Lymphoid-primed Multipotent Progenitors Research Articles

Dissection of Complementary Vs Unique Cntributions of Kit, Flt3 and IL-7R Signaling to Lymphohematopoietic Development in Vivo

Lymphohematopoietic development requires partially complementary cytokine signals mediated by the receptor tyrosine kinases Kit and Flt3, while lymphoid development also depends on the Type I cytokine receptor IL-7R. Common lymphoid progenitors (CLP) and the earliest thymic progenitors (ETP) uniquely express all three receptors. Complicating our understanding of the roles of these three cytokine pathways are synergistic cross-receptor interactions by which the RTK can activate non-cognate Type I cytokine receptors, e.g., IL-7R. To unravel the complex interplay between these three pathways, particularly in lymphopoiesis, we analyzed marrow, thymus and spleen populations in mice with loss of function of each of these pathways, alone or in combination (KitW41/W41 mutant, Flt3L-/-, and IL-7-/- knockouts (KO) so that the respective receptors Kit, Flt3 and IL-7R could be used for phenotypic analyses of progenitor populations). As expected, mice with mutations of Kit, alone or in combination with Flt3L and/or IL-7 KO, showed ~85% loss of both long-term (LT) and short-term (ST) HSC, and numbers of subsequent marrow and thymic progenitor populations (multipotent progenitor (MPP) 2, MPP3, CD62L+ lymphoid primed MPP (LMPP), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), megakaryocyte-erythroid progenitor (MEP), CLP, ETP) decreased proportionately. The earliest effects of Flt3L loss occurred at the MPP3 stage (~80% loss); surprisingly, the number of myeloid progenitors was increased and CLP decreased. IL-7 KO showed declines in lymphoid committed cells at the LMPP through the CLP and ETP stages. Again unexpectedly, IL-7 loss resulted in increased CMP. Mice deficient for both Kit and Flt3L were viable, had further additive reduction in MPP2 numbers, but showed profound, synergistic reductions in MPP3, LMPP, GMP, CLP, and ETP populations. The combination of IL-7 KO with either KitW41/W41 or Flt3L KO had no effect on the MPP2, but caused additive declines in LMPP and CLP. Mice triply deficient for Kit, Flt3L and IL-7 (“TKO” mice) were also viable, but showed profound (>99%) loss of all lymphoid progenitors (LMPP, CLP and ETP). To account for the downstream effects of the reduced HSC compartment in KitW41/W41 mice, the committed progenitor numbers were normalized to the input HSC numbers in the respective mouse strains. In the absence of Kit signaling, Flt3L and IL-7 loss had ~2-3 log greater effects on lymphopoiesis than myelopoiesis. Consistent with the analyses of numbers of lymphoid vs myeloid progenitors, gene expression analyses of sorted progenitors also showed that in the absence of either Kit and Flt3L, Kit and IL-7, or Kit, Flt3L, and IL-7, MPP3 showed a global shift away from expression of lymphoid developmental pathways and towards myeloid pathways. In addition to expression by CLP and ETP, a subset of MPP3 and LMPP also expressed IL-7R at low levels. These results demonstrate that while Kit has global effects on HSC and their progeny, and Flt3 and IL-7R signals are required only by committed progenitors, 1) Flt3 and IL-7R synergize with and complement Kit in directing lymphoid commitment and/or differentiation of MPP; and 2) that Flt3 and IL-7R signal at the MPP3 or LMPP stages to promote lymphoid development, i.e., earlier than phenotypic CLP. These results can be explained by the co-expression of the RTKs and signaling competent IL-7R in LMPP prior to the phenotypic CLP stage, and the previously described RTK-mediated activation of the IL-7R by non-cognate cross-receptor interactions. Analyses of these complex cytokine interactions may be useful in the elucidation of lymphoid committed progenitor subpopulations and lineage fate decisions among MPP, and the development of therapeutic strategies for expansion of specific progenitor cell populations. DisclosuresBrown:Cellerant Therapeutics: Consultancy, Equity Ownership, Patents & Royalties.

