Two different types of CpG motif-containing oligonucleotides (CpG ODN) have been described: CpG-A with high induction of IFN- § in plasmacytoid dendritic cells; and CpG-B with little induction of IFN- § , but potent activation of B cells. In this study, we demonstrate that CpG-A fail to activate B cells unless plasmacytoid dendritic cells are present. We identified a new set of CpG ODN sequences which induces high levels of IFN- § in plasmacytoid dendritic cells but remains capable of directly activating B cells. These new CpG ODN (termed CpG-C) are more potent stimulants of B cells than CpG-B due to their ability of directly and indirectly (via plasmacytoid dendritic cells) activating B cells. The sequence of CpG-C combines structural elements of both CpG-A and CpG-B. The most potent sequence, M362, contains a 5'-end 'TCGTCG-motif' and a 'GTCGTT-motif', both of which are present in CpG-B (ODN 2006); a palindromic sequence characteristic for CpG-A (ODN 2216); but no poly G motif required for CpG-A. In conclusion, we defined the first CpG-containing sequences that potently activate both TLR9-expressing immune cell subsets in humans, the plasmacytoid dendritic cell and the B cell. CpG-C may allow for improved therapeutic immuno-modulation in vivo.

Two different CpG oligonucleotides (ODN) were used to study the regulation of type I IFN in human plasmacytoid dendritic cells (PDC): ODN 2216, a CpG-A ODN, known to induce high amounts of IFN-α in PDC, and ODN 2006, a CpG-B ODN, which is potent at stimulating B cells. CpG-A ODN showed higher and prolonged kinetics of type I IFN production compared with that of CpG-B ODN. In contrast, CpG-B ODN was more active than CpG-A ODN in stimulating IL-8 production and increasing costimulatory and Ag-presenting molecules, suggesting that CpG-A and CpG-B trigger distinct regulatory pathways in PDC. Indeed, CpG-A ODN, but not CpG-B ODN, activated the type I IFNR-mediated autocrine feedback loop. PDC were found to express high constitutive levels of IFN regulatory factor (IRF)7. IRF7 and STAT1, but not IRF3, were equally up-regulated by both CpG-A and CpG-B. CD40 ligand synergistically increased CpG-B-induced IFN-α independent of the IFNR but did not affect CpG-B-induced IFN-β. In conclusion, our studies provide evidence for the existence of two distinct regulatory pathways of type I IFN synthesis in human PDC, one dependent on and one independent of the IFNR-mediated feedback loop. The alternate use of these pathways is based on the type of stimulus rather than the quantity of IFN-αβ available to trigger the IFNR. Constitutive expression of IRF7 and the ability to produce considerable amounts of IFN-α independent of the IFNR seem to represent characteristic features of PDC.

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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...

Human B cells and plasmacytoid dendritic cells recognize CpG motifs within microbial DNA via Toll-like receptor 9. Two functionally distinct types of CpG motif containing oligonucleotides (CpG ODN) have been described, CpG-A and CpG-B. In contrast to CpG-B, CpG-A induces high amounts of type I IFN (IFN-α and IFN-β) in plasmacytoid dendritic cells. In the present study, we examined the effects of CpG-A on human primary monocytes. In PBMC stimulated with CpG-A and GM-CSF, monocytes showed excellent survival, increased in size and granularity, and within 3 days developed a dendritic cell-like phenotype that was characterized by down-regulation of CD14, partial up-regulation of CCR7, and an increased surface expression of costimulatory and Ag-presenting molecules. This effect could be inhibited by a combination of blocking Abs to type I IFN, and no such CpG-A-induced changes were observed in purified monocytes. Although IL-12 production by this dendritic cell-like phenotype required additional stimulation with CD40 ligand, this cell type spontaneously up-regulated IL-15 expression. Consistent with the known effect of IL-15 on effector and memory CD8 T cells, the frequency of CCR7−/CD45RA− CD8 T cells was selectively increased in allogeneic T cell assays. Furthermore, this dendritic cell type was more potent to support both the generation and the IFN-γ production of autologous influenza matrix peptide-specific memory CD8 T cells as compared with dendritic cells generated in the presence of GM-CSF and IL-4. In conclusion, monocytes exposed to the cytokine milieu provided by CpG-A rapidly develop a dendritic cell-like phenotype that is well equipped to support CD8 T cell responses.