Common Lymphoid Progenitors Research Articles

IMiD compounds affect CD34+ cell fate and maturation via CRBN-induced IKZF1 degradation

AbstractWe have previously shown that immunomodulatory drug (IMiD) compounds induce a shift into immature myeloid precursors with a maturational arrest and subsequent neutropenia. The mechanism of action is unknown. Here we found that IMiD compounds cause selective ubiquitination and degradation of the transcription factor IKZF1 in CD34+ cells by the Cereblon (CRBN) E3 ubiquitin ligase. Loss of IKZF1 is associated with a decrease of the IKZF1-dependent transcription factor PU.1, critical for the development and maturation of neutrophils. Using a thalidomide analog bead pull-down assay, we showed that IMiD compounds directly bind CRBN in CD34+ cells. Knockdown of CRBN in CD34+ cells resulted in resistance to POM-induced IKZF1 downregulation and reversed the POM-induced lineage shift in colony-formation assays, suggesting that the POM-induced degradation of IKZF1 in CD34+ cells requires CRBN. Chromatin immunoprecipitation assays revealed that IKZF1 binds to the promoter region of PU.1, suggesting that PU.1 is a direct downstream target of IKZF1 in CD34+ cells. POM failed to induce IKZF1 degradation in IKZF1-Q146H-OE CD34+ cells, indicating that CRBN binding to IKZF1 and subsequent IKZF1 ubiquitination is critical in this process. Using the NOD/SCID/γ-c KO mouse model, we confirmed the induction of myeloid progenitor cells by IMiD compounds at the expense of common lymphoid progenitors. These results demonstrate a novel mechanism of action of IMiD compounds in hematopoietic progenitor cells, leading to selective degradation of transcription factors critical for myeloid maturation, and explain the occurrence of neutropenia associated with treatment by IMiD compounds.

The pathogenic role of MEF2D-SS18 fusion gene in B-cell acute lymphoblastic leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is characterized by various fusion genes resulted from chromosome translocations, and MEF2D related fusions are recently identified in a B-ALL subtype with relatively worse survival. In this study, we investigated the pathogenic role of MEF2D-SS18 fusion in B-ALL. The recombinant retrovirus and lentivirus plasmids containing the MEF2D-SS18 transcript were constructed for functional studies. Immuno-fluorescent staining presented the nuclear localization of MEF2D-SS18. In vitro B cell differentiation assay showed MEF2D-SS18 significantly inhibited the differentiation of mouse common lymphoid progenitors into CD19 positive B cells. The data of RBMT mouse model also showed B cell developmental arrest. In REH cells overexpressing MEF2D-SS18, genes related to B cell development were down-regulated, while genes related to drug sensitivity and B cell survival were up-regulated. In conclusion, MEF2D-SS18 fusion gene blocked the differentiation of B cells, thus exerted a funding role in the pathogenesis and prognosis of B-ALL.

Human Innate lymphoid cells

Innate lymphoid cells (ILCs) are a heterogeneous population of immune cells with two defining characteristics: 1) they have lymphoid origin, differentiating from the common lymphoid progenitor (CLP) and dependent on IL-2Rgc signaling. 2) They lack antigen-specific receptors and therefore do not require the RAG proteins for development. Although ILCs have been extensively studied in model organisms, little is known about these "first responders" in humans, especially their lineage and functional kinships to cytokine-secreting T helper (Th) cell counterparts. We investigated gene regulatory circuitries for four human ILC-Th counterparts derived from mucosal environments. We found that each ILC subset diverges as a distinct lineage from Th and circulating natural killer cells but shares circuitry devoted to functional polarization with their Th counterparts. Super-enhancers demarcate cohorts of cell-identity genes in each lineage, uncovering new modes of regulation for signature cytokines, new molecules that likely impart important functions to ILCs, and potential mechanisms for autoimmune disease SNP associations within ILC-Th subsets.

Wnt Signaling in Hematological Malignancies.

