Ewa Sitnicka

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.

Upregulation of Flt3 Expression within the Bone Marrow Lin−Sca1+c-kit+ Stem Cell Compartment Is Accompanied by Loss of Self-Renewal Capacity

Flt3 has emerged as a potential regulator of hematopoietic stem cells (HSC). Sixty percent of cells in the mouse marrow Lin(-)Sca1(+)c-kit(+) HSC pool expressed flt3. Although single cell cloning showed comparable high proliferative, myeloid, B, and T cell potentials of Lin(-)Sca1(+)c-kit(+)flt3(+) and Lin(-)Sca1(+)c-kit(+)flt3(-) cells, only Lin(-)Sca1(+)c-kit(+)flt3(-) cells supported sustained multilineage reconstitution. In striking contrast, Lin(-)Sca1(+)c-kit(+)flt3(+) cells rapidly and efficiently reconstituted B and T lymphopoiesis, whereas myeloid reconstitution was exclusively short term. Unlike c-kit, activation of flt3 failed to support survival of HSC, whereas only flt3 mediated survival of Lin(-)Sca1(+)c-kit(+)flt3(+) reconstituting cells. Phenotypic and functional analysis support that Lin(-)Sca1(+)c-kit(+)flt3(+) cells are progenitors for the common lymphoid progenitor. Thus, upregulation of flt3 expression on Lin(-)Sca1(+)c-kit(+) HSC cells is accompanied by loss of self-renewal capacity but sustained lymphoid-restricted reconstitution potential.

Key Role of flt3 Ligand in Regulation of the Common Lymphoid Progenitor but Not in Maintenance of the Hematopoietic Stem Cell Pool

The first lineage commitment step of hematopoietic stem cells (HSC) results in separation into distinct lymphoid and myeloid differentiation pathways, reflected in the generation of common lymphoid and myeloid progenitors (CLP and CMP, respectively). In this report we present the first evidence for a nonredundant regulator of this process, in that adult mice deficient in expression of the flt3 ligand (FL) have severely (10-fold) reduced levels of the CLP, accompanied by reductions in the earliest identifiable B and T cell progenitors. In contrast, CMP and HSC are unaffected in FL-deficient mice. Noteworthy, CLP express high levels of both the flt3 receptor and ligand, indicating a potential autocrine role of FL in regulation of the earliest lymphoid commitment step from HSC.

Complementary signaling through flt3 and interleukin-7 receptor alpha is indispensable for fetal and adult B cell genesis

Extensive studies of mice deficient in one or several cytokine receptors have failed to support an indispensable role of cytokines in development of multiple blood cell lineages. Whereas B1 B cells and Igs are sustained at normal levels throughout life of mice deficient in IL-7, IL-7Rα, common cytokine receptor gamma chain, or flt3 ligand (FL), we report here that adult mice double deficient in IL-7Rα and FL completely lack visible LNs, conventional IgM+ B cells, IgA+ plasma cells, and B1 cells, and consequently produce no Igs. All stages of committed B cell progenitors are undetectable in FL−/− × IL-7Rα−/− BM that also lacks expression of the B cell commitment factor Pax5 and its direct target genes. Furthermore, in contrast to IL-7Rα−/− mice, FL−/− × IL-7Rα−/− mice also lack mature B cells and detectable committed B cell progenitors during fetal development. Thus, signaling through the cytokine tyrosine kinase receptor flt3 and IL-7Rα are indispensable for fetal and adult B cell development.

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.

IFN-{gamma} Negatively Modulates Self-Renewal of Repopulating Human Hemopoietic Stem Cells.

Whereas multiple growth-promoting cytokines have been demonstrated to be involved in regulation of the hemopoietic stem cell (HSC) pool, the potential role of negative regulators is less clear. However, IFN-γ, if overexpressed, can mediate bone marrow suppression and has been directly implicated in a number of bone marrow failure syndromes, including graft-vs-host disease. Whether IFN-γ might directly affect the function of repopulating HSCs has, however, not been investigated. In the present study, we used in vitro conditions promoting self-renewing divisions of human HSCs to investigate the effect of IFN-γ on HSC maintenance and function. Although purified cord blood CD34+CD38− cells underwent cell divisions in the presence of IFN-γ, cycling HSCs exposed to IFN-γ in vitro were severely compromised in their ability to reconstitute long-term cultures in vitro and multilineage engraft NOD-SCID mice in vivo (>90% reduced activity in both HSC assays). In vitro studies suggested that IFN-γ accelerated differentiation of targeted human stem and progenitor cells. These results demonstrate that IFN-γ can negatively affect human HSC self-renewal.

Distinct myeloid progenitor-differentiation pathways identified through single-cell RNA sequencing

According to current models of hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs) (Lin−Sca-1+c-Kit+CD34+Flt3hi) and common myeloid progenitors (CMPs) (Lin−Sca-1+c-Kit+CD34+CD41hi) establish an early branch point for separate lineage-commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloid-restricted pre–granulocyte-macrophage progenitor (pre-GM) (Lin−Sca-1−c-Kit+CD41−FcγRII/III−CD150−CD105−). By single-cell transcriptome profiling of pre-GMs, we identified distinct myeloid differentiation pathways: a pathway expressing the gene encoding the transcription factor GATA-1 generated mast cells, eosinophils, megakaryocytes and erythroid cells, and a pathway lacking expression of that gene generated monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic-lineage bifurcation that separates the myeloid lineages before their segregation from other hematopoietic-lineage potential.

Identification of a NK/T cell restricted progenitor in adult bone marrow contributing to bone marrow and thymic-dependent NK cells

Although bone marrow (BM) is the main site of natural killer (NK)-cell development in adult mice, recent studies have identified a distinct thymic-dependent NK pathway, implicating a possible close link between NK- and T-cell development in adult hematopoiesis. To investigate whether a potential NK-/T-lineage restriction of multipotent progenitors might take place already in the BM, we tested the full lineage potentials of NK-cell progenitors in adult BM. Notably, although Lin(-)CD122(+)NK1.1(-)DX5(-) NK-cell progenitors failed to commit to the B and myeloid lineages, they sustained a combined NK- and T-cell potential in vivo and in vitro at the single-cell level. Whereas T-cell development from NK/T progenitors is Notch-dependent, their contribution to thymic and BM NK cells remains Notch-independent. These findings demonstrate the existence of bipotent NK-/T-cell progenitors in adult BM.

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.

FLT3-ITDs Instruct a Myeloid Differentiation and Transformation Bias in Lymphomyeloid Multipotent Progenitors.

Summary Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies.