CpG Oligodeoxynucleotides as TLR9 Agonists

Toll-like receptors (TLRs) are part of the innate immune system, and they belong to the pattern recognition receptors (PRR) family. The PRR family is designed to recognize and bind conserved pathogen-associated molecular patterns, which are not generated by the host and are restricted and essential to microorganisms. TLR9, which recognizes unmethylated CpG (cytosine guanosine dinucleotide), is a very promising target for therapeutic activation. Stimulation of TLR9 activates human plasmacytoid dendritic cells and B cells, and results in potent T helper-1 (T_h1)-type immune responses and antitumor responses in mouse tumor models and in patients. Several pharmaceutical companies, such as Pfizer, Idera, and Dynavax, are developing CpG oligodeoxynucleotides (ODNs) for the treatment of cancer, along with other conditions, such as infections and allergy. CpG ODNs have shown promising results as vaccine adjuvants and in combination with cancer immunotherapy. Several TLR9 agonists are being developed and have entered clinical trials to evaluate their safety and efficacy for the treatment of several hematopoietic and solid tumors. In this review, we discuss the use of CpG ODNs in several phase I and II clinical trials for the treatment of NHL, renal cell carcinoma, melanoma, and non-small cell lung cancer, either alone or in combination with other agents.     

CpG Oligodeoxynucleotides as TLR9 Agonists

Unmethylated cytosine-phosphate-guanine (CpG) dinucleotides in microbial DNA sequences activate Toll-like receptor (TLR) 9, and previous studies have shown that oligodeoxynucleotides (ODNs) containing CpG in specific base sequence motifs (CpG ODNs) can reiterate the majority of the immunomodulatory effects produced by bacterial DNA. Many of the manifestations in allergic diseases are primarily due to T helper (T_h)-2 cell-type responses. CpG ODNs can induce T_h1 and T-regulatory (T_reg) cell-type cytokines that can suppress the T_h2 response. The therapeutic application of TLR9 has been explored extensively in recent years, and many studies are being conducted to assess the safety and efficacy of TLR9 agonists in various diseases, including atopic and infectious diseases, and cancer. Studies in murine models have shown that the development of atopic airway disease can be prevented by treatment with CpG ODNs. Various clinical trials are currently ongoing to determine the efficacy of CpG ODNs as a therapeutic tool for atopic diseases. In this review, we discuss the therapeutic application of CpG ODNs in allergy and asthma. CpG ODNs may be used alone or as an adjuvant to immunotherapy to treat these disorders.     

Immune Mechanisms and Therapeutic Potential of CpG Oligodeoxynucleotides

Unmethylated CpG motifs in bacterial DNA and synthetic oligodeoxynucleotides activate immune cells that express Toll-like Receptor 9. Activation through this receptor triggers cellular signaling that leads to production of a proinflammatory and a Th1-type, antigen-specific immune response. The immunostimulatory effects of CpG oligodeoxynucleotides confer protection against infectious disease, allergy and cancer in animal models, and clinical trials have been initiated. However, CpG oligodeoxynucleotides may exacerbate disease in some situations. We will review current concepts in the mechanisms of activating Toll-like Receptor 9 with CpG oligodeoxynucleotides and highlight opportunities for using large animal models to better determine the mechanisms of action.     

Repeated Peripheral Administrations of CpG Oligodeoxynucleotides Lead to Sustained CNS Immune Activation

Bacterial DNA containing CpG motifs activates cells of the innate immune system. In this study, we examined the effects of multiple peripheral bacterial DNA-mediated CNS innate immune stimulation. To study this issue, we repeatedly peripherally administered synthetic CpG-oligodeoxynucleotides (CpG-ODN) and assayed effects on CNS-associated TNF-alpha (TNFα) and C1q mRNA levels. We for the first time accounted for frequency of CpG-ODN administration and time kinetics of mRNA expression. We were able show that multiple intraperitoneal CpG-ODN administrations have a sustainable effect on immune effectors of the brain and stimulate TNFα mRNA secretion even up to 7 days after the last CpG-ODN application. This could on the one hand indicate a depot effect after multiple peripheral CpG-ODN administrations, however, it could also indicate that the cell producing TNFα mRNA remains activated for the indicated time period. Furthermore, elevated mRNA levels of C1q were observed, possibly indicating microglial activation after multiple peripheral bacterial DNA administrations. In this study, we have correlated frequency of CpG-ODN administrations with CNS-associated TNFα mRNA levels and show that multiple peripheral administrations of CpG-ODN lead to a sustained level of a Th1-associated cytokine in the brain. These findings indicate that the repeated peripherial administration of CpG oligodeoxynucleotides offer a therapeutical possibility for CNS-associated infections and tumors.     