Leukemia and lymphoma are a wide encompassing term for a diverse set of blood malignancies that affect people of all ages and result in approximately 23,000 deaths in the United States per year (Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7-30.). Hematopoietic stem cells (HSCs) are tissue-specific stem cells at the apex of the hierarchy that gives rise to all of the terminally differentiated blood cells, through progressively restricted progenitor populations, a process that is known to be Wnt-responsive. In particular, the progenitor populations are subject to uncontrolled expansion during oncogenic processes, namely the common myeloid progenitor and common lymphoid progenitor, as well as the myeloblast and lymphoblast. Unregulated growth of these cell-types leads to mainly three types of blood cancers (i.e., leukemia, lymphoma, and myeloma), which frequently exhibit deregulation of the Wnt signaling pathway. Generally, leukemia is caused by the expansion of myeloid progenitors, leading to an overproduction of white blood cells; as such, patients are unable to make sufficient numbers of red blood cells and platelets. Likewise, an overproduction of lymphocytes leads to clogging of the lymph system and impairment of the immune system in lymphomas. Finally, cancer of the plasma cells in the blood is called myeloma, which also leads to immune system failure. Within each of these three types of blood cancers, there are multiple subtypes, usually characterized by their timeline of onset and their cell type of origin. Of these, 85% of leukemias are encompassed by the four most common diseases, that is, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL); AML accounts for the majority of leukemia-related deaths (Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7-30.). Through understanding how HSCs are normally developed and maintained, we can understand how the normal functions of these pathways are disrupted during blood cancer progression; the Wnt pathway is important in regulation of both normal and malignant hematopoiesis. In this chapter, we will discuss the role of Wnt signaling in normal and aberrant hematopoiesis. Our understanding the relationship between Wnt and HSCs will provide novel insights into therapeutic targets.

IL-33 promotes the egress of group 2 innate lymphoid cells from the bone marrow.

Group 2 innate lymphoid cells (ILC2s) are effector cells within the mucosa and key participants in type 2 immune responses in the context of allergic inflammation and infection. ILC2s develop in the bone marrow from common lymphoid progenitor cells, but little is known about how ILC2s egress from the bone marrow for hematogenous trafficking. In this study, we identified a critical role for IL-33, a hallmark peripheral ILC2-activating cytokine, in promoting the egress of ILC2 lineage cells from the bone marrow. Mice lacking IL-33 signaling had normal development of ILC2s but retained significantly more ILC2 progenitors in the bone marrow via augmented expression of CXCR4. Intravenous injection of IL-33 or pulmonary fungal allergen challenge mobilized ILC2 progenitors to exit the bone marrow. Finally, IL-33 enhanced ILC2 trafficking to the lungs in a parabiosis mouse model of tissue disruption and repopulation. Collectively, these data demonstrate that IL-33 plays a critical role in promoting ILC2 egress from the bone marrow.

