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-alpha 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-alpha 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-alpha. 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 Th1 adjuvants. 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.     

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-alpha-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.     

Toll-like receptor agonists induce apoptosis in mouse B-cell lymphoma cells by altering NF-κB activation

Toll-like receptor 9 (TLR9) recognizes microbial DNA containing unmethylated cytosyl guanosyl (CpG) sequences, induces innate immune responses, and facilitates antigen-specific adaptive immunity. Recent studies report that in addition to stimulating innate immunity, TLR9 ligands induce apoptosis of TLR9 expressing cancer cells. To understand the mechanism of TLR9-induced apoptosis, we compared the effects of CpG containing oligodeoxynucleotides (CpG ODN) on a mouse B-cell lymphoma line, CH27, with those on mouse splenic B cells. CpG ODN inhibited constitutive proliferation and induced apoptosis in the CH27 B-cell lymphoma line. In contrast, CpG ODN-treated primary B cells were stimulated to proliferate and were rescued from spontaneous apoptosis. The induction of apoptosis required the ODNs to contain the CpG motif and the expression of TLR9 in lymphoma B cells. A decrease in Bcl-xl expression and an increase in Fas and Fas ligand expression accompanied lymphoma B-cell apoptosis. Treatment with the Fas ligand-neutralizing antibody inhibited CpG ODN-induced apoptosis. CpG ODN triggered a transient NF-κB activation in the B-cell lymphoma cell line, which constitutively expresses a high level of c-Myc, while CpG ODN induced sustained increases in NF-κB activation and c-Myc expression in primary B cells. Furthermore, an NF-κB inhibitor inhibited the proliferation of the CH27 B-cell lymphoma line. Our data suggest that the differential responses of lymphoma and primary B cells to CpG ODN are the result of differences in NF-κB activation. The impaired NF-κB activation in the CpG ODN-treated B-cell lymphoma cell line alters the balance between NF-κB and c-Myc, which induces Fas/Fas ligand-dependent apoptosis.     

Synergy between CpG- or non-CpG DNA and specific antigen for B cell activation

DNA or oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG DNA) activate antigen-presenting cells and switch on T(h)1 immunity to antigen. B cells are synergistically activated by CpG DNA in combination with non-physiologic B cell stimulators such as polyclonal mitogen and surface Ig cross-linkers. This study shows the unexpected finding that not only CpG ODN, but also non-CpG and methylated ODN synergize with specific antigen, hen egg lysozyme (HEL), in stimulating HEL-specific B cells to proliferate, to express the early activation marker CD69 and to activate the NF-kappaB pathway. In vivo, non-CpG and methylated CpG ODN also enhanced anti-HEL antibody production in HEL-immunized mice, with a bias towards the production of T(h)1-associated isotypes. The synergy with all ODN to enhance B cell immune function was epitope-specific since neither denatured HEL nor other antigens enhanced the ODN effect on HEL-specific B cells. Furthermore, the synergy was independent of whether the ODN backbone was phosphorothioate or phosphodiester, or whether natural vertebrate genomic DNA was used. In all functional analyses, non-CpG and methylated CpG ODN showed lower activity than CpG ODN. These studies demonstrate that the presence of specific physiologic antigen might broaden the spectrum of DNA/ODN that stimulate B cells, with potential implications for the initiation and regulation of normal and pathologic immune responses.     

含CpG基序的寡核苷酸对树突状细胞的作用及其机制

树突状细胞(dendriticcells,DC)是目前所知功能最强的抗原提呈细胞(antigenpresentingcells,APC),其最大特点在于能激活初始型T细胞(naiveTcell),是机体免疫应答的始动者,在免疫应答中起着重要的作用。DC的成熟、活化是其发挥免疫作用的先决条件,因而成为研究的焦点。目前已发现刺激DC成熟的多种因素,包括肿瘤坏死因子-α(tumornecrosis factor-α,TNF-α)、CD40-CD40L、脂多糖(lipopolysaccharide,LPS)和含有非甲基化胞嘧啶鸟嘌呤二核苷酸(cytosinephosphate-guanosine,CpG)基序的寡核苷酸(CpGmotif-containingoligodeoxynucle …     

Cross-talk between toll-like receptors 5 and 9 on activation of human immune responses

