Christian Schetter

Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities.

Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl‐deoxyguanosine (CpG) dinucleotides (CpG ODN) mimic the immunostimulatory activity of bacterial DNA and are recognized by the Toll‐like receptor 9 (TLR9). CpG ODN of the B‐Class stimulate strong B cell and NK cell activation and cytokine production. The highest degrees of NK stimulation as well as IFN‐α secretion by plasmacytoid DC were found to occur only with A‐Class ODN. A third class of CpG ODN combines the immune effects of A‐ and B‐Class CpG ODN. C‐Class ODN strongly stimulate B cell or NK cell activation and IFN‐α production. In contrast to the A‐Class, the C‐Class is wholly phosphorothioate, has no poly‐G stretches, but has palindromic sequences combined with stimulatory CpG motifs. All classes stimulate TLR9‐dependent signaling, but with strikingly different dose‐response relationships that are quite in contrast to those observed for IFN‐α. Effects similar to those on human cells were observed on mouse splenocytes. In contrast, splenocytes from TLR9‐deficient mice did not show any response to the three CpG ODN classes. In vivo studies demonstrate that C‐Class ODN are very potent Th1adjuvants. C‐Class ODN may represent new therapeutic drugs that combine the effects of A‐ and B‐Class ODN for broad applications in infectious disease or cancer therapy.

Oligodeoxynucleotides lacking CpG dinucleotides mediate Toll-like receptor 9 dependent T helper type 2 biased immune stimulation

Oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides mimic the immune stimulatory activity of bacterial DNA in vertebrates and are recognized by Toll‐like receptor 9 (TLR9). It is also possible to detect immune activation with certain phosphorothioate sequences that lack CpG motifs. These ODN are less potent than CpG ODN and the mechanism by which they stimulate mammalian leucocytes is not understood. We here provide several lines of evidence demonstrating that the effects induced by non‐CpG ODN are mediated by TLR9. First, non‐CpG ODN could not stimulate cytokine secretion from the splenocytes of TLR9‐deficient (TLR9–/–) mice. Second, immunization of TLR9+/+ but not TLR9–/– mice with non‐CpG ODN enhanced antigen‐specific antibody responses, although these were T helper type 2 (Th2)‐biased. Third, reactivity to non‐CpG ODN could be reconstituted by transfection of human TLR9 into non‐responsive cells. In addition, we define a new efficient immune stimulatory motif aside from the CpG dinucleotide that consists of a 5′‐TC dinucleotide in a thymidine‐rich background. Non‐CpG ODN containing this motif induced activation of human B cells, but lacked stimulation of Th1‐like cytokines and chemokines. Our study indicates that TLR9 can mediate either efficient Th1‐ or Th2‐dominated effects depending on whether it is stimulated by CpG or certain non‐CpG ODN.

Modulating responsiveness of human TLR7 and 8 to small molecule ligands with T-rich phosphorothiate oligodeoxynucleotides

Toll‐like receptors (TLR) 7 and 8 are closely related members of the TLR family of pathogen‐associated molecular pattern recognition receptors and have an important function in activation of innate immune responses upon viral infection. TLR7 can be activated selectively by the guanosine analogue loxoribine, whereas the imidazoquinoline derivative Resiquimod (R‐848) activates both TLR7 and TLR8. We demonstrate that co‐incubation of R‐848 with thymidine homopolymer oligodeoxynucleotides (ODN) significantly increased activity of R‐848 on TLR8‐expressing HEK 293 cells, but abolished TLR7‐mediated signaling. Similarly, the combination of loxoribine and thymidine ODN redirected the stimulatory effect of loxoribine away from TLR7, and toward TLR8. This alteration in ligand specificity was demonstrated both in TLR‐transfected HEK cells, and also in human PBMC, with a corresponding change in cytokine production away from IFN‐α secretion by TLR7‐expressing plasmacytoid DC and toward IL‐12, TNF‐α and IFN‐γ secretion by TLR8‐expressing monocytes and NK cells. These results demonstrate an unexpected plasticity in the ligand specificities of TLR7 and TLR8, and suggest a novel sequence‐selective interaction between these receptors and synthetic phosphorothioate ODN.