Stag2 regulates Hematopoietic Differentiation and Self-Renewal

Cohesin is a multimeric protein complex, which has been initially implicated in the cohesion of replicated sister chromatids but more recently shown to be involved in the long-range regulation of gene expression by stabilizing 3-dememsional structure of the genomic DNA. Recently, multiple components of the cohesin complex has been shown to be a recurrent target of somatic mutations in various myeloid malignancies, in which STAG2 is most frequently mutated and undergoes loss-of-function. However, the leukemogenic mechanism of mutated- STAG2 has not been fully understood, although recent studies have reported deregulated differentiation and enhanced self-renewal of STAG2 -mutated stem cells.To investigate the functional role of STAG2 in leukemogenesis as well as normal hematopoiesis, we generated Stag2 conditional knockout (cKO) mice with an Mx1 -cre allele, in which Stag2 deletion was induced by polyIC injection. When assessed in the peripheral blood of 12 to 20 week-old mice, the complete blood count showed no significant changes between Stag2 cKO mice and controls. No morphological alterations were observed in the peripheral blood as well as in the bone marrow. However, in the spleen, the extramedullary hematopoiesis was evident, exhibiting increased myeloid progenitors and erythroblasts. In repeated methylcellulose cultures, Stag2 deficient BM cells showed an enhanced serial replating capacity, suggesting an increased self-renewal potential in Stag2 deficient hematopoietic stem cells (HSCs) in vitro.Flow cytometry of bone marrow cells revealed increased numbers of hematopoietic stem and progenitor cells (HSPCs) defined as Lineage−Sca-1+Kit+(LSK) cells in Stag2 cKO mice compared with controls. Within the myeloid progenitor (MP) compartment, we observed increased common myeloid progenitors (CMPs), while decreased megakaryocyte/erythroid (MEPs) and common lymphoid progenitors (CLPs), compared to controls. Moreover, CD11b+Gr-1+ mature myeloid cells were significantly increased in the bone marrow of Stag2 cKO mice. These results suggest that Stag2 deficiency causes myeloid skewing.In competitive bone marrow transplantation assays assessing the reconstitution potential, Stag2 cKO-derived cells showed reduced chimerism in the peripheral blood compared to wild-type mice-derived cells. However, the reduced chimerism of Stag2 cKO-derived cells was largely confined to B-lymphocytes showing a severe reduction, while the chimerism of Stag2 cKO-derived myeloid cells was not affected compared to controls. In the bone marrow, by contrast, the chimerism of Stag2 cKO cells was not significantly changed, but rather tended to show increased numbers in the LSK, CMP and granulocyte/macrophage progenitor (GMP) fractions, while the MEP and CLP fractions were reduced. These results suggest that Stag2 deficiency could enhance the self-renewal capacity of HSCs in vivo, but Stag2 -deleted cells may not uniformly contribute to all hematopoietic cell fractions probably due to the impaired differentiation of these HSCs.Next, we assessed the effect of Stag2 -deficiency on gene expression, where RNA sequencing of Stag2 -deleted HSPCs revealed a subset of genes differentially expressed between Stag2 WT and cKO cells. Including key myeloid-specific regulators, these genes were considered to be potential gene targets of Stag2, deregulation of which is implicated in the abnormal hematopoiesis of Stag2 cKO mice. Given that the cohesin complex is known to be involved in the establishing and maintaining DNA accessibility, we also assessed the global chromatin accessibility by assays for transposase accessible chromatin with sequencing (ATAC-seq) of Stag2 -deleted HSPCs. Of interest, chromatin accessibility in Stag2 -deleted cells was increased for genes implicated in myeloid differentiation, whereas reduced for those in lymphoid differentiation. Motif analysis of more accessible regions in Stag2 -deleted cells revealed an enrichment of the binding site for the transcription factor Runx1, which is known to regulate HSC differentiation.Our results demonstrate that Stag2 loss leads to the impaired hematopoietic differentiation and enhances the self-renewal potential of HSCs through the modulation of chromatin accessibility and consequent abnormal gene expression, possibly contributing to leukemogenesis. DisclosuresTakaori-Kondo:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Research Funding; Janssen Pharma: Honoraria; Pfizer​: Honoraria.

Junctional Adhesion Molecule 2 Intensifies T Lymphopoiesis of Hematopoietic Stem Cells By Facilitating Notch/Delta Signaling

