Lymphoid-primed Multipotent Progenitors Research Articles

Diverse and heritable lineage imprinting of early haematopoietic progenitors

Haematopoietic stem cells (HSCs) and their subsequent progenitors produce blood cells, but the precise nature and kinetics of this production is a contentious issue. In one model, lymphoid and myeloid production branch after the lymphoid-primed multipotent progenitor (LMPP), with both branches subsequently producing dendritic cells. However, this model is based mainly on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single-cell complexity. Here we avoid these issues by using a new quantitative version of 'cellular barcoding' to trace the in vivo fate of hundreds of LMPPs and HSCs at the single-cell level. These data demonstrate that LMPPs are highly heterogeneous in the cell types that they produce, separating into combinations of lymphoid-, myeloid- and dendritic-cell-biased producers. Conversely, although we observe a known lineage bias of some HSCs, most cellular output is derived from a small number of HSCs that each generates all cell types. Crucially, in vivo analysis of the output of sibling cells derived from single LMPPs shows that they often share a similar fate, suggesting that the fate of these progenitors was imprinted. Furthermore, as this imprinting is also observed for dendritic-cell-biased LMPPs, dendritic cells may be considered a distinct lineage on the basis of separate ancestry. These data suggest a 'graded commitment' model of haematopoiesis, in which heritable and diverse lineage imprinting occurs earlier than previously thought.

Proliferation-Dependent Alterations of the DNA Methylation Landscape Underlie Hematopoietic Stem Cell Aging

The functional potential of hematopoietic stem cells (HSCs) declines during aging, and in doing so, significantly contributes to hematopoietic pathophysiology in the elderly. To explore the relationship between age-associated HSC decline and the epigenome, we examined global DNA methylation of HSCs during ontogeny in combination with functional analysis. Although the DNA methylome is generally stable during aging, site-specific alterations of DNA methylation occur at genomic regions associated with hematopoietic lineage potential and selectively target genes expressed in downstream progenitor and effector cells. We found that age-associated HSC decline, replicative limits, and DNA methylation are largely dependent on the proliferative history of HSCs, yet appear to be telomere-length independent. Physiological aging and experimentally enforced proliferation of HSCs both led to DNA hypermethylation of genes regulated by Polycomb Repressive Complex 2. Our results provide evidence that epigenomic alterations of the DNA methylation landscape contribute to the functional decline of HSCs during aging.

Pbx1 restrains myeloid maturation while preserving lymphoid potential in hematopoietic progenitors

The capacity of the hematopoietic system to promptly respond to peripheral demands relies on adequate pools of progenitors able to transiently proliferate and differentiate in a regulated manner. However, little is known about factors that may restrain progenitor maturation to maintain their reservoirs. Conditional knockout mice for the Pbx1 proto-oncogene have a significant reduction in lineage-restricted progenitors in addition to a profound defect in hematopoietic stem cell (HSC) self-renewal. Through analysis of purified progenitor proliferation, differentiation capacity and transcriptional profiling, we demonstrate that Pbx1 regulates the lineage-specific output of multipotent and oligopotent progenitors. In the absence of Pbx1 multipotent progenitor (MPP) and common myeloid progenitor (CMP) pools are reduced due to aberrantly rapid myeloid maturation. This is associated with premature expression of myeloid differentiation genes and decreased maintenance of proto-oncogene transcriptional pathways, including reduced expression of Meis1, a Pbx1 dimerization partner, and its subordinate transcriptional program. Conversely, Pbx1 maintains the lymphoid differentiation potential of lymphoid-primed MPPs (LMPPs) and common lymphoid progenitors (CLPs), whose reduction in the absence of Pbx1 is associated with a defect in lymphoid priming that is also present in CMPs, which persistently express lymphoid and HSC genes underlying a previously unappreciated lineage promiscuity that is maintained by Pbx1. These results demonstrate a role for Pbx1 in restraining myeloid maturation while maintaining lymphoid potential to appropriately regulate progenitor reservoirs.

Prospective Isolation of the Earliest Common Myeloid Progenitor in Adult Hematopoiesis

