Antigen shared by human hemopoietic precursor cells and T and B lineage cells

In this study, we investigated antigens present at the surface of acute myeloblastic leukemia (AML) cells by using the monoclonal antibody (MAb) approach. The MAb AGF43 reacted with acute myeloid and lymphoid leukemia cells and chronic lymphocytic leukemia cells and was unreactive against chronic myeloid leukemia cells. A large proportion of AML blasts showing minimal or no differentiation (AML-M1) were intensely labeled by AGF43 in contrast to a smaller percentage of blasts showing partial differentiation (AML-M2 and acute myelomonocytic leukemia). The AGF43 antigen is expressed by bone marrow lymphoid (TdT+) and myeloid (CFU-GM) progenitor cells, 95% of B cells and 65% of T cells in the blood and absent from monocytes. Only 17% of normal myeloblasts were weakly stained by AGF43. Sections of tonsil and spleen were used to confirm that, unlike antibodies to MHC class II antigens, AGF43 stained a majority of T cells and macrophages were unreactive. In conclusion, the MAb AGF43 identifies a new precursor cell antigen. The distribution of this antigen during normal myelopoiesis and on AML cells support the suggestion that acute myeloid leukemias originate in pluripotent or closely related myeloid stem cells.     

Generation of B lymphocytes from a single hemopoietic progenitor cell in vitro

The first stages of the pathway by which lymphocytes differentiate from hemopoietic stem cells were studied at a clonal level. When 211 interleukin 3 (IL-3)-induced blast colonies shown to be capable of differentiating into a variety of hemopoietic cells were individually transferred into wells containing a monolayer of stromal cells, growth in granulocyte, macrophage, megakaryocyte, or mast cell lineages was observed in 192 wells. In seven of these 192 wells, lymphoid cell growth also was seen. The lymphoid cells were proved to be B lymphocytes by phenotype and immunoglobulin gene rearrangement analyses and by demonstration of surface expression of IgM. The clonal origin of myeloid and B lymphocyte lineage cells was further confirmed by the generation of both myeloid and B lymphoid cells in the same well following FACS clone-sorting of IL-3 induced blast cells. These results provide in vitro evidence that cells of B lymphoid and myeloid lineage can originate clonally from single primitive hemopoietic stem cells.     

The splenic pool of mouse stem cells: in vitro differentiation and expression of the BP-1 alloantigen on cells of the lymphoid and myeloid lineages

The relative paucity of data about the development of the stem cell pool present in the spleen prompted this study. During in vitro cultures of B-enriched lymphocytes from mouse spleens and in the presence of a culture supernatant of WEHI-3 cells (WEHI-SUP), a population of cells expressing the BP-1 antigen appears progressively, reaches an optimal size 8 days after initiation of the culture, and disappears on day 28. In 8-day-old cultures, a minor population of cells bearing both BP-1 and B220 can be detected. The growth of this cell population, with characteristics of the B lymphoid lineage (pro-B), is strictly dependent on the presence of WEHI-SUP in the medium. After 2 weeks of culture, the BP-1 antigen is expressed on a cell population, which is essentially constituted of B220-, polynuclear cells. The BP-1 antigen, which is considered as characteristic of early cells of the B lymphoid lineage, can therefore also be expressed on cells of the myeloid lineage. The injection of BP-1+ or B220+ cells in irradiated mice can hardly reconstitute their B cell pool, whereas BP-1- and B220- cells are much more efficient in vivo progenitors of this cell lineage.     

Liver sinusoidal endothelial cells promote lymphohematopoietic differentiation from murine embryonic stem cells: Role of soluble factors