Phenotypically described hematopoietic stem cells (HSCs) represent a functionally heterogeneous pool of primitive cells with conceivable potential to replenish and maintain the whole hematopoietic system. The diverse lineage potential of HSCs is supposed to play a significant role in the response to different kinds of hematopoietic stress. Since subcategorization of HSCs biased towards specific lineage(s) highly relies on the retrospective information, e.g. transplantation assay, exploring additional markers will allow us to understand further molecular mechanisms of HSC regulation such as activation and lineage choice but also the degree of correlation between them.Here, we show that the cell surface protein Junctional adhesion molecule 2 (Jam2) serves as an amplifier of the Notch/Delta signal thereby representing the higher T cell potential of HSCs. Flow cytometry analyses revealed that a subset of CD150+CD48-KSL cells in mouse bone marrow (BM) were positive for Jam2 (Jam2+HSC, 36.6 ±13.0%), while other Jam family member, Jam1 (F11r), was expressed on all HSCs and Jam3 was not detected. Transplantation assay using 30 Jam2+ or Jam2-HSCs revealed that Jam2+HSCs reconstituted lethally irradiated mice more efficiently than Jam2-HSCs (77.5 ±15.9 and 51.7 ±29.3% in peripheral blood, respectively). Lineage analyses revealed that Jam2+HSCs have a greater potential in lymphoid cell reconstitution, particularly T cells, whereas the chimerism in myeloid cells was not significantly different from Jam2-HSCs. This tendency of higher contribution to the T cell development was even more pronounced in the secondary transplantation experiments, where the contribution of Jam2+HSCs in T cells was close to 100%. Of note, most of Jam2+HSCs were in a dormant state, suggesting that the T cell potential of Jam2+HSCs is independent of the cell cycle progression.Jam2 has been reported to mediate the Notch signaling through an interaction with Jam1 (Kobayashi et al., Nature, 2014). In addition, Jam2+HSCs express Notch1 at a higher level than Jam2-HSCs (23.6 ±6.7 and 9.05 ±5.8%, respectively). We therefore analyzed the functional role of Jam2 in the Notch/Delta-oriented T cell production using a competitive feeder-free T cell culture system. At a low concentration of DLL1, that is insufficient to promote T cell production by itself, Jam2+HSCs effectively produced T cell lineages only in the presence of recombinant Jam1 protein, but not Jam2 or Jam3. In contrast, Jam2+HSCs did not require Jam1 protein with a higher concentration of DLL1. These differences were not observed with Jam2-HSCs, indicating that Jam2/Jam1 interaction amplifies Notch signal transduction and is crucial for the subsequent T cell specification of Jam2+HSCs.To elucidate the molecular signature of Jam2+HSCs, gene expression profiling was performed using a microarray analysis. Gene set enrichment analysis (GSEA) observed that Jam2+HSCs were significantly enriched for common lymphoid progenitor (CLP) and early T cell gene expression. Of note, Jam2+HSCs were also enriched for E2F target genes, G2M checkpoint genes and glycolysis related genes, which potentially explains the reason why Jam2+HSCs display a bivalent phenotype: being more dormant compared to Jam2-HSCs at the steady state but at the same time having the capacity to reconstitute more actively upon engraftment.Since Jam2 positivity correlates to T cell potential, we asked if altered T lymphopoietic environment affects the proportion of Jam2+HSCs. In vivo T cell depletion resulted in significantly higher frequency of Jam2+HSCs but not upon other stress inducers, such as 5-FU treatment, suggesting that the increase in Jam2+HSC pool was specifically due to the T cell deficiency. These findings indicate that the lack of T cells, which also means a requirement for immediate T cell replenishment, leads to an increase of Jam2+HSC fraction.Our findings suggest that Jam2 is the key protein that controls T lymphopoiesis by enhancing the Notch/Delta signal transduction via interaction with Jam1. It also means that the lineage balance particularly towards T lymphopoiesis might be regulated at a higher stage of hematopoietic hierarchy than currently understood. Thus, Jam2 is a new marker representing the T lymphocyte potential of HSCs, as the frequency of Jam2+HSCs sensitively reflects the state of the T cell environment. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic-Specific CSNK2B Loss in Mice Causes Impaired Erythropoiesis

CK2 (Csnk2, casein kinase 2) is a Ser-Thr kinase composed by two catalytic (α) and two regulatory (β) subunits and involved in the regulation of various signaling cascades, which are critical for stem cell biology and hematopoietic development. However, a direct role for CK2 during blood cell differentiation is still undefined. Here, we examined the function of CK2 in erythropoiesis by using a hematopoietic-specific conditional knockout mouse model of the β regulatory subunit (Vav1-CRE x Csnk2β f/f mice). Since CK2β knockout mice died in utero, the study was carried out during gestation collecting fetuses from 12.5 to 17.5 days post conception (dpc) and performing the analysis on fetal liver. CK2β knockout fetuses were pale and hydropic, displayed a smaller liver, disarrayed vascularization and haemorrhages. Lack of CK2β caused depletion of hematopoietic/precursor cells, in particular of common lymphoid progenitors and megakaryocyte-erythrocyte progenitors. CK2β loss resulted to affect both early and late erythroid maturation and red cell viability. CK2β knockout contained lower numbers of TER119 positive cells, which displayed a down modulation of the surface expression of transferrin receptor (CD71) and an increased spontaneous apoptosis. Erythroid cells showed alterations in morphology compatible with myelodysplastic changes. Loss of CK2β caused alterations of erythroid cell proliferation, which was different depending on the stage of erythroid maturation: indeed, BrdU and 7AAD staining showed that less mature erythroid cells (CD71+Ter119-) had a lower rate of proliferation but a normal viability; on the contrary, more mature (CD71-Ter119+) erythroid cells suffered in part of apoptosis and in part accumulated in the S phase. RNA seq analysis performed on purified Ter119+ cells revealed upregulation of TP53 -associated genes as well as of Cdkn1a (p21); on the contrary, there was a down-modulation of Stat5 (an erythropoietin receptor down-stream effector) and genes involved in red cell survival and differentiation in particular c-kit and genes associated to the PI3/Akt pathway. The expression of adhesion molecules and surface carriers for inorganic cations/anionsimportant for the osmotic equilibrium and cell membrane integrity was also found markedly dysregulated. Real time quantitative PCR and Western Blot (WB) analyses confirmed the expression data of Cdkn1a, c-Kit, Bcl-xL, Jak-Stat5 as well as of Akt-Gata-1 axis. Gata-1, the key transcription factor for definitive erythropoiesis, was reduced in CK2β knockout mice as were its downstream target genes such as Alas-2, Lrf, Eklf, Epo-R, β-globin. Immature fetal globins accumulated. In order to find a molecular mechanism, we used an in vitro model of erythroid differentiation based on G1ER cells, an estrogen inducible GATA-1 null murine erythroblast cell line; the combined treatment of β-estradiol and inhibition of CK2 through the chemical inhibitor CX-4945 or RNA interference against CK2β confirmed the negative effect on differentiation. Western blot analysis indicated a potential role of the kinase in the regulation of Akt, Gata-1 and Stat5 protein stability. Moreover, the blockade or down modulation of CK2 caused changes in Gata-1 nuclear distribution with loss of the speckled pattern induced by β-estradiol. Thus, CK2 is a likely essential controller of GATA-1 transcriptional function. Altogether, our work demonstrates that CK2 is a master regulator of erythroid development, by impinging on Stat5, Akt and Gata-1 signaling and influencing red cell viability, bioenergetics, proliferation and maturation. DisclosuresNo relevant conflicts of interest to declare.

