Charlotta Böiers

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.

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.

Downregulation 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.

Impact of gene dosage, loss of wild-type allele, and FLT3 ligand on Flt3-ITD-induced myeloproliferation

Acquisition of homozygous activating growth factor receptor mutations might accelerate cancer progression through a simple gene-dosage effect. Internal tandem duplications (ITDs) of FLT3 occur in approximately 25% cases of acute myeloid leukemia and induce ligand-independent constitutive signaling. Homozygous FLT3-ITDs confer an adverse prognosis and are frequently detected at relapse. Using a mouse knockin model of Flt3-internal tandem duplication (Flt3-ITD)-induced myeloproliferation, we herein demonstrate that the enhanced myeloid phenotype and expansion of granulocyte-monocyte and primitive Lin(-)Sca1(+)c-Kit(+) progenitors in Flt3-ITD homozygous mice can in part be mediated through the loss of the second wild-type allele. Further, whereas autocrine FLT3 ligand production has been implicated in FLT3-ITD myeloid malignancies and resistance to FLT3 inhibitors, we demonstrate here that the mouse Flt3(ITD/ITD) myeloid phenotype is FLT3 ligand-independent.

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.

Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice

Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19(+) B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R(+)CD19(+) ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R(+) myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R(+) myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported.

TSLP-mediated fetal B lymphopoiesis?

To the editor: In the August 2003 issue of Nature Immunology1, as well as in later studies2, Voβhenrich et al. presented experimental data on the cytokine requirements of developing B cells, which led them to conclude that “TSLP [thymic stromal lymphopoietin] is the factor responsible for most of the fetal and perinatal B cell production that takes place when the IL-7–γc [interleukin 7–common γ-chain] signaling pathway is disrupted.”1 Although the data reported were technically sound and compatible with such a conclusion, the authors did not provide direct evidence to support (or exclude) the idea of a critical function for TSLP in IL-7-independent fetal B lymphopoiesis. The conclusions of Voβhenrich et al. were based on the demonstration that B lymphopoiesis was much more affected (tenfold more) in mice deficient in IL-7 receptor α-chain, essential for IL-7 as well as TSLP signaling, than in mice deficient in the common γ-chain (γc), required for IL-7 but not TSLP-mediated signaling3,4. However, these data could at best be considered strong indirect support for the idea of TSLP as the main cytokine driving IL-7-independent fetal B lymphopoiesis, as there could be other reasons for a difference in the phenotypes of γcdeficient mice and those deficient in the IL-7 receptor α-chain. Furthermore, Voβhenrich et al. used bone marrow of mice 4–12 weeks of age, not fetal liver, for their comparative in vivo analysis of B lymphopoiesis in these mice1,2. Instead, the extrapolation to the idea that TSLP is key to the fetal stages of B lymphopoiesis was based on the finding that fetal but not adult pro–B cells were responsive to TSLP in vitro1,2. In contrast, a lack of an important function for TSLP in adult B lymphopoiesis has been indicated by studies of TSLP receptor–deficient (Tpte2–/–) mice5,6. As fetal lymphopoiesis had not been examined in singly deficient Tslp–/– or Tpte2–/– mice, we investigated B lymphopoiesis in the livers of Tpte2–/– mice at embryonic day 17.5 but found no deficiency in Tpte2–/– fetuses at any stage of B cell development (Fig. 1a and Supplementary Fig. 1 online). Furthermore, when comparing B lymphopoiesis in the fetal livers of Il7–/– and Il7–/–Tpte2–/– mice, we obtained no evidence for substantial involvement of TSLP in IL-7independent regulation of fetal pro–B cells or pre–B cells, whereas we noted a slight additional reduction in the number of immature B cells in Il7–/–Tpte2–/– fetuses relative to that in Il7–/– fetuses (Fig. 1b and Supplementary Fig. 1). Thus, although Voβhenrich et al. provided compelling evidence that fetal pro–B cells are highly responsive to TSLP1, our studies of Tpte2–/– and Il7–/–Tpte2–/– fetuses fail to support their claim that TSLP is the most important cytokine promoting IL-7independent fetal B lymphopoiesis. Instead, although Voβhenrich et al. also concluded that “Flk-2 is involved, but TSLP is the main factor driving IL-7-independent fetal and perinatal lymphopoiesis,”1 we have done additional studies of mice deficient in the cytokine Flt3L (also called Flk-2 ligand) and IL-7 (Flt3l–/–Il7–/– mice) and of Flt3l–/–Tpte2–/– mice and have found that the reported complete loss of B-1 as well as B-2 B lymphopoiesis in Flt3l–/–Il7r–/– mice7 and Flk2–/–Il7r–/– mice1 is entirely due to the simultaneous loss of function of IL-7 and Flt3L (C.T.J. and S.E.W.J., unpublished observations). Collectively, our findings suggest that Flt3L rather than TSLP is the key regulator of IL-7-independent B lymphopoiesis and that intact TSLP function is insufficient to restore any detectable B lymphopoiesis in the absence of these two critical regulators of B cell progenitors.

LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR11. Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2–8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9–11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death inxa0Hoil-1−/− (also known as Rbck1−/−) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3−/−Casp8−/−Hoil-1−/− embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.The HOIL-1 component of the LUBAC ubiquitin ligase complex is required for LUBAC activity, which prevents lethality during embryogenesis by preventing aberrant TNFR1-mediated endothelial cell death and RIPK1-mediated defects in haematopoiesis.

Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell–restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs.

Single-Cell Analysis Identifies Distinct Stages of Human Endothelial-to-Hematopoietic Transition

During development, hematopoietic cells originate from endothelium in a process known as endothelial-to-hematopoietic transition (EHT). To study human EHT, we coupled flow cytometry and single-cell transcriptional analyses of human pluripotent stem cell-derived CD34+ cells. The resulting transcriptional hierarchy showed a continuum of endothelial and hematopoietic signatures. At the interface of these two signatures, a unique group of cells displayed both an endothelial signature and high levels of key hematopoietic stem cell-associated genes. This interphase group was validated via sort and subculture as an immediate precursor to hematopoietic cells. Differential expression analyses further divided this population into subgroups, which, upon subculture, showed distinct hematopoietic lineage differentiation potentials. We therefore propose that immediate precursors to hematopoietic cells already have their hematopoietic lineage restrictions defined prior to complete downregulation of the endothelial signature. These findings increase our understanding of the processes of de novo hematopoietic cell generation in the human developmental context.