Human thymic epithelial cells inhibit IL-15- and IL-2-driven differentiation of NK cells from the early human thymic progenitors

T/NK progenitors are present in the thymus; however, the thymus predominantly promotes T cell development. In this study, we demonstrated that human thymic epithelial cells (TEC) inhibit NK cell development. Most ex vivo human thymocytes express CD1a, indicating that thymic progenitors are predominantly committed to the T cell lineage. In contrast, the CD1a(-)CD3(-)CD56(+) NK population comprises only 0.2% (n = 7) of thymocytes. However, we observed increases in the percentage (20- to 25-fold) and absolute number (13- to 71-fold) of NK cells when thymocytes were cultured with mixtures of either IL-2, IL-7, and stem cell factor or IL-15, IL-7, and stem cell factor. TEC, when present in the cultures, inhibited the increases in the percentage (3- to 10-fold) and absolute number (3- to 25-fold) of NK cells. Furthermore, we show that TEC-derived soluble factors inhibit generation of NK-CFU and inhibit IL15- or IL2-driven NK cell differentiation from thymic CD34(+) triple-negative thymocytes. The inhibitory activity was found to be associated with a 8,000- to 30,000 Da fraction. Thus, our data demonstrate that TEC inhibit NK cell development from T/NK CD34(+) triple negative progenitors via soluble factor(s), suggesting that the human thymic microenvironment not only actively promotes T cell maturation but also controls the development of non-T lineage cells such as the NK lineage.     

Broad distribution of colony-forming cells with erythroid, myeloid, dendritic cell, and NK cell potential among CD34(++) fetal liver cells.

The generation of erythroid, myeloid, and lymphoid cells from human fetal liver progenitors was studied in colony-forming cell (CFC) assays. CD38(-) and CD38(+) progenitors that expressed high levels of CD34 were grown in serum-deprived medium supplemented with kit ligand, flk2/flt3 ligand, GM-CSF, c-mpl ligand, erythropoietin, and IL-15. The resulting colonies were individually analyzed by flow cytometry. CD56(+) NK cells were detected in 21.9 and 9.9% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. NK cells were detected in mostly large CD14(+)/CD15(+) myeloid colonies that also, in some cases, contained red cells. NK cells were rarely detected in erythroid colonies, suggesting an early split between the erythroid and the NK cell lineages. CD1a(+) dendritic cells were also present in three-quarters of the colonies grown from CD38(-) and CD38(+) progenitors. Multilineage colonies containing erythrocytes, myeloid cells, and NK cells were present in 13.7 and 2.7% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. High proliferative-potential CFCs that generated multilineage colonies were also detected among both populations of progenitors. The total number of high proliferative-potential CFCs with erythroid, myeloid, and NK cell potential was estimated to be 2-fold higher in the CD38(+) fraction compared with the CD38(-) fraction because of the higher frequency of CD38(+) cells among CD34(++) cells. The broad distribution of multipotent CFCs among CD38(-) and CD38(+) progenitors suggests that the segregation of the erythroid, myeloid, and lymphoid lineages may not always be an early event in hemopoiesis. Alternatively, some stem cells may be present among CD38(+) cells.     

Age-related changes in human hematopoietic stem/progenitor cells

Adult stem cells are critical for maintaining cellular homeostasis throughout life, yet the effects of age on their regenerative capacity are poorly understood. All lymphoid and myeloid blood cell lineages are continuously generated from hematopoietic stem cells present in human bone marrow. With age, significant changes in the function and composition of mature blood cells are observed. In this study, we report that age-related changes also occur in the human hematopoietic stem cell compartment. We find that the proportion of multipotent CD34(+) CD38(-) cells increases in the bone marrow of elderly (>70 years) individuals. CD34(+) CD38(+) CD90(-) CD45RA(+/-) CD10(-) and CD34(+) CD33(+) myeloid progenitors persist at the same level in the bone marrow, while the frequency of early CD34(+) CD38(+) CD90(-) CD45RA(+) CD10(+) and committed CD34(+) CD19(+) B-lymphoid progenitors decreases with age. In contrast to mice models of aging, transplantation experiments with immunodeficient NOD/SCID/IL-2Rγ null (NSG) mice showed that the frequency of NSG repopulating cells does not change significantly with age, and there is a decrease in myeloid lineage reconstitution. An age-related decrease in the capacity of CD34(+) cells to generate myeloid cells was also seen in colony-forming assays in vitro. Thus, with increasing age, human hematopoietic stem/progenitor cells undergo quantitative changes as well as functional modifications.     

Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells

In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin(-)CD34(+)CD43(+)CD45(+) multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5-19 d of culture with cytokines, depending on the cell type.     

Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems

Embryonic stem (ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decisions and to develop scalable methods of cell production. We compared four standard ES cell differentiation culture systems by measuring aspects of embryoid body (EB) formation efficiency and cell proliferation, and by tracking development of a specific differentiated tissue type-blood-using functional (colony-forming cell) and phenotypic (Flk-1 and CD34 expression) assays. We report that individual murine ES cells form EBs with an efficiency of 42 +/- 9%, but this value is rarely obtained because of EB aggregation-a process whereby two or more individual ES cells or EBs fuse to form a single, larger cell aggregate. Regardless of whether EBs were generated from a single ES cell in methylcellulose or liquid suspension culture, or aggregates of ES cells in hanging drop culture, they grew to a similar maximum cell number of 28,000 +/- 9,000 cells per EB. Among the three methods for EB generation in suspension culture there were no differences in the kinetics or frequency of hematopoietic development. Thus, initiating EBs with a single ES cell and preventing EB aggregation should allow for maximum yield of differentiated cells in the EB system. EB differentiation cultures were also compared to attached differentiation culture using the same outputs. Attached colonies were not similarly limited in cell number; however, hematopoietic development in attached culture was impaired. The percentage of early Flk-1 and CD34 expressing cells was dramatically lower than in EBs cultured in suspension, whereas hematopoietic colony formation was almost completely inhibited. These results provide a foundation for development of efficient, scalable bioprocesses for ES cell differentiation, and inform novel methods for the production of hematopoietic tissues.     

Detection and characterization of hemopoietic stem cells in the adult human small intestine

The concept of lymphoid differentiation in the human gastrointestinal tract is controversial but is the focus of this study, which examined adult human small intestinal tissue for the presence of CD34(+)CD45(+) hemopoietic stem cells (HSCs) and lymphoid progenitors. Flow cytometry demonstrated that over 5% of leukocytes (CD45(+) cells) isolated from human gut were HSCs coexpressing CD34, a significantly higher incidence than in matched peripheral blood or control bone marrow. HSCs were detected in cell preparations from both the epithelium and lamina propria of all samples tested and localized to the intestinal villous and crypt regions using immunofluorescence. A high proportion of gut HSCs expressed the activation marker CD45RA, and few expressed c-kit, indicating ongoing differentiation. The vast majority of intestinal HSCs coexpressed the T cell Ag, CD7 (92% in the epithelium, 80% in the lamina propria) whereas <10% coexpressed the myeloid Ag CD33, suggesting that gut HSCs are a relatively mature population committed to the lymphoid lineage. Interestingly, almost 50% of epithelial layer HSCs coexpressed CD56, the NK cell Ag, compared with only 10% of the lamina propria HSC population, suggesting that the epithelium may be a preferential site of NKR(+) lymphoid differentiation. In contrast, bone marrow HSCs displayed low coexpression of CD56 and CD7 but high coexpression of CD33. The phenotype of intestinal HSCs, which differs significantly from circulating or bone marrow HSCs, is consistent with a role in local lymphoid development.     

Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and, (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes (alpha-globin, betaH-1 globin, beta-major globin, epsilon -globin, and zeta-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, epsilon-globin, gamma-globin, betaH1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of secondary EB origin cultured in media containing dexamethasone showed a down-regulation of GATA-3 and an up-regulation of GATA-1, Flk-1, and Epo-R in comparison to the two other cytokines and growth factor combinations containing media. The secondary differentiation also showed an enhanced production of erythrocytic precursors in dexamethasone containing media in comparison to that in the control media. Our results indicate that dexamethasone can prove to be an effective agent which can be employed to enhance differentiation towards erythrocytic progenitors from ES cells.     

Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors

Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.     

