to the marrow have not been completely elucidated and are still an object of intense study. One important aspect is the modification of expression and affinity of adhesion molecules by stem and progenitor cells which are required both for stem cell retention, migration and development. Adhesion is regulated by expression of the adhesion molecules, their affinity and avidity. Affinity regulation is related to the molecular binding recognition and bond strength. Here, we describe the in vitro FACS assay used in our research to explore the expression, affinity and function of the integrin α4β1 (also termed VLA-4) for murine bone marrow retained EPCR+ long term repopulation HSC (LT-HSC) (Gur- Cohen et al., 2015 ).

Human Hematopoietic Stem/Progenitor Cells (HSPCs) and Mesenchymal Stromal Cells (MSCs) Express Several Functional Pituitary and Gonadal Sex Hormone Receptors - Identification of Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) As New Growth Factors for HSPCs and MSCs

Background. Evidence has accumulated that murine hematopoietic stem/progenitor cells (HSPCs) share several markers with the germline, a connection supported by our recent reports that pituitary and gonadal sex hormones (SexHs) regulate development of murine HSPCs (Stem Cells & Develop. 2015, 24, 927-937). In contrast to mice, relatively little is known about the role of SexHs in human hematopoiesis, except that androgens have been employed successfully to treat certain cases of bone marrow (BM) failure. In particular, no studies have been performed to study the roles of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in this process. It is known that the blood level of FSH increases with age and is high in older patients, which correlates with increased risk of hematopoietic malignancies.Aim of the study. To better address the effects of SexHs, and particularly pituitary SexHs, on human hematopoiesis, we tested human HSPCs purified from umbilical cord blood (UCB) and BM for expression of receptors for pituitary SexHs, including FSH, LH, and prolactin (PRL), as well as the receptors for gonadal SexHs, including progesterone, estrogens, and androgen. We then tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. In parallel, we tested the effect of SexHs on human mesenchymal stromal cells (MSCs). Finally, based on our observation that at least some of the UCB-derived, CD45- very small embryonic-like stem cells become specified into CD45+ HSPCs (Leukemia 2011;25,1278-1285), we also evaluated the expression of pituitary and gonadal SexH receptors on these cells.Results. We report for the first time that, like their murine counterparts, human HSPCs, small CD45- very small embryonic-like stem cells endowed with hematopoietic specification potential, and MSCs expressed functional pituitary and gonadal SexH receptors at the mRNA and protein level and responded by MAPKp42/44 and AKT phosphorylation to SexH stimulation. Most importantly, human HSPCs proliferated in vitro in response to SexH stimulation, as did MSCs. Finally, FSH and LH also chemoattracted MSCs in a Transwell migration assay and stimulated these cells to secrete several factors that enhanced in vitro angiogenesis.Conclusions. We report for the first time that FSH, LH, estrogens, and progesterone stimulate growth of human HSPCs and MSCs. In addition, we confirmed that androgens and prolactin stimulate proliferation of human HSPCs. These results are important for understanding the interplay of SexHs in the development and aging of BM, as plasma levels of some of these hormones change with age. Finally, these results further support a developmental link between hematopoiesis and the germline as previously proposed by us and others. DisclosuresNo relevant conflicts of interest to declare.

Eukaryotic translation initiation factor 6 is a novel regulator of reactive oxygen species‐dependent megakaryocyte maturation

Ribosomopathies constitute a class of inherited disorders characterized by defects in ribosome biogenesis and function. Classically, bone marrow (BM) failure is a clinical symptom shared between these syndromes, including Shwachman-Bodian-Diamond syndrome (SBDS). Eukaryotic translation initiation factor 6 (eIF6) is a critical translation factor that rescues the quasilethal effect of the loss of the SBDS protein. To determine whether eIF6 activity is necessary for BM development. We used eIF6(+/-) mice and primary BM megakaryocytes to investigate the involvement of eIF6 in the regulation of hematopoiesis. We provide evidence that reduced eIF6 expression negatively impacts on megakaryopoiesis. We show that inhibition of eIF6 leads to a reduction in cell size and mean ploidy level of megakaryocytes and a delay in megakaryocyte maturation by blocking the G1 /S transition. Consistent with this phenotype, only few megakaryocyte-forming proplatelets were found in eIF6(+/-) cells. We also discovered that, in eIF6(+/-) cells, the steady-state abundance of mitochondrial respiratory chain complex I-encoding mRNAs is decreased, resulting in decreased reactive oxygen species (ROS) production. Intriguingly, connectivity map analysis showed that eIF6-mediated changes overlap with specific translational inhibitors. eIF6 is a translation factor acting downstream of insulin/phorbol 12-myristate 13-acetate (PMA) stimulation. PMA treatment significantly restored eIF6(+/-) megakaryocyte maturation, indicating that activation of eIF6 is essential for the rescue of the phenotype. Taken together, our results show a role for eIF6-driven translation in megakaryocyte development, and unveil the novel connection between translational control and ROS production in this cell subset.