Adjuvant Activity of CpG Oligodeoxynucleotides

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs directly stimulate human B cells and plasmacytoid dendritic cells (pDCs), thereby promoting the production of Th1 and proinflammatory cytokines and the maturation/activation of professional antigen-presenting cells. These activities enable CpG ODNs to act as immune adjuvants, accelerating and boosting antigen-specific immune responses by 5- to 500-fold. The CpG motifs present in bacterial DNA plasmids may contribute to the immunogenicity of DNA vaccines. Ongoing clinical studies indicate that CpG ODNs are safe and well tolerated when administered as adjuvants to humans and can improve vaccine-induced immune responses.     

Repeated Administration of Synthetic Oligodeoxynucleotides Expressing CpG Motifs Provides Long-Term Protection against Bacterial Infection

Synthetic oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs stimulate an innate immune response characterized by the production of polyreactive immunoglobulin M antibodies and immunomodulatory cytokines. This immune response has been shown to protect mice from challenge by Listeria monocytogenes and Francisella tularensis for up to 2 weeks. By repeatedly administering CpG ODN two to four times/month, we found that this protection could be maintained indefinitely. Protection was associated with a significant increase in the number of spleen cells that could be triggered by subsequent pathogen exposure to secrete gamma interferon and interleukin-6 in vivo (P < 0.01). ODN-treated animals remained healthy and developed neither macroscopic nor microscopic evidence of tissue damage or inflammation. Thus, repeated administration of CpG ODN may provide a safe means of conferring long-term protection against infectious pathogens.     

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.     

Immunomodulatory Effects of CpG Oligodeoxynucleotides on Established Th2 Responses

CpG oligodeoxynucleotides (CpG ODNs) are known to induce type 1 T-helper-cell (Th1) responses. We have previously demonstrated that CpG ODNs administered during sensitization prevent Th2-mediated eosinophilic airway inflammation in vivo. We also reported that key Th1 cytokines, gamma interferon (IFN-γ) and interleukin 12 (IL-12), are not necessary for this protection. Recent in vivo data suggest that CpG ODNs might also reverse established pulmonary eosinophilia. In order to clarify how CpG ODNs can inhibit established Th2 responses, we evaluated the cytokine production from splenocytes from antigen- and alum-immunized mice. Restimulation with antigen induced IL-5, which was clearly inhibited by coculture with CpG ODNs in a concentration-dependent manner. CpG ODNs also induced IFN-γ, but in a concentration-independent manner. The inhibition of IL-5 production was not mediated through natural killer cells or via CD8⁺ T lymphocytes. Although IFN-γ plays an important role in inhibition of antigen-induced IL-5 production by CpG ODNs, IFN-γ was not the sole factor in IL-5 inhibition. CpG ODNs also induced IL-10, and this induction correlated well with IL-5 inhibition. Elimination of IL-10 reduced the anti-IL-5 effect of CpG ODNs, although incompletely. This may be because IFN-γ, induced by CpG ODNs, is also inhibited by IL-10, serving as a homeostatic mechanism for the Th1-Th2 balance. Overproduction of IFN-γ was downregulated by CpG ODN-induced IL-10 via modulation of IL-12 production. These data suggest that CpG ODNs may inhibit established Th2 immune responses through IFN-γ and IL-10 production, the latter serving to regulate excessive Th1 bias. These properties of CpG ODNs might be a useful feature in the development of immunotherapy adjuvants against allergic diseases such as asthma.     

CpG motif-independent activation of TLR9 upon endosomal translocation of "natural" phosphodiester DNA

Endosomally translocated host (self) DNA activates Toll-like receptor 9 (TLR9), while extracellular self-DNA does not. This inconsistency reflects poor endosomal DNA translocation but also implies that host DNA contains DNA sequences that function as ligands for TLR9. Herein we report that contrary to phosphorothioate (PS)-stabilized oligonucleotides (ODN), "natural" phosphodiester (PD) ODN lacking CpG motifs activate TLR9. CpG motif-independent TLR9 activation of Flt3-L-induced dendritic cells (DC) was dependent on enforced endosomal translocation and triggered upregulation of CD40 and CD69 as well as production of IL-6 and IFN-alpha. Binding studies utilizing surface plasmon resonance technology (Biacore) revealed low TLR9 binding to single-stranded (ss) PD-ODN lacking CpG motifs. At higher concentrations their TLR9 binding activity compared well with TLR9 binding of canonical ss PD CpG-ODN. These results imply that both the chemical modification of the DNA backbone as well as the amount of endosomally translocated DNA represent determining factors that allow CpG motif-independent activation of TLR9 by ss PD-DNA.     

TLR9 signals after translocating from the ER to CpG DNA in the lysosome

Microbial DNA sequences containing unmethylated CpG dinucleotides activate Toll-like receptor 9 (TLR9). We have found that TLR9 is localized to the endoplasmic reticulum (ER) of dendritic cells (DCs) and macrophages. Because there is no precedent for immune receptor signaling in the ER, we investigated how TLR9 is activated. We show that CpG DNA binds directly to TLR9 in ligand-binding studies. CpG DNA moves into early endosomes and is subsequently transported to a tubular lysosomal compartment. Concurrent with the movement of CpG DNA in cells, TLR9 redistributes from the ER to CpG DNA-containing structures, which also accumulate MyD88. Our data indicate a previously unknown mechanism of cellular activation involving the recruitment of TLR9 from the ER to sites of CpG DNA uptake, where signal transduction is initiated.     

Multiple Administrations of Oligodeoxynucleotides Containing CpG Motifs Influence Ig Isotype Production

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) activate cells of the innate immune system. Recent studies have shown that sole CpG-ODN administration induces resistance against infection and tumors. Effects of CpG-ODN administration are rapidly induced, and regarding infections only short-term protection was seen. One conceivable strategy to prolong protective effects is multiple administrations of CpG-ODN. However, inappropriate immune activation via CpG motifs has been implicated in septic shock and autoimmunity. To investigate effects of multiple CpG-ODN administrations, we analyzed Th1- and Th-2-associated Ig antibody levels, during and after multiple treatment with CpG-ODN. Our results show that multiple administrations of CpG-ODN lead to an increase in total IgG2c levels in CpG-ODN-treated mice in comparison to controls with distinct time and frequency correlation, in the absence of additional stimuli. This indicates a humoral Th1 bias based on stimulation of Th1-Ig isotype-producing B cells. These effects could account for observed anti-infection and anti-tumor properties of multiple CpG-ODN administrations; on the other hand, they might cause autoimmune disease.     

Competition by inhibitory oligonucleotides prevents binding of CpG to C‐terminal TLR9

TLR9 recognizes unmethylated CpG‐containing DNA commonly found in bacteria. Synthetic oligonucleotides containing CpG‐motifs (CpG ODNs) recapitulate the activation of TLR9 by microbial DNA, whereas inversion of the CG dinucleotide within the CpG motif to GC (GpC ODNs) renders such ODNs inactive. This difference cannot be attributed to binding of ODNs to the full‐length TLR9 ectodomain, as both CpG and GpC ODNs bind comparably. Activation of murine TLR9 requires cleavage into an active C‐terminal fragment, which binds CpG robustly. We therefore compared the ability of CpG and GpC ODNs to bind to full‐length and C‐terminal TLR9, and their impact on the cleavage of TLR9. We found that CpG binds better to C‐terminal TLR9 when compared with GpC, despite comparably low binding of both ODNs to full‐length TLR9. Neither CpG nor GpC ODNs affected TLR9 cleavage in murine RAW 264.7 cells stably expressing TLR9‐Myc. Inhibitory ODNs (IN‐ODNs) block TLR9 signaling, but how they do so remains unclear. We show here that inhibitory ODNs do not impede TLR9 cleavage but bind to C‐terminal TLR9 preferentially, and thereby compete for CpG ODN binding both in RAW cells and in TLR9‐deficient cells transduced with TLR9‐Myc. Ligand binding to C‐terminal fragment thus determines the outcome of activation through TLR9.     

Differential signaling by CpG DNA in DCs and B cells: not just TLR9

CpG-containing oligodeoxynucleotides (CpG ODNs) act on Toll-like receptor 9 (TLR9) that is expressed on B cells and plasmacytoid dendritic cells (pDCs) to stimulate the innate immune system, however, different types of CpG ODNs induce distinct responses. Recent papers suggest some CpG ODNs could require a second receptor or cofactor to signal. The different signaling complexes assembled might impact on the affinity with which CpG ODNs signal to TLR9 or activate additional pathways that lead to distinct immune responses.     

Specific siRNA downregulated TLR9 and altered cytokine expression pattern in macrophage after CpG DNA stimulation

Bacterial CpG DNA or synthetic oligonucleotides(ODNs)that contain unmethylated CpG motifs(CpG ODN)candirectly activate antigen-presenting cells(APCs)to secrete various cytokines through the intraceilular receptorTLR9.Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial tothe subsequent immune responses.To date,cytokine profiles in APCs upon CpG ODN stimulation in vitro are notfully investigated.In the present study,vector-based siRNA was used to downregulate TLR9 expression.Cytokineprofiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid uponCpG ODN stimulation.We found that not all the cytokine expressions by the macrophage were decreased whileTLR9 was downregulated. IL-12, TNF-α, IFN-γ and IL-1β expressions were significantly decreased,but IL-6,IFN-β and IL-10 expressions were not affected.Interestingly,the level of IFN-α was even increased.This alterationof cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role ofCpG DNA is more complicated in the pathogenesis and prevention of diseases.Cellular & Molecular Immunology.2005;2(2):130-135.     

Down-Regulation of TLR9 Expression Affects the Maturation and Function of Murine Bone Marrow-Derived Dendritic Cells Induced by CpG

Toll-like receptor 9 （TLR9） is expressed intracellularly by dendritic cells （DCs） and specifically recognizes unmethylated CpG motif. Recognition of TLR9 to CpG DNA can induce DC maturation followed by the subsequent immune responses. Here, RNA interference （RNAi） was used to identify the effect of CpG DNA signaling on DC function. The results showed that transfection of DCs with siRNA specific for TLR9 gene significantly down-regulated TLR9 expression. Immature DCs transfected with TLR9 siRNA did not differentiate into mature DCs with exposure to CpG. TLR9 siRNA-treated DCs expressed low levels of MHC II and CD40 without reducing endocytosis. Furthermore, TLR9 siRNA-transfected DCs exhibited a decreased allostimulatory capacity in a lymphocyte proliferation assay and attenuated Thl responses by decreasing IL-12p70 production. Our findings indicate that siRNA in silencing TLR9 gene in DCs may offer a potential tool to study the TLR9-CpG pathway.     

Modulation of NK cell activity by CpG oligodeoxynucleotides

Oligodeoxynucleotides (ODN) with hypomethylated CpG motifs have been found to be potent stimulators of various aspects of innate and adaptive immunity. One of their major effects is the activation of natural killer (NK) killing activity in vitro and in vivo. There are several categories of CpG classified as type A, type B, and type C, although another category with inhibitory activity is being characterized further. CpG type A (CpG-A) is the most potent at activating NK cells. Examination of the cells and soluble mediators involved in this activation has led to an understanding of an interesting cascade of events. It appears that CpG activates dendritic cells (DC) which in turn activate NK-cells. This is not surprising since NK-cells do not seem to express TLR9, the CpG receptor. Of the various cytokines involved in NK-cell activation, it appears that type 1 interferon plays a pivotal role. Having activated NK-cells, DC themselves appear to become susceptible to lysis by the NK-cells they activated but with a delayed time kinetic. CpG ODN have been examined as monotherapeutic agents in murine tumor models. In one model, B16 melanoma, CpG ODN were very effective and NK cells were both necessary and sufficient for that effect. In another model, EL4 lymphoma, NK cells were necessary but not sufficient. Moreover, CpG were able to induce long-term survival in mice with established tumor.Studies in humans show similar results with potent activation in vitro. In a limited Phase I dose escalation study it also appeared that CpG ODN induce NK cell activation in humans in vivo.     