Sox4 Is Required for the Formation and Maintenance of Multipotent Progenitors

IntroductionHematopoiesis is maintained by a hierarchical system, whereas aberrant control of hematopoiesis is the underlying cause of many diseases. Within the hematopoietic hierarchy, hematopoietic stem cells (HSCs) give rise to multipotent progenitors that have lost their self-renewal capacity but remain multipotent to differentiate into mature blood cells. However, the precise molecular mechanisms that modulate this transition are not fully understood yet.ResultsWe recently discovered that genetic ablation of SRY sex determining region Y-box 4 gene (Sox4) in the murine hematopoietic system resulted in dramatic loss of multipotent progenitor population (CD48+CD150-Lin-kit+Sca1+, or CD48+CD150-LSK) both relatively (to the total LSK population) and in absolute number. Interestingly, the absolute number of HSCs (CD48-CD150+Lin-kit+Sca1+, or SLAM+LSK) in these conditional Sox4-deficient mice was comparable to their wild-type counterparts. Transcriptional factor Sox4 belongs to the high-mobility group (HMG) domain superfamily which also includes other Sox proteins, TCF-1 (T-cell factor 1) and LEF-1 (lymphoid enhancer factor 1). Sox4 has been implicated in leukemogenesis and may potentially contribute to stem cell properties. Nevertheless, the precise roles of Sox4 in hematopoietic stem/progenitor cells and the underlying mechanisms have not been defined yet. Further analysis of stem/progenitor compartment defined by Flt3 and CD34 expression demonstrated a major loss in lymphoid-primed multipotent progenitors (LMPPs) (CD34+Flt3+LSK) with relatively normal formation of LT-HSCs (CD34-Flt3-LSK) and ST-HSCs (CD34+Flt3-LSK) upon the loss of Sox4, suggesting that Sox4 is essential for the development from HSCs to multipotent progenitors. Such observation is in line with the expression pattern of Sox4. Quantitative PCR (qPCR) analysis of wild-type mice revealed that expression of Sox4 increased from HSCs to multipotent progenitors which expressed Sox4 at the highest level among all the hematopoietic compartments.Studies of biological behaviors further indicateed that disruption of Sox4 had no effect on proliferative capacity of HSCs and multipotent progenitors, as evidenced by BrdU incorporation assay. However, Annexin V/propidium iodide staining revealed an increased frequency of apoptotic multipotent progenitors, but not that of HSCs upon the ablation of Sox4. In a transplantation setting, although Sox4-deficient LSKs homed appropriately to the bone marrow, they exhibited severely impaired ability to give rise to multipotent progenitors, but contributed normally to HSCs compared to the wild-type donors.Among a set of genes crucial to the biological properties of stem/progenitor cells, qPCR analysis revealed that upon the loss of Sox4, only the levels of Ikaros1 and Ikaros2, the two major Ikaros isoforms in stem/progenitor cells, were downregulated specifically in multipotent progenitors, but remained normal in HSCs. Intriguingly, in a reminiscent manner of Sox4-deficient mice, mice lacking both Ikaros 1 and Ikaros 2 proteins, also exhibited disrupted B cell development and selectively impaired LMPPs. Previous study identified an enhancer of Ikaros locus as the only cis-regulatory element that was capable of stimulating reporter expression in the LMPPs. Our sequence analysis revealed a highly conserved Sox4 binding motif within this enhancer, therefore potentially connecting Sox4 with the known regulatory networks that modulate the differentiation of HSCs. Currently, we are working on (1) confirming the direct transcriptional regulation of Ikaros by Sox4; (2) assessing whether Ikaros mediates the functions of Sox4 in the formation or maintenance of the multipotent progenitors population in vivo; and (3) delineating the downstream regulatory network of Sox4 in stem/progenitor cells.ConclusionIn summary, out study reveals a novel role for Sox4 gene in early hematopoiesis and brings important insights into the regulatory mechanisms underlying the commitment of HSCs toward multipotent progenitors. DisclosuresNo relevant conflicts of interest to declare.

Defects in Early Lymphopoiesis Contribute to Sepsis-Induced Thymic Atrophy

Emergency hematopoiesis is important for the host to replenish the loss of immune cells and meet the enhanced demand for appropriate immune cells during acute infection. Previous studies have explored several intrathymic mechanisms that negatively affect thymopoiesis and subsequentlycontribute to sepsis-associated immunosuppression. However, the extrathymic mechanisms remain poorly understood.In line with previous studies, the mice subjected to cecal ligation and puncture developed severe T-cell lymphopenia. Moreover, lower numbers of T cell receptor circles excision circles (TRECs) were detected in septic mice when compared with control mice, which suggested a decrease in T-cell output from the thymus. Gross and histologic examinations and total thymus cellularity were in agreement with decreased thymic output. Next, we investigated whether sepsis affected intrathymic progenitors. There was a dramatic decline in the proportions as well as absolute numbers of early T-lineage progenitors (ETPs, Lin-CD117+CD44-CD25-CD127-) in the thymi of septic mice (Figure 1 A). Notably, the percentage of ETPs correlated positively with the number of thymocytes (r=0.6481, p=0.0005, Figure 1B).However, septic mice did not show a decrease in the number of bone marrow (BM) precursor cells. Instead, transwell migration assay exhibited that compared with cells from control mice, lymphoid-primed multipotent progenitors (LMPPs) and common lymphoid progenitors (CLPs) from septic mice exhibited significantly reduced chemotaxis towards chemokine gradients, including CCL19, CCL25, and P-selectin. In line with these results, real-time PCR revealed decreased mRNA levels of CCR7 (receptor of CCL19 and CCL21), CCR9 (receptor of CCL25), and P-selectin glycoprotein ligand (PSGL)1 in BM LMPPs and CLPs from septic mice when compared with control mice. In contrast, thymic tissues of septic mice expressed higher levels of mRNA for CCL19, CCL21 and CCL25. Thus, decreased expression of chemokine receptors might contribute to impaired chemotaxis of BM lymphoid progenitors in septic mice.To investigate whether sepsis affected the differentiation program of hematopoietic stem and progenitor cells (HSPCs), we compared the mRNA expression profiles of key regulators of lymphoid differentiation in HSPCs from control and septic mice. hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), LMPPs and CLPs from mice challenged with cecal ligation and puncture exhibited downregulated expression of several important genes for lymphoid lineage commitment, including Notch1, IL-7R, Flt-3, Egr-1, Ikaros and Rag-1. Unlike a universal decrease in the genes related to lymphoid development, the genes involved in myeloid lineage commitment displayed distinct patterns of kinetic changes in different progenitors, including HSCs and MPPs, following cecal ligation and puncture.Next, we wondered whether altered gene expression occurred in BM HSCs and MPPs disturbed the homeostasis between myeloid and lymphoid progenitors. In accordance with in vivo observations, we found that a significantly higher number of myeloid colonies were elicited by GM-CSF and a combination of IL-3, IL-6 and SCF from BM cells of septic mice than sham controls. However, despite an enhanced myelopoiesis in CLP mice, we found a defect in vitro T cell development by culturing LSK cells on OP9-DL1 cells. These observations further indicated that LPS might switch in the program controlling lineage commitment favoring myepoiesis.Thus, a depletion of ETPs in septic mice might be a consequence of an impaired migration of BM progenitors to the thymus, as well as a defect in lymphoid lineage commitment. Our findings present a novel view of how sepsis induces lymphopenia. Given that the disturbed hematopoiesis occurs at the level of stem cells and primitive progenitors, the defects might be amplified at more mature stages of several cell types, and hosts need more time to recover from disturbed immune homeostasis. These mechanisms might be involved in various pathological conditions of infection, therefore, such insights might further provide a rationale for manipulating immune responses and designing effective immunotherapeutic strategies for a variety of inflammatory diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterization of the Hematopoietic Stem and Progenitor Cell Hierarchy in Myelodysplastic Syndromes Patients with Monosomy 7 As the Sole Cytogenetic Abnormality

Deletions involving chromosome 7 constitute the second most common cytogenetic abnormality in myelodysplastic syndromes (MDS), associated with an overall poor outcome and high risk of transformation to acute myeloid leukemia (AML). Previous studies focusing on low to intermediate risk MDS, including cases with del(5q) as the only cytogenetic abnormality, have revealed a remarkably preserved stem and myeloid progenitor cell hierarchy, more characteristic of normal hematopoiesis than that of AML. Herein we explored this in a group of seven higher-risk MDS cases, all with an isolated deletion of the entire chromosome 7 (-7; monosomy 7), as the only cytogenetic abnormality.In all investigated isolated monosomy 7 cases, the expression of cell surface markers used to define the normal hematopoietic stem and myeloid progenitor cell hierarchy in bone marrow resembled more the expression pattern observed in AML than in low-risk MDS, irrespectively of the -7 MDS patients blast counts. Specifically, when compared to healthy age-matched controls, we observed a 15-fold reduction of LIN-CD34+CD38-CD45RA-CD90+ cells (hematopoietic stem cells; HSCs, p=0.010), a 15-fold increase in LIN-CD34+CD38+CD123+CD45RA+ cells (Granulocyte-macrophage progenitors; GMPs, p=0.038) and a 300-fold increase of LIN-CD34+CD38-CD90-CD45RA+ cells (lymphoid-primed multipotent progenitors; LMPPs, p=0.050) within total bone marrow mononuclear cells of MDS patients with isolated monosomy 7. Fluorescence in situ hybridization (FISH) analysis revealed high -7 clonal involvement (>80%) for all investigated progenitor populations, whereas it was much lower (<20%) in the HSC compartment. Functional assays (colony forming unit assays, long term culture initiating cell assays and mouse xenotransplantation in-vivo) revealed dramatic reductions in stem and progenitor activities in the investigated -7 patients, more resembling that previously reported in AML than in low risk MDS cases. Various differentiation assays supported that the -7 clone was much less affected in its myeloid than erythroid differentiation pathway. Similar to -7, also identifiedconcomitant recurrent driver mutations showed high clonal involvement. Notably, while a 3- to 4-fold reduction of -7 clonal involvement was observed in total bone marrow cells in patients achieving good partial remissions in response to azacytidine, the treatment failed to significantly impact on the high -7 clonal involvement in expanded GMPs and LMPPs.In conclusion, investigations of the stem and progenitor cell compartments of MDS patients with loss of chromosome 7 as the sole cytogenetic abnormality, is associated with a loss of HSC phenotype and function, and an expansion of cells with a GMP and LMPP phenotype, remarkably similar to what is frequently observed in AML, and in contrast to reported low-risk MDS cases. While azacytidine treatment significantly reduces the overall clonal involvement in bone marrow, it fails to significantly affect the -7 clonal involvement in notably expanded progenitor cell populations, providing an explanation for the typically short duration of remissions in these patients, and identifying distinct and potentially important target cells for future treatments. DisclosuresNo relevant conflicts of interest to declare.