Immature B Cell Egress from Bone Marrow Is SOCS3 Independent.

Suppressor of cytokine signaling (SOCS)-3 has been suggested to regulate CXCR4 signaling in a variety of human cell lines. In mice, conditional SOCS3 inactivation in hematopoietic cells including B-lineage lymphocytes has been reported to exacerbate CXCR4-signaling and focal adhesion kinase phosphorylation, which resulted in altered immature B cell distribution in bone marrow (BM) due to sustained α4β1 integrin-mediated adhesion to the extracellular matrix. However, a recent study examining conditional SOCS3 deletion specifically in B-lineage cells failed to detect significant roles in B-lineage cell retention in BM. In this study we carefully examined the role played by SOCS3 in CXCR4 signaling in developing B cell subsets. We show that in mice conditionally deficient in SOCS3 exclusively in B cells (Socs3 fl/fl Mb1 cre/+) there was no detectable difference in B cell development in BM and in periphery. We show that SOCS3 deficient and sufficient immature B cell subsets are similarly distributed between BM parenchyma and sinusoids, and are equally competent at exiting BM into peripheral blood. Furthermore, we found no significant differences in CXCR4 desensitization upon ligand exposure in developing B lymphocyte subsets. Consequently, SOCS3-deficient and sufficient B-lineage cell migration towards CXCL12 in vitro was undistinguishable, and B-lineage cell amoeboid motility within BM parenchyma was also unaffected by SOCS3-deficiency. Thus we conclude that SOCS3 has no detectable influence on biological processes known to be controlled by CXCR4 signaling.

Dual-regulated Lentiviral Vector for Gene Therapy of X-linked Chronic Granulomatosis

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.

High Fat Diet Rapidly Suppresses B Lymphopoiesis by Disrupting the Supportive Capacity of the Bone Marrow Niche

The bone marrow (BM) niche is the primary site of hematopoiesis, and cues from this microenvironment are critical to maintain hematopoiesis. Obesity increases lifetime susceptibility to a host of chronic diseases, and has been linked to defective leukogenesis. The pressures obesity exerts on hematopoietic tissues led us to study the effects of a high fat diet (HFD: 60% Kcal from fat) on B cell development in BM. Seven week old male C57Bl/6J mice were fed either a high fat (HFD) or regular chow (RD) diet for periods of 2 days, 1 week and 6 weeks. B-cell populations (B220+) were not altered after 2 d of HFD, within 1 w B-cell proportions were reduced by −10%, and by 6 w by −25% as compared to RD (p<0.05). BM RNA was extracted to track the expression of B-cell development markers Il-7, Ebf-1 and Pax-5. At 2 d, the expression of Il-7 and Ebf-1 were reduced by −20% (p = 0.08) and −11% (p = 0.06) whereas Pax-5 was not significantly impacted. At one week, however, the expressions of Il-7, Ebf-1, and Pax-5 in HFD mice fell by -19%, −20% and −16%, and by six weeks were further reduced to −23%, −29% and −34% as compared to RD (p<0.05 for all), a suppression paralleled by a +363% increase in adipose encroachment within the marrow space (p<0.01). Il-7 is a critical factor in the early B-cell lineage which is secreted by supportive cells in the BM niche, and is necessary for B-cell commitment. These data indicate that BM Il-7 expression, and by extension B-cell differentiation, are rapidly impaired by HFD. The trend towards suppressed expression of Il-7 following only 2 d of HFD demonstrates how susceptible the BM niche, and the cells which rely on it, are to diet, which ultimately could contribute to disease susceptibility in metabolic disorders such as obesity.

