PyNTTTTGT and CpG Immunostimulatory Oligonucleotides: Effect on Granulocyte/Monocyte Colony-Stimulating Factor (GM-CSF) Secretion by Human CD56+ (NK and NKT) Cells

CD56⁺ cells have been recognized as being involved in bridging the innate and acquired immune systems. Herein, we assessed the effect of two major classes of immunostimulatory oligonucleotides (ODNs), PyNTTTTGT and CpG, on CD56⁺ cells. Incubation of human peripheral blood mononuclear cells (hPBMC) with some of these ODNs led to secretion of significant amounts of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and granulocyte/monocyte colony-stimulating factor (GM-CSF), but only if interleukin 2 (IL2) was present. IMT504, the prototype of the PyNTTTTGT ODN class, was the most active. GM-CSF secretion was very efficient when non-CpG ODNs with high T content and PyNTTTTGT motifs lacking CpGs were used. On the other hand, CpG ODNs and IFNα inhibited this GM-CSF secretion. Selective cell type removal from hPBMC indicated that CD56⁺ cells were responsible for GM-CSF secretion and that plasmacytoid dendritic cells (PDCs) regulate this process. In addition, PyNTTTTGT ODNs inhibited the IFNα secretion induced by CpG ODNs in PDCs by interference with the TLR9 signaling pathway. Since IFNα is essential for CD56⁺ stimulation by CpG ODNs, there is a reciprocal interference of CpG and PyNTTTTGT ODNs when acting on this cell population. This suggests that these synthetic ODNs mimic different natural alarm signals for activation of the immune system.     

Immunostimulatory PyNTTTTGT oligodeoxynucleotides: structural properties and refinement of the active motif

It is well known that synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides within a given context stimulate B lymphocytes and plasmacytoid dendritic cells (PDCs) of the vertebrate immune system. We have reported that B lymphocyte and PDC stimulation in humans could also be efficiently achieved by using non-CpG ODNs bearing the immunostimulatory sequence (motif) PyNTTTTGT, wherein Py is C or T and N is any deoxyribonucleotide. We are now reporting a series of studies that gives further precision regarding the composition of this immunostimulatory motif. The analysis of hundreds of ODNs led us to the conclusion that the motif for optimal CpG-independent immune stimulation can be represented by a sequence of the following general formula: PyN(T/A)(T/C/G)(T/C/G)(T/G)GT, wherein Py is C or T and N is any deoxyribonucleotide and wherein at least two of the positions represented within parentheses are Ts. Requirements for optimal ODN activity are as follows: (1) at least one of the versions of the general motif must be located near the central portion of the ODN; and (2) the ODN must be 20 or more nucleotides long. PyN(T/A)(T/C/G)(T/C/G)(T/G)GT ODNs are active in a phosphorothioate or in a phosphodiester backbone. In a phosphodiester backbone a canonical motif is strongly required whereas in a phosphorothioate backbone specificity is, within certain limits, less strict. On the other hand, PyN(T/A)(T/C/G)(T/C/G)(T/G)GT oligonucleotides are inactive as a double chain or as a modified (phosphorothioate or hydroxyl-methyl modified) or unmodified RNA backbone.     

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.     

Requirements for effective inhibition of immunostimulatory CpG motifs by neutralizing motifs

The DNA of bacteria and many viruses contain unmethylated CpG dinucleotides in particular sequence contexts that activate vertebrate immune cells. A subset of these CpG motifs was previously found to oppose the effects of immunostimulatory (CpG-S) motifs and has been termed neutralizing (CpG-N) motifs. Here we show that oligodeoxynucleotides (ODNs) composed of clusters of CpG-N motifs could partially inhibit the induction of interleukin-12 (IK-12) from mouse spleen cells by ODN containing CpG-S motifs. However, non-CpG-containing ODN were also inhibitory, suggesting that neutralization of CpG-S ODNs by CpG-N ODNs in trans was nonspecific. Neutralization of CpG-S motifs by CpG-N motifs in cis was specific, but the degree of inhibition was strongly dependent on the particular CpG-S motif being neutralized, with motifs having an A residue 5' to the CG being much more resistant to inhibition than motifs having a T residue 5' to the CG. The degree of inhibition was dependent on the spacing between the CpG-S and CpG-N motifs, with the ability to neutralize inversely correlating with distance. In addition, whereas ODNs containing extended clusters of CpG-N motifs were nonstimulatory, isolated CpG-N motifs remained stimulatory in most sequence contexts. Finally, CpG-N ODNs were shown to be nonstimulatory when instilled into the lungs of BALB/c mice, but the ability of CpG-N motifs to neutralize CpG-S motifs in cis was not observed. These results show that there are precise and fairly complex interactions between immunostimulatory and inhibitory sequence motifs that govern whether a given DNA is able to activate the vertebrate immune system.     

