The number of cells produced in the cultures increased substantially and peaked earlier, whereas the clusters increased a little in both number and size (Fig

The number of cells produced in the cultures increased substantially and peaked earlier, whereas the clusters increased a little in both number and size (Fig. development from these thymic precursors did not include granulocyte/macrophage colony stimulating factor (GM-CSF), usually required for DC development in culture. The addition of antiCGM-CSF antibody or the use of precursors from GM-CSFCdeficient mice did not prevent DC development. Addition of GM-CSF was without effect on DC yield when interleukin (IL) Cot inhibitor-1 3 and IL-7 were present, although some stimulation by GM-CSF was noted in their absence. In contrast, DC development was enhanced by addition of the Flt3/Flk2 ligand, in line with the effects of the administration of this cytokine in vivo. The results indicate that the development of a particular lineage of DC, probably those of lymphoid precursor origin, may be independent of the myeloid Cot inhibitor-1 hormone GM-CSF. Dendritic cells (DC)1 (1) are generally considered as relatives of monocytes and macrophages and to be of myeloid origin. Strong support for this view comes from many studies on the outgrowth of DC in culture, induced principally by GM-CSF, usually in association with other cytokines including TNF- (2C11; for review see reference 12). The DC appear to derive from a progenitor also capable of forming granulocytes and macrophages (13), although more recently a committed DC progenitor, possibly a downstream precursor, has been identified (14). The myeloid nature of DC is emphasized by the direct development of a form of DC from blood monocytes (9, 15C17; for review see reference Cot inhibitor-1 12). All these lines of evidence for a myeloid origin of DC derive from culture studies using GM-CSF. In contrast to these studies, we FBW7 have used adoptive transfer of highly purified precursor cells isolated from the mouse thymus to demonstrate that certain types of DC are related to the lymphoid lineage. The earliest T precursor population isolated from the adult mouse thymus, the low CD4 precursor, was unable to form detectable erythroid or myeloid cells, yet had the potential to form T cells, B cells, NK cells, and DC (18C23; for review see reference 24). A progenitor cell with similar developmental potential has since been isolated from human bone marrow (25). T cells and DC bearing CD8, a characteristic of murine thymic Cot inhibitor-1 DC (26), developed in parallel when this low CD4 precursor was transferred directly into a recipient thymus (21). We have recently found that a downstream thymic precursor (CD4?8?44+25+c-kit+), now no longer able to form B cells or NK cells, still retains full capacity to form DC as well as T cells, suggesting a strong relationship between the two lineages (27). These thymic precursors also formed CD8+ DC in the spleen after intravenous transfer, suggesting that the CD8+ DC normally found in peripheral lymphoid tissues might also be of lymphoid origin. These CD8+ splenic DC appear to have a regulatory role in T cell responses (28). Our initial attempts to grow the thymic low CD4 precursors in culture under the influence of multiple combinations of up to three cytokines were unsuccessful. Some growth and development was obtained using an underlay of a thymic epithelial cell line; under these conditions, a limited production of DC was evident (29). However, more extensive growth, with development into DC rather than T cell progeny, was evident once a more complex cocktail of cytokines was used. It was notable that GMCSF was not required for this DC development in culture. Materials and Methods Mice. The mice used for isolation of thymic low CD4 precursors, or for isolation of thymic DC, were usually 5C7 wk-old C57BL/6J Wehi females, bred under specific pathogenCfree conditions at The Walter and Eliza Hall Institute animal facility. The GM-CSFCnull mice, produced at the Ludwig Institute (30), were originally on a C57BL/6 129 background but had been backcrossed for five generations onto C57BL/6J mice; 5C9-wk-old males and females were used..