B Lymphocyte Specification Is Preceded by Extensive Epigenetic Priming in Multipotent Progenitors.

B lymphocyte development is dependent on the interplay between the chromatin landscape and lineage-specific transcription factors. It has been suggested that B lineage commitment is associated with major changes in the nuclear chromatin environment, proposing a critical role for lineage-specific transcription factors in the formation of the epigenetic landscape. In this report, we have used chromosome conformation capture in combination with assay for transposase-accessible chromatin sequencing analysis to enable highly efficient annotation of both proximal and distal transcriptional control elements to genes activated in B lineage specification in mice. A large majority of these genes were annotated to at least one regulatory element with an accessible chromatin configuration in multipotent progenitors. Furthermore, the majority of binding sites for the key regulators of B lineage specification, EBF1 and PAX5, occurred in already accessible regions. EBF1 did, however, cause a dynamic change in assay for transposase-accessible chromatin accessibility and was critical for an increase in distal promoter-enhancer interactions. Our data unravel an extensive epigenetic priming at regulatory elements annotated to lineage-restricted genes and provide insight into the interplay between the epigenetic landscape and transcription factors in cell specification.

EBF1 and PAX5 control pro-B cell expansion via opposing regulation of the Myc gene.

Genes encoding B lineage-restricted transcription factors are frequently mutated in B-lymphoid leukemias, suggesting a close link between normal and malignant B-cell development. One of these transcription factors is early B-cell factor 1 (EBF1), a protein of critical importance for lineage specification and survival of B-lymphoid progenitors. Here, we report that impaired EBF1 function in mouse B-cell progenitors results in reduced expression of Myc. Ectopic expression of MYC partially rescued B-cell expansion in the absence of EBF1 both in vivo and in vitro. Using chromosome conformation analysis in combination with ATAC-sequencing, chromatin immunoprecipitation-sequencing, and reporter gene assays, six EBF1-responsive enhancer elements were identified within the Myc locus. CRISPR-Cas9-mediated targeting of EBF1-binding sites identified one element of key importance for Myc expression and pro-B cell expansion. These data provide evidence that Myc is a direct target of EBF1. Furthermore, chromatin immunoprecipitation-sequencing analysis revealed that several regulatory elements in the Myc locus are targets of PAX5. However, ectopic expression of PAX5 in EBF1-deficient cells inhibits the cell cycle and reduces Myc expression, suggesting that EBF1 and PAX5 act in an opposing manner to regulate Myc levels. This hypothesis is further substantiated by the finding that Pax5 inactivation reduces requirements for EBF1 in pro-B-cell expansion. The binding of EBF1 and PAX5 to regulatory elements in the human MYC gene in a B-cell acute lymphoblastic leukemia cell line indicates that the EBF1:PAX5:MYC regulatory loop is conserved and may control both normal and malignant B-cell development.

Lin28b controls a neonatal to adult switch in B cell positive selection.

The ability of B-1 cells to become positively selected into the mature B cell pool, despite being weakly self-reactive, has puzzled the field since its initial discovery. Here, we explore changes in B cell positive selection as a function of developmental time by exploiting a link between CD5 surface levels and the natural occurrence of self-reactive B cell receptors (BCRs) in BCR wild-type mice. We show that the heterochronic RNA binding protein Lin28b potentiates a neonatal mode of B cell selection characterized by enhanced overall positive selection in general and the developmental progression of CD5+ immature B cells in particular. Lin28b achieves this by amplifying the CD19/PI3K/c-Myc positive feedback loop, and ectopic Lin28b expression restores both positive selection and mature B cell numbers in CD19-/- adult mice. Thus, the temporally restricted expression of Lin28b relaxes the rules for B cell selection during ontogeny by modulating tonic signaling. We propose that this neonatal mode of B cell selection represents a cell-intrinsic cue to accelerate the de novo establishment of the adaptive immune system and incorporate a layer of natural antibody-mediated immunity throughout life.