Constitutively active beta-catenin promotes expansion of multipotent hematopoietic progenitors in culture

This study was designed to investigate one component of the Wnt/beta-catenin signaling pathway that has been implicated in stem cell self-renewal. Retroviral-mediated introduction of stable beta-catenin to primitive murine bone marrow cells allowed the expansion of multipotential c-Kit(low)Sca-1(low/-)CD19(-) CD11b/Mac-1(-)Flk-2(-)CD43(+)AA4.1(+)NK1.1(-)CD3(-)CD11c(-)Gr-1(-)CD45R/B220(+) cells in the presence of stromal cells and cytokines. They generated myeloid, T, and B lineage lymphoid cells in culture, but had no T lymphopoietic potential when transplanted. Stem cell factor and IL-6 were found to be minimal requirements for long-term, stromal-free propagation, and a beta-catenin-transduced cell line was maintained for 5 mo with these defined conditions. Although multipotential and responsive to many normal stimuli in culture, it was unable to engraft several types of irradiated recipients. These findings support previous studies that have implicated the canonical Wnt pathway signaling in regulation of multipotent progenitors. In addition, we demonstrate how it may be experimentally manipulated to generate valuable cell lines.     

Expression of alpha(4)beta(7) integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells

During embryogenesis, the Peyer's patch anlagen are induced by a cell population that produces lymphotoxin (LT) alpha(1)beta(2) following stimulation of IL-7Ralpha. In this study, we show that the LT-producing cell is localized within the IL-7Ralpha(+) and integrin alpha(4)beta(7) (alpha(4)beta(7))(+) population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of alpha(4)beta(7). Before expression of alpha(4)beta(7), the potential of IL-7Ralpha(+) population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7Ralpha(+)alpha(4)beta(7)(+) cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7Ralpha(+)alpha(4)beta(7)(+) population migrates to the intestine, where it induces the Peyer's patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7Ralpha(+)alpha(4)beta(7)(+) population can differentiate into fully competent NK1.1(+) NK cells or CD11c(+) APCs. Expression of alpha(4)beta(7) is lost during differentiation of both lineages; IL-7Ralpha expression is lost during NK1.1(+) cells differentiation. A newly discovered lineage(-)IL-7Ralpha(+)c-Kit(+)alpha(4)beta(7)(+) population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.     

Distinct hemogenic potential of endothelial cells and CD41+ cells in mouse embryos

Definitive hematopoietic progenitor cells have been thought to develop from the vascular endothelium located in the aorta-gonad-mesonephros region of the mouse embryo. However, several recent findings have suggested that most hematopoietic progenitors are derived from non-endothelial precursor cells expressing CD41. We characterized two distinct precursor populations of definitive hematopoietic cell lineages, vascular endothelial (VE)-cadherin(+) CD41(-) CD45(-) endothelial cells and CD41(+) CD45(-) non-endothelial progenitors, both of which are derived from lateral mesoderm. VE-cadherin(+) endothelial cells obtained from cultures of differentiating embryonic stem cells possessed hematopoietic potential encompassing erythroid, myeloid and B lymphoid lineages, whereas CD41(+) progenitors lacked the B lymphopoietic potential. VE-cadherin(+) endothelial cells in the lower trunk of the embryo proper showed a significant potential for initiating B lymphopoiesis in cultures, while endothelial cells in the yolk sac appeared to have a bias for myeloerythropoietic differentiation. CD41(+) progenitors isolated from yolk sac and embryo proper were capable of generating multiple hematopoietic lineages, although mast cell precursors were exclusively enriched in CD41(+) progenitors in the yolk sac. These results suggest that hemogenic endothelial cells and CD41(+) progenitors possess distinct hematopoietic potential depending on the tissues in which they reside.     

Expression of stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4, on CD34+ human bone marrow cells is a phenotypic alteration for committed lymphoid progenitors.

We found that the stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4 (CXCR4), is expressed on human CD34+ bone marrow (BM) cells. Stringently FACS-sorted CD34+CXCR4+ BM cells completely lack myeloid, erythroid, megakaryocytic, and mixed colony-forming potential (myeloid progenitors), but give rise to B and T lymphoid progenitors, whereas CD34+CXCR4- BM cells can generate colonies formed by myeloid progenitors and can also develop into these lymphoid progenitors. Therefore, expression of CXCR4 on CD34+ BM cells can allow lymphoid progenitors to be discriminated from myeloid progenitors. Because CD34+CXCR4+ cells are differentiated from CD34+CXCR4- cells, multipotential progenitors located in the BM are likely to be negative for CXCR4 expression. CXCR4 seems to be expressed earlier than the IL-7R and terminal deoxynucleotidyl transferase during early lymphohemopoiesis. These results suggest that the expression of CXCR4 on CD34+ BM cells is one of the phenotypic alterations for committed lymphoid progenitors.     