CpG oligodeoxynucleotides protect normal and SIV-infected macaques from Leishmania infection

Oligodeoxynucleotides containing CpG motifs (CpG ODNs) mimic microbial DNA and activate effectors of the innate immune response, which limits the spread of pathogens and promotes an adaptive immune response. CpG ODNs have been shown to protect mice from infection with intracellular pathogens. Unfortunately, CpG motifs that optimally stimulate humans are only weakly active in mice, mandating the use of nonhuman primates to monitor the activity and safety of "human" CpG ODNs in vivo. This study demonstrates that CpG ODN treatment of rhesus macaques significantly reduces the severity of the lesions caused by a challenge with Leishmania: Leishmania superinfection is common in immunocompromised hosts, particularly those infected with HIV. This study shows that PBMCs from HIV-infected subjects respond to stimulation with CpG ODNs. To determine whether CpG ODNs can protect retrovirus-infected primates, SIV-infected macaques were treated with CpG ODNs and then challenged with Leishmania: Both lesion size and parasite load were significantly reduced in the CpG-treated animals. These findings support the clinical development of CpG ODNs as immunoprotective agents in normal and HIV-infected patients.     

Stimulation of thymocyte proliferation by phosphorothioate DNA oligonucleotides

DNA is a complex macromolecule the immunological properties of which depend on short sequence motifs called CpG motifs or immunostimulatory sequences (ISS). These sequences are mitogenic for B cells and can stimulate macrophage cytokine production. While these sequences do not directly activate T cells, they can augment effects of stimulation via the TCR. Furthermore, ISS can affect T cells because of macrophage production of IL-12 and IFN-alpha/beta. In these studies, we further evaluated the immune effects of DNA on T cells, testing the possibility that certain T cell populations can respond directly to this stimulus. We therefore tested the in vitro responses of thymocytes to a series of phosphodiester (Po) and phosphorothioate (Ps) oligonucleotides (ODNs) varying in sequence. In in vitro cultures, phosphorothioate ODNs (sODNs) containing CpG motifs induced significant proliferation of murine thymocytes, although phosphodiester compounds lacked activity. The magnitude of stimulation varied with sequences flanking the CpG motifs, as both dA and dT sequences enhanced the stimulatory capacity of the CpG motif. Furthermore, CpG sODNs were strong costimulators of anti-CD3-mediated thymocyte activation, increasing proliferation compared to anti-CD3 in the absence of DNA. This activation was only partially inhibited by cyclosporine A and was not dependent on a calcium influx. Together, these results indicate that phosphorothioate oligonucleotides containing CpG motifs can directly induce thymocyte proliferation as well as augment TCR activation. These observations thus extend the range of actions of CpG DNA and suggest additional mechanisms for its function as an immunomodulatory agent or adjuvant.     

Safety of CpG oligodeoxynucleotides in veterinary species

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in particular sequence contexts (CpG ODN) are recognized as a danger signal by the innate immune system of vertebrates. For this reason, CpG ODNs have a potential application as both an adjuvant and nonspecific immune modulator and are currently being evaluated in a number of human and veterinary clinical trials. Given their potent immunostimulatory activity, CpG ODNs could possibly induce adverse reactions. As all adjuvants and immune modulators must be nontoxic to meet safety requirements, it was essential to address the safety aspects of CpG ODNs. The current review summarizes experiments carried out to date to establish the safety of CpG ODNs in animals.     