B-cell development and functions and therapeutic options in adenosine deaminase–deficient patients

Adenosine deaminase (ADA) deficiency causes severe cellular and humoral immune defects and dysregulation because of metabolic toxicity. Alterations in B-cell development and function have been poorly studied. Enzyme replacement therapy (ERT) and hematopoietic stem cell (HSC) gene therapy (GT) are therapeutic options for patients lacking a suitable bone marrow (BM) transplant donor. We sought to study alterations in B-cell development in ADA-deficient patients and investigate the ability of ERT and HSC-GT to restore normal B-cell differentiation and function. Flow cytometry was used to characterize B-cell development in BM and the periphery. The percentage of gene-corrected B cells was measured by using quantitative PCR. B cells were assessed for their capacity to proliferate and release IgM after stimulation. Despite the severe peripheral B-cell lymphopenia, patients with ADA-deficient severe combined immunodeficiency showed a partial block in central BM development. Treatment with ERT or HSC-GT reverted most BM alterations, but ERT led to immature B-cell expansion. In the periphery transitional B cells accumulated under ERT, and the defect in maturation persisted long-term. HSC-GT led to a progressive improvement in B-cell numbers and development, along with increased levels of gene correction. The strongest selective advantage for ADA-transduced cells occurred at the transition from immature to naive cells. B-cell proliferative responses and differentiation to immunoglobulin secreting IgM after B-cell receptor and Toll-like receptor triggering were severely impaired after ERT and improved significantly after HSC-GT. ADA-deficient patients show specific defects in B-cell development and functions that are differently corrected after ERT and HSC-GT.

Abstract 5448: Genome-wide ChIP-seq of C/EBPε reveals networks controlling myelopoiesis .

Abstract CAAT/enhancer-binding protein epsilon (C/EBPε) is known to be a critical transcription factor mediating terminal differentiation of granulocytes and a target to boost killing of bacteria by the innate immune system. However, our data found that ∼9 month old C/EBPε knockout (KO) mice developed a novel phenotype similar to the myeloproliferative disorder. In bone marrow (BM) of C/EBPε KO mice, colonies of granulocyte-macrophage progenitor (CFU-GM) were increased (143±21 vs. 59±13, P&amp;lt;0.01) while erythroid progenitors (BFU-E) were decreased (8±2 vs.14±4, P&amp;lt;0.01) as compared to age- and sex- matched wild type controls. Flow data showed that the percentage of BM immature myeloid (Mac1+Gr1int) were much higher (48%±3% vs. 9%±5%, P&amp;lt;0.01) while Ter119+ erythrocytes were less (13%±4% vs. 47%±1%, P&amp;lt;0.01) than control. In addition, the ratio of spleen/ body weight in C/EBPε KO mice was augmented (0.81%±0.16% vs. 0.25%±0.03%, P&amp;lt;0.01) and spleen H&amp;E staining showed extramedullary hemopoiesis. Compared to spleen samples in control mice, CFU-GM (97±16 vs. 9±2, P&amp;lt;0.01), BFU-E (20±5 vs. 4±1, P&amp;lt;0.01) and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitors (CFU-GEMM, 11±3 vs. 2±1, P&amp;lt;0.01) in C/EBPε KO mice were significantly increased. Consistent with spleen CFU assay, the percentage of Mac1+Gr1int immature myeloid cells (18%±8% vs. 3%±1%, P&amp;lt;0.05) and Ter119+ erythrocytes (38%±1% vs. 12%±2%, P&amp;lt;0.01) were raised in KO than in control mice by flow cytometry analysis. Taken together, C/EBPε deletion in mice blocks myeloid differentiation and impairs erythoid development in BM as well as leads to extramedullary hemopoiesis in spleen. To investigate regulatory networks controlled by C/EBPε, we performed genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq) in wild type and C/EBPε KO mice BM using N- and C-terminal C/EBPε antibodies. We have identified ∼15000 significant C/EBPε binding sites and as with the majority of transcription factors, C/EBPε targets predominantly distal intergenic and intronic regulatory regions. However, 15% of significant binding sites are localized within gene promoters and some of them are known to play a critical role in hematopoietic lineage differentiation such as PU.1, RUNX1 and SCL. Interestingly, C/EBPε also binds known distal regulatory regions as the PU.1 enhancer, and our data has highlighted novel potential intergenic and intronic enhancers for RUNX1. The ChIP-PCR results of these transcription factors are consistent with ChIP-seq. In summary, our study suggests that C/EBPε can have a profound effect on myeloid development by regulating key genes and its deregulation can produce a myeloproliferative abnormality. Citation Format: Tong Yin, Touati Benoukraf, Phillip H. Koeffler. Genome-wide ChIP-seq of C/EBPε reveals networks controlling myelopoiesis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5448. doi:10.1158/1538-7445.AM2013-5448 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Differential requirement for Hoxa9 in the development and differentiation of B, NK, and DC-lineage cells from Flt3+ multipotential progenitors