IL-3 increases production of B lymphoid progenitors from human CD34+CD38- cells

The effect of IL-3 on the B lymphoid potential of human hemopoietic stem cells is controversial. Murine studies suggest that B cell differentiation from uncommitted progenitors is completely prevented after short-term exposure to IL-3. We studied B lymphopoiesis after IL-3 stimulation of uncommitted human CD34+CD38- cells, using the stromal cell line S17 to assay the B lymphoid potential of stimulated cells. In contrast to the murine studies, production of CD19+ B cells from human CD34+CD38- cells was significantly increased by a 3-day exposure to IL-3 (p < 0.001). IL-3, however, did not increase B lymphopoiesis from more mature progenitors (CD34+CD38+ cells) or from committed CD34-CD19+ B cells. B cell production was increased whether CD34+CD38- cells were stimulated with IL-3 during cocultivation on S17 stroma, on fibronectin, or in suspension. IL-3Ralpha expression was studied in CD34+ populations by RT-PCR and FACS. High IL-3Ralpha protein expression was largely restricted to myeloid progenitors. CD34+CD38- cells had low to undetectable levels of IL-3Ralpha by FACS. IL-3-responsive B lymphopoiesis was specifically found in CD34+ cells with low or undetectable IL-3Ralpha protein expression. IL-3 acted directly on progenitor cells; single cell analysis showed that short-term exposure of CD34+CD38- cells to IL-3 increased the subsequent cloning efficiency of B lymphoid and B lymphomyeloid progenitors. We conclude that short-term exposure to IL-3 significantly increases human B cell production by inducing proliferation and/or maintaining the survival of primitive human progenitors with B lymphoid potential.     

The regulatory role of Hyper-IL-6 in the differentiation of myeloid and erythroid progenitors derived from human cord blood

This study was designed to investigate the regulatory role of soluble interleukin-6 receptor (sIL-6R) and interleukin-6 (IL-6) fusion protein (Hyper-IL-6) in the differentiation of human myeloid and erythroid progenitors by a serum-free liquid suspension culture system, using the human cord blood-derived CD34(+)CD38(-) cells as a target. We found that Hyper-IL-6 promoted the generation of CD15(+) granulocytic and CD14(+) monocytic cells and suppressed that of CD14(-)CD1a(+) dendritic cells from CD36(-)CD15(-)CD14(-)CD1a(-)IL-6R(+) myeloid progenitors. Conversely, CD34(+)CD38(-) cell-derived early erythroid progenitors were negative for IL-6R expression. Hyper-IL-6 potentiated the generation of CD36(+)glycophorinA(high) mature erythroid cells from the IL-6R(-) early erythroid progenitors. Our results indicate that Hyper-IL-6 augments the generation of CD15(+) granulocytic, CD14(+) monocytic and CD36(+)glycophorinA(high) cell and suppresses that of CD14(-)CD1a(+) dendritic cells.     

Determination of lymphoid cell fate is dependent on the expression status of the IL-7 receptor

Signaling through the IL-7 receptor (IL-7R) is necessary for the development of the earliest B- and T-lineage cells. IL-7R is first expressed on common lymphoid progenitor cells and is not detected on primitive common myeloid progenitors. In this study, we show that enforced expression of IL-7R on multipotential stem cells does not influence lymphoid versus myeloid cell fate. T cell development was compatible with sustained IL-7R expression; however, we observed a near complete block in B cell development at the onset of B-lineage commitment. Unlike pre-proB cells from control animals, developmentally-arrested IL-7R(+)B220(+)CD19(-)NK1.1(-)Ly-6C(-) cells failed to express EBF and Pax5. These results suggest that transient downregulation of IL-7R signaling is a necessary event for induction of EBF and Pax5 expression and B-lymphocyte commitment.     

