Proliferation assays & ELISPOT analyses of long peptide vaccines; measurement of regulatory T cells

We have obtained valuable data on chemically well-defined TLR ligand conjugates and their effectiveness to induce a strong immune response via the cross-presentation pathway.

We have studied long peptides which were covalently conjugated to either the TLR2 ligand or TLR9 ligand, Pam3CysSK4 or CpG, respectively have been used to study the uptake, antigen presentation, and induction of specific T-cells. A pronounced enhancement in antigen presentation by DC in vitro was facilitated by the conjugated peptides compared to peptide alone, or peptide mixed with the free TLR ligand. Importantly, direct injection of the conjugates or pre-loading of the conjugates onto dendritic cells followed by injection into naïve mice, lead to a robust induction of specific CD8 T-cells, as detected by tetramers staining, compared to mice injected with the peptide mixed with the free TLR ligand.

With regard to TLR2 ligands, we have investigated the behaviour of two diastereomers of the TLR-2 ligand Pam3CSK4 (Pam) derivatives, namely the R- and S-epimers at C-2 of the glycerol moiety. In this respect we have chemically coupled either the R-epimer of the Pam3CSK4 or the S-epimer of the Pam3CSK4 to long peptides containing a CTL epitope. We found that the Pam(R)-conjugates lead to better activation of dendritic cells than the Pam(S)-conjugates as judged by higher IL-12 secretion, up-regulation 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.

These results show that chemically well-defined TLR ligand conjugates induce a strong immune response, significantly more effective than combining the free peptide and the TLR ligand. By combining efficient antigen targeting and DC maturation, TLR ligand conjugates are promising vehicles to deliver antigens via the cross-presentation pathway.

• molecule type
• peptide,
• CD8,
• Pam3CysSK4,
• CpG,
• Toll like receptor 2,
• Toll like receptor 9

• organism type
• Mus musculus

• cell type
• dendritic cell,
• regulatory t cell

• experimental factor type
• Cell type factor

created over 16 years ago (2 March 2009)    last modified over 13 years ago (28 September 2011)   [ RDF ]   [ RelFinder ]