Abstract 768In murine hematopoiesis, hematopoietic stem cells (HSCs) and multi-potent progenitors (MPPs) have been identified within the LSK (Lin− Sca-1+ c-kit+) fraction of bone marrow cells (Morrison SJ and Weissman IL Immunity 1994). We have proposed that the first commitment step at the myeloid versus lymphoid bifurcation occurred outside the LSK fraction, where myeloid or lymphoid lineage-committed progenitors such as common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) are prospectively isolated (Akashi et al Nature 2000). However, recent studies revealed that the initial commitment step might occur within LSK fraction. Adolfsson et al showed that a fraction of LSK cells expressing Flt3 at a high level have lost megakaryocyte/erythroid (MegE) potential and were largely primed for granulocyte/monocyte (GM) and lymphoid cells. This population was named lymphoid-primed multipotent progenitors (LMPPs) (Adolfsson et al Cell 2005). In our hands, by utilizing mice harboring a fluorescent reporter for GATA-1 transcription factor, we demonstrated that the initial upregulation of GATA-1 transcription factor was observed within MPP population, which was defined as CD34+ LSK fraction, and GATA-1+ MPPs were capable of generating only myelo-erythroid cells but lacked lymphoid potential (Arinobu et al Cell Stem Cell 2007). This result strongly suggested that the initial myelo-erythroid commitment occurs at the MPP stage.To isolate the earliest myelo-erythroid LSK progenitors in normal mice without utilizing the GATA-1 reporter system, we conducted expression profiling of GATA-1+ MPP population by cDNA microarray analyses and following validation by FACS analyses. We found that a cell-surface antigen CD41 was specifically expressed at a high level in GATA-1+ MPP cells, indicating that CD41hi MPP cells might be the corresponding population to GATA-1+ MPP cells.CD41hi MPPs gave rise exclusively to GM and MegE colonies in vitro, but no lymphoid colonies were observed even under lymphoid-inducible conditions such as co-culture with OP9 or OP9-DL1 stromal cell layer. In vivo, CD41hi MPPs showed differentiation potential into GM cells, erythroid cells and platelets, lacking B or T lymphoid cell-producing potential in congenic transplantation assay. While the GM cell-reconstitution potential of LMPPs and CMPs peaked around day10 and day4 after transplantation, respectively, CD41hi MPPs had their peak around 3 weeks and relatively long-lasting reconstitution potential, presumably reflecting their immaturity. To further evaluate differentiation potential more directly and quantitatively, CD41hi MPPs with LMPPs or CMPs were injected together into one individual recipient mouse. In this head-to-head competitive assay system, CD41hi MPPs generated a plenty of mature GM cells, whose numbers were nearly ten times of those produced from the original CMPs or LMPPs in a reconstitution setting. Thus CD41hi MPPs possess potent restricted-lineage differentiation potential into GM and MegE lineages.In addition, the gene expression analysis by single-cell quantitative real-time PCR and cDNA microarray revealed that the CD41hi MPP population expresses both GM- and MegE-affiliated genes, but not lymphoid genes, reflecting their lineage restriction. More importantly, each single CD41hi MPP cell expresses either one or both of GATA-1 and PU.1 at a low level, suggesting that the promiscuous expression of these transcription factors might play a critical role in myelo-erythroid lineage commitment (Miyamoto et al Developmental Cell 2002).Furthermore, to evaluate the functional importance of CD41hi MPP population, we created the systemic infectious model mice, mimicking the severe peritonitis. CD41hi MPP pool as well as GMP expands dramatically in response to an increased demand for GM cells, while LMPP does not; thus, CD41hi MPPs compose physiologically important pathway in myelo-erythropoiesis.Accordingly, CD41hi MPP population might represent the earliest branch point for myelo-erythroid development, which resides upstream of the conventional CMP. Based on these data, we propose to redefine the true CMP as CD41hi MPP population. This CD41hi earliest myelo-erythroid progenitor should be useful to investigate the molecular mechanisms for hematopoietic lineage fate decision. Disclosures:No relevant conflicts of interest to declare.

STAT5 Promotes Hematopoietic Stem Cell Maintenance by Increasing Quiescence-Associated Gene Expression and Suppressing Lymphoid Commitment

AbstractAbstract 851Signal transducer and activator of transcription 5 (STAT5) is a critical regulator of lympho-myeloid hematopoiesis through activation downstream of early-acting cytokines, their receptors (e.g. c-Mpl), and janus kinases (JAKs). We have previously reported that hematopoietic stem cells (HSC) in pI:pC treated Mx1-Cre conditional STAT5 knockout mice, like c-Mpl deficient HSC, are persistently more actively cycling and become apoptotic, reducing long-term HSC number. Since down-regulation of c-Mpl is associated with HSC heterogeneity defined by differential CD150/Flk2 expression and skewing toward lymphoid-primed multipotential progenitors (LMPP), we set to investigate whether STAT5-deficient cycling KLS cells are similarly lymphoid-primed. First, we generated novel Vav1-Cre/+STAT5abfl/fl hematopoietic-specific conditional knockout mice with 98% deletion efficiency in sorted KLS cells as determined by real-time PCR (N=3 mice). The majority of Vav1-Cre/+STAT5abfl/fl mice were viable with significantly reduced white blood cell counts (1.84±1.01 x103/μl, N=17 vs 8.46±2.26 x103/μl, N=27), red blood cell counts (7.53±0.78 x106/μl vs 10.19±0.80 x106/μl) and hematocrits (33.2±4.3% vs 47.1±3.2%) relative to wild-type. Competitive repopulation was 16-fold reduced CD45.2 donor chimerism with Vav-Cre/+STAT5abfl/fl (3.3±0.7%; N=5) compared to wild-type (55±2%; N=5) in a 1:1 mix with wild-type Boy J bone marrow cells. In non-ablative bone marrow transplantation, injection of a single dose of 5 million donor bone marrow cells gave rise to high levels of engraftment (84±8%, N=5) in the peripheral blood of congenic adult Vav-Cre/+STAT5abfl/fl recipients compared to no detectable engraftment controls. The KLS fraction was analyzed using CD34 and FLK2 markers. Despite severe reduction in the long-term repopulating HSC CD34-FLK2-KLS (N=3, p<0.001) and short-term repopulating HSC CD34+FLK2-KLS (p=0.03) fractions, the CD34+FLK2+KLS fraction enriched for LMPP was relatively maintained. We obtained similar results using CD150/CD48 in both Vav-Cre/+STAT5abfl/fl and STAT5ab null/null fetal liver transplanted mice. Using Affymetrix mouse gene chips to compare global gene expression between CD45.2+ KLS cells from STAT5ab null/null vs. wild-type fetal liver transplanted mice, several HSC-related genes were among those decreased whereas the majority of increased genes were associated with B-lineage commitment and immunoglobulin production. Furthermore, single cell gene expression analysis on the Fluidigm BioMark 48.48 chip (20 individual cells each genotype) was consistent with the microarray profiling. Half of wild-type KLS cells expressed high levels of HSC transcripts such as c-Mpl and Gfi1b and low levels of lymphoid transcripts whereas half were the opposite. In contrast, all STAT5 deficient KLS cells were lymphoid-primed and deficient in HSC transcripts. Due to the STAT5-dependent block at the pre-pro-B-cell stage, we are unable to functionally readout the donor B-lymphoid cells in the peripheral blood of transplanted mice. Since the combination of bcl-2 and myc induces aggressive B-cell acute lymphoblastic leukemia, we set out to use both bcl-2 and myc transgenic mouse models to test whether lymphoid-primed KLS cells lacking STAT5 would promote B cell development through a non-canonical pathway. Transgenic myc driven from an immunoglobulin H chain enhancer (Eμ-Myc) combined with H2K-bcl-2 induced faster B-ALL in STAT5ab null/null vs. wild type control fetal liver transplanted mice. The latency was significantly reduced from a median survival of 91 days in the wild type control to a median survival of 44 days in lethally-irradiated recipients (P<0.001; N range from 8–14 mice/group). Similar results were obtained in sub-lethally irradiated recipients (P=0.007; N range 10–20 mice/group). Loss of STAT5 promoted the accumulation of B-lineage committed progenitor cells in combination with pre-pro-B blockage. We also found a recently described Sca1+c-Kit-Lin- alternative lymphoid progenitor population to be expanded only in the STAT5ab null/null genotype expressing myc and bcl-2. Whether this population possesses the leukemia-initiating potential is still under investigation. Overall, we have found that STAT5 plays an unexpected role in hematopoietic stem cell differentiation by limiting early lymphoid lineage commitment. Disclosures:No relevant conflicts of interest to declare.

Adaptor Protein Lnk Binds to and Inhibits Normal and Leukemic FLT3

AbstractAbstract 1312 BackgroundThe production and lineage commitment of hematopoietic cells is controlled by the actions of a complex network of signaling pathways. Mutations and translocations of tyrosine kinases within these pathways lead to constitutive signaling and enhanced proliferation. Classic examples are BCR-ABL in CML, Janus kinase 2 (JAK2) mutations in MPN, Fms-like tyrosine kinase 3 (FLT3) and c-KIT mutations in AML.FLT3 is a receptor tyrosine kinase with important roles in hematopoietic progenitor cell survival and proliferation. It is mutated in about 1/3 of AML patients, mostly by internal tandem duplications (ITD). Adaptor protein Lnk is expressed in hematopoietic cells and is an important negative regulator in cytokine signaling and hematopoiesis. Previously, we and others have shown that Lnk interacts with the JXM domain of c-KIT, PDGFRA, PDGFRB and FMS, all of which share a similar sequence in this domain. The fact that FLT3 harbors a conserved JXM domain prompted us to investigate whether Lnk interacts with FLT3. Methods and ResultsCo-immunoprecipitation and GST-pulldown assay showed that Lnk physically interacts with both wild-type FLT3 (FLT3-WT) and FLT3-ITD through its SH2 domain in multiple types of hematopoietic cells. Through affinity fishing assay with immobilized peptides, we identified the tyrosine residues 572, 591 and 919 of FLT3 as phosphorylation sites involved in direct binding to Lnk. Importantly, Lnk itself was tyrosine-phosphorylated by both FLT3 ligand (FL)-activated FLT3-WT and constitutively activated FLT3-ITD. Functionally, both shRNA-mediated depletion and ectopic expression of Lnk demonstrated that activation signals emanating from both forms of FLT3 are under negative regulation by Lnk. Consequently, Lnk inhibited 32D cell proliferation driven by different FLT3 oncogenic variants. Moreover, analysis of primary bone marrow cells from Lnk−/−mice showed that Lnk suppresses the expansion of FL-stimulated hematopoietic progenitors, including lymphoid-primed multipotent progenitors, mainly through inhibiting MAPK-ERK activation by FL. ConclusionsThis study reveals that through direct binding to FLT3-WT and FLT3-ITD, Lnk constrains FLT3-WT/ITD-dependent signaling pathways involved in the proliferation and expansion of hematopoietic cells as well as related leukemic cells. Modulation of Lnk expression levels may provide a unique therapeutic approach for FLT3-ITD-associated hematopoietic diseases. Disclosures:No relevant conflicts of interest to declare.