CCR7 ligands, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are chemoattractants for CD56(+)CD16(-) NK cells and late stage lymphoid progenitors

Two human CC chemokines, SLC/6Ckine/Exodus2/TCA4 and CKbeta-11/MIP-3beta/ELC, are previously reported as efficacious chemoattractants for T- and B-cells and dendritic cells. SLC and CKbeta-11 share only 32% amino acid identity, but are ligands for the same chemokine receptor, CCR7. In this study, we examined chemotactic activity of SLC and CKbeta-11 for NK cells and lymphoid progenitors in bone marrow and thymus. It was found that these two CCR7 ligands are chemoattractants for neonatal cord blood and adult peripheral blood NK cells and cell lines. SLC and CKbeta-11 preferentially attract the CD56(+)CD16(-) NK cell subset over CD56(+)CD16(+) NK cells. SLC and CKbeta-11 also demonstrate selective chemotactic activity on late stage CD34(-)CD19(+)IgM- B-cell progenitors and CD4(+) and CD8(+) single-positive thymocytes, but not early stage progenitors. It was noted that SLC is an efficient desensitizer of CKbeta-11-dependent NK cell chemotaxis, while CKbeta-11 is a weak desensitizer of SLC-dependent chemotaxis. Taken together, these results suggest that SLC and CKbeta-11 have the potential to control trafficking of NK cell subsets and late stage lymphoid progenitors in bone marrow and thymus.     

Differentiation of monocytic cell clones into CD8 alpha+ dendritic cells (DC) suggests that monocytes can be direct precursors for both CD8 alpha+ and CD8 alpha- DC in the mouse

Dendritic cells (DC) are the professional APCs that initiate T cell immune responses. DC can develop from both myeloid and lymphoid progenitors. In the mouse, the CD8alpha(+) DC had been designated as "lymphoid" DC, and CD8alpha(-) DC as "myeloid" DC until recently when it was demonstrated that common myeloid progenitors can also give rise to CD8alpha(+) DC in bone marrow chimera mice. However, it is still not clear which committed myeloid lineages differentiate into CD8alpha(+) DC. Because monocytes can differentiate into DC in vivo, the simplest hypothesis is that the CD8alpha(+) DC can be derived from the monocyte/macrophage. In this study we show that cell clones, isolated from CD8alpha(+) DC lymphoma but with a monocytic phenotype (CD11c(low/-)D11b(high)CD8alpha(-)I-A(low)), can redifferentiate into CD8alpha(+) DC either when stimulated by LPS and CD40L or when they migrate into the lymphoid organs. Maturation of DC in vivo correlated with strong priming of allogeneic T cells. Moreover, the monocytes from cultured splenocytes or peritoneal exudates macrophages of wild-type mice are also capable of differentiating into CD11c(+)CD8alpha(+) DC after their migration into the draining lymph nodes. Our results suggest that monocytes can be direct precursors for CD11c(+)CD8alpha(+) DC in vivo. In addition, the monocyte clones described in this study may be valuable for studying the differentiation and function of CD8alpha(+) DC that mediate cross-presentation of Ag to CD8 T cells specific for cell-associate Ags.     

Production and differentiation of NK lineage cells in long-term bone marrow cultures in the absence of exogenous growth factors.

Neither lytic NK cells nor IL-2-responsive NK precursors were produced in myeloid (Dexter) long-term bone marrow cultures (LTBMC). However, when myeloid LTBMC were switched to lymphoid (Whitlock-Witte) conditions and reseeded ("recharged") with fresh bone marrow cells (BMC), nonadherent cells with NK lytic activity and NK 1.1+ phenotype were produced within 1-2 weeks without the addition of exogenous IL-2 to the cultures. NK- and T cell-depleted BMC proliferated extensively in switched cultures and in 2 weeks generated cells that lysed the NK target YAC-1 but not the LAK target P815. The presence of NK precursors in the cultures was confirmed by reculturing nonadherent cells harvested from recharged LTBMC in fresh medium containing 50 U rIL-2/ml. High levels of NK lytic activity were generated. Sequential expression of NK 1.1 and IL-2 responsiveness followed by lytic activity was demonstrated by harvesting cells early after recharge, prior to the appearance of lytic cells. Elimination of NK 1.1+ cells depleted the ability to respond to IL-2 in secondary culture. Our studies demonstrate that myeloid-to-lymphoid switched LTBMC support the proliferation and differentiation of NK lineage cells from their NK 1.1-, nonlytic progenitors in the absence of an exogenous source of growth factors.