Foxp3+ regulatory T cells ensure B lymphopoiesis by inhibiting the granulopoietic activity of effector T cells in mouse bone marrow.

Foxp3(+) Treg cells are crucial for maintaining T-cell homeostasis, but their role in B-cell homeostasis remains unclear. Here, we found that Foxp3 mutant scurfy mice had fewer B-lineage cells and progenitors, including common lymphoid progenitors and lymphoid-primed multipotent progenitors, but higher myeloid-lineage cell numbers in BM compared with WT littermates. Homeostasis within the HSC compartment was also compromised with apparent expansion of long- and short-term HSCs. This abnormality was due to the lack of Treg cells, but not to the Treg-cell extrinsic functions of Foxp3 or cell-autonomous defects. Among cytokines enriched in the BM of scurfy mice, IFN-γ affected only B lymphopoiesis, but GM-CSF, TNF, and IL-6 collectively promoted granulopoiesis at the expense of B lymphopoiesis. Neutralization of these three cytokines reversed the hematopoietic defects on early B-cell progenitors in scurfy mice. Treg cells ensured B lymphopoiesis by reducing the production of these cytokines by effector T cells, but not by directly affecting B lymphopoiesis. These results suggest that Treg cells occupy an important niche in the BM to protect B-lineage progenitor cells from excessive exposure to a lymphopoiesis-regulating milieu.

Requirement for ssbp2 in hematopoietic stem cell maintenance and stress response.

Transcriptional mechanisms governing hematopoietic stem cell (HSC) quiescence, self-renewal, and differentiation are not fully understood. Sequence-specific ssDNA-binding protein 2 (SSBP2) is a candidate acute myelogenous leukemia (AML) suppressor gene located at chromosome 5q14. SSBP2 binds the transcriptional adaptor protein Lim domain-binding protein 1 (LDB1) and enhances LDB1 stability to regulate gene expression. Notably, Ldb1 is essential for HSC specification during early development and maintenance in adults. We previously reported shortened lifespan and greater susceptibility to B cell lymphomas and carcinomas in Ssbp2(-/-) mice. However, whether Ssbp2 plays a regulatory role in normal HSC function and leukemogenesis is unknown. In this study, we provide several lines of evidence to demonstrate a requirement for Ssbp2 in the function and transcriptional program of hematopoietic stem and progenitor cells (HSPCs) in vivo. We found that hematopoietic tissues were hypoplastic in Ssbp2(-/-) mice, and the frequency of lymphoid-primed multipotent progenitor cells in bone marrow was reduced. Other significant features of these mice were delayed recovery from 5-fluorouracil treatment and diminished multilineage reconstitution in lethally irradiated bone marrow recipients. Dramatic reduction of Notch1 transcripts and increased expression of transcripts encoding the transcription factor E2a and its downstream target Cdkn1a also distinguished Ssbp2(-/-) HSPCs from wild-type HSPCs. Finally, a tendency toward coordinated expression of SSBP2 and the AML suppressor NOTCH1 in a subset of the Cancer Genome Atlas AML cases suggested a role for SSBP2 in AML pathogenesis. Collectively, our results uncovered a critical regulatory function for SSBP2 in HSPC gene expression and function.