Liver sinusoid endothelial cells (LSEC) constitute an in vitro and in vivo microenvironment for the proliferation and differentiation of hematopoietic stem cells (HSC). Previously, we have shown that LSEC support the survival and growth of murine embryonic stem cells (ESC). In this study, we investigated the capacity of LSEC to promote hematopoietic differentiation from the murine ESC cell line, CGR8. Undifferentiated ESC were cultured on LSEC monolayers in the absence of exogenous cytokines. After 10 and 20 days, cells were harvested and examined by their morphology, phenotype and capacity of hematopoietic colony formation. Microscopic observation of LSEC/ESC cocultures showed the presence of cobblestone areas formation, which indicates active hematopoiesis. Morphological analysis of cell from these foci showed the presence of hematopoietic cells at different stages of differentiation. Cells expressing B lymphoid markers (B220 and CD19) were detected by flow cytometry, and clonogenic assays showed the formation of CFU-pre B colonies. Similar results were observed when ESC were cultured with LSEC conditioned media. Myeloid precursors were also detected by the presence of CFU-GM colonies and cells expressing myeloid markers. These results indicate that LSEC provided an in vitro microenvironment mainly for B cell development, but also myeloid differentiation from ESC. Coculture of ESC with LSEC may constitute a very powerful tool to study the mechanisms involved in B cell generation from ESC.     

The Polyclonal B Cell Activator Dextran-Sulphate Induces Formation of Colony Stimulating Activity

The effect of dextran-sulphate (DS), a polyclonal activator known to stimulate immature murine B-cells, was assayed in a culture system allowing the growth of myeloid cells. It was known that DS induced the production of a myeloid colony stimulating factor (CSF) by cells from both spleen and bone marrow. Nylonwool purified mouse spleen cells, enriched for T cells, showed a dimished CSF production in response to DS, while CSF production in response to Con A was increased. Furthermore, DS induced CSF in both spleen and bone marrow cells from nude mice. Removal of macrophages did not affect CSF production. The CSF induced was non-dialysable and no small molecular weight or lipoprotein inhibitors could be demonstrated. The results suggest that DS activates cells other than T cells or macrophages (possibly B cells or null cells) to produce a myeloid stem cell stimulating substance. These results indicate that interactions between lymphoid and myeloid cells can take place during differentiation.     

Generation of functionally distinct B lymphocytes from common myeloid progenitors

Current models of adult haematopoiesis propose that haematopoietic stem cells (HSCs) differentiate into common lymphoid (CLP) and common myeloid (CMP) progenitors and establish an early separation between myeloid and lymphoid lineages. Nevertheless, the developmental potential of CMP-associated B cells suggests the existence of alternate pathways for B lymphopoesis. The aim of this study was to compare the developmental and functional properties of CMP- and CLP-derived B cells. While both populations matured through pro-B cell and transitional B cell intermediates in the bone marrow and spleen, respectively, following transfer into irradiated mice, mature CMP- and CLP-derived B cells exhibit distinct functional responses. Specifically, CMP-derived B cells did not respond to mitogenic stimulation to the same degree as their CLP-derived counterparts and secrete lower levels of IgM and the inflammatory cytokines such as interleukin (IL)-6 and IL-10. Together, these data suggest the existence of multiple pathways for generating functionally distinct B cells from bone marrow precursors.     

miR‐221 redirects precursor B cells to the BM and regulates their residence

Pluripotent hematopoietic stem cells and multipotent myeloid/lymphoid progenitors express miR‐221 and miR‐222. When Pax5 expression commits these progenitors to monopotent pre‐B lymphocytes the two microRNAs (miRNAs) are downregulated. Upon transplantation, stem cells and progenitors can reside in the BM, while pre‐B cells, after their commitment, no longer do so. Retrovirally transduced, doxycycline‐induced overexpression of either miR‐221 or miR‐222 in pre‐B‐I cells does not revert their monopotency to multipotency. However, upon transplantation miR‐221, but not miR‐222, transduced pre‐B‐I cells regain the capacity to reside in the BM. Upon subsequent termination of miR‐221‐expression by removal of doxycycline, the transplanted cells leave the BM again. Microarray analyses identified 25 downregulated miR‐221‐target genes, which could function to localize phases of B‐lymphocyte development in BM before and after commitment.     

Unraveling the origin of lymphocyte progenitors

Lymphocytes are ultimately derived from hematopoietic stem cells, however the intervening steps that give rise to lymphocyte progenitors are still under intense investigation. In particular, whether a common lymphocyte progenitor serves exclusively as a lymphoid precursor, or whether this cell retains limited myeloid progenitor potential are addressed by Rolink and colleagues. This issue is highlighted by their identification within the bone marrow of early progenitors with lymphoid and myeloid developmental potential or EPLMs. How these cells fit into our current understanding of lymphocyte development, and how this new subset helps to further refine our view of the common lymphocyte progenitor are discussed.     

