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Human Valpha24+ Vbeta11+ natural killer T cells (NKT cells) are "natural memory" T cells that detect glycolipid antigens such as alpha-galactosylceramide (alpha-GalCer) presented on CD1d. In the present study we found that highly purified Valpha24+ 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 alpha-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 alpha-GalCer and CpG ODN. While PDC were responsible for the stimulation of NKT cells with CpG ODN, MDC but not PDC presented alpha-GalCer via CD1d. Partial activation of NKT cells was mediated by PDC-derived IFN-alpha, whereas full activation of NKT cells as indicated by IFN-gamma production required cell-to-cell contact of PDC and NKT cells in addition to IFN-alpha; OX40 was involved in this interaction. We conclude that CpG-activated PDC enhance alpha-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.

The detection of microbial molecules via Toll-like receptors (TLR) in B cells is not well characterized. In this study, we found that both naive and memory B cells lack TLR4 (receptor for LPS) but express TLR9 (receptor for CpG motifs) and produce IL-6, TNF-alpha, and IL-10 upon stimulation with CpG oligonucleotides (ODN), synthetic mimics of microbial DNA. Consistent with the lack of TLR4, purified B cells failed to respond to LPS. Similar to CpG ODN, CD40 ligand (CD40L) alone induced IL-6, TNF-alpha, and IL-10. Production of these cytokines as well as IgM synthesis was synergistically increased when both CpG ODN and CD40L were combined. Unlike IL-6, TNF-alpha, and IL-10, the Th1 cytokine IL-12p70 was detected only when both CpG ODN and CD40L were present, and its induction was independent of B cell receptor cross-linking. CpG ODN did not increase the capacity of CD40L-activated B cells to induce proliferation of naive T cells. However, B cells activated with CpG ODN and CD40L strongly enhanced IFN-gamma production in developing CD4 T cells via IL-12. Together, these results demonstrate that IL-12p70 production in human B cells is under the dual control of microbial stimulation and T cell help. Our findings provide a molecular basis for the potent adjuvant activity of CpG ODN to support humoral immune responses observed in vivo, and for the limited value of LPS.

Two different types of CpG motif-containing oligonucleotides (CpG ODN) have been described: CpG-A with high induction of IFN-alpha in plasmacytoid dendritic cells; and CpG-B with little induction of IFN-alpha, 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-alpha 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.

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-alpha and IFN-beta) 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-gamma 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.

Oligodeoxynucleotides with CpG motifs (CpG ODN) mimic microbial DNA and activate effectors of innate immunity including NK cells. Human gamma delta T cells (Vgamma9/Vdelta2) are antigen specific "natural memory" T cells in a preactivated stage, which respond to common non-protein phosphoantigens. Among several CpG ODN tested, distinct CpG ODN sequences characterized by inducing high amounts of IFN-alpha/-beta in PBMC elicited strong gamma delta T cell and NK cell responses, as determined by CD69 expression, IFN-gamma production, perforin content and lytic activity. These CpG ODN activated gamma delta T cells and NK cells in the absence of an additional stimulus and synergistically increased responsiveness to cell-type-specific antigens like isopentenylpyrophosphate for gamma delta T cells and NK-sensitive tumor cells for NK cells. NK cells and gamma delta T cells were activated via IFN-alpha/-beta released by CpG ODN-stimulated PBMC. Purified gamma delta T cells and NK cells did not respond to CpG ODN but to recombinant IFN-alpha/-beta. In conclusion, CpG ODN sequences were identified which, based on their ability to induce high amounts of IFN-alpha/-beta, represent strong adjuvants for "natural memory" cells including responses of gamma delta T cells to non-protein antigens. Early IFN-alpha/-beta dependent stimulation of IFN-gamma synthesis in NK cells and gamma delta T cells may contribute to the CpG ODN-induced Th1 bias of an evolving immune response.