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ABSTRACT: The synthesis of an array of 2-azidoalkoxy substituted 7-hydro-8-oxoadenines is described. The relation of the structure of these compounds and their ability to induce maturation of dendritic cells is evaluated.
Bioorganic & medicinal chemistry letters 05/2009; 19(8):2249-51. · 2.65 Impact Factor
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Nadine van Montfoort,
Marcel G Camps,
Selina Khan,
Dmitri V Filippov, Jimmy J Weterings,
Janice M Griffith,
Hans J Geuze,
Thorbald van Hall,
J Sjef Verbeek,
Cornelis J Melief,
Ferry Ossendorp
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ABSTRACT: Dendritic cells (DCs) are crucial for priming of naive CD8(+) T lymphocytes to exogenous antigens, so-called "cross-priming." We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte cross-priming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained cross-presentation by DCs, despite the short-lived expression of MHC class I-peptide complexes at the cell surface.
Proceedings of the National Academy of Sciences 05/2009; 106(16):6730-5. · 9.68 Impact Factor
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Advances in experimental medicine and biology 02/2009; 611:141-2. · 1.09 Impact Factor
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Selina Khan, Jimmy J Weterings,
Cedrik M Britten,
Ana R de Jong,
Dirk Graafland,
Cornelis J M Melief,
Sjoerd H van der Burg,
Gijs van der Marel,
Hermen S Overkleeft,
Dmitri V Filippov,
Ferry Ossendorp
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ABSTRACT: Covalent conjugation of synthetic Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides provides well-defined constructs that have significantly improved capacity to induce efficient priming of CD8(+) T lymphocytes in vivo. We have recently explored the cellular mechanisms underlying the efficient induction of a CD8(+) cytotoxic T lymphocyte response by such synthetic model vaccines [Khan, S., Bijker, M.S., Weterings, J.J., Tanke, H.J., Adema, G.J., van, H.T., Drijfhout, J.W., Melief, C.J., Overkleeft, H.S., van der Marel, G.A., Filippov, D.V., van der Burg, S.H., Ossendorp, F., 2007. Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells. J. Biol. Chem. 282, 21145-21159.]. In the current study we have investigated the behaviour of two diastereomers of the TLR-2 ligand Pam(3)CSK(4) (Pam) derivatives, namely the R- and S-epimers at C-2 of the glycerol moiety. Other studies have shown that the Pam(3)Cys based lipopeptides of R-configuration (Pam(R)) in the glycerol moiety enhanced macrophage and B-cell activation compared to those with S-configuration (Pam(S)). Here we report that Pam(R)-conjugates lead to better activation of dendritic cells than the Pam(S)-conjugates as judged by higher IL-12 secretion, upregulation of relevant markers for dendritic cell maturation. In contrast both epimers were internalized equally efficient in a clathrin-dependent manner indicating no qualitative difference in the uptake of the two stereoisomeric Pam-conjugates. We conclude that the enhanced DC activation is due to enhanced TLR-2 triggering by the Pam(R)-conjugate in contrast to the Pam(S)-conjugate. Importantly, induction of specific CD8(+) T-cells was significantly higher in mice injected with the Pam(R)-conjugates compared to mice injected with the Pam(S)-conjugate. In summary we show that the favourable effects of the Pam(R)-configuration of TLR-2 ligand can be attributed to direct effects on dendritic cells resulting in enhancement of CD8(+) T-cell responses.
Molecular Immunology 12/2008; 46(6):1084-91. · 2.90 Impact Factor
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Selina Khan,
Martijn S Bijker, Jimmy J Weterings,
Hans J Tanke,
Gosse J Adema,
Thorbald van Hall,
Jan W Drijfhout,
Cornelis J M Melief,
Hermen S Overkleeft,
Gijsbert A van der Marel,
Dmitri V Filippov,
Sjoerd H van der Burg,
Ferry Ossendorp
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ABSTRACT: Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.
Journal of Biological Chemistry 08/2007; 282(29):21145-59. · 4.77 Impact Factor
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ABSTRACT: The preparation of three different 2-alkoxy-8-hydroxyadenylpeptide conjugates has been accomplished by solid-phase synthesis combined with 'on-resin' Cu(I) catalyzed Huisgen cycloaddition. The immunogenicity of the compounds has been evaluated in IL-12 production and antigen presentation assays.
Bioorganic & Medicinal Chemistry Letters 07/2006; 16(12):3258-61. · 2.55 Impact Factor