Suppression of SRCAP chromatin remodelling complex and restriction of lymphoid lineage commitment by Pcid2

Lymphoid lineage commitment is an important process in haematopoiesis, which forms the immune system to protect the host from pathogen invasion. However, how multipotent progenitors (MPP) switch into common lymphoid progenitors (CLP) or common myeloid progenitors (CMP) during this process remains elusive. Here we show that PCI domain-containing protein 2 (Pcid2) is highly expressed in MPPs. Pcid2 deletion in the haematopoietic system causes skewed lymphoid lineage specification. In MPPs, Pcid2 interacts with the Zinc finger HIT-type containing 1 (ZNHIT1) to block Snf2-related CREBBP activator protein (SRCAP) activity and prevents the deposition of histone variant H2A.Z and transcription factor PU.1 to key lymphoid fate regulator genes. Furthermore, Znhit1 deletion also abrogates H2A/H2A.Z exchange in MPPs. Thus Pcid2 controls lymphoid lineage commitment through the regulation of SRCAP remodelling activity.

Altered B-lymphopoiesis in mice with deregulated thrombopoietin signaling

Thrombopoietin (TPO) is the master cytokine regulator of megakaryopoiesis. In addition to regulation of megakaryocyte and platelet number, TPO is important for maintaining proper hematopoietic stem cell (HSC) function. It was previously shown that a number of lymphoid genes were upregulated in HSCs from Tpo−/− mice. We investigated if absent or enhanced TPO signaling would influence normal B-lymphopoiesis. Absent TPO signaling in Mpl−/− mice led to enrichment of a common lymphoid progenitor (CLP) signature in multipotential lineage-negative Sca-1+c-Kit+ (LSK) cells and an increase in CLP formation. Moreover, Mpl−/− mice exhibited increased numbers of PreB2 and immature B-cells in bone marrow and spleen, with an increased proportion of B-lymphoid cells in the G1 phase of the cell cycle. Conversely, elevated TPO signaling in TpoTg mice was associated with reduced B-lymphopoiesis. Although at steady state, peripheral blood lymphocyte counts were normal in both models, Mpl−/− Eµ-myc mice showed an enhanced preneoplastic phase with increased numbers of splenic PreB2 and immature B-cells, a reduced quiescent fraction, and augmented blood lymphocyte counts. Thus, although Mpl is not expressed on lymphoid cells, TPO signaling may indirectly influence B-lymphopoiesis and the preneoplastic state in Myc-driven B-cell lymphomagenesis by lineage priming in multipotential progenitor cells.

Molecular synergy underlies the co-occurrence patterns and phenotype of NPM1-mutant acute myeloid leukemia

AbstractNPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandem duplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound Npm1cA/+;NrasG12D/+ or Npm1cA;Flt3ITD share a number of features: Hox gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in Npm1cA/+;NrasG12D/+ mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD, but not Npm1cA/+;NrasG12D/+, progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/+;NrasG12D/+. During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/+;NrasG12D/+ mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRAS-G12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML.