Stimulating capacity of fresh and cultured human leukaemic lymphoid and myeloid cells in `one-way'' mixed lymphocyte reaction

Fresh normal peripheral blood B lymphocytes possess a strong stimulating capacity while fresh thymus cells or fresh peripheral T lymphocytes possess a weak, but significant stimulating capacity on allogeneic lymphocytes in `one-way' mixed lymphocyte reaction. Fresh leukaemic T lymphoid cells from patients with T-cell ALL or T-cell CLL exert little or no stimulation on allogeneic lymphocytes. Fresh leukaemic B lymphoid cells from patients with B-cell CLL or B-cell HCL, on the other hand, exert a lesser stimulation on allogeneic lymphocytes, as compared to that of normal B lymphocytes. Leukaemic myeloblasts from patients with AML or Ph<sup>1</sup>(+) CML-BP exert significantly higher stimulation than leukaemic lymphoid cells in `one-way' mixed lymphocyte reaction (P<0.05). Cultured leukaemic T lymphoid cells (MOLT-4) possess no stimulating capacity, cultured leukaemic B lymphoid cells (BALM-2) possess a moderate degree of stimulating capacity and cultured leukaemic, possibly myeloid, cells (NALM-1 and K562) possess vigorous stimulation on allogeneic lymphocytes. The stimulating capacity of NALM-1 or K562 cells is significantly higher than that of BALM-2 cells (P<0.01 or P<0.05, respectively) and that of MOLT-4 cells (P<0.001). These observations suggest that the stimulating capacity of leukaemic T or B lymphoid cells may have been completely or partially lost during the process of leukaemogenesis. Since we do not have an opportunity to study the stimulating capacity of normal myeloblasts, it is not known whether the stimulating capacity of leukaemic myeloblasts, which is found to be very strong on allogeneic lymphocytes, may have been modified during the process of leukaemogenesis.     

Derivation of dendritic cells from myeloid and lymphoid precursors

The antigen presenting dendritic cells (DC) found in mouse and human lymphoid tissues are heterogeneous. Several subsets of mature DC have been described and these may correspond to distinct lineages. In this review, we present evidence obtained from a series of studies on the lineage origin of DC. This evidence points to the existence of at least three pathways for DC development, namely one from myeloid progenitors, a second from lymphoid progenitors and the third for Langerhans cells from precursors whose relationship to myeloid or lymphoid cell types is not yet clearly defined.     

An interleukin-4-dependent precursor clone is an intermediate of the differentiation pathway from an interleukin-3-dependent precursor clone into myeloid cells as well as B lymphocytes

An interleukin-3-dependent progenitor clone LyD9 and its interleukin-4-dependent derivative clone K-4 were shown to differentiate into myeloid cells as well as B lymphocytes by coculture with bone marrow stroma cells. The K-4 clone is an intermediate between myeloid/lymphoid cells and the LyD9 clone that requires interleukin-4 for differentiation into B lymphocytes and myeloid cells. Granulocyte-macrophage colony stimulating factor-dependent derivatives (LS-1 and K-GM) were also established from induced LyD9 cells. LS-1 and K-GM were myeloid-committed cells.     

The normal flora may contribute to the quantitative preponderance of myeloid cells under physiological conditions

Under physiological conditions, the innate immune cells derived from myeloid lineage absolutely outnumber the lymphoid cells. At present, two theories are attributed to the maintenance of haemopoiesis: the asymmetric cell division and the bone marrow hematopoietic microenvironment or "niche". However, the former only explains the self-renewal of haemopoietic stem cell (HSC) and the start of haemopoietic differentiation but fails to address the inducers of cell fate decisions; the latter has to admit that the hematopoietic cytokines, despite their significance in the maintenance of haemopoiesis, have no specific effect on lineage commitment. Given these flaws, the advantageous mechanism of myeloid haemopoiesis has not yet been uncovered in the current theories. The discoveries that bacterial components (lipopolysaccharide, LPS) and intestinal decontamination affect the mobilization of HSC trigger the interest in normal flora, which together with their components may have an effect on haemopoiesis. In the experiments in dogs and mice, researchers documented that the generation of myeloid cells has undergone changes in the bone marrow and periphery when antibiotics are used to regulate the normal intestinal flora and the concentration of its components. However, the same changes are not involved in lymphoid cells. Therefore, we hypothesize that in human body normal flora and its components are a driving force to maintain myeloid haemopoiesis under physiological conditions. To account for the selectiveness in haemopoiesis, these facts should be taken into consideration, such as HSC and mesenchymal stem cells (MSC) functionally expressed pattern recognition receptors (PRR), and both of them can self-migrate or be recruited by normal flora or its components into periphery. Dynamically monitoring the myeloid haemopoiesis may provide an important complementary program that precludes the abuse of antibiotics, which prevents diseases triggered by the imbalance of normal flora. Meanwhile, the regulation of normal flora and the use of purified microecological modulator may serve as valuable auxiliary treatments to mobilize HSC prior to the HSC transplantation as well as to promote hematopoietic recovery after transplantation or chemotherapy in the blood diseases.     

Developmental plasticity of lymphoid progenitors

The identification of the common lymphoid progenitors in mouse bone marrow allows us to directly assess the regulatory mechanisms of lymphoid lineage commitment. The unexpected finding of a latent myeloid differentiation potential in lymphoid progenitors sheds light on the importance of cytokine receptor expression at this stage. We will discuss the biological nature of common lymphoid progenitors as a model of differentiation from multipotent to lineage committed progenitors. Elucidation of this hidden differentiation potential in progenitors will help further our understanding of the molecular mechanisms that control the cell fate determination of not only common lymphoid progenitors, but also their ancestors, hematopoietic stem cells, and their descendents such as committed T and B cell progenitors.     

Endotoxemia down-regulates bone marrow lymphopoiesis but stimulates myelopoiesis: the effect of G6PD deficiency

Bone marrow (BM) dysfunction is an important component of immunomodulation. This study investigated alterations in cell content, apoptotic responses, and cell proliferation in BM, blood, and spleen in endotoxemic mice (LPS from Escherichia coli). As the decreased antioxidant status associated with glucose-6-phosphate dehydrogenase (G6PD) deficiency has been shown to modulate the innate immune response, we also tested whether a G6PD mutation (80% decrease in cellular enzyme activity) alters BM responses during endotoxemia. LPS decreased BM myeloid (CD45(+)CD11b(+)) and B lymphoid (CD45(+)CD19(+)CD11b(-)) cell content compared with controls. In contrast, LPS increased CD11b(+) myeloid but decreased T and B cell counts in the circulation. Endotoxemia inhibited spontaneous, heat shock, and H(2)O(2)-induced apoptosis as well as proliferative activity in BM lymphoid cells. In contrast, BM myeloid cell apoptosis was not altered, and their proliferative activity was increased during endotoxemia. Following LPS, splenic myeloid cell content was increased, and T and B cell content was unchanged; furthermore, splenocytes showed increased apoptosis compared with controls. BM cell content, including lymphoid and myeloid cells, was greater in G6PD mutant than wild-type (WT) mice, and LPS decreased BM cell counts to a greater degree in mutant than WT mice. Endotoxemia caused widespread inhibition of BM cytokine and chemokine production; however, IL-6 production was increased compared with controls. LPS-induced IL-6 production was decreased in G6PD mutant animals compared with WT. This study indicates that endotoxin inversely affects BM myeloid and lymphoid cell production. LPS-induced down-regulation of B cell production contributes to the generalized lymphopenia and lymphocyte dysfunction observed following nonspecific immune challenges.     

Key role of flt3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool

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

Constitutively active beta-catenin confers multilineage differentiation potential on lymphoid and myeloid progenitors

Beta-catenin-mediated Wnt signaling may contribute to the self-renewal of hematopoietic stem cells and proliferation in some malignancies. We now show that expression of constitutively active beta-catenin in normal lymphoid or myeloid progenitors generated uncommitted cells with multilineage differentiation potential. Inappropriate gene expression occurred in cells destined to produce either cell type and caused corresponding changes in their characteristics. For example, forced activation of beta-catenin quickly increased C/EBPalpha while reducing EBF and Pax-5 in lymphoid progenitors that then generated myeloid cells. Inversely, EBF dramatically increased in transduced myeloid progenitors and lymphocytes were produced. The results indicate that ectopic activation of beta-catenin destabilizes lineage fate decisions and confers some, but not all, stem cell properties on committed progenitors.     