Dissection of progenitor compartments resolves developmental trajectories in B-lymphopoiesis.

To understand the developmental trajectories in early lymphocyte differentiation, we identified differentially expressed surface markers on lineage-negative lymphoid progenitors (LPs). Single-cell polymerase chain reaction experiments allowed us to link surface marker expression to that of lineage-associated transcription factors (TFs) and identify GFRA2 and BST1 as markers of early B cells. Functional analyses in vitro and in vivo as well as single-cell gene expression analyses supported that surface expression of these proteins defined distinct subpopulations that include cells from both the classical common LPs (CLPs) and Fraction A compartments. The formation of the GFRA2-expressing stages of development depended on the TF EBF1, critical both for the activation of stage-specific target genes and modulation of the epigenetic landscape. Our data show that consecutive expression of Ly6D, GFRA2, and BST1 defines a developmental trajectory linking the CLP to the CD19+ progenitor compartment.

Identification of Stage-Specific Surface Markers in Early B Cell Development Provides Novel Tools for Identification of Progenitor Populations.

Whereas the characterization of B lymphoid progenitors has been facilitated by the identification of lineage- and stage-specific surface markers, the continued identification of differentially expressed proteins increases our capacity to explore normal and malignant B cell development. To identify novel surface markers with stage-specific expression patterns, we explored the reactivity of CD19(+) B cell progenitor cells to Abs targeted to 176 surface proteins. Markers with stage-specific expression were identified using a transgenic reporter gene system subdividing the B cell progenitors into four surface IgM(-) stages. This approach affirmed the utility of known stage-specific markers, as well as identifying additional proteins that selectively marked defined stages of B cell development. Among the stage-specific markers were the cell adhesion proteins CD49E, CD11A, and CD54 that are highly expressed selectively on the most immature progenitors. This work identifies a set of novel stage-specific surface markers that can be used as a complement to the classical staining protocols to explore B lymphocyte development.

Exploring the multifaceted nature of the common lymphoid progenitor compartment.

While the common lymphoid progenitor compartment was originally thought to be a rather homogenous cell population, it has become increasingly clear that this compartment is highly heterogeneous both with regard to phenotypic and functional features. The exploration of this cellular complexity has generated novel molecular insights into regulatory events in lymphoid lineage restriction and provided support for the idea that multiple lineage restriction events occur at this developmental stage. Furthermore, the identification of multiple lineage-restricted progenitors with mixed lineage potential challenges a strictly hierarchical model for lymphoid development. Instead we propose a model based on competence windows during which cell fates are established through the action of lineage determining factors.

Lymphomyeloid contribution of an immune-restricted progenitor emerging prior to definitive hematopoietic stem cells.

In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis.

Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.

The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding--a common and life-threatening side effect of many cancer therapies--and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output.

Expression and role of FLT3 in regulation of the earliest stage of normal granulocyte-monocyte progenitor development.

Mice deficient in c-fms-like tyrosine kinase 3 (FLT3) signaling have reductions in early multipotent and lymphoid progenitors, whereas no evident myeloid phenotype has been reported. However, activating mutations of Flt3 are among the most common genetic events in acute myeloid leukemia and mice harboring internal tandem duplications within Flt3 (Flt3-ITD) develop myeloproliferative disease, with characteristic expansion of granulocyte-monocyte (GM) progenitors (GMP), possibly compatible with FLT3-ITD promoting a myeloid fate of multipotent progenitors. Alternatively, FLT3 might be expressed at the earliest stages of GM development. Herein, we investigated the expression, function, and role of FLT3 in recently identified early GMPs. Flt3-cre fate-mapping established that most progenitors and mature progeny of the GM lineage are derived from Flt3-expressing progenitors. A higher expression of FLT3 was found in preGMP compared with GMP, and preGMPs were more responsive to stimulation with FLT3 ligand (FL). Whereas preGMPs and GMPs were reduced in Fl(-/-) mice, megakaryocyte-erythroid progenitors were unaffected and lacked FLT3 expression. Notably, mice deficient in both thrombopoietin (THPO) and FL had a more pronounced GMP phenotype than Thpo(-/-) mice, establishing a role of FL in THPO-dependent and -independent regulation of GMPs, of likely significance for myeloid malignancies with Flt3-ITD mutations.