Adaptor protein Lnk binds to and inhibits normal and leukemic FLT3

AbstractFms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase with important roles in hematopoietic progenitor cell survival and proliferation. It is mutated in approximately one-third of AML patients, mostly by internal tandem duplications (ITDs). Adaptor protein Lnk is a negative regulator of hematopoietic cytokine signaling. In the present study, we show that Lnk interacts physically with both wild-type FLT3 (FLT3-WT) and FLT3-ITD through the SH2 domains. We have identified the tyrosine residues 572, 591, and 919 of FLT3 as phosphorylation sites involved in direct binding to Lnk. Lnk itself was tyrosine phosphorylated by both FLT3 ligand (FL)–activated FLT3-WT and constitutively activated FLT3-ITD. Both shRNA-mediated depletion and forced overexpression of Lnk demonstrated that activation signals emanating from both forms of FLT3 are under negative regulation by Lnk. Moreover, Lnk inhibited 32D cell proliferation driven by different FLT3 variants. Analysis of primary BM cells from Lnk-knockout mice showed that Lnk suppresses the expansion of FL-stimulated hematopoietic progenitors, including lymphoid-primed multipotent progenitors. The results of the present study show that through direct binding to FLT3, Lnk suppresses FLT3-WT/ITD–dependent signaling pathways involved in the proliferation of hematopoietic cells. Therefore, modulation of Lnk expression levels may provide a unique therapeutic approach for FLT3-ITD–associated hematopoietic disease.

Perturbation of fetal liver hematopoietic stem and progenitor cell development by trisomy 21

The 40-fold increase in childhood megakaryocyte-erythroid and B-cell leukemia in Down syndrome implicates trisomy 21 (T21) in perturbing fetal hematopoiesis. Here, we show that compared with primary disomic controls, primary T21 fetal liver (FL) hematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors are markedly increased, whereas granulocyte-macrophage progenitors are reduced. Commensurately, HSC and megakaryocyte-erythroid progenitors show higher clonogenicity, with increased megakaryocyte, megakaryocyte-erythroid, and replatable blast colonies. Biased megakaryocyte-erythroid-primed gene expression was detected as early as the HSC compartment. In lymphopoiesis, T21 FL lymphoid-primed multipotential progenitors and early lymphoid progenitor numbers are maintained, but there was a 10-fold reduction in committed PreproB-lymphoid progenitors and the functional B-cell potential of HSC and early lymphoid progenitor is severely impaired, in tandem with reduced early lymphoid gene expression. The same pattern was seen in all T21 FL samples and no samples had GATA1 mutations. Therefore, T21 itself causes multiple distinct defects in FL myelo- and lymphopoiesis.

The transcription factor Lyl-1 regulates lymphoid specification and the maintenance of early T lineage progenitors

Thymopoiesis depends on recruitment and expansion of bone marrow-derived progenitors, tight regulation of which is required to maintain T-lineage homeostasis. Lyl1, a transcription factor regulating hematopoietic progenitors, is expressed in thymocyte progenitors until T cell commitment. Here we demonstrate a requirement for Lyl1 in lymphoid specification and the maintenance of early T lineage progenitors (ETPs). Lyl1 deficiency resulted in profound defects in generation of lymphoid primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and ETPs. Lyl1-deficient ETPs and DN2 thymocyte progenitors showed increased apoptosis, blocked differentiation and impaired expansion. We identified Gfi1 as a critical transcriptional target of Lyl1-mediated T-lymphopoiesis. Thus, Lyl1 is a pivotal component of a transcriptional program that controls lymphoid specification and maintenance of ETPs.

IRF-8 extinguishes neutrophil production and promotes dendritic cell lineage commitment in both myeloid and lymphoid mouse progenitors

AbstractWhile most blood lineages are assumed to mature through a single cellular and developmental route downstream of HSCs, dendritic cells (DCs) can be derived from both myeloid and lymphoid progenitors in vivo. To determine how distinct progenitors can generate similar downstream lineages, we examined the transcriptional changes that accompany loss of in vivo myeloid potential as common myeloid progenitors differentiate into common DC progenitors (CDPs), and as lymphoid-primed multipotent progenitors (LMPPs) differentiate into all lymphoid progenitors (ALPs). Microarray studies revealed that IFN regulatory factor 8 (IRF-8) expression increased during each of these transitions. Competitive reconstitutions using Irf8−/− BM demonstrated cell-intrinsic defects in the formation of CDPs and all splenic DC subsets. Irf8−/− common myeloid progenitors and, unexpectedly, Irf8−/− ALPs produced more neutrophils in vivo than their wild-type counterparts at the expense of DCs. Retroviral expression of IRF-8 in multiple progenitors led to reduced neutrophil production and increased numbers of DCs, even in the granulocyte-macrophage progenitor (GMP), which does not normally possess conventional DC potential. These data suggest that IRF-8 represses a neutrophil module of development and promotes convergent DC development from multiple lymphoid and myeloid progenitors autonomously of cellular context.