Accessory-Cell-Mediated Activation of Porcine NK Cells by Toll-Like Receptor 7 (TLR7) and TLR8 Agonists

The induction of innate immune responses by toll-like receptor (TLR) agonists is the subject of intense investigation. In large part, this reflects the potential of such compounds to be effective vaccine adjuvants. For that reason, we analyzed the activation of innate cells in swine by TLR7 and TLR8 agonists. These agonists activated porcine NK cells by increasing gamma interferon (IFN-γ) expression and perforin storage. The activation of porcine NK cells was mediated by accessory cells, since their depletion resulted in reduced cytotoxicity toward target cells. Accessory cells were stimulated to produce interleukin 12 (IL-12), IL-15, IL-18, and IFN-α after treatment with TLR7 or TLR8 agonists. Neutralization of these cytokines reduced but did not completely inhibit the induction of NK cell cytotoxicity. Direct stimulation of NK cells with TLR7 or TLR8 agonists resulted in minimal cytotoxicity but levels of IFN-γ equivalent to those detected in the presence of accessory cells. Porcine NK cells express both TLR7 and TLR8 mRNAs, and treatment with these TLR agonists induced higher mRNA expression levels of TRAIL and IL-15Rα, which may contribute to the activity of NK cells. These data indicate that TLR7 and TLR8 agonists indirectly or directly activate porcine NK cells but that optimum levels of activation require cytokine secretion by accessory cells activated by these compounds. Interestingly, NK cells activated by TLR7 or TLR8 agonists were cytotoxic against foot-and-mouth disease virus (FMDV)-infected cells in vitro, indicating that these TLR agonists may be beneficial as adjuvants to stimulate the innate immunity against FMDV.Toll-like receptors (TLRs) are pathogen-associated molecular pattern recognition receptors responsible for signaling intrusion by pathogens. These receptors are expressed by cells mediating innate responses. Pathogens are recognized directly by the binding of pathogen-associated molecules. For example, TLR3 recognizes double-stranded RNA during virus replication; TLR5 binds bacterial flagellin; TLR9 detects nonmethylated, CG-rich prokaryotic DNA, i.e., CpG; and TLR7 and TLR8 recognize single-stranded RNA (44), although recognition by TLR8 may be species specific, as demonstrated recently by Forsbach et al. (11). The consequence of engaging TLRs is the induction of signals that lead to the expression of proinflammatory cytokines, antimicrobial and antiviral effector molecules, and costimulatory molecules on macrophages (Mφ) and dendritic cells (DCs) (25, 39). Overall, such events affect the activation and functional status of innate immune cells, such as natural killer (NK) cells and DCs, and further influence the organization of adaptive immune responses.NK cells perform a critical role in innate immunity, leading to protection against various pathogens well before the adaptive immune responses develop. NK cells are lymphocyte-derived cells that engage nonspecific target recognition mechanisms to eliminate malignant or virus-infected cells (10). However, it has recently been shown that some receptors on NK cells engage viral gene products. For example, Ly49H recognizes m157 of murine cytomegalovirus in mice, while NKp44 and NKp46 bind influenza virus hemagglutinin (4, 27). Moreover, NK cells express both inhibitory and activating receptors, which directly influence the outcome of NK cell activation. Besides the expression of such receptors (22), NK cells have other mechanisms that enhance their function as natural spontaneous effector cells (24). Such mechanisms include the expression of TLRs, which possibly allow NK cells to respond to the presence of pathogens by direct activation via these receptors.NK cells express TLR9 in mice (26) and TLR1 to TLR10 in humans (12, 17, 23). However, not much information is currently available on the expression of these receptors on immune cells of domestic livestock species, such as porcine or bovine species. Direct stimulation of human NK cells through TLR2, TLR3, TLR7, and TLR8 leads