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Synthesis and biological activity of alpha-L-fucosyl ceramides, analogues of the potent agonist, alpha-D-galactosyl ceramide KRN7000.

Veerapen N, Reddington F, Bricard G, Porcelli SA, Besra GS - Bioorg. Med. Chem. Lett. (2010)

Bottom Line: Activation of iNKT cells is known to lead to the production of cytokines that can help alleviate or exacerbate these conditions. alpha-Galactosyl ceramide (alpha-GalCer) is a known agonist of iNKT cells and it is believed that its fucosyl counterpart might have similar immunogenic properties.The key challenge in the synthesis of the target molecules involved the stereoselective synthesis of the alpha-glycosidic linkage.Of the methods examined, the per-TMS-protected glycosyl iodide donor was completely alpha-selective, and could be scaled up to provide gram quantities of the azide precursor 11, from which a range of N-acylated alpha-L-fucosyl ceramides were readily obtained and evaluated for ex vivo expansion of human iNKT cells.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

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Related in: MedlinePlus

Ex vivo expansion of human iNKT cells by α-l-fucosylceramides. PBMC from four different donors were stimulated with the indicated glycolipids at a concentration of 250 nM in the presence of low levels of exogenous IL-2 and IL-7. At day 8, cultures were harvested and analysed by flow cytometry using monoclonal antibodies specific for CD3 and for the invariant TCRα chain expressed by iNKT cells (6B11). (A) Dot plots showing relative levels of CD3+ 6B11+iNKT cells are shown for one representative donor. Numbers in upper right quadrant indicate percentages of total lymphocytes that are iNKT cells. (B) Absolute numbers of iNKT cells in the cultures were determined by flow cytometry using fluorescent counting beads, and the values of iNKT cell fold expansion were determined by dividing by the input number of iNKT cells.
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fig1: Ex vivo expansion of human iNKT cells by α-l-fucosylceramides. PBMC from four different donors were stimulated with the indicated glycolipids at a concentration of 250 nM in the presence of low levels of exogenous IL-2 and IL-7. At day 8, cultures were harvested and analysed by flow cytometry using monoclonal antibodies specific for CD3 and for the invariant TCRα chain expressed by iNKT cells (6B11). (A) Dot plots showing relative levels of CD3+ 6B11+iNKT cells are shown for one representative donor. Numbers in upper right quadrant indicate percentages of total lymphocytes that are iNKT cells. (B) Absolute numbers of iNKT cells in the cultures were determined by flow cytometry using fluorescent counting beads, and the values of iNKT cell fold expansion were determined by dividing by the input number of iNKT cells.

Mentions: To assess the biological activity of the α-l-fucosylceramides and compare these to α-GalCer (KRN7000, 2), we assessed the ability of each compound to induce the expansion of iNKT cells in samples of human peripheral blood mononuclear cells (PBMC) during an eight-day in vitro culture.39 The results showed that both the percentages and absolute numbers of iNKT cells in cultures were increased by stimulation with α-l-fucosylceramides with C26:0 (4d) > C18:0 (OH) (4b) > C24:0 (4c). The α-l-fucosylceramide containing a C26:0 fatty acid (4d) was the most active of the fucosyl series, and stimulated iNKT cell expansions in some donors that approached those seen with the prototype iNKT cell agonist KRN7000 (2). In contrast, the α-l-fucosylceramide containing the C20:2 fatty acid (4a) was found to lack detectable iNKT cell stimulating activity in any of the donors tested (Fig. 1B). Representative profiles obtained by flow cytometry of cultures from one normal blood donor are shown in Figure 1A. This analysis was carried out with PBMC from four separate donors (Fig. 1B). Although, differences were observed for the levels of iNKT cell expansion between different donors, all donors responded significantly to two of the α-l-fucosylceramide analogues (4b and 4d).


Synthesis and biological activity of alpha-L-fucosyl ceramides, analogues of the potent agonist, alpha-D-galactosyl ceramide KRN7000.

Veerapen N, Reddington F, Bricard G, Porcelli SA, Besra GS - Bioorg. Med. Chem. Lett. (2010)

Ex vivo expansion of human iNKT cells by α-l-fucosylceramides. PBMC from four different donors were stimulated with the indicated glycolipids at a concentration of 250 nM in the presence of low levels of exogenous IL-2 and IL-7. At day 8, cultures were harvested and analysed by flow cytometry using monoclonal antibodies specific for CD3 and for the invariant TCRα chain expressed by iNKT cells (6B11). (A) Dot plots showing relative levels of CD3+ 6B11+iNKT cells are shown for one representative donor. Numbers in upper right quadrant indicate percentages of total lymphocytes that are iNKT cells. (B) Absolute numbers of iNKT cells in the cultures were determined by flow cytometry using fluorescent counting beads, and the values of iNKT cell fold expansion were determined by dividing by the input number of iNKT cells.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2957807&req=5

fig1: Ex vivo expansion of human iNKT cells by α-l-fucosylceramides. PBMC from four different donors were stimulated with the indicated glycolipids at a concentration of 250 nM in the presence of low levels of exogenous IL-2 and IL-7. At day 8, cultures were harvested and analysed by flow cytometry using monoclonal antibodies specific for CD3 and for the invariant TCRα chain expressed by iNKT cells (6B11). (A) Dot plots showing relative levels of CD3+ 6B11+iNKT cells are shown for one representative donor. Numbers in upper right quadrant indicate percentages of total lymphocytes that are iNKT cells. (B) Absolute numbers of iNKT cells in the cultures were determined by flow cytometry using fluorescent counting beads, and the values of iNKT cell fold expansion were determined by dividing by the input number of iNKT cells.
Mentions: To assess the biological activity of the α-l-fucosylceramides and compare these to α-GalCer (KRN7000, 2), we assessed the ability of each compound to induce the expansion of iNKT cells in samples of human peripheral blood mononuclear cells (PBMC) during an eight-day in vitro culture.39 The results showed that both the percentages and absolute numbers of iNKT cells in cultures were increased by stimulation with α-l-fucosylceramides with C26:0 (4d) > C18:0 (OH) (4b) > C24:0 (4c). The α-l-fucosylceramide containing a C26:0 fatty acid (4d) was the most active of the fucosyl series, and stimulated iNKT cell expansions in some donors that approached those seen with the prototype iNKT cell agonist KRN7000 (2). In contrast, the α-l-fucosylceramide containing the C20:2 fatty acid (4a) was found to lack detectable iNKT cell stimulating activity in any of the donors tested (Fig. 1B). Representative profiles obtained by flow cytometry of cultures from one normal blood donor are shown in Figure 1A. This analysis was carried out with PBMC from four separate donors (Fig. 1B). Although, differences were observed for the levels of iNKT cell expansion between different donors, all donors responded significantly to two of the α-l-fucosylceramide analogues (4b and 4d).

Bottom Line: Activation of iNKT cells is known to lead to the production of cytokines that can help alleviate or exacerbate these conditions. alpha-Galactosyl ceramide (alpha-GalCer) is a known agonist of iNKT cells and it is believed that its fucosyl counterpart might have similar immunogenic properties.The key challenge in the synthesis of the target molecules involved the stereoselective synthesis of the alpha-glycosidic linkage.Of the methods examined, the per-TMS-protected glycosyl iodide donor was completely alpha-selective, and could be scaled up to provide gram quantities of the azide precursor 11, from which a range of N-acylated alpha-L-fucosyl ceramides were readily obtained and evaluated for ex vivo expansion of human iNKT cells.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.

Show MeSH
Related in: MedlinePlus