Multipotent Hematopoietic Progenitors Divide Asymmetrically to Create Progenitors of the Lymphomyeloid and Erythromyeloid Lineages

SummaryHematopoietic stem and progenitor cells (HSPCs) can self-renew and create committed progenitors, a process supposed to involve asymmetric cell divisions (ACDs). Previously, we had linked the kinetics of CD133 expression with ACDs but failed to detect asymmetric segregation of classical CD133 epitopes on fixed, mitotic HSPCs. Now, by using a novel anti-CD133 antibody (HC7), we confirmed the occurrence of asymmetric CD133 segregation on paraformaldehyde-fixed and living HSPCs. After showing that HC7 binding does not recognizably affect biological features of human HSPCs, we studied ACDs in different HSPC subtypes and determined the developmental potential of arising daughter cells at the single-cell level. Approximately 70% of the HSPCs of the multipotent progenitor (MPP) fraction studied performed ACDs, and about 25% generated lymphoid-primed multipotent progenitor (LMPP) as wells as erythromyeloid progenitor (EMP) daughter cells. Since MPPs hardly created daughter cells maintaining MPP characteristics, our data suggest that under conventional culture conditions, ACDs are lineage instructive rather than self-renewing.

CD11a is essential for normal development of hematopoietic intermediates.

The process of lymphopoiesis begins in the bone marrow (BM) and requires multiple cellular intermediates. For T cell production, lymphoid progenitors exit the BM and home to the thymus where maturation and selection ensue. These processes are dependent on a number of factors, including chemokines and adhesion molecules. Although the β2 integrin CD11a plays an important role in the migration of lymphocytes to lymph nodes, the role of CD11a in T cell development is largely undefined. Our studies now show that, in CD11a(-/-) mice, thymic cellularity was decreased and early T cell development was partially impaired. Remarkably, CD11a was critical for generation of common lymphoid progenitors (CLPs) and lymphoid-primed multipotent progenitors. However, in intact CD11a(-/-) mice, peripheral B and T cell subsets were only modestly altered, suggesting that compensatory mechanisms were operating. In contrast, competitive BM-reconstitution assays revealed an essential role for CD11a in the generation of thymocytes and mature T and B cells. This defect was linked to the requirement for CD11a in the development of CLPs. Furthermore, our results identified CLPs, and not lymphoid-primed multipotent progenitors, as the requisite CD11a-dependent precursor for lymphocyte development. Thus, these findings established a key role for CD11a in lymphopoiesis.

Chemokine treatment rescues profound T-lineage progenitor homing defect after bone marrow transplant conditioning in mice

AbstractDevelopment of T cells in the thymus requires continuous importation of T-lineage progenitors from the bone marrow via the circulation. Following bone marrow transplant, recovery of a normal peripheral T-cell pool depends on production of naïve T cells in the thymus; however, delivery of progenitors to the thymus limits T-lineage reconstitution. Here, we examine homing of intravenously delivered progenitors to the thymus following irradiation and bone marrow reconstitution. Surprisingly, following host conditioning by irradiation, we find that homing of lymphoid-primed multipotent progenitors and common lymphoid progenitors to the thymus decreases more than 10-fold relative to unirradiated mice. The reduction in thymic homing in irradiated mice is accompanied by a significant reduction in CCL25, an important chemokine ligand for thymic homing. We show that pretreatment of bone marrow progenitors with CCL25 and CCL21 corrects the defect in thymic homing after irradiation and promotes thymic reconstitution. These data suggest new therapeutic approaches to promote T-cell regeneration.

Unique progenitors in adult mouse bone marrow expressing CD127 (IL-7Rα) and high level of cKit efficiently develop into all lymphocytes including T cells and innate lymphoid cells (HEM2P.266)

Abstract All lymphocytes are thought to develop from common lymphoid progenitors (CLPs) that are found in the Lin−Sca-1locKitloCD127+Flt3hi bone marrow (BM) cell population. However, only small numbers of T cells and innate lymphoid cells (ILCs) are generated from CLPs transplanted into irradiated mice, suggesting that they might mainly develop from alternative progenitors such as lymphoid-primed multipotent progenitors (LMPPs). We now have identified novel progenitors in adult mouse BM that efficiently develop into all lymphocytes including T cells and ILC2s. The novel progenitors are similar to LMPPs and found in the cKithiSca-1+Flt3+ BM cell subset, but unlike LMPPs they express the lymphoid lineage marker CD127, thus resembling early thymic progenitors (ETPs). Upon transplantation into irradiated mice, CD127+ LMPP-like cells robustly engraft and develop into T, B, NK cells and ILC2s. They are more efficient than LMPPs and CLPs in generating T cells and ILC2s. Similar to LMPPs, they also retain myeloid potentials. Flt3L-deficient mice have greatly reduced LMPPs and CLPs but have almost normal numbers of CD127+ LMPP-like progenitors, pre-ILC2 in the BM and mature ILC2 in the lung. While the exact relationship between CD127+ LMPP-like progenitors, CD127- LMPPs and CLPs are yet to be clarified, these results suggest that CD127+ LMPP-like BM cells are important progenitors for T cells and ILC2s independent of CLPs.

Notch signals are required for in vitro but not in vivo maintenance of human hematopoietic stem cells and delay the appearance of multipotent progenitors

AbstractAll blood cell lineages start from hematopoietic stem cells (HSCs), which were recently shown to represent a heterogeneous group of cells. In mice, Notch signaling promotes the maintenance of “stemness” as well as the expansion of self-renewing HSCs in vitro. Additionally, human CD34+ cells were shown to expand in vitro in response to Notch signals. However, it is unclear whether Notch directly affects all HSCs, and whether this role is relevant in vivo. Here, we developed culture conditions that support the maintenance of CD34+CD133+CD90lowCD38−CD7−CD10−CD45RA− (CD90low) cells, phenotypically defined HSCs, as well as 2 early progenitor cells (CD34+CD38−CD7−CD10−CD45RAint [RAint] and CD34+CD38−CD7−CD10−CD45RAhi [RAhi]) that were functionally equivalent to multipotent progenitor-2 and lymphoid-primed multipotent progenitor, respectively, found in cord blood. Using a genetic approach, we show that Notch signals were required for HSC preservation, with cultured HSCs being equal to ex vivo HSC cells in their ability to reconstitute immunodeficient mice; however, dnMaml-transduced HSCs were not maintained in vitro. Interestingly, Notch signaling did not appear to be required for the self-renewal of human HSCs in vivo. Our findings support the notion that Notch signals maintain human HSCs in vitro that have hematopoietic-reconstituting ability in vivo and delay the appearance of 2 newly described early progenitor cells.

Granulocyte-Macrophage Progenitors (GMPs) Express Low Adhesive Potential and High CXCR-4 Levels

Hematopoietic stem and progenitor cells (HSPCs) reside in a particular and specialized microenvironmental niche. Interaction of HSPCs with their niche is fundamental for cell proliferation, differentiation and quiescence. While alterations of HSPC-niche interactions have been shown to play an important role in animal models, methods to study the human HSPC niche are lacking. In order to characterize the adhesion of various HSPC subsets to various extracellular matrix proteins found in the bone marrow, we used high-throughput fluorescent microscopy to study cell adhesion by quantifying the ratio of adherent versus non-adherent cells being detached by gravity. This allowed the analysis of low cell numbers making the study of clinical samples feasible. Multiparameter flow cytometry was used to analyze both the adherent and non-adherent fractions to study the major subsets of CD34+ cells, evaluating CD34, CD38, CD45RA, CD123 and CD90 surface levels.More than 80% of plated CD34+ cells adhered to fibronectin, 70% to VCAM-1, while only a minor proportion adhered to laminin, osteopontin and collagen-1 (11%-19%). Inhibitory antibodies against α4 and α5 integrin were used to inhibit adhesion of CD34+ cells to fibronectin and VCAM-1. α4 integrin antibody significantly decrease the number of adherent CD34+ cells to fibronectin from 65% to 38% (p< 0.0001) and on VCAM-1 from 51% to 12% (p< 0.0001). No effect was obtained by α5 integrin antibody. When comparing the major subsets of CD34+ cells, megakaryocyte-erythroid progenitors (MEPs) were the subpopulation that adhered strongest to each substrate followed by common myeloid progenitors (CMPs) and hematopoietic stem cells (HSCs). By contrast, granulocyte-macrophage progenitors (GMPs) adhered less to each of the studied matrix proteins (Fig. 1a-1b). Expression levels of CD34 were higher in adherent than in non-adherent cells, while CD45RA+ cells showed the opposite pattern (Fig. 1c-1d). These data suggest that the level of differentiation influences the adhesion properties and that more immature cells have a higher affinity to niche elements. We then analyzed the expression of adhesion markers in the different CD34+ cells subsets. Unexpectedly, GMPs, the least adhesive population, showed higher expression of CXCR4, a key chemokine receptor involved in HSPCs' homing and differentiation. GMPs have been described to constitute the leukemic stem cell population in AML. GMPs also showed a high expression of CD44, a glycoprotein required for migration, cell interactions and adhesion. HSCs showed higher levels of α2 integrin while MEPs expressed more α4 integrin. The other subpopulations, common myeloid progenitors (CMPs), multipotent progenitors (MPPs) and lymphoid-primed multipotent progenitors (LMPPs) showed lower levels of all studied adhesion markers.In conclusion, we present a method to quantify the adhesion of different human HPSC subpopulations. The assay is suitable for analysis of human myeloid neoplasms and may elucidate aberrant patterns as well as reveal potential targets for anti-adhesive therapy inhibiting the niche«s ability to sustain the leukemic stem cell population. [Display omitted] MEPs show more adhesiveness to all the substrates studied, followed by CMPs and HSCs. HSCs exhibits a preference for fibronectin compared to the other proteins tested. The less adhesive seems to be GMPs (a,b,c). Difference between GMPs and MEPs adhesiveness is statistically validated (p< 0.01). CD34 expression is higher on adherent cells; on the contrary, CD45RA is more expressed on non adherent cells (d,e). [Display omitted] GMPs express more CXCR-4 and CD44 than the other subsets, MEPs exhibit high levels of α4 integrin, explaining why this population seems more adhesive to all the substrates. α4 and α5 integrins are important also for GMPs (a).Expression of CXCR-4 in all the subpopulations (b). Disclosures:No relevant conflicts of interest to declare.

Early B-cell Factor 1 Regulates the Expansion of B-cell Progenitors in a Dose-dependent Manner

Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1(+/-) pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development.

Transcriptional regulation of the Ikzf1 locus

AbstractIkaros is a critical regulator of lymphocyte development and homeostasis; thus, understanding its transcriptional regulation is important from both developmental and clinical perspectives. Using a mouse transgenic reporter approach, we functionally characterized a network of highly conserved cis-acting elements at the Ikzf1 locus. We attribute B-cell and myeloid but not T-cell specificity to the main Ikzf1 promoter. Although this promoter was unable to counter local chromatin silencing effects, each of the 6 highly conserved Ikzf1 intronic enhancers alleviated silencing. Working together, the Ikzf1 enhancers provided locus control region activity, allowing reporter expression in a position and copy-independent manner. Only 1 of the Ikzf1 enhancers was responsible for the progressive upregulation of Ikaros expression from hematopoietic stem cells to lymphoid-primed multipotent progenitors to T-cell precursors, which are stages of differentiation dependent on Ikaros for normal outcome. Thus, Ikzf1 is regulated by both epigenetic and transcriptional factors that target its enhancers in both redundant and specific fashions to provide an expression profile supportive of normal lymphoid lineage progression and homeostasis. Mutations in the Ikzf1 regulatory elements and their interacting factors are likely to have adverse effects on lymphopoiesis and contribute to leukemogenesis.

Growth factor independent-1 Maintains Notch1-Dependent Transcriptional Programming of Lymphoid Precursors

Growth factor independent 1 (Gfi1) is a transcriptional repressor originally identified as a gene activated in T-cell leukemias induced by Moloney-murine-leukemia virus infection. Notch1 is a transmembrane receptor that is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL). Gfi1 is an important factor in the initiation and maintenance of lymphoid leukemias and its deficiency significantly impedes Notch dependent initiation of T-ALL in animal models. Here, we show that immature hematopoietic cells require Gfi1 to competently integrate Notch-activated signaling. Notch1 activation coupled with Gfi1 deficiency early in T-lineage specification leads to a dramatic loss of T-cells, whereas activation in later stages leaves development unaffected. In Gfi1 deficient multipotent precursors, Notch activation induces lethality and is cell autonomous. Further, without Gfi1, multipotent progenitors do not maintain Notch1-activated global expression profiles typical for T-lineage precursors. In agreement with this, we find that both lymphoid-primed multipotent progenitors (LMPP) and early T lineage progenitors (ETP) do not properly form or function in Gfi1−/− mice. These defects correlate with an inability of Gfi1−/− progenitors to activate lymphoid genes, including IL7R, Rag1, Flt3 and Notch1. Our data indicate that Gfi1 is required for hematopoietic precursors to withstand Notch1 activation and to maintain Notch1 dependent transcriptional programming to determine early T-lymphoid lineage identity.

