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Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor.

Saunders D, Lucas K, Ismaili J, Wu L, Maraskovsky E, Dunn A, Shortman K - J. Exp. Med. (1996)

Bottom Line: The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures.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 hormone GM-CSF.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

ABSTRACT
The earliest lymphoid precursor population in the adult mouse thymus had previously been shown to produce not only T cells, but also dendritic cell (DC) progeny on transfer to irradiated recipients. In this study, culture of these isolated thymic precursors with a mixture of cytokines induced them to proliferate and to differentiate to DC, but not to T lineage cells. At least 70% of the individual precursors had the capacity to form DC. The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures. The cultured DC also expressed high levels of class I and class II major histocompatibility complex, together with CD11c, DEC-205, CD80, and CD86, markers characteristic of mature DC in general. However, they did not express CD8 alpha or BP-1, markers characteristic of normal thymic DC. The optimized mixture of five to seven cytokines required for DC 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 anti-GM-CSF antibody or the use of precursors from GM-CSF-deficient mice did not prevent DC development. Addition of GM-CSF was without effect on DC yield when interleukin (IL) 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 hormone GM-CSF.

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The expression of typical thymic DC surface markers on the  DC developing in culture of thymic low CD4 precursors. The precursors  (20,000) were grown for 4 d in 0.1 ml medium in 96-well, flat-bottomed  culture trays, in the presence of IL-1β, TNF-α, IL-3, IL-7, and SCF,  alone (left) or together with Flt3L and the mAb FGK45.5 reactive with  CD40 (right). The cultured were harvested, pooled, dissociated by EDTA  treatment, and then stained with mAb, generally in two fluorescent colors. Full details are given in Materials and Methods. Dead or damaged  cells, ∼15% of the total, were excluded from analysis by propidium iodide staining and forward light scatter characteristics; otherwise, all cells  growing in the cultures are represented in fluorescence distribution profiles. The broken line gives the background staining, omitting the relevant  mAb. Each result is representative of two to five such analyses.
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Figure 7: The expression of typical thymic DC surface markers on the DC developing in culture of thymic low CD4 precursors. The precursors (20,000) were grown for 4 d in 0.1 ml medium in 96-well, flat-bottomed culture trays, in the presence of IL-1β, TNF-α, IL-3, IL-7, and SCF, alone (left) or together with Flt3L and the mAb FGK45.5 reactive with CD40 (right). The cultured were harvested, pooled, dissociated by EDTA treatment, and then stained with mAb, generally in two fluorescent colors. Full details are given in Materials and Methods. Dead or damaged cells, ∼15% of the total, were excluded from analysis by propidium iodide staining and forward light scatter characteristics; otherwise, all cells growing in the cultures are represented in fluorescence distribution profiles. The broken line gives the background staining, omitting the relevant mAb. Each result is representative of two to five such analyses.

Mentions: Immunofluorescent staining and flow cytometry was used to analyze the surface antigens on the cultured cells. Figs. 7 and 8 give results for day 4 cultures of low CD4 precursors grown in the mix of TNF-α, IL-1α, IL-3, IL-7, and SCF, together with the results for cultures grown in these five cytokines together with Flt3L and the anti-CD40 mAb. The surface phenotype of the DC was similar under these two conditions. It was also largely unchanged when day 6 cultures were compared with day 4 cultures.


Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor.

Saunders D, Lucas K, Ismaili J, Wu L, Maraskovsky E, Dunn A, Shortman K - J. Exp. Med. (1996)

The expression of typical thymic DC surface markers on the  DC developing in culture of thymic low CD4 precursors. The precursors  (20,000) were grown for 4 d in 0.1 ml medium in 96-well, flat-bottomed  culture trays, in the presence of IL-1β, TNF-α, IL-3, IL-7, and SCF,  alone (left) or together with Flt3L and the mAb FGK45.5 reactive with  CD40 (right). The cultured were harvested, pooled, dissociated by EDTA  treatment, and then stained with mAb, generally in two fluorescent colors. Full details are given in Materials and Methods. Dead or damaged  cells, ∼15% of the total, were excluded from analysis by propidium iodide staining and forward light scatter characteristics; otherwise, all cells  growing in the cultures are represented in fluorescence distribution profiles. The broken line gives the background staining, omitting the relevant  mAb. Each result is representative of two to five such analyses.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196371&req=5

Figure 7: The expression of typical thymic DC surface markers on the DC developing in culture of thymic low CD4 precursors. The precursors (20,000) were grown for 4 d in 0.1 ml medium in 96-well, flat-bottomed culture trays, in the presence of IL-1β, TNF-α, IL-3, IL-7, and SCF, alone (left) or together with Flt3L and the mAb FGK45.5 reactive with CD40 (right). The cultured were harvested, pooled, dissociated by EDTA treatment, and then stained with mAb, generally in two fluorescent colors. Full details are given in Materials and Methods. Dead or damaged cells, ∼15% of the total, were excluded from analysis by propidium iodide staining and forward light scatter characteristics; otherwise, all cells growing in the cultures are represented in fluorescence distribution profiles. The broken line gives the background staining, omitting the relevant mAb. Each result is representative of two to five such analyses.
Mentions: Immunofluorescent staining and flow cytometry was used to analyze the surface antigens on the cultured cells. Figs. 7 and 8 give results for day 4 cultures of low CD4 precursors grown in the mix of TNF-α, IL-1α, IL-3, IL-7, and SCF, together with the results for cultures grown in these five cytokines together with Flt3L and the anti-CD40 mAb. The surface phenotype of the DC was similar under these two conditions. It was also largely unchanged when day 6 cultures were compared with day 4 cultures.

Bottom Line: The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures.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 hormone GM-CSF.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

ABSTRACT
The earliest lymphoid precursor population in the adult mouse thymus had previously been shown to produce not only T cells, but also dendritic cell (DC) progeny on transfer to irradiated recipients. In this study, culture of these isolated thymic precursors with a mixture of cytokines induced them to proliferate and to differentiate to DC, but not to T lineage cells. At least 70% of the individual precursors had the capacity to form DC. The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures. The cultured DC also expressed high levels of class I and class II major histocompatibility complex, together with CD11c, DEC-205, CD80, and CD86, markers characteristic of mature DC in general. However, they did not express CD8 alpha or BP-1, markers characteristic of normal thymic DC. The optimized mixture of five to seven cytokines required for DC 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 anti-GM-CSF antibody or the use of precursors from GM-CSF-deficient mice did not prevent DC development. Addition of GM-CSF was without effect on DC yield when interleukin (IL) 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 hormone GM-CSF.

Show MeSH
Related in: MedlinePlus