Molecular and Functional Characterization of Lymphoid Progenitor Subsets Reveals a Bipartite Architecture of Human Lymphopoiesis

The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127- and CD127+ early lymphoid progenitors (ELPs). Combining molecular analyses with invitro and invivo functional assays, we demonstrated that CD127- and CD127+ ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127- ELPs comprised precursors of Tcells, marginal zone Bcells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127+ ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis.

Asynchronous lineage priming determines commitment to T cell and B cell lineages in fetal liver.

The molecular events that initiate lymphoid-lineage specification remain unidentified because the stages of differentiation during which lineage commitment occurs are difficult to characterize. We isolated fetal liver progenitor cells undergoing restriction of their differentiation potential toward the T cell-innate lymphoid cell lineage or the B cell lineage. Transcripts that defined the molecular signatures of these two subsets were sequentially upregulated in lympho-myeloid precursor cells and in common lymphoid progenitor cells, respectively, and this preceded lineage restriction; this indicates that T cell-versus-B cell commitment is not a binary fate 'decision'. The T cell-bias and B cell-bias transcriptional programs were frequently co-expressed in common lymphoid progenitor cells and were segregated in subsets biased toward T cell differentiation or B cell differentiation, after interleukin 7 (IL-7) signaling that controlled the number of progenitor cells engaging in T cell differentiation versus B cell differentiation.

IL-7R\u03b1 glutamylation and activation of transcription factor Sall3 promote group 3 ILC development

Group 3 innate lymphoid cells (ILC3) promote lymphoid organogenesis and potentiate immune responses against bacterial infection. However, how ILC3 cells are developed and maintained is still unclear. Here, we show that carboxypeptidase CCP2 is highly expressed in common helper-like innate lymphoid progenitors, the progenitor of innate lymphoid cells, and CCP2 deficiency increases ILC3 numbers. Interleukin-7 receptor subunit alpha (IL-7Rα) is identified as a substrate of CCP2 for deglutamylation, and IL-7Rα polyglutamylation is catalyzed by polyglutamylases TTLL4 and TTLL13 in common helper-like innate lymphoid progenitors. IL-7Rα polyglutamylation triggers STAT5 activation to initiate transcription factor Sall3 expression in common helper-like innate lymphoid progenitors, which drives ILC3 cell differentiation. Moreover, Ttll4−/− or Ttll13−/− mice have reduced IL-7Rα polyglutamylation and Sall3 expression in common helper-like innate lymphoid progenitors. Importantly, mice with IL-7Rα E446A mutation have reduced Sall3 expression and ILC3 population. Thus, polyglutamylation and deglutamylation of IL-7Rα tightly controls the development and effector functions of ILC3s.

CCCTC-binding factor is essential to the maintenance and quiescence of hematopoietic stem cells in mice

Hematopoiesis involves a series of lineage differentiation programs initiated in hematopoietic stem cells (HSCs) found in bone marrow (BM). To ensure lifelong hematopoiesis, various molecular mechanisms are needed to maintain the HSC pool. CCCTC-binding factor (CTCF) is a DNA-binding, zinc-finger protein that regulates the expression of its target gene by organizing higher order chromatin structures. Currently, the role of CTCF in controlling HSC homeostasis is unknown. Using a tamoxifen-inducible CTCF conditional knockout mouse system, we aimed to determine whether CTCF regulates the homeostatic maintenance of HSCs. In adult mice, acute systemic CTCF ablation led to severe BM failure and the rapid shrinkage of multiple c-Kithi progenitor populations, including Sca-1+ HSCs. Similarly, hematopoietic system-confined CTCF depletion caused an acute loss of HSCs and highly increased mortality. Mixed BM chimeras reconstituted with supporting BM demonstrated that CTCF deficiency-mediated HSC depletion has both cell-extrinsic and cell-intrinsic effects. Although c-Kithi myeloid progenitor cell populations were severely reduced after ablating Ctcf, c-Kitint common lymphoid progenitors and their progenies were less affected by the lack of CTCF. Whole-transcriptome microarray and cell cycle analyses indicated that CTCF deficiency results in the enhanced expression of the cell cycle-promoting program, and that CTCF-depleted HSCs express higher levels of reactive oxygen species (ROS). Importantly, in vivo treatment with an antioxidant partially rescued c-Kithi cell populations and their quiescence. Altogether, our results suggest that CTCF is indispensable for maintaining adult HSC pools, likely by regulating ROS-dependent HSC quiescence.