FLT3 ligand and not TSLP is the key regulator of IL-7-independent B-1 and B-2 B lymphopoiesis.

Phenotypically and functionally distinct progenitors and developmental pathways have been proposed to exist for fetally derived B-1 and conventional B-2 cells. Although IL-7 appears to be the primary cytokine regulator of fetal and adult B lymphopoiesis in mice, considerable fetal B lymphopoiesis and postnatal B cells are sustained in the absence of IL-7; in humans, B-cell generation is suggested to be largely IL-7-independent, as severe combined immune-deficient patients with IL-7 deficiency appear to have normal B-cell numbers. However, the role of other cytokines in IL-7-independent B lymphopoiesis remains to be established. Although thymic stromal lymphopoietin (TSLP) has been proposed to be the main factor driving IL-7-independent B lymphopoiesis and to distinguish fetal from adult B-cell progenitor development in mice, recent studies failed to support a primary role of TSLP in IL-7-independent fetal B-cell development. However, the role of TSLP in IL-7-independent adult B lymphopoiesis and in particular in regulation of B-1 cells remains to be established. Here we demonstrate that, rather than TSLP, IL-7 and FLT3 ligand are combined responsible for all B-cell generation in mice, including recently identified B-1-specified cell progenitors. Thus, the same IL-7- and FLT3 ligand-mediated signal-ing regulates alternative pathways of fetal and adult B-1 and B-2 lymphopoiesis.

Kit regulates maintenance of quiescent hematopoietic stem cells.

Hematopoietic stem cell (HSC) numbers are tightly regulated and maintained in postnatal hematopoiesis. Extensive studies have supported a role of the cytokine tyrosine kinase receptor Kit in sustaining cycling HSCs when competing with wild-type HSCs posttransplantation, but not in maintenance of quiescent HSCs in steady state adult bone marrow. In this study, we investigated HSC regulation in White Spotting 41 (Kit(W41/W41)) mice, with a partial loss of function of Kit. Although the extensive fetal HSC expansion was Kit-independent, adult Kit(W41/W41) mice had an almost 2-fold reduction in long-term HSCs, reflecting a loss of roughly 10,000 Lin(-)Sca-1(+)Kit(high) (LSK)CD34(-)Flt3(-) long-term HSCs by 12 wk of age, whereas LSKCD34(+)Flt3(-) short-term HSCs and LSKCD34(+)Flt3(+) multipotent progenitors were less affected. Whereas homing and initial reconstitution of Kit(W41/W41) bone marrow cells in myeloablated recipients were close to normal, self-renewing Kit(W41/W41) HSCs were progressively depleted in not only competitive but also noncompetitive transplantation assays. Overexpression of the anti-apoptotic regulator BCL-2 partially rescued the posttransplantation Kit(W41/W41) HSC deficiency, suggesting that Kit might at least in the posttransplantation setting in part sustain HSC numbers by promoting HSC survival. Most notably, accelerated in vivo BrdU incorporation and cell cycle kinetics implicated a previously unrecognized role of Kit in maintaining quiescent HSCs in steady state adult hematopoiesis.

Down-regulation of Mpl marks the transition to lymphoid-primed multipotent progenitors with gradual loss of granulocyte-monocyte potential.