The recognition of pathogen-associated molecular patterns by TLRs triggers the activation of innate and adaptive immune responses. Flagellin, the agonist of TLR5, is expressed by prokaryotes and eukaryotes, and DNA sequences containing unmethylated CpG dinucleotides, agonists of TLR9, are present essentially in prokaryotes. To test the potential modulating effects of simultaneous activation of different TLRs on the immune response, we compared the outcomes in different immune cell compartments induced by triggering TLR5 and TLR9 individually and in combination. PBMCs, monocytes, and monocyte-derived DC (MoDC) secreted high levels of IL-10 in response to flagellin, whereas oligodeoxynucleotides (ODN) containing the CpG sequence (CpG-ODN), synthetic ligands of TLR9, did not induce IL-10 secretion in any of the three cell types but synergized with flagellin in this induction. In contrast, PBMC production of IFN-alpha induced by CpG-ODN was strongly inhibited by flagellin. Conversely, CpG-ODN did not enhance the up-regulation of activation markers in MoDC induced to mature in the presence of flagellin. Flagellin-matured, but not CpG-ODN-matured, MoDC stimulated the expansion of allogeneic CD4+CD25+ T cells, and the extent of expansion induced by MoDC, matured in the presence of flagellin and CpG-ODN, was similar to that induced by flagellin-matured MoDC. Moreover, flagellin and CpG-ODN differentially affected NK-mediated cytotoxicity, and flagellin completely abrogated the NK-mediated immune response induced by CpG-ODN stimulation. Together, these results suggest that flagellin inhibits the TLR9-induced cell activation and cytokine production, which favor Th1-type immune responses, possibly because the signals evoked by flagellin to indicate the presence of extracellular pathogens must favor a Th2-polarized response. Thus, TLR5 and TLR9, alerted by the presence of microorganisms, influence each other to mount the more efficient and appropriate immune response to contain the infection of a specific pathogen.     

The immunostimulatory activity of phosphorothioate CpG oligonucleotides is affected by distal sequence changes

CpG motifs in bacterial DNA activate innate immune cells via toll like receptor 9 (TLR9). Short synthetic oligodeoxynucleotides (ODN) containing a six base CpG motif can mimic the immunostimulatory activity of bacterial DNA. Phosphorothioate (PS) modification of the backbone of ODN makes them more resistant to nuclease degradation and consequently preferable for therapeutic use. Previous studies have shown that the sequence requirements for PS-ODN to have maximal stimulatory activity are more stringent than for normal phosphodiester (PO) ODN. Here we show small sequence changes distal to the CpG motif can affect the activity of PS-ODN whilst having no effect on the activity of PO-ODN. The addition of terminal dG residues and other minor changes to the potently immunostimulatory PS-ODN 1668S caused delayed signalling. The reduction in immunostimulatory activity of PS-ODN was associated with a delay in the activation of MAP kinases.     

Defect of toll-like receptor 9-mediated activation in NC/Nga mouse macrophages

Toll-like receptors (TLRs) control activation of adaptive immune responses by antigen-presenting cells (APCs). In this study, we examined TLR9-mediated activation in NC/Nga mice, an animal model for human atopic dermatitis. NC/Nga mouse macrophages produced significantly less TNF-alpha than did BALB/c mouse macrophages in response to CpG oligonucleotide (ODN). In addition to defective TLR9-mediated TNF-alpha production, phosphorylation of ERK1,2 and p38 was rapidly diminished after 60 min of CpG ODN stimulation, whereas phosphorylation of these molecules was sustained until 60 min in BALB/c mice. Furthermore, phosphorylation of c-Jun N-terminal kinase (JNK) was not observed in NC/Nga mouse macrophages. In contrast, B cells and dendritic cells (DCs) from NC/Nga mice showed normal responses to CpG ODN stimulation. The expression level of TLR9 in NC/Nga mouse macrophages was significantly lower than that in BALB/c mouse macrophages, whereas levels of TLR9 expression in B cells and DCs in NC/Nga mice were the same as those in BALB/c mice. These results suggest that defective TLR9-mediated activation in NC/Nga mouse macrophages contributes to the reduction of TLR9 expression levels.     

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.     

A human microsatellite DNA-mimicking oligodeoxynucleotide with CCT repeats negatively regulates TLR7/9-mediated innate immune responses via selected TLR pathways

A human microsatellite DNA-mimicking ODN (MS ODN) composed of CCT repeats, designated as SAT05f, has been studied for its capacity of negatively regulating innate immunity induced by TLR7/TLR9 agonists in vitro and in mice. The result showed that SAT05f could down-regulate TLR7/9-dependent IFN-α production in cultured human PBMC stimulated by inactivated Flu virus PR8 or HSV-1 or CpG ODN or imiquimod, protect d-GalN-treated mice from lethal shock induced by TLR9 agonist, not by TLR3/4 agonist. In addition, SAT05f significantly inhibit IFN-α production from purified human plasmacytoid cells (pDCs) stimulated by CpG ODN. