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Results: 5

1.
Fig. 3

Fig. 3. From: CD1a expression defines an interleukin-12 producing population of human dendritic cells.

Expression of CD1 isoforms and maturation markers on CD1a+ and CD1a dendritic cells (DC) subsets derived from CD34+ haematopoetic precursors. (a) Cell surface expression of CD1b, CD1c and CD1d on the CD1a+ and CD1a DC subsets was analysed by flow cytometry. In contrast to monocyte-derived DC that had similar expression levels of all the CD1 isoforms, only the CD1a+ DC subset derived from CD34+ haematopoetic precursors had significant expression of the other CD1 isoforms. (b) Both the CD1a+ and CD1a DC subsets derived from CD34+ haematopoetic precursors expressed the maturation markers CD86 and HLA-DR. The CD1a+ subset expressed higher cell surface levels of these markers. The percentage positive for each quadrant of the gated population is presented in bold type and the mean fluorescence intensity is listed underneath in italics.

M Cernadas, et al. Clin Exp Immunol. 2009 March;155(3):523-533.
2.
Fig. 5

Fig. 5. From: CD1a expression defines an interleukin-12 producing population of human dendritic cells.

CD34+ haematopoetic progenitor-derived CD1a+ and CD1a dendritic cells (DC) subsets have a similar functional phenotype to monocyte-derived DC. CD1a+ and CD1a subsets of CD34+ progenitor-derived DC were separated by sorting and cultured with CD40L L cell transfectants, lipopolysaccharide (LPS) or media alone for 24 h. Supernatants were assayed for production of IL-12p70, IL-10 and IL-6. (a) Similar to CD1a+ DC obtained from monocyte-derived DC, the CD1a+ subset of CD34+ progenitor-derived DC also produces significantly more IL-12p70 than its CD1a counterpart. IL-6 production was similar between the subsets. There was significant variability of IL-10 production between donors (data not shown). Enzyme-linked immunosorbent assay results are representative of four individual experiments; **P < 0·05. (b) CD1a+ CD34+ progenitor-derived DC polarize naive CD4+ T cells to a Th1 phenotype. Interferon-γ production by restimulated allogeneic naive T cells cultured with CD1a+ CD34+ progenitor-derived DC was enhanced significantly compared with CD1a DC; **P < 0·001.

M Cernadas, et al. Clin Exp Immunol. 2009 March;155(3):523-533.
3.
Fig. 2

Fig. 2. From: CD1a expression defines an interleukin-12 producing population of human dendritic cells.

(a) CD1a+ and CD1a monocyte-derived dendritic cells (DC) cytospins were prepared from DC sorted by flow cytometry. Slides were stained with haematoxylin and eosin and imaged by light microscopy. There were no significant morphological differences between the DC subsets. Both subsets exhibited dendritic cell morphology, with dendrites distributed homogeneously around the cell. Some DC were noted to have a veiled appearance in both subsets. Representative images were obtained at 400×. (b) CD14+ monocytes isolated from normal blood donors were cultured in RPMI-1640 complete media + IL-4 and GM-CSF. Cells were analysed at days 0–14 for expression of CD1a, CD14, CD9, CD16 and CD32. CD14 expression was promptly lost on both subsets. Other than the CD1a subset expressing higher levels of CD32 initially, there were no consistent differences among the CD1a subsets in this or any of the other donors studied. The cell surface expression of CD9 and CD32 changed significantly over the culture period. Other cell surface markers which were analysed, however, did not show significant differences between the groups and are not displayed. This experiment is representative of four independent experiments with different donors.

M Cernadas, et al. Clin Exp Immunol. 2009 March;155(3):523-533.
4.
Fig. 4

Fig. 4. From: CD1a expression defines an interleukin-12 producing population of human dendritic cells.

(a) CD1a expression identifies a monocyte-derived dendritic cells (DC) subset that produces interleukin IL-12 upon stimulation. The CD1a+ subset of monocyte-derived DC stimulated with lipopolysaccharide or CD40L L cell transfectants for 24 h produces significantly greater amounts of IL-12p70 compared with its CD1a negative counterpart among all donors studied. (b) Similar levels of IL-10 were produced by both subsets. This pattern was also observed with all donors. (c) Although there were differences in IL-6 production with the donor presented, there was significant variability in IL-6 production among the donors studied. (d) Summary of individual donor DC subsets IL-6, IL-10 and IL-12p70 production data. Data are presented as the ratio of the absolute cytokine production of CD1a+/CD1a DC. The median CD1a+/CD1a DC cytokine production ratio is indicated by the horizontal bar, n = 8–12 individual donors; **P < 0·05 (n.d.: not detected). (e) CD1a+ monocyte-derived DC polarize naive CD4+ T cells to a Th1 phenotype. Naive CD4+ T cells cultured with the CD1a+ DC subset, shown above to produce significant levels of IL-12p70, had a Th1 phenotype upon restimulation. CD4+ T cells cultured with the CD1a DC did not produce significant amounts of either Th1 or Th2 cytokines. The IL-4 and IL-13 data are not shown, given that these cytokines were not detectable for any sample. The data presented are representative of four independent experiments using different monocyte donors; **P < 0·05.

M Cernadas, et al. Clin Exp Immunol. 2009 March;155(3):523-533.
5.
Fig. 1

Fig. 1. From: CD1a expression defines an interleukin-12 producing population of human dendritic cells.

(a) Cell surface expression of the CD1b, c and d isoforms on monocyte-derived CD1a dendritic cell (DC) subsets was determined by flow cytometry. The CD1c and CD1d isoforms were generally expressed at similar levels on both the CD1a+ and CD1a DC subsets. In the case of CD1b, the CD1a DC subset expressed higher cell surface levels of CD1b. Flow cytometry profiles from two donors are shown and are representative of data obtained from six individual donors in independent experiments. (b) The percentage of monocyte-derived DC that were CD1a+ varied among individual donors and ranged from 36·8% to 81% (n = 20 individual donors). (c) Expression of maturation markers on monocyte-derived DC. Flow cytometry was performed on monocyte-derived DC incubated with and without 10 ng/ml lipopolysaccharide (LPS) for 24 h. The CD1a DC subset expressed higher levels of CD86 and MHC class II. Both subsets up-regulated CD80, CD86 and MHC class II in response to LPS. The percentage of CD1a expressing DC did not change with maturation. Flow cytometry plots are representative of four independent experiments. (d) CD1a expression is not induced on monocyte-derived CD1a DC with activation. CD1a DC isolated by sorting were incubated with LPS 100 ng/ml for 24 h. Cell surface expression of CD1a (green) and HLA-DR (green) were then analysed after staining with directly conjugated monoclonal antibody and compared with the appropriate isotype control (red). There was no induction of CD1a expression on CD1a DC upon activation. All the cells were MHC class II positive, consistent with their DC phenotype. This experiment is representative of three independent experiments using different donors.

M Cernadas, et al. Clin Exp Immunol. 2009 March;155(3):523-533.

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