Antitumor mechanisms of oligodeoxynucleotides with CpG and polyG motifs in murine prostate cancer cells: decrease of NF-kappaB and AP-1 binding activities and induction of apoptosis

Previous studies have shown that CpG oligodeoxynucleotides (ODNs) have substantial immunostimulatory effects with anticancer applications. The antitumor applications that have been described previously are mediated through the CpG-induced activation of the host immune system, not through direct antitumor effects. Using cytostasis and cell proliferation assays, we demonstrated that specific ODNs inhibit the proliferation of RM-1 cells, a murine prostate cancer cell line. Flow cytometry analysis using propidium iodide (PI) nuclear staining confirmed the direct proapoptotic effect of ODNs on prostate cancer cells. This effect was dose dependent. Further studies using Western blot analysis and electrophoresis mobility shift assay (EMSA) revealed that the treatment of prostate cancer cells with specific ODNs activated the caspase pathway(s) and decreased the binding activities of AP-1 and NF-kappaB in a time-dependent manner. Evaluation of a panel of ODNs containing different DNA motifs demonstrated that the optimal proapoptotic sequences required polyG sequences but that CpG motifs were not essential. Finally, in vivo antitumor studies showed that the proapoptotic polyG motifs significantly inhibited prostate tumor growth. PolyG motifs inhibited tumor growth, and the effects were enhanced by CpG immune activating sequences. ODN containing both polyG and CpG motifs may have enhanced efficacy in tumor therapy through multiple mechanisms of action, including direct antitumor activities and immune activation.     

Reconstruction of SEA-B7.1 double signals on human hepatocellular carcinoma cells and analysis of its immunological effect

Since transfection of established tumors with immunostimulatory genes, such as superantigens (SAg), a family of bacterial and viral proteins with strong immunostimulatory properties, can elicit antitumor immunity, direct transfection of tumors with genes of staphylococcal enterotoxin A (SEA) could probably set up a new way of immunological pathway. In our study, human hepatocellular carcinoma (HCC) cell lines stably transduced with SEA and B7.1/SEA fused genes, HHCCSEA and HHCCBS, were obtained by using the method of retroviral mediated gene transduction. The results showed that human HCC cells could express SEA gene. Although a tiny quantity of expression was detected, a robust immune response was promoted. The cytotoxicity of CTL on HHCCBS was the same as that on HHCCSEA. But the K(m) value of the reaction of the former was lower than that of HHCCSEA. Furthermore, the activity assay of T cells by ELISPOT demonstrated that HHCCBS could elicit more CTL activity than HHCCSEA and HHCCB7.1. It suggested that the affinity of T cells to HHCCBS was higher and the maxim velocity of reaction could be attained at an early stage of the reaction. Transduced HCC cells were also analyzed for HLA expression, and it was found that a majority of the cells expressed HLA-I molecules but no HLA-DR molecules. After blocking the HLA-I molecules by HLA-I mAb, the cytotoxicity of T lymphocytes dropped remarkably. The results suggested that SEA were mainly presented by HLA-I molecules, and that B7.1 and SEA could have synergistic action at the early stage of the reaction, but the relationship between them in the consequent process needs to be clarified.     

G3139 and other CpG-containing immunostimulatory phosphorothioate oligodeoxynucleotides are potent suppressors of the growth of human tumor xenografts in nude mice

Several phosphorothioate antisense oligodeoxynucleotides (ODN) are developed to target factors potentially involved in tumor growth and apoptosis suppression. Among them, the 18-mer G3139 (Oblimersen), which targets Bcl-2, is currently being tested in phase II and phase III clinical trials for various tumors in combination with chemotherapy. On the other hand, ODNs containing CpG dinucleotides (CpG-ODN) within specific-sequence contexts (CpG motifs) have been shown to activate rodent or primate immune cells via toll-like receptor 9 (TLR9) and have demonstrated remarkable T cell-dependent antitumor efficacy in a series of murine tumor models. However, immune cell activation by CpG-ODN is largely diminished upon C-5 methylation at CpG cytosine. As G3139 contains CpG motifs, we questioned whether the antitumor effects seen in human tumor xenografts might be abrogated by cytosine C-5 methylation of G3139, which retained the ability of G3139 to suppress Bcl-2 expression in tissue culture, or by similar derivatization of other phosphorothioate ODNs developed for the immune activation of rodent or human cells. The in vivo antitumor efficacy of the immunostimulatory H1826 and H2006 ODNs was compared with that of G3139. Bcl-2 suppression achieved by G3139 purportedly sensitizes tumor cells toward cytotoxic agents, and some of the experiments employed combinations of ODN with such drugs as cisplatin or etoposide. H1826, H2006, and G3139 all produced similar, striking, growth inhibitory effects on either H69 SCLC, A2780 ovarian carcinoma, or A549 lung adenocarcinoma human tumor xenografts at doses of 0.3 mg/kg and 1 mg/kg (H1826, H2006) or 12 mg/kg (G3139) per day. In contrast, the H2006-mC (1 mg/kg) or G3139-mC (12 mg/kg) derivatives demonstrated no significant antitumor effects. The combination of G3139 (12 mg/kg) with cisplatin produced some additive antitumor efficacy, which was not seen in combinations of G3139-mC (12 mg/kg) or H1826 (1 mg/kg) with cisplatin. G3139, at a dose of 12 mg/kg, alone induced extensive enlargement of the spleen. Immunostimulation was evaluated in vitro by flow cytometric measurements of the CD80 and CD86 activation markers found on CD19+ murine splenocytes. The CpG-ODN producing strong antitumor effects in vivo also induced these activation markers in vitro, in contrast to the in vivo inactive G3139-mC. Our data indicate a significant contribution of the immunostimulatory properties of CpG-ODN (including G3139) to the antitumor effects observed in nude mouse xenograft models. This is in contrast to previous data presented by other authors indicating that the activity of G3139 in human tumor xenografts was Bcl-2 specific. Furthermore, as nude mice are devoid of T cells, a T cell-mediated immune response apparently is not required for the potent antitumor responses observed here; innate immune responses are sufficient.     

A GpC-Rich Oligonucleotide Acts on Plasmacytoid Dendritic Cells To Promote Immune Suppression

Short synthetic oligodeoxynucleotides (ODNs) rich in CpG or GpG motifs have been considered as potential modulators of immunity in clinical settings. In this study, we show that a synthetic GpC-ODN conferred highly suppressive activity on mouse splenic plasmacytoid dendritic cells, demonstrable in vivo in a skin test assay. The underlying mechanism involved signaling by noncanonical NF-κB family members and TGF-β-dependent expression of the immunoregulatory enzyme IDO. Unlike CpG-ODNs, the effects of GpC-ODN required TLR7/TRIF-mediated but not TLR9/MyD88-mediated events, as do sensing of viral ssRNA and the drug imiquimod. Induction of IDO by a GpC-containing ODN could also be demonstrated in human dendritic cells, allowing those cells to assist FOXP3(+) T cell generation in vitro. Among potentially therapeutic ODNs, this study identifies GpC-rich sequences as novel activators of TLR7-mediated, IDO-dependent regulatory responses.     

Identification of immunostimulatory oligodeoxynucleotide from Escherichia coli genomic DNA

Bacterial DNA containing immunostimulatory CpG motifs can stimulate antigen-presenting cells to express costimulatory molecules and to produce various cytokines in vivo and in vitro. In this study, we fragmented macromolecular E.coli genomic DNA with DNase I, and analyzed the ability of the resulting DNA fragments to induce the NF-kappaB activation and humoral immune response. Furthermore, using computational analysis and luciferase assay for synthetic ODNs based on the sequence of the immunostimulatory DNA fragments (DF-ODNs), an active component of DF-ODNs sequences was investigated. Experimental results demonstrated that DF-ODN is optimal for the NF-kappaB-responsive promoter activation in the mouse macrophage cell line and the humoral immune response in vivo. In agreement with the activity of the DFODNs processed by DNase I, a synthetic ODN based on the DF-ODN sequences is potent at inducing IL-12 mRNA expression in primary dendritic cells. These results suggest that the discovery and characterization of a highly active natural CpG-ODN may be achieved by the analyses of bacterial DNA fragments generated by a nuclease activity.     