The Earliest Thymic T Cell Progenitors Sustain B Cell and Myeloid Lineage Potentials

Abstract 2335The stepwise lineage-commitment from hematopoietic stem cells (HSCs) in the bone marrow (BM) to T-lymphocyte-restricted progenitors in the thymus represents a paradigm for how distinct stages of lineage restriction from a multipotent to a lineage-restricted progenitor require different extrinsic cues. However, the commitment stage at which progenitors migrate from the BM to the thymus remains unclear. Previous studies demonstrated the existence of adult early thymic progenitors (ETPs) restricted to T lymphocyte and granulocyte-monocyte (GM) fates (Bell and Bhandoola, Nature 2008; Wada et al., Nature 2008). The challenge remains to couple this thymic T–GM restricted progenitor to candidate thymus seeding progenitors (TSPs) identified in the BM, such as common lymphoid progenitors, lymphoid-primed multipotent progenitors (LMPPs) and HSCs, which all also possess B cell potential.Using high resolution FACS and sensitive clonal assays, we have identified the most primitive ETPs in the neonatal thymus and demonstrate at the single cell level that rare Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs possess combined granulocyte-monocyte (GM), T and B lymphocyte but not megakaryocyte-erythroid (MkE) lineage potentials, identical to closely molecularly related thymus-seeding progenitors in the BM.Moreover, global molecular profiling demonstrates that neonatal Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs cluster closest to multipotent progenitors in the BM, rather than subsequent (DN1 and DN2) progenitor stages in the thymus. Finally, in support of Lin−CD4−CD8a−CD25−KIThiFLT3hi ETPs with combined T, B and GM lineage potentials, being the most multipotent progenitors in the thymus, phenotypic and functional studies demonstrate that the neonatal thymus is not seeded by HSCs or MPPs with MkE potential. These findings establish for the first time a distinct lineage commitment stage for the transition of T-lineage commitment from the BM to the remote thymus. Disclosures:No relevant conflicts of interest to declare.

FLT3-ITDs Introduce a Myeloid Differentiation and Transformation Bias to Multipotent Lympho-Myeloid Progenitors