Different subsets of haematopoietic cells and immune cells in bone marrow between young and older donors

Young donors are reported to be associated with better transplant outcomes than older donors in allogeneic hematopoietic stem cell transplantation (allo‐HSCT), but the mechanism is still unclear. The current study compared the different subsets of haematopoietic stem cells (HSCs) and their progenitors as well as immune cells in bone marrow (BM) between young and older donors. The frequencies of HSCs, multipotent progenitors (MPPs) and myeloid progenitors, including common myeloid progenitors (CMPs) and megakaryocyte–erythroid progenitors (MEPs), were decreased, whereas those of lymphoid progenitors, including multi‐potent lymphoid progenitors (MLPs) and common lymphoid progenitors (CLPs), were increased in the BM of young donors compared with in that of older donors. Lower reactive oxygen species (ROS) levels were observed in BM HSCs and six progenitor lines in young donors. Furthermore, young donors demonstrated higher frequencies of naive T cells and immune suppressor cells, such as alternative macrophages (M2) and lower frequencies of memory T cells and immune effectors, including T helper‐1 and T cytotoxic‐1 cells, in BM than older donors. Multivariate analysis demonstrated that donor age was independently correlated with BM HSC frequency. Although further validation is required, our results suggest that the differences in the frequency and immune differentiation potential of HSCs in BM between young donors and older donors may partly explain the different outcomes of allo‐HSCT.     

Regulation of murine hematopoiesis by arachidonic acid metabolites

Arachidonic acid metabolites have been shown to exert a variety of regulatory effects on cellular activation and proliferation. Recently, a role for these products as regulators of hematopoiesis was suggested and evidence provided that products of the lipoxygenase pathway, specifically leukotrienes, are essential for human myeloid colony formation in vitro. In this report the broader role of these metabolites in hematopoiesis was examined using murine bone marrow stem cell assays for both myeloid and lymphoid cell lines. The effects of lipoxygenase and/or cyclooxygenase pathway inhibitors on stem cell colony formation were evaluated and compared to qualitative and quantitative changes in arachidonic acid metabolism that occurred in similarly treated bone marrow cell cultures. Interruption of the lipoxygenase pathway by esculetin or nordihydroguaiaretic acid resulted in decreased colony formation in both lymphoid and myeloid stem cells. This inhibition of colony growth was partly reversed by the addition of leukotrienes and was particularly evident in B-cell progenitor cultures to which was added LTB4. Inhibition of the cyclooxygenase pathway by indomethacin or ibuprofen had a slight stimulatory effect on myeloid colony formation, while slightly inhibiting the formation of lymphoid colonies. These results support a direct role for lipoxygenase products in myeloid colony formation and lymphoid stem cell proliferation. A more complex role for cyclooxygenase metabolites in the hematopoietic process appears probable.     

Myeloid dendritic cells.

There is considerable but as yet incomplete evidence for two developmental lineages of dendritic cells: a myeloid lineage shared with phagocytes and a lymphoid lineage shared with T cells. The two corresponding functional states, which may not require the existence of two formal lineages, are that myeloid dendritic cells capture antigens in the periphery and then migrate to the lymphoid organs to initiate immunity, whereas lymphoid dendritic cells are found in the thymic medulla and lymph node T cell areas and are responsible for tolerance. The latter may occur through immune regulation and/or deletion. Myeloid dendritic cells undergo many activities that contribute to the initiation of immunity. These are summarized here and include mobilization from progenitors and precursors in the blood and marrow, maturation from immature cells in the skin and interstitial spaces, formation of MHC-peptide complexes in MHC class II compartments or MIICs, migration to the T cell areas, and finally, mortality. The death of migratory dendritic cells seems to be accompanied by their phagocytosis and processing by other dendritic cells in the T area. We speculate that this transfer of antigens, including self-peptides captured by the uptake of apoptotic cells in peripheral tissues, is an important prelude to the regulatory function of resident or lymphoid dendritic cells in the T cell area. These features of dendritic cell biology provide targets to manipulate the immune response in vivo.