Superior activity of the type C class of ISS in vitro and in vivo across multiple species

CpG-C are a novel class of CpG motif-containing immunostimulatory sequences (ISS) that includes both a 5'-TCG element and a CpG-containing palindrome. CpG-C drive all known ISS activities and, in particular, are potent enhancers of IFN-alpha from plasmacytoid dendritic cells (PDCs). In our examination of CpG-C sequence requirements, we determined that optimal IFN-alpha-inducing activity could be achieved with longer palindromes. Longer palindromes also correlated with maintenance of the double-stranded (ds) form despite concentration and pH changes, indicating a preference for ds oligodeoxynucleotides (ODNs) by the ISS-induced signaling mechanism for IFN-alpha synthesis. This correlation did not hold for all arms of the ISS-induced immune response, since we did not observe increased B cell activity with the longer palindrome CpG-C ODNs. We further demonstrated that CpG-C retained activity in an in vitro primate system and induced the expression of several cytokines and IFN-alpha-inducible genes when CpG-C were administered in vivo to mice and primates. In conclusion, we have shown CpG-C to exert several types of immune functions across multiple species, and this novel class is thus an attractive candidate for ISS-based therapeutic strategies.     

Oligodeoxynucleotides differentially modulate activation of TLR7 and TLR8 by imidazoquinolines

Among the 11 human TLRs, a subfamily TLR7, TLR8, and TLR9 display similarities in structure and endosomal localization. Natural agonists consisting of nucleic acids, such as ssRNA or DNA with CpG motifs, activate the innate immune cells through these TLRs. Immune response modifiers (IRMs) of imidazoquinoline class compounds 3M-001, 3M-002, and 3M-003 have been shown to activate the innate immune system via TLR7, TLR8, and TLR7/8, respectively. In looking at the effect of the agonists of the TLR7, TLR8, and TLR9 on the activation of NF-kappaB of transfected HEK cells, we discovered that some oligodeoxynucleotides (ODNs) could modulate imidazoquinoline effects in a negative or positive manner. In this study we demonstrate that poly(T) ODNs can inhibit TLR7 and enhance TLR8 signaling events involving NF-kappaB activation in HEK cells and cytokine production (IFN-alpha, TNF, and IL-12) by human primary PBMC. In contrast, TLR3 agonist poly(I:C) does not affect imidazoquinoline-induced responses. The modulation of TLR7 and TLR8 responses is independent of CpG motifs or the nature of the ODN backbone structure. Furthermore, we show that to be an effective modulator, the ODNs need to be in the cell at the same time with either of the TLR7 or TLR8 agonist. We have also demonstrated that there is a physical interaction between IRMs and ODNs. The cross-talk between ODNs, IRMs, and TLR7 and TLR8 uncovered by this study may have practical implications in the field of microbial infections, vaccination, and tumor therapy.     

Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein

We have analyzed the influence of codon usage modifications on the expression levels and immunogenicity of DNA vaccines, encoding the human immunodeficiency virus type 1 (HIV-1) group-specific antigen (Gag). In the presence of Rev, an expression vector containing the wild-type (wt) gag gene flanked by essential cis-acting sites such as the 5'-untranslated region and 3'-Rev response element supported substantial Gag protein expression and secretion in human H1299 and monkey COS-7 cells. However, only weak Gag production was observed from the murine muscle cell line C2C12. In contrast, optimization of the Gag coding sequence to that of highly expressed mammalian genes (syngag) resulted in an obvious increase in the G+C content and a Rev-independent expression and secretion of Gag in all tested mammalian cell lines, including murine C2C12 muscle cells. Mice immunized intramuscularly with the syngag plasmid showed Th1-driven humoral and cellular responses that were substantially higher than those obtained after injection of the Rev-dependent wild-type (wt) gag vector system. In contrast, intradermal immunization of both wt gag and syngag vector systems with the particle gun induced a Th2-biased antibody response and no cytotoxic T lymphocytes. Deletion analysis demonstrated that the CpG motifs generated within syngag by codon optimization do not contribute significantly to the high immunogenicity of the syngag plasmid. Moreover, low doses of coadministered stimulatory phosphorothioate oligodeoxynucleotides (ODNs) had only a weak effect on antibody production, whereas at higher doses immunostimulatory and nonstimulatory ODNs showed a dose-dependent suppression of humoral responses. These results suggest that increased Gag expression, rather than modulation of CpG-driven vector immunity, is responsible for the enhanced immunogenicity of the syngag DNA vaccine.     

Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.     

An immunomodulatory GpG oligonucleotide for the treatment of autoimmunity via the innate and adaptive immune systems

Bacterial DNA and immunostimulatory CpG oligodeoxynucleotides (ODNs) activate the innate immune system to produce proinflammatory cytokines. Shown to be potent Th1-like adjuvants, stimulatory CpG motifs are currently used as effective therapeutic vaccines for various animal models of infectious diseases, tumors, allergies, and autoimmune diseases. In this study, we show that the application of an immunomodulatory GpG ODN, with a single base switch from CpG to GpG, can effectively inhibit the activation of Th1 T cells associated with autoimmune disease. Moreover, this immunomodulatory GpG ODN suppresses the severity of experimental autoimmune encephalomyelitis in mice, a prototypic Th1-mediated animal disease model for multiple sclerosis.     

Oligodeoxynucleotides without CpG motifs work as adjuvant for the induction of Th2 differentiation in a sequence-independent manner

The outcomes of immune responses are regulated by various parameters including how Ags are handled by APCs. In this study, we describe the intrinsic immunomodulatory characteristics of oligodeoxynucleotides (ODNs) that improve the Ag presentation by APCs. ODNs (20-mer) containing CpG motifs induced strong Th1-skewed responses. In contrast, those without CpG motifs enhanced cytokine production by effector Th cells without particular skewing toward Th1 responses or induced the differentiation of unprimed CD4(+) T cells toward Th2 cells. These functional features were prominently envisaged when ODNs were conjugated to the Ag, and were underlain by the facilitated binding of ODN-conjugated Ag to Ia(+) cells. Despite the functional differences between ODNs with CpG motifs and those without CpG motifs, both ODNs bound to Ia(+) cells with similar affinity and kinetics. Immunoenhancing activities of the ODNs were not sequence-dependent; the characteristics, including the facilitation of Ag capture, enhancement of effector Th cell responses, and induction of Th2 cells, were shared by randomly synthesized ODNs conjugated to Ag. This is the first study suggesting that ODNs, independent of the sequences, enhance immune responses through the promoted capture of ODN-conjugated Ag by APCs.     

Necessity of oligonucleotide aggregation for toll-like receptor 9 activation

Toll-like receptor 9 (TLR9), a member of the interleukin-1 (IL-1) family of pathogen-associated molecular pattern receptors, is activated by unmethylated CpG-containing sequences in bacterial DNA or synthetic oligonucleotides (ODNs) in the endosomal compartment. The stimulation of an IL-1 response is thought to require the aggregation of its receptor. By analogy, we postulated that the potency of a TLR9 ligand should depend first on its ability to enter cells and gain access to TLR9 and second on its capacity to form a multimeric complex capable of cross-linking these receptors. Previously, we selected from a random library a series of phosphodiester ODNs with enhanced ability to permeate cells. Here, we studied the structural requirements for these penetrating ODNs to elicit a functional TLR9 response, as assessed by cytokine production from bone marrow-derived mouse mononuclear cells. The presence of a prototypic murine immunostimulatory DNA hexameric sequence (purine-purine-CG-pyrimidine-pyrimidine) in the ODNs was not sufficient for stimulation. In addition, the TLR9-activating ODNs had to have the ability to form aggregates and often to form secondary structures near the core CpG motifs. Multimerization was promoted by the presence of a guanine-rich 3'-terminus. The phosphodiester ODNs with CpG motifs that did not aggregate antagonized the effects of the multimeric TLR9 activators. These findings suggest that an optimal TLR9 agonist needs to contain a spatially distinct multimerization domain and a receptor binding CpG domain. This concept may prove useful for the design of new TLR9-modulating agents.