Abstract 1380Although the growth factor receptor (GFR) FLT3 has a crucial role in normal early B- and T-lymphoid development, constitutively activating internal tandem duplications (ITDs) of FLT3 are almost entirely restricted to patients with adverse-risk acute myeloid leukemia. We used a murine knock-in model of FLT3-ITD myeloproliferative disease (MPD) to gain a better understanding of the cellular and molecular basis for the myeloid-bias of FLT3-ITD-induced hematological malignancies.As Flt3 cell surface expression is lacking in homozygous Flt3-itd mice, we used CD48 and CD150 expression to investigate the distribution of multipotent progenitors (MPPs) and hematopoietic stem cells (HSCs) within the primitive Lin-Sca1+Kit+ (LSK) compartment. Notably, phenotypic (LSKCD150+CD48-) and functional HSCs were markedly reduced in adult Flt3-itd mice. Competitive transplantation experiments using fetal liver confirmed that HSC numbers were reduced (20-fold reduction) by Flt3-ITDs, in a cell-extrinsic manner. Rather, LSKCD48+150- cells (MPPs) were expanded 2.7-fold in Flt3-itd mice comprising >90% of LSK cells. Similarly to Flt3high wild type (WT) lymphoid-primed multipotent progenitors (LMPPs), nanofluidic gene-expression analysis demonstrated that WT MPPs and Flt3-itd MPPs were myeloid-primed (Csf1r, Csf2r, Cebpa, Mpo) with loss of megakaryocyte and erythroid (MkE) priming (Eklf, Epor, Vwf, Gata1). In contrast, the lymphoid (Il7r, Rag1, sIgH) transcriptional priming of WT MPPs was downregulated in Flt3-itd MPPs. In agreement with this, Flt3-itd MPPs sustained extensive GM potential in vitro, with no MkE potential and, unlike WT MPPs, considerably reduced lymphoid potential. Furthermore, microarray analysis demonstrated global upregulation of the myeloid program in Flt3-itd MPPs. These findings demonstrate that primitive lympho-myeloid MPPs, are expanded and biased towards myeloid development by Flt3-ITDs.In agreement with reduced lymphoid-priming of Flt3-itd MPPs, analysis of early thymic development demonstrated a 10-fold reduction of early thymic progenitors (DN1 Kit+) in Flt3-itd mice. Subsequent stages of thymic development were also reduced, as was overall thymic cellularity. Interestingly, expression of the chemokine receptor CCR9 was 5.5-fold reduced in Flt3-itd MPPs suggesting that thymic seeding progenitors in the bone marrow are suppressed by FLT3-ITDs.Previous studies have suggested that the earliest stage of B-cell development, pre-pro-B cells, retain both B-cell and myeloid potential. Lin-CD19-CD24-AA4.1+CD43+B220+ pre-pro-B cells were expanded 13.7-fold in Flt3-itd mice, whereas subsequent stages of CD19+ B-lymphopoiesis were all reduced. The expanded pre-pro-B cells in Flt3-itd mice were myeloid biased at the transcriptional level with markedly reduced expression of lymphoid genes.Pu1 is a master-regulator of myeloid commitment in early hematopoiesis and a STAT3 target gene. As FLT3-ITDs are known to activate STAT3, unlike WT FLT3, we therefore investigated Pu1 expression in Flt3-itd mice using a Pu1-YFP reporter. Expression of Pu1 was significantly increased in LSK cells (1.4 fold) and in pre-pro-B cells (2.6 fold) in Flt3-itd mice. Furthermore, other STAT3 target genes (Cish, Id1, Pim1, Socs1, Junb) were also upregulated in these cell populations in Flt3-itd mice. Moreover, gene-set enrichment analysis in MPPs demonstrated upregulation of Pu1 target genes in Flt3-itd mice, thus providing a link between aberrant ITD signaling and the observed myeloid bias.In order to determine the functional relevance of this myeloid-bias of Flt3-itd MPPs for disease transformation, we targeted a conditional Aml1-ETO fusion-gene to the earliest B-cell progenitors in Flt3-itd mice using Mb1-Cre. Expression of AML1-ETO in WT mice did not induce any phenotype. However, Mb1-Cre induced AML1-ETO expression in Flt3-itd mice led to a high-penetrance, short latency acute leukaemia. All leukaemias expressed myeloid markers (Mac1 and Gr1) but lacked CD19 and B220 expression.These data demonstrate that Flt3-ITDs expand primitive MPPs with a myeloid lineage bias at the molecular and cellular level, at the expense of HSCs and early lymphoid development. This provides insight into the mechanisms by which mutations resulting in activation of a GFR introduce a lineage bias of resulting hematological malignancies. Disclosures:No relevant conflicts of interest to declare.

All Hematopoietic Cells Develop from Hematopoietic Stem Cells through Flk2/Flt3-Positive Progenitor Cells

While it is clear that a single hematopoietic stem cell(HSC) is capable of giving rise to all other hematopoietic cell types, the differentiation paths beyond HSC remain controversial. Contradictory reports onthe lineage potential of progenitor populations have questioned their physiological contribution of progenitor populations to multilineage differentiation. Here, we established a lineage tracing mouse model that enabled direct assessment of differentiation pathways invivo. We provide definitive evidence that differentiation into all hematopoietic lineages, including megakaryocyte/erythroid cell types, involves Flk2-expressing non-self-renewing progenitors. A Flk2+ stage was used during steady-state hematopoiesis, after irradiation-induced stress and upon HSC transplantation. In contrast, HSC origin and maintenance do not include a Flk2+ stage. These data demonstrate that HSC specification and maintenance are Flk2 independent, and that hematopoietic lineage separation occurs downstream of Flk2 upregulation.

Asynchronous and modular repression of neutrophil production by IRF8 leads to the development of progenitors with partially overlapping lineage potentials. (153.24)

Abstract Most previous studies have assumed that the maturation of a particular blood cell type downstream of hematopoietic stem cells follows a singular developmental route; therefore the identification of distinct hematopoietic progenitors with partially overlapping lineage potentials has led to considerable controversy. For example, common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) both generate dendritic cells upon adoptive transfer in vivo. To determine the molecular mechanisms by which progenitors with partially overlapping lineage potentials arise, we examined the transcriptional changes that occur as lymphoid-primed multipotent progenitors (LMPPs) differentiate into CLPs, and as CMPs differentiate into common dendritic cell progenitors (CDPs). In both of these independent pathways, we determined that the expression of the transcription factor IRF8 was strongly induced. IRF8-/- mice showed reduced numbers of both CLPs and CDPs. Competitive bone marrow reconstitutions also demonstrated cell-intrinsic defects in the generation of CD8a- dendritic cells, previously reported to be normal in IRF8-/- mice. Microarray comparisons between dendritic cells derived from either wild type CMPs or CLPs demonstrated that these cells are identical. Thus, the asynchronous expression of IRF8 leads to the formation of progenitors with partially overlapping developmental potentials, which can resolve themselves to ultimately generate the same mature cell type.

CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adult mice

AbstractNatural killer (NK) cells are granular lymphocytes that are generated from hematopoietic stem cells and play vital roles in the innate immune response against tumors and viral infection. Generation of NK cells is known to require several cytokines, including interleukin-15 (IL-15) and Fms-like tyrosine kinase 3 ligand, but not IL-2 or IL-7. Here we investigated the in vivo role of CXC chemokine ligand-12 (CXCL12) and its primary receptor CXCR4 in NK-cell development. The numbers of NK cells appeared normal in embryos lacking CXCL12 or CXCR4; however, the numbers of functional NK cells were severely reduced in the bone marrow, spleen, and peripheral blood from adult CXCR4 conditionally deficient mice compared with control animals, probably resulting from cell-intrinsic CXCR4 deficiency. In culture, CXCL12 enhanced the generation of NK cells from lymphoid-primed multipotent progenitors and immature NK cells. In the bone marrow, expression of IL-15 mRNA was considerably higher in CXCL12-abundant reticular (CAR) cells than in other marrow cells, and most NK cells were in contact with the processes of CAR cells. Thus, CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adults, and CAR cells might function as a niche for NK cells in bone marrow.

Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia

The relationships between normal and leukemic stem/progenitor cells are unclear. We show that in ∼80% of primary human CD34+ acute myeloid leukemia (AML), two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. Both populations have leukemic stem cell (LSC) activity and are hierarchically ordered; one LSC population gives rise to the other. Global gene expression profiling shows the LSC populations are molecularly distinct and resemble normal progenitors but not stem cells. The more mature LSC population most closely mirrors normal granulocyte-macrophage progenitors (GMP) and the immature LSC population a previously uncharacterized progenitor functionally similar to lymphoid-primed multipotential progenitors (LMPPs). This suggests that in most cases primary CD34+ AML is a progenitor disease where LSCs acquire abnormal self-renewal potential.

A Chromatin Modifier SATB1 Promotes Lymphocyte Production From Primitive Hematopoietic Stem/Progenitor Cells

AbstractAbstract 836Although hematopoietic stem cells (HSC) are capable for reconstituting all hematopoietic lineages in serial transplantation experiments, they undergo substantial age-related changes. Indeed, many studies have suggested that the immune system qualitatively and quantitatively changes along aging, including the selective loss of lymphopoietic potential. Lymphocyte progenitors explosively expand in fetal liver, but after birth their production sifts to bone marrow (BM) and becomes stable. Thereafter, replenishment of the adaptive immune system declines with age. If key inducers priming lymphoid lineage in HSC can be identified, they would be practical for expanding lymphocytes in culture for clinical purposes. Additionally, exogenous manipulation of their expression might boost the immune systems of immuno-compromised and/or elderly people.A primary aim of the present work was to describe molecular signatures of early stages of lymphopoiesis. Our previous studies developed a method to isolate Rag1+ early lymphocyte progenitors (ELP) from the Lineage marker- Sca-1+ c-kitHi (LSK) HSC-enriched fraction using Rag1/GFP reporter mice. We exploited the method and performed microarray analyses to compare gene expression patterns between HSC and ELP. While many molecules specific for the lymphoid lineage including T cell receptor and immunoglobulin heavy chain were highly induced at the ELP stage, some lymphoid-related genes were already expressed in the HSC-enriched fraction. In addition, our search identified many signal transduction kinases of whose function in lymphopoiesis is unknown. We then sought candidate genes governing the early lymphoid program with the goal of discovering molecules involved in the HSC to lymphoid lineage transition. Chromatin modifiers particularly drew our attention because of their ability to control spatial and temporal expression of essential genes. As a result, we identified Special AT-rich Sequence Binding 1 (SATB1) whose expression was faint in HSC and markedly up-regulated in ELP. SATB1, originally identified as a protein binding to the enhancer region of the IgH gene, is recently attracting considerable attention as a global transcription organizer for various genes.Our real-time RT-PCR analyses revealed that Satb1 expression remarkably increases along the differentiation of HSC to lymphoid-primed multipotent progenitors and ELP. In contrast, it was significantly down-regulated in the Lineage marker- Sca-1- c-kitHi myeloid progenitors. Interestingly, our analyses also found approximately 2-fold reduction of Satb1 transcripts in the Rag1/GFP- LSK cells of aged BM. In functional assays, adult BM-derived Flt3-LSK cells were retrovirally transduced with SATB1 and cultured in various conditions. In stromal cell co-cultures that support the growth of B lymphocytes and myeloid cells, the SATB1 transduction gave rise to 50–300 fold more CD19+ CD45R/B220+ Mac1- B lineage cells. In stromal-free cultures containing SCF, Flt3-ligand and IL7, SATB1 transduction also strongly induced B lineage cell production. On the other hand, methylcellulose colony assays showed 2-fold reduction of CFU-GM colony formation from SATB1-transduced cells compared to Mock-transduced cells. Next we examined if the exogenous expression of SATB1 promotes B lymphocyte growth from embryonic stem (ES) cells. SATB1 overexpression effectively promoted B1-B cell growth from mouse ES cells in OP9 co-culture system. Furthermore, we found that exogenous SATB1 expression partly restored the lymphopoietic potential in aged BM HSC that are compromised in lymphopoietic activity. Finally, we performed microarray analyses comparing SATB1- and Mock-transduced Flt3- LSK cells to seek candidate genes involved in the SATB1 function. While neither Pu.1, Ikaros, E2a, nor Notch1 was affected, some lymphoid-related genes, such as Sp4, Maf and Rag1, were increased in SATB1-overexpressed HSC. In conclusion, our results indicate that SATB1 plays critical roles in producing lymphoid lineage from primitive stem/progenitor cells. The findings are providing important information regarding how the earliest step of lymphopoiesis is epigenetically regulated and how modulation of the process might be useful to induce or rejuvenate the immune system. Disclosures:No relevant conflicts of interest to declare.

