Natsuki Hoshino

A novel role for Notch ligand Delta-1 as a regulator of human Langerhans cell development from blood monocytes.

Human Langerhans cells (LCs) are of hematopoietic origin, but cytokine regulation of their development is not fully understood. Notch ligand Delta‐1 is expressed in a proportion of the skin. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and transforming growth factor‐β1 (TGF‐β1) are also secreted in the skin. We report here that Delta‐1, in concert with GM‐CSF and TGF‐β1, induces the differentiation of human CD14+ blood monocytes into cells that express LC markers: CD1a, Langerin, cutaneous lymphocyte‐associated antigen, CC chemokine receptor 6, E‐cadherin, and Birbeck granules. The resulting cells display phagocytic activity and chemotaxis to macrophage inflammatory protein‐1α (MIP‐1α). In response to CD40 ligand and tumor necrosis factor α, the cells acquire a mature phenotype of dendritic cells that is characterized by up‐regulation of human leukocyte antigen (HLA)‐ABC, HLA‐DR, CD80, CD86, CD40, and CD54 and appearance of CD83. These cells in turn show chemotaxis toward MIP‐1β and elicit activation of CD8+ T cells and T helper cell type 1 polarization of CD4+ T cells. Thus, blood monocytes can give rise to LCs upon exposure to the skin cytokine environment consisting of Delta‐1, GM‐CSF, and TGF‐β1, which may be, in part, relevant to the development of human epidermal LCs. Our results extend the functional scope of Notch ligand δ‐1 in human hematopoiesis.

The Soluble Notch Ligand, Jagged-1, Inhibits Proliferation of CD34 + Macrophage Progenitors

The Notch/Notch ligand system controls diverse cellular processes. The proteolytic cleavage generates transmembrane and soluble forms of Notch ligands.We examined the effect of a soluble Notch ligand, human Jagged-1, on human cord blood (CB) CD34+ cells, under serum-deprived conditions, using soluble human Jagged-1—immunoglobulin G1 chimera protein (hJagged-1). Soluble hJagged-1 inhibited myeloid colony formation but not erythroid-mix or erythroid colony formation, in the presence of stem cell factor (SCF), interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, thrombopoietin, and erythropoietin. Cytological analysis revealed that the decrease in myeloid colonies resulted mainly from the inhibition of macrophage colony formation. Furthermore, soluble hJagged-1 led to the inhibition of macrophage colony formation supported by M-CSF plus SCF and GM-CSF plus SCF. Delayed-addition experiments and the analysis of colony sizes demonstrated that soluble hJagged-1 inhibited the growth of macrophage progenitors by acting in the early stage of macrophage development. The direct action of hJagged-1 was confirmed by the enhanced expression of the HES-1 (hairy enhancer of the split-1) gene. These results suggest that soluble hJagged-1 may regulate human hematopoiesis in the monocyte/macrophage lineage.

Efficient ex vivo generation of dendritic cells from CD14+ blood monocytes in the presence of human serum albumin for use in clinical vaccine trials

Dendritic cells (DC) with the potential to induce anti‐tumour immunity represent one of the promising candidates for cancer vaccines. Efficiency of ex vivo DC generation depends on culture conditions, especially protein components in the plasma or serum used. Using human serum albumin (HSA), we devised a constant and reproducible culture method for DC generation from peripheral blood CD14+ cells. The number of DC obtained with 2% HSA‐supplemented cultures containing granulocyte‐macrophage colony‐stimulating factor and interleukin 4 were consistently higher than in cultures with various concentrations of autologous plasma or serum. The concentrations and time points tested for plasma or serum considerably affected the number of DC recovered. DC prepared with HSA acquired the ability to uptake dextran, and expressed high levels of major histocompatibility (MHC) and co‐stimulatory molecules similar to DC cultured with autologous plasma or serum. Although DC cultured with autologous plasma or serum consisted of CD1a+ and CD1a− populations, DC differentiated in the presence of HSA expressed CD1a. DC obtained with HSA primed and induced immunogenic peptide‐specific cytotoxic T lymphocytes against a tumour rejection antigen, HER2. These findings suggest that our method for preparation of DC with HSA should prove valuable in DC generation for immunotherapy.

Two Independent Clones in Myelodysplastic Syndrome Following Treatment of Acute Myeloid Leukemia

We describe a 55-year-old Japanese woman with therapy-related myelodysplastic syndrome (t-MDS) with 2 independent clones, t(1;2)(p36;p21) and t(11;12)(p15;q13). She was diagnosed with acute myeloid leukemia (AML) with cytological features of the bone marrow and peripheral blood. Cytogenetic evaluation revealed a 46,XX karyotype. She received chemotherapy and achieved complete remission (CR). Despite maintenance chemotherapy, she suffered a relapse. Chromosomal analysis showed t(1;2)(p36;p21) in 2 of 20 metaphases. At second CR, this clone transiently disappeared. Nine months later, t(1;2) (p36;p21) was detected again in 3 of 20 metaphases while the patient remained in CR. Six months later, bone marrow examination disclosed trilineage dysplasia without an excess of blasts, suggesting MDS. t(1;2)(p36;p21) was observed in 16 of 20 metaphases. The clinical course and serial cytogenetic findings were diagnostic of t-MDS. The duration of t-MDS was 6 years. During this period, persistent t(1;2)(p36;p21) and transient t(11;12)(p15;q13) were found.When t-MDS evolved to AML, cytogenetic evaluation revealed 46,XX,t(1;2)(p36;p21),del(7)(q22),add(19)(p13).

Efficient Ex Vivo Generation of Human Dendritic Cells from Mobilized CD34p+ Peripheral Blood Progenitors

We tried to efficiently generate human dendritic cells (DCs) from CD34p+ peripheral blood hematopoietic progenitor cells mobilized by high-dose chemotherapy and subsequent administration of granulocyte colony-stimulating factor, using a liquid suspension culture system. Among various combinations, the combination ofc-kit ligand,flt-3 ligand,c-mpl ligand (TPO), and interleukin (IL)-4 most potently generated the number of CD1ap+CD14p- DCs in cultures containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor α (TNF-α). The delayed addition of IL-4 on day 6 of culture gave rise to an additional increase in the yield of CD1ap+CD14p- DCs that were characterized by the expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. The majority of the sorted CD1ap-CD14p+ cells derived from 6-day culture of CD34p+cells gave rise to CD1ap+CD14p- DCs and CD1a-CD14p+ macrophages on day 12 of culture in the presence and absence of IL-4, respectively. These findings suggest that IL-4 promotes the differentiation of CD1ap-CD14p+ cells derived from mobilized CD34p+ peripheral blood hematopoietic progenitors to CD1ap+CD14p- DCs. The majority of these DCs expressed CD68 but not the Langerhans-associated granule antigen, a finding that suggests they emerge through the monocyte differentiation pathway. The addition of TPO and IL-4 to cultures did not affect the potential of DCs to stimulate the primary allogeneic T-cell response. These findings demonstrated that the combination ofc-kit ligand plusflt-3 ligand plus TPO with GM-CSF plus TNF-α, followed by IL-4, is useful for ex vivo generation of human DCs from mobilized CD34p+ peripheral blood progenitors.