Highly Immunostimulatory CpG-Free Oligodeoxynucleotides for Activation of Human Leukocytes

Synthetic oligodeoxynucleotides (ODNs) bearing CpG dinucleotides can mimic the immunostimulatory effects of bacterial DNA in vertebrates. Besides the known CpG motifs, no other sequence motif has been shown to have independent immunostimulatory effects. Several past investigators have demonstrated that the nucleotide content or the phosphorothioate (PS) backbone may have effects independently of the sequence. However, the effect of both nucleotide content and PS backbone to stimulate human leukocytes is not well understood. We investigated the immunostimulatory activity of 34 PS-ODNs with different nucleotide contents, lengths, and methylation status on human leukocytes. The thymidine content showed strong CpG-independent contribution to immunostimulation. In contrast, ODNs rich in other nucleotides (guanosine, cytosine, or adenosine) induced no or much lower levels of immunostimulation. The observed effects were highly dependent on the PS backbone chemistry. In addition to the base content and the backbone chemistry, the length of the PS-ODN was directly related to the magnitude of its stimulatory effects, especially on B cells. In addition, methylation of CpG dinucleotides did not always cause an abrogation of the immunostimulation. Immunostimulatory effects could be observed with methylated CpG ODNs, specifically as the ODN length was increased from 18 to 24 or more nucleotides (nt). In contrast, PS-ODNs with inverted CpG dinucleotides showed some but only weak immunostimulation. Our results demonstrate that non-CpG ODNs rich in thymidine or ODNs with methylated CpG motifs have length-dependent immunostimulatory effects. Such ODNs can induce effects similar to those seen with CpG ODNs but are much less efficient in stimulating human immune cells.

CpG oligodeoxynucleotides stimulate IFN-γ-inducible protein-10 production in human B cells

Several classes of CpG oligodeoxynucleotides (ODNs) with different immune stimulatory profiles were recently identified: the A-, B- and C-classes. In this study, we investigated the CpG-dependent stimulation of IFN-γ-inducible protein 10 (IP-10 or CXCL10) in different human immune cell types. CpG ODNs induced IP-10 in monocytes, pDCs and in B cells. Purified B cells as well as RPMI 8226 cells responded to CpG stimulation by IP-10 production. Treatment with exogenous IFN-α2b sensitized PBMCs, purified B cells as well as RPMI 8226 cells to respond more efficiently to stimulation with CpG ODNs by IP-10 production. IP-10 signaling could be directly stimulated via TLR9 in CpG-unresponsive HEK293 cells transfected with human TLR9 and an IP-10 reporter construct. Therefore, CpG-mediated IP-10 production is stimulated through IFN-α in cells that express the IFN-α receptor, a second pathway for IP-10 induction exists in TLR9-expressing B cells and pDCs where IP-10 is stimulated directly upon CpG-mediated TLR9 signaling. Our data provide a better understanding of the mechanisms through which CpG ODNs induce efficient Th1 responses.

Immunopharmacology of CpG Oligodeoxynucleotides and Ribavirin

ABSTRACT To investigate their potential mechanisms of action, the nucleoside analogue ribavirin and a TLR9 agonist were compared. The CpG oligodeoxynucleotides (ODN) demonstrated strong TLR9-related Th1-type effects, and ribavirin appeared only to mediate signaling in TLR-transfected cells. CpG ODN represent a promising new type of therapeutic drug for hepatitis C or other infectious diseases.

Impact of modifications of heterocyclic bases in CpG dinucleotides on their immune-modulatory activity

Synthetic phosphorothioate oligodeoxynucleotides (ODN) bearing unmethylated CpG motifs can mimic the immune‐stimulatory effects of bacterial DNA and are recognized by Toll‐like receptor 9 (TLR9). Past studies have demonstrated that nucleotide modifications at positions at or near the CpG dinucleotides can severely affect immune modulation. However, the effect of nucleotide modifications to stimulate human leukocytes and the mechanism by which chemically modified CpG ODN induce this stimulation are not well understood. We investigated the effects of CpG deoxyguanosine substitutions on the signaling mediated by human TLR9 transfected into nonresponsive cells. ODN incorporating most of these substitutions stimulated detectable TLR9‐dependent signaling, but this was markedly weaker than that induced by an unmodified CpG ODN. One of the most active ODN tested contained deoxyinosine for deoxyguanosine substitutions (CpI ODN), but its relative activity to induce cytokine secretion on mouse cells was much weaker than on human cells. The activity was dependent on TLR9, as splenocytes from mice genetically deficient in TLR9 did not respond to CpI ODN stimulation. It is surprising that CpI ODN were nearly as strong as CpG ODN for induction of human B cell stimulation but were inferior to CpG ODN in their ability to induce T helper cell type 1 effects. These data indicate that certain deoxyguanosine substitutions in CpG dinucleotides are tolerated to stimulate a TLR9‐mediated immune response, but this response is insufficient to induce optimal interferon‐α‐mediated effects, which depend on the presence of an unmodified CpG dinucleotide. These studies provide a structure‐activity relationship for TLR9 agonist compounds with diverse immune effects.