Hoxa9 is a homeodomain transcription factor important for the generation of Flt3+hiIL-7R- lymphoid biased-multipotential progenitors, Flt3+IL-7R+ common lymphoid progenitors (CLPs), and B cell precursors (BCP) in bone marrow (BM). In addition to B-cell, Flt3+IL-7R+ CLPs possess NK and DC developmental potentials, although DCs arise from Flt3+IL-7R- myeloid progenitors as well. In this study, we investigated the requirement for Hoxa9, from Flt3+ or Flt3- progenitor subsets, in the development of NK and DC lineage cells in BM. Flt3+IL-7R+Ly6D- CLPs and their Flt3+IL-7R+Ly6D+ B lineage-restricted progeny (BLP) were significantly reduced in hoxa9−/− mice. Interestingly, the reduction in Flt3+IL-7R+ CLPs in hoxa9−/− mice had no impact on the generation of NK precursor (NKP) subsets, the differentiation of NKP into mature NK cells, or NK homeostasis. Similarly, percentages and numbers of common dendritic progenitors (CDP), as well as their plasmacytoid or conventional dendritic cell progeny in hoxa9−/− mice were comparable to wildtype. These findings reveal distinct requirements for Hoxa9 or Hoxa9/Flt3 molecular circuits in regulation of B versus NK and DC development in BM.

Dietary supplementation with probiotics improves hematopoiesis in malnourished mice.

Background Lactobacillus rhamnosus CRL1505 (Lr) administered during the repletion of immunocompromised-malnourished mice improves the resistance against intestinal and respiratory infections. This effect is associated with an increase in the number and functionality of immune cells, indicating that Lr could have some influence on myeloid and lymphoid cell production and maturation.ObjectiveThis study analyzed the extent of the damage caused by malnutrition on myeloid and lymphoid cell development in the spleen and bone marrow (BM). We also evaluated the impact of immunobiotics on the recovery of hematopoiesis affected in malnourished mice.MethodsProtein malnourished mice were fed on a balanced conventional diet for 7 or 14 consecutive d with or without supplemental Lr or fermented goat's milk (FGM). Malnourished mice and well-nourished mice were used as controls. Histological and flow cytometry studies were carried out in BM and spleen to study myeloid and lymphoid cells.ResultsMalnutrition induced quantitative alterations in spleen B and T cells; however, no alteration was observed in the ability of splenic B cells to produce immunoglobulins after challenge with LPS or CpG. The analysis of BM B cell subsets based on B220, CD24, IgM and IgD expression showed that malnutrition affected B cell development. In addition, BM myeloid cells decreased in malnourished mice. On the contrary, protein deprivation increased BM T cell number. These alterations were reverted with Lr or FGM repletion treatments since normal numbers of BM myeloid, T and B cells were observed in these groups.ConclusionsProtein malnutrition significantly alters B cell development in BM. The treatment of malnourished mice with L. rhamnosus CRL1505 was able to induce a recovery of B cells that would explain its ability to increase immunity against infections. This work highlights the possibility of using immunobiotics to accelerate the recovery of lymphopoyesis in immunocompromised-malnourished hosts.

Role of Bruton’s tyrosine kinase in macrophage apoptosis

Macrophages and polymorphonuclear cells (PMNs) rapidly respond to microbial and immune inflammatory stimuli and die during these responses. We have shown earlier that many macrophage and PMN functions are compromised in x-linked immunodeficient (Xid) mice with functional deficiency in Bruton's tyrosine kinase (Btk). We now report that Btk-deficient macrophages show enhanced susceptibility to apoptotic death on exposure to the microbial and immune inflammatory signals bacterial lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in vitro. In vivo in mixed bone marrow (BM) chimeras Btk deficiency leads primarily to loss of peripheral macrophage numbers without affecting BM development, suggesting a role of inflammation-induced apoptosis in regulating macrophage life span. Surprisingly, Btk deficiency does not affect macrophage apoptosis induced by DNA damage or CD95 engagement. Reactive nitrogen and oxygen species also do not contribute to inflammation-induced apoptosis, but apoptotic process involves loss of mitochondrial potential, shows increased activation of caspase 9 and enhanced loss of Bcl-xL. The lack of pro-survival signaling through the Btk-phosphotidylinositol 3-kinase-Akt pathway, and persistent MEK signaling, lead to enhanced death in Btk-deficient macrophages only downstream of inflammatory triggers. These data underline the complex role of Btk in the regulation of macrophage survival and function.