title: Chirality of TLR-2 ligand Pam3CysSK4 in fully synthetic peptide conjugates critically influences the induction of specific CD8+ T-cells.
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.
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, PO Box 9600, 2300 RC Leiden, The Netherlands. s.khan@lumc.nl
authors.forename: Selina
authors.initials: S
authors.lastname: Khan
authors.forename: Jimmy J
authors.initials: JJ
authors.lastname: Weterings
authors.forename: Cedrik M
authors.initials: CM
authors.lastname: Britten
authors.forename: Ana R
authors.initials: AR
authors.lastname: de Jong
authors.forename: Dirk
authors.initials: D
authors.lastname: Graafland
authors.forename: Cornelis J M
authors.initials: CJ
authors.lastname: Melief
authors.forename: Sjoerd H
authors.initials: SH
authors.lastname: van der Burg
authors.forename: Gijs
authors.initials: G
authors.lastname: van der Marel
authors.forename: Hermen S
authors.initials: HS
authors.lastname: Overkleeft
authors.forename: Dmitri V
authors.initials: DV
authors.lastname: Filippov
authors.forename: Ferry
authors.initials: F
authors.lastname: Ossendorp
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002460:Cell Line; D003713:Dendritic Cells / Q000276:immunology; D018664:Interleukin-12 / Q000276:immunology; D055666:Lipopeptides / Q000737:chemistry / Q000276:immunology*; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D013237:Stereoisomerism; D051195:Toll-Like Receptor 2 / Q000276:immunology*
chemical_list: D055666:Lipopeptides; C508075:Pam(3)CSK(4) peptide; C493486:Tlr2 protein, mouse; D051195:Toll-Like Receptor 2; D018664:Interleukin-12