DNA damage tolerance in hematopoietic stem and progenitor cells in mice

DNA damage tolerance (DDT) enables bypassing of DNA lesions during replication, thereby preventing fork stalling, replication stress, and secondary DNA damage related to fork stalling. Three modes of DDT have been documented: translesion synthesis (TLS), template switching (TS), and repriming. TLS and TS depend on site-specific PCNA K164 monoubiquitination and polyubiquitination, respectively. To investigate the role of DDT in maintaining hematopoietic stem cells (HSCs) and progenitors, we used PcnaK164R/K164R mice as a unique DDT-defective mouse model. Analysis of the composition of HSCs and HSC-derived multipotent progenitors (MPPs) revealed a significantly reduced number of HSCs, likely owing to increased differentiation of HSCs toward myeloid/erythroid-associated MPP2s. This skewing came at the expense of the number of lymphoid-primed MPP4s, which appeared to be compensated for by increased MPP4 proliferation. Furthermore, defective DDT decreased the numbers of MPP-derived common lymphoid progenitor (CLP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), and granulocyte-macrophage progenitor (GMP) cells, accompanied by increased cell cycle arrest in CMPs. The HSC and MPP phenotypes are reminiscent of premature aging and stressed hematopoiesis, and indeed progressed with age and were exacerbated on cisplatin exposure. Bone marrow transplantations revealed a strong cell intrinsic defect of DDT-deficient HSCs in reconstituting lethally irradiated mice and a strong competitive disadvantage when cotransplanted with wild-type HSCs. These findings indicate a critical role of DDT in maintaining HSCs and progenitor cells, and in preventing premature aging.

The miR-23a~27a~24-2 microRNA cluster buffers transcription and signaling pathways during hematopoiesis.

MicroRNA cluster mirn23a has previously been shown to promote myeloid development at the expense of lymphoid development in overexpression and knockout mouse models. This polarization is observed early in hematopoietic development, with an increase in common lymphoid progenitors (CLPs) and a decrease in all myeloid progenitor subsets in adult bone marrow. The pool size of multipotential progenitors (MPPs) is unchanged; however, in this report we observe by flow cytometry that polarized subsets of MPPs are changed in the absence of mirn23a. Additionally, in vitro culture of MPPs and sorted MPP transplants showed that these cells have decreased myeloid and increased lymphoid potential in vitro and in vivo. We investigated the mechanism by which mirn23a regulates hematopoietic differentiation and observed that mirn23a promotes myeloid development of hematopoietic progenitors through regulation of hematopoietic transcription factors and signaling pathways. Early transcription factors that direct the commitment of MPPs to CLPs (Ikzf1, Runx1, Satb1, Bach1 and Bach2) are increased in the absence of mirn23a miRNAs as well as factors that commit the CLP to the B cell lineage (FoxO1, Ebf1, and Pax5). Mirn23a appears to buffer transcription factor levels so that they do not stochastically reach a threshold level to direct differentiation. Intriguingly, mirn23a also inversely regulates the PI3 kinase (PI3K)/Akt and BMP/Smad signaling pathways. Pharmacological inhibitor studies, coupled with dominant active/dominant negative biochemical experiments, show that both signaling pathways are critical to mirn23a’s regulation of hematopoietic differentiation. Lastly, consistent with mirn23a being a physiological inhibitor of B cell development, we observed that the essential B cell transcription factor EBF1 represses expression of mirn23a. In summary, our data demonstrates that mirn23a regulates a complex array of transcription and signaling pathways to modulate adult hematopoiesis.

Research Highlights

Research Highlights

Hhex Regulates Hematopoietic Stem Cell Self-Renewal and Stress Hematopoiesis via Repression of Cdkn2a.