E proteins and the regulation of early lymphocyte development

Lymphopoiesis generates mature B, T, and NK lymphocytes from hematopoietic stem cells via a series of increasingly restricted developmental intermediates. The transcriptional networks that regulate these fate choices are composed of both common and lineage-specific components, which combine to create a cellular context that informs the developmental response to external signals. E proteins are an important factor during lymphopoiesis, and E2A in particular is required for normal T- and B-cell development. Although the other E proteins, HEB and E2-2, are expressed during lymphopoiesis and can compensate for some of E2A's activity, E2A proteins have non-redundant functions during early T-cell development and at multiple checkpoints throughout B lymphopoiesis. More recently, a role for E2A has been demonstrated in the generation of lymphoid-primed multipotent progenitors and shown to favor their specification toward lymphoid over myeloid lineages. This review summarizes both our current understanding of the wide-ranging functions of E proteins during the development of adaptive lymphocytes and the novel functions of E2A in orchestrating a lymphoid-biased cellular context in early multipotent progenitors.

PU.1 - c-Jun Interactions Are Crucial for PU.1 Function in Myeloid Development.

Abstract 3651Poster Board III-587The Ets transcription factor PU.1 is a master regulator absolutely required for the differentiation of monocytes, macrophages, and B cells in the fetal liver and in the adult bone marrow. PU.1 drives hematopoietic differentiation partly through direct protein-protein interactions with other transcription factors, such as the AP-1 transcription factor c-Jun. We have shown that c-Jun can be recruited to promoters which do not include AP-1 binding sites, such as the MCSFR promoter, and act as a PU.1-dependent co-activator. To address the functional importance of this interaction, we identified and studied PU.1 point mutants that lost the capability to physically interact with c-Jun while retaining normal DNA binding affinity. These mutants failed to efficiently transactivate a PU.1 target reporter, and, more importantly, were unable to induce monocyte/macrophage differentiation of the PU.1-deficient immature myeloid 503 cell line.Subsequently, we have generated knock-in mouse models harboring these single point mutations by means of homologous recombination. The mutant mice phenotypically resemble PU.1-deficient mice, have an early block in hematopoiesis, and die perinatally. We show that the mutant PU.1 mRNA and protein is expressed in long-term and short-term hematopoietic stem cells, but that the maturation into lymphoid primed multipotent progenitor (LMPP) and later progenitor populations is severely blocked, leading to an almost complete loss of mature B, T and myeloid cells. Collectively, our data strongly suggest that the PU.1-c-Jun interaction is crucial for normal PU.1 function in vivo during murine fetal hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Obesity accelerates thymic aging

AbstractAs the expanding obese population grows older, their successful immunologic aging will be critical to enhancing the health span. Obesity increases risk of infections and cancer, suggesting adverse effects on immune surveillance. Here, we report that obesity compromises the mechanisms regulating T-cell generation by inducing premature thymic involution. Diet-induced obesity reduced thymocyte counts and significantly increased apoptosis of developing T-cell populations. Obesity accelerated the age-related reduction of T-cell receptor (TCR) excision circle bearing peripheral lymphocytes, an index of recently generated T cells from thymus. Consistent with reduced thymopoiesis, dietary obesity led to reduction in peripheral naive T cells with increased frequency of effector-memory cells. Defects in thymopoiesis in obese mice were related with decrease in the lymphoid-primed multipotent progenitor (Lin−Sca1+Kit+ Flt3+) as well as common lymphoid progenitor (Lin−Sca1+CD117loCD127+) pools. The TCR spectratyping analysis showed that obesity compromised V-β TCR repertoire diversity. Furthermore, the obesity induced by melanocortin 4 receptor deficiency also constricted the T-cell repertoire diversity, recapitulating the thymic defects observed with diet-induced obesity. In middle-aged humans, progressive adiposity with or without type 2 diabetes also compromised thymic output. Collectively, these findings establish that obesity constricts T-cell diversity by accelerating age-related thymic involution.