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Synthesis and biological activity of alpha-glucosyl C24:0 and C20:2 ceramides.

Jervis PJ, Veerapen N, Bricard G, Cox LR, Porcelli SA, Besra GS - Bioorg. Med. Chem. Lett. (2010)

Bottom Line: Alpha-glucosyl ceramides 4 and 5 have been synthesised and evaluated for their ability to stimulate the activation and expansion of human iNKT cells.The key challenge in the synthesis of both target molecules was the stereoselective synthesis of the alpha-glycosidic linkage.Of the methods examined, glycosylation using per-TMS-protected glucosyl iodide 16 was completely alpha-selective and provided gram quantities of amine 11, from which alpha-glucosyl ceramides 4 and 5 were obtained by N-acylation. alpha-GlcCer 4, containing a C24 saturated acyl chain, stimulated a marked proliferation and expansion of human circulating iNKT cells in short-term cultures. alpha-GlcCer 5, which contains a C20 11,14-cis-diene acyl chain (C20:2), induced extremely similar levels of iNKT cell activation and expansion.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.

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Ex vivo expansion of human iNKT cells by α-GlcCer and α-GalCer analogues. Peripheral blood mononuclear cells (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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fig3: Ex vivo expansion of human iNKT cells by α-GlcCer and α-GalCer analogues. Peripheral blood mononuclear cells (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-glucosyl ceramides 4 and 5 and compare these to KRN7000 1 and the α-galactosyl ceramide analogues 2 (C24:0) and 3 (C20:2), 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.32 The results showed that both the percentages and absolute numbers of iNKT cells in the cultures were markedly increased to similar levels by stimulation with both of the α-GlcCer analogues 4 and 5 (Fig. 3). The level of iNKT cell expansion, at least with a relatively high concentration of the glycolipids (250 nM), was comparable for both of the N-acyl variants of α-GlcCer and very similar to levels obtained with the related α-GalCer analogues (2 (C24:0) and 3 (C20:2)) and with the prototypical iNKT cell activator KRN7000 (1 (C26:0)). Representative profiles obtained by flow cytometry of cultures from one normal blood donor are shown in Figure 3A. This analysis was carried out with PBMC from four separate donors (Fig. 3B). Although differences were observed for the levels of iNKT cell expansion between different donors, all donors responded well to the two α-GlcCer analogues. In all cases, these responses were similar to those generated by the analogous α-GalCer compounds.


Synthesis and biological activity of alpha-glucosyl C24:0 and C20:2 ceramides.

Jervis PJ, Veerapen N, Bricard G, Cox LR, Porcelli SA, Besra GS - Bioorg. Med. Chem. Lett. (2010)

Ex vivo expansion of human iNKT cells by α-GlcCer and α-GalCer analogues. Peripheral blood mononuclear cells (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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fig3: Ex vivo expansion of human iNKT cells by α-GlcCer and α-GalCer analogues. Peripheral blood mononuclear cells (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-glucosyl ceramides 4 and 5 and compare these to KRN7000 1 and the α-galactosyl ceramide analogues 2 (C24:0) and 3 (C20:2), 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.32 The results showed that both the percentages and absolute numbers of iNKT cells in the cultures were markedly increased to similar levels by stimulation with both of the α-GlcCer analogues 4 and 5 (Fig. 3). The level of iNKT cell expansion, at least with a relatively high concentration of the glycolipids (250 nM), was comparable for both of the N-acyl variants of α-GlcCer and very similar to levels obtained with the related α-GalCer analogues (2 (C24:0) and 3 (C20:2)) and with the prototypical iNKT cell activator KRN7000 (1 (C26:0)). Representative profiles obtained by flow cytometry of cultures from one normal blood donor are shown in Figure 3A. This analysis was carried out with PBMC from four separate donors (Fig. 3B). Although differences were observed for the levels of iNKT cell expansion between different donors, all donors responded well to the two α-GlcCer analogues. In all cases, these responses were similar to those generated by the analogous α-GalCer compounds.

Bottom Line: Alpha-glucosyl ceramides 4 and 5 have been synthesised and evaluated for their ability to stimulate the activation and expansion of human iNKT cells.The key challenge in the synthesis of both target molecules was the stereoselective synthesis of the alpha-glycosidic linkage.Of the methods examined, glycosylation using per-TMS-protected glucosyl iodide 16 was completely alpha-selective and provided gram quantities of amine 11, from which alpha-glucosyl ceramides 4 and 5 were obtained by N-acylation. alpha-GlcCer 4, containing a C24 saturated acyl chain, stimulated a marked proliferation and expansion of human circulating iNKT cells in short-term cultures. alpha-GlcCer 5, which contains a C20 11,14-cis-diene acyl chain (C20:2), induced extremely similar levels of iNKT cell activation and expansion.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.

Show MeSH