The hematopoietically expressed homeobox transcription factor (Hhex) is important for the maturation of definitive hematopoietic progenitors and B-cells during development. We have recently shown that in adult hematopoiesis, Hhex is dispensable for maintenance of hematopoietic stem cells (HSCs) and myeloid lineages but essential for the commitment of common lymphoid progenitors (CLPs) to lymphoid lineages. Here, we show that during serial bone marrow transplantation, Hhex-deleted HSCs are progressively lost, revealing an intrinsic defect in HSC self-renewal. Moreover, Hhex-deleted mice show markedly impaired hematopoietic recovery following myeloablation, due to a failure of progenitor expansion. In vitro, Hhex-null blast colonies were incapable of replating, implying a specific requirement for Hhex in immature progenitors. Transcriptome analysis of Hhex-null Lin- Sca+ Kit+ cells showed that Hhex deletion leads to derepression of polycomb repressive complex 2 (PRC2) and PRC1 target genes, including the Cdkn2a locus encoding the tumor suppressors p16Ink 4a and p19Arf . Indeed, loss of Cdkn2a restored the capacity of Hhex-null blast colonies to generate myeloid progenitors in vitro, as well as hematopoietic reconstitution following myeloablation in vivo. Thus, HSCs require Hhex to promote PRC2-mediated Cdkn2a repression to enable continued self-renewal and response to hematopoietic stress. Stem Cells 2017;35:1948-1957.

Phenotype and Function of Human Gut Hematopoietic Stem Cells and Progenitors

Lin−CD45+CD34+ cells have been reported in human small intestine and liver, but further phenotypic or functional analysis was not described[1][2]. We utilized flow cytometry and cytometry by time of flight (CyTOF) to study the composition and phenotype of human gut hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPs) from deceased donors. Colony-forming-cell (CFC) assays, long-term-culture-initiating-cell (LTC-IC) assays and humanized mouse models were established to further evaluate the differentiation potential of gut HSCs/HPs. HSCs and multiple types of HPs, including multipotent progenitors (MPP), lymphoid-primed multipotent progenitors (LMPP), common lymphoid progenitors (CLP), myeloid progenitors (MP), and unclassified progenitors co-expressing CD56, were detected in human intestinal mucosa and mesenteric lymph nodes (MLNs). Ileum lamina propria lymphocytes (LPL) included significantly higher percentages of HSCs than intraepithelial lymphocytes (IEL). Both IEL and LPL showed significantly lower percentages of CLP compared to MLNs. From jejunum or ileum specimens of ITx patients collected from their stoma revision/closure (POD47- 1544), variable levels of Lin−CD45+CD34+ cells were detected (Figure 1A). However, when gating on HSCs, donor cells (>90%) predominated over recipient cells in four patients with chimerism, regardless of their ages or early clinical outcomes (Figure 1B-C). Lin−CD45+CD34+ cells from ileum LP and bone marrow (BM) demonstrated similar properties in viSNE plots from CyTOF analysis (Figure 2), suggesting similar functional phenotypes. Both ileum LP and BM Lin−CD45+CD34+ cells contained subpopulations expressing the thymus-homing marker CCR9, and T/NK lineage polarized progenitors coexpressing CD45RA and CD7. Additionally, CFC assays showed that both ileum LP and BM Lin−CD34-enriched cells can form myeloid/erythroid colonies, although ileum LP CD34 cells were less efficient than BM CD34 cells. LTC-IC assay using single cell sorted ileum LP HSC co-culture with murine feeder cells suggested myeloid/lymphoid differentiation potential of human gut HSCs. Experiments using human gut Lin−CD45+CD34+ cells to reconstitute humanized mice are ongoing.FIGURE 1: (A) Lin−CD45+CD34+ cells were detected in human intestinal mucosa long-term post-Tx (POD47-1544) (B) Donor-derived hematopoietic stemcells (HSCs) and/or multiple types of hematopoietic progenitors (HPs)were detected in transplanted small bowel specimens. (C) Composition of HSCs/HPs when gating on donor (upper panel) or recipient (lower panel) Lin−CD45+CD34+ cells.FIGURE 2: Lin−CD45+CD34+ cells from bone marrow (BM) and ileum lamina propria (LP) located in the same region of the viSNE plot from CyTOF analysis of individual organ donor, suggesting the similarity of their phenotype and origin.Taken together, our data suggest that human gut HSCs/HPs may be functionally similar to BM HSCs/HPs that have thymus-homing ability and can give rise to multiple lineages, which likely contribute to the long-persistent mixed chimerism post-ITx. Research reported in this publication was performed in the Columbia Center for Translational Immunology (CCTI) Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. CyTOF study was performed in collaboration with the Human Immune Monitoring Core (HIMC) of Icahn School of Medicine at Mount Sinai.