Diverse and heritable lineage imprinting of early hematopoietic progenitors

Hematopoietic stem cells (HSCs) and their subsequent progenitors produce blood cells, but the precise nature and kinetics of this production is contentious. In one model, lymphoid and myeloid production split after the lymphoid-primed multipotent progenitor (LMPP), with both branches subsequently producing dendritic cells (DCs). However, this model is largely based on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single cell complexity. Here we avoid these pitfalls using a new quantitative version of

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.

Hoxa9 and Flt3 Signaling Synergistically Regulate an Early Checkpoint in Lymphopoiesis

Hoxa9 and Flt3 signaling are individually important for the generation of lymphoid lineage precursors from multipotent hematopoietic progenitors (MPP) in bone marrow. Mice deficient for Hoxa9, Flt3, or Flt3 ligand (FL) have reduced numbers of lymphoid-primed multipotential progenitors (LMPP), common lymphoid progenitors (CLP), and B/T cell precursors. Hoxa9 regulates lymphoid development, in part, through transcriptional regulation of Flt3. However, it was unclear whether Hoxa9 has functions in lymphopoiesis independent of, or alternatively, synergistically with Flt3 signaling. In this study, we show that Hoxa9(-/-)Flt3l(-/-) mice have more severe deficiencies in all B lineage cells, CLP, LMPP, and total Flt3(+) MPP in bone marrow than the single knockouts. Although LMPP and Flt3(+) CLP contain precursors for NK and dendritic cell lineage cells, no deficiencies in these lineages beyond that in Flt3l(-/-) mice was found. Thymocyte cellularity was significantly reduced in the compound knockout, although peripheral T cell numbers mirrored Flt3l(-/-) mice. Analysis of the hematopoietic progenitor compartment revealed elevated numbers of CD150(+hi)CD34(-)CD41(+) myeloid-biased stem cells in Hoxa9(-/-)Flt3l(-/-) mice. In contrast, CD150(-) MPP enriched for lymphoid potential were synergistically reduced, suggesting Hoxa9 and Flt3 signaling function coordinately to regulate lymphopoiesis at a very early stage. Real-time PCR analysis of CD150(-)Flt3(+) cells from wild-type control, Hoxa9(-/-), and Flt3l(-/-) single knockouts revealed decreased lymphoid transcripts, corroborating the importance of these regulators in lymphoid development. Taken together, these studies reveal a very early checkpoint in lymphopoiesis dependent on the combinatorial activities of Hoxa9 function and Flt3 signaling.

Hematopoietic progenitor cell lines with myeloid and lymphoid potential

Investigation of immune cell differentiation and function is limited by shortcomings of suitable and scalable experimental systems. Here we show that an estrogen–regulated form of HOXB8 that is retrovirally delivered into mouse bone marrow cells can be used along with FLT3 ligand to conditionally immortalize early hematopoietic progenitor cells (Hoxb8–FL). Hoxb8–FL cells have lost self–renewal capacity and megakaryocyte/ erythroid lineage potential, but sustain myeloid and lymphoid potential. Hoxb8–FL cells differentiate in vitro and in vivo into different myeloid and lymphoid cell types, including macrophages, granulocytes, dendritic cells and B– and T–lymphocytes, which are phenotypically and functionally indistinguishable from their primary counterparts. Quantitative in vitro cell lineage potential assays implicate that myeloid and B–cell potential of Hoxb8–FL cells is comparable to primary lymphoid–primed multipotent progenitors, while T–cell potential is comparatively reduced. Given the simplicity and unlimited proliferative capacity of Hoxb8–FL cells, this system provides unique opportunities to investigate cell differentiation and immune cell functions.

A Clonogenic Progenitor with Prominent Plasmacytoid Dendritic Cell Developmental Potential

Macrophage and dendritic cell (DC) progenitors (MDPs) and common DC progenitors (CDPs) are bone marrow (BM) progenitors with DC differentiation potential. However, both MDPs and CDPs give rise to large numbers of conventional DCs (cDCs) and few plasmacytoid DCs (pDCs), implying that more dedicated pDC progenitors remain to be identified. Here we have described DC progenitors with a prominent pDC differentiation potential. Although both MDPs and CDPs express the macrophage colony stimulating factor (M-CSF) receptor (M-CSFR), the progenitors were confined to a M-CSFR(-) fraction, identified as Lin(-)c-Kit(int/lo)Flt3(+)M-CSFR(-), and expressed high amounts of E2-2 (also known as Tcf4) an essential transcription factor for pDC development. Importantly, they appeared to be directly derived from either CDPs or lymphoid-primed multipotent progenitors (LMPPs). Collectively, our findings provide insight into DC differentiation pathways and may lead to progenitor-based therapeutic applications for infection and autoimmune disease.