Evidence for a novel route of adult hematopoietic stem-cell lineage commitment through Lin-Sca-1+Kit+Flt3hi (LSKFlt3hi) lymphoid-primed multipotent progenitors (LMPPs) with granulocyte/monocyte (GM) and lymphoid but little or no megakaryocyte/erythroid (MkE) potential was recently challenged, as LSKFlt3hi cells were reported to possess MkE potential. Herein, residual (1%-2%) MkE potential segregated almost entirely with LSKFlt3hi cells expressing the thrombopoietin receptor (Mpl), whereas LSKFlt3hiMpl- LMPPs lacked significant MkE potential in vitro and in vivo, but sustained combined GM and lymphoid potentials, and coexpressed GM and lymphoid but not MkE transcriptional lineage programs. Gradually increased transcriptional lymphoid priming in single LMPPs from Rag1GFP mice was shown to occur in the presence of maintained GM lineage priming, but gradually reduced GM lineage potential. These functional and molecular findings reinforce the existence of GM/lymphoid-restricted progenitors with dramatically down-regulated probability for committing toward MkE fates, and support that lineage restriction occurs through gradual rather than abrupt changes in specific lineage potentials.

Permissive roles of hematopoietin and cytokine tyrosine kinase receptors in early T-cell development.

Although several cytokines have been demonstrated to be critical regulators of development of multiple blood cell lineages, it remains disputed to what degree they act through instructive or permissive mechanisms. Signaling through the FMS-like tyrosine kinase 3 (FLT3) receptor and the hematopoietin IL-7 receptor alpha (IL-7Ralpha) has been demonstrated to be of critical importance for sustained thymopoiesis. Signaling triggered by IL-7 and thymic stromal lymphopoietin (TSLP) is dependent on IL-7Ralpha, and both ligands have been implicated in T-cell development. However, we demonstrate that, whereas thymopoiesis is abolished in adult mice doubly deficient in IL-7 and FLT3 ligand (FLT3L), TSLP does not play a key role in IL-7-independent or FLT3L-independent T lymphopoiesis. Furthermore, whereas previous studies implicated that the role of other cytokine tyrosine kinase receptors in T lymphopoiesis might not involve permissive actions, we demonstrate that ectopic expression of BCL2 is sufficient not only to partially correct the T-cell phenotype of Flt3l(-/-) mice but also to rescue the virtually complete loss of all discernable stages of early T lymphopoiesis in Flt3l(-/-)Il7r(-/-) mice. These findings implicate a permissive role of cytokine receptors of the hematopoietin and tyrosine kinase families in early T lymphopoiesis.

Critical role of FLT3 ligand in IL-7 receptor independent T lymphopoiesis and regulation of lymphoid-primed multipotent progenitors.

The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow (BM) stem/progenitor cells that continuously replace thymic progenitors remain largely unknown. Herein, we show that fms-like tyrosine kinase 3 (Flt3) ligand (Fl)-deficient mice have distinct reductions in the earliest thymic progenitors in fetal, postnatal, and adult thymus. A critical role of FL in thymopoiesis was particularly evident in the absence of interleukin-7 receptor alpha (IL-7Ralpha) signaling. Fl-/-Il-7r-/- mice have extensive reductions in fetal and postnatal thymic progenitors that result in a loss of active thymopoiesis in adult mice, demonstrating an indispensable role of FL in IL-7Ralpha-independent fetal and adult T lymphopoiesis. Moreover, we establish a unique and critical role of FL, distinct from that of IL-7Ralpha, in regulation of the earliest lineage-negative (Lin(-)) Lin(-)SCA1+KIT+ (LSK) FLT3(hi) lymphoid-primed multipotent progenitors in BM, demonstrating a key role of FLT3 signaling in regulating the very earliest stages of lymphoid progenitors.

Ectopic expression of PAX5 promotes maintenance of biphenotypic myeloid progenitors coexpressing myeloid and B-cell lineage-associated genes.

