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Human Va24 + Vb11 + natural killer T cells (NKT cells) are "natural memory" T cells that detect glycolipid antigens such as a-galactosylceramide (a-GalCer) presented on CD1d.In the present study we found that highly purified Va24 + NKT cells lack TLR9 mRNA, and thus are not sensitive towards stimulation with CpG oligodeoxynucleotides (ODN). Within PBMC, however, CpG ODN synergistically activated NKT cells stimulated with their cognate antigen a-GalCer. Depletion of plasmacytoid dendritic cells (PDC) or myeloid dendritic cells (MDC) revealed that both DC subsets were necessary for the synergistic activation of NKT cells by a-GalCer and CpG ODN. While PDC were responsible for the stimulation of NKT cells with CpG ODN, MDC but not PDC presented a-GalCer via CD1d. Partial activation of NKT cells was mediated by PDC-derived IFN-a, whereas full activation of NKT cells as indicated by IFN-c production required cell-tocell contact of PDC and NKT cells in addition to IFN-a; OX40 was involved in this interaction. We conclude that CpG-activated PDC enhance a-GalCer-specific NKT cell activation, and bias activated NKT cells towards a Th1 phenotype. Our results lead to a novel concept of PDC function: to regulate effector activity of antigen-stimulated T cells in a cell contact-dependent manner without the need of simultaneous presentation of the cognate T cell antigen. IntroductionNatural killer T (NKT) cells represent a small subset of non-conventional innate T cells with a restricted TCR repertoire for the recognition of glycolipid antigens presented on CD1d, an MHC class I-like molecule. NKT cells express markers of NK cells and exhibit an activated memory phenotype. Depending on the microenvironment and the type of stimulation, they can release large amounts of both Th1 and Th2 cytokines (especially IL-4 and IFN-c) and show cytotoxic activity in vitro (reviewed in [1][2][3]). In both mice and man NKT cells express a homologous semi-invariant TCR that in humans consists of a Va24 chain preferentially paired with a Vb11 chain [4]. In mice, NKT cells are most frequent in the liver (about 30% of hepatic T cells), bone marrow and thymus (reviewed in [5]). Smaller NKT cell populations are present in spleen and blood (reviewed in [5]). Similar to their murine counterparts, human NKT cells preferen-A. M., S. R. and V. H. equally contributed to this manuscript. tially accumulate in the liver though with a far lower frequency than in mice (4% of hepatic T cells) ([6, 7] and reviewed in [1,5]). Due to the conserved TCR, both mouse and human NKT cells recognise the same specific glycolipid antigen a-galactosylceramide (a-GalCer) when presented by CD1d molecules on antigen-presenting cells (APC) [8][9][10]. Originally isolated from a marine sponge, aGalCer was first described as an agent with strong antimetastatic activity in mice and later found to specifically activate NKT cells in a CD1d-restricted manner [8,11,12]. Glycolipid antigens similar to a-GalCer have been detected in certain bacteria and under some abnormal conditions...

The aim of this paper was to investigate if the immunostimulatory effects of CpG-oligonucleotides (CpG-ODN) can be enhanced by the use of biodegradable protamine nanoparticles (proticles). We analyzed size, surface charge, and morphology of protamine nanoparticles containing CpG-ODN with photon correlation spectroscopy and transmission electron microscopy. Immunostimulatory effects of these nanoparticles on B cells, plasmacytoid dendritic cells (PDC), peripheral blood mononuclear cells, and whole blood were studied. Cytokine production, activation of the cells in terms of upregulation of surface molecules and uptake of nanoparticles were examined. We found that the use of protamine nanoparticles significantly increased (20-fold) CpG-ODN mediated interferon (IFN)-alpha production of PDC. ODN uptake in PDC was only marginally enhanced. CpG-ODN mediated IP-10 production in whole blood was strongly enhanced by the use of nanoparticles. Apart from a slight increase in CpG-ODN-induced interleukin (IL)-6 production in B cells, other parameters like the CpG-mediated activation of B cells and PDC as well as tumor necrosis factor (TNF)-alpha production of PDC remained largely unchanged. The use of control ODN indicated that the protamine nanoparticles themselves have no immunostimulatory properties. These results strongly support the use of particulate delivery systems like biodegradable protamine nanoparticles for the development of CpG-ODN-based therapeutics.

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