to the upregulation of gamma interferon (IFN-γ) secretion, although in some instances this response requires the presence of interleukin 12 (IL-12) (6, 12, 17). Furthermore, activation via TLR5 is reported to stimulate NK cell proliferation but not IFN-γ production (45). Additionally, stimulation via TLR2 or TLR7 induces chemokines such as CCL3, CCL4, and CCL5 (36). Although TLR9 is expressed in NK cells, it does not induce IFN-γ production directly unless the NK cells are presented with antibody-coated target cells or are cultured on plates with an immobilized antibody against immunoglobulin G (35). Therefore, TLR expression in NK cells may be involved in the differential regulation of these vital cells of the innate response. However, not much is known about the direct effect of TLR stimulation on the expression of NK cell effector molecules, such as perforin, granzymes, and cytokines.Using this class of molecules, it is now possible to formulate vaccine adjuvants that prime cell-mediated immunity. Engaging TLR receptors with specific synthetic agonists introduces a new way of inducing early innate responses as well as increasing the potency of adaptive immunity. The importance of such an approach is exemplified by several clinical studies currently under way. TLR9 and TLR4 agonist are being tested as vaccine adjuvants, and a TLR7 agonist is being tested in the treatment of genital warts caused by herpes simplex virus (28). In addition, TLR4 is being tested for the treatment of allergies, endotoxemia, and liver disease, TLR7 for cancer treatment, and TLR9 as a treatment for melanoma (reviewed by Ulevitch [46]).Foot-and-mouth disease virus (FMDV) infects cloven-hoofed animals, leading to devastating economic consequences (16). This is a highly contagious viral infection that causes a very acute disease. Clinical symptoms are detected within 1 or 2 days of exposure and resolve within a week to 10 days. Viral clearance may be mediated in part by antibodies, but under controlled experimental conditions, viremia is gone by day 3 or 4 after infection when anti-FMDV immunoglobulin M is barely detectable (2, 13). These results strongly suggest that other antiviral mechanisms contribute to the elimination of the virus in vivo. Innate responses, including the activation of DCs and NK cells, are likely involved.Therapeutic approaches involving TLR stimulation via synthetic agonists can augment innate responses, indicating that such therapeutics have the potential to induce early protection against foot-and-mouth disease (29, 30). Therefore, we have studied the effects of TLR agonists on porcine NK cells in vitro. We report that TLR7 and TLR8 agonists and a combined TLR7/8 agonist activate porcine CD2⁺ CD8⁺ CD3⁻ NK cells through accessory-cell-mediated mechanisms, such as the secretion of cytokines, including IFN-α, IL-12, IL-15, and IL-18. In addition, porcine NK cells are partially activated by the direct interaction of TLR7 and TLR8 agonists through these receptors expressed by NK cells. Activated cells show enhanced secretion of IFN-γ and storage of perforin granules and can effectively lyse tumor or FMDV-infected targets. These results are discussed in the context of rational approaches to antiviral measures against FMDV.     

Immune mechanisms and therapeutic potential of CpG oligodeoxynucleotides

Unmethylated CpG motifs in bacterial DNA and synthetic oligodeoxynucleotides activate immune cells that express Toll-like Receptor 9. Activation through this receptor triggers cellular signaling that leads to production of a proinflammatory and a Th1-type, antigen-specific immune response. The immunostimulatory effects of CpG oligodeoxynucleotides confer protection against infectious disease, allergy and cancer in animal models, and clinical trials have been initiated. However, CpG oligodeoxynucleotides may exacerbate disease in some situations. We will review current concepts in the mechanisms of activating Toll-like Receptor 9 with CpG oligodeoxynucleotides and highlight opportunities for using large animal models to better determine the mechanisms of action.