The transcription factor PAX5 is a critical regulator of B-cell commitment and development. Although normally not expressed in myeloid progenitors, PAX5 has recently been shown to be frequently expressed in myeloid malignancies and to suppress expression of myeloid differentiation genes, compatible with an effect on the differentiation or maintenance of myeloid progenitors. However, previous studies in which PAX5 was ectopically expressed in normal myeloid progenitors in vivo and in vitro provided conflicting results as to the effect of PAX5 on myeloid development. Herein, we demonstrate that on ectopic expression of PAX5 in bone marrow multipotent stem/progenitor cells, cells with a biphenotypic B220(+)GR-1/MAC-1(+) phenotype are produced. These remain cytokine-dependent, but unlike control-transduced cells they sustain long-term generation of myeloid progenitors in vitro and remain capable of myeloid differentiation. Notably, PAX5(+)B220(+)GR-1/MAC-1(+) myeloid progenitors coexpress, at the single-cell level, myeloid genes and otherwise B-cell-specific PAX5 target genes. These findings establish that ectopic expression of PAX5 introduces extensive self-renewal properties in otherwise short-lived myeloid progenitors. Along with the established ectopic expression of PAX5 in acute myeloid leukemia, this motivates a careful investigation of the potential involvement of ectopic PAX5 expression in myeloid and biphenotypic leukemias.

Critical role of thrombopoietin in maintaining adult quiescent hematopoietic stem cells.

The role of cytokines in regulation of hematopoietic stem cells (HSCs) remains poorly understood. Herein we demonstrate that thrombopoietin (THPO) and its receptor, MPL, are critically involved in postnatal steady-state HSC maintenance, reflected in a 150-fold reduction of HSCs in adult Thpo(-/-) mice. Further, whereas THPO and MPL proved not required for fetal HSC expansion, HSC expansion posttransplantation was highly MPL and THPO dependent. The distinct role of THPO in postnatal HSC maintenance is accompanied by accelerated HSC cell-cycle kinetics in Thpo(-/-) mice and reduced expression of the cyclin-dependent kinase inhibitors p57(Kip2) and p19(INK4D) as well as multiple Hox transcription factors. Although also predicted to be an HSC viability factor, BCL2 failed to rescue the HSC deficiency of Thpo(-/-) mice. Thus, THPO regulates posttransplantation HSC expansion as well as the maintenance of adult quiescent HSCs, of critical importance to avoid postnatal HSC exhaustion.

Cytokines regulate postnatal hematopoietic stem cell expansion: opposing roles of thrombopoietin and LNK.

The role of cytokines as regulators of hematopoietic stem cell (HSC) expansion remains elusive. Herein, we identify thrombopoietin (THPO) and the cytokine signaling inhibitor LNK, as opposing physiological regulators of HSC expansion. Lnk(-/-) HSCs continue to expand postnatally, up to 24-fold above normal by 6 mo of age. Within the stem cell compartment, this expansion is highly selective for self-renewing long-term HSCs (LT-HSCs), which show enhanced THPO responsiveness. Lnk(-/-) HSC expansion is dependent on THPO, and 12-wk-old Lnk(-/-)Thpo(-/-) mice have 65-fold fewer LT-HSCs than Lnk(-/-) mice. Expansions of multiple myeloid, but not lymphoid, progenitors in Lnk(-/-) mice also proved THPO-dependent.

Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment.

All blood cell lineages derive from a common hematopoietic stem cell (HSC). The current model implicates that the first lineage commitment step of adult pluripotent HSCs results in a strict separation into common lymphoid and common myeloid precursors. We present evidence for a population of cells which, although sustaining a high proliferative and combined lympho-myeloid differentiation potential, have lost the ability to adopt erythroid and megakaryocyte lineage fates. Cells in the Lin-Sca-1+c-kit+ HSC compartment coexpressing high levels of the tyrosine kinase receptor Flt3 sustain granulocyte, monocyte, and B and T cell potentials but in contrast to Lin-Sca-1+c-kit+Flt3- HSCs fail to produce significant erythroid and megakaryocytic progeny. This distinct lineage restriction site is accompanied by downregulation of genes for regulators of erythroid and megakaryocyte development. In agreement with representing a lymphoid primed progenitor, Lin-Sca-1+c-kit+CD34+Flt3+ cells display upregulated IL-7 receptor gene expression. Based on these observations, we propose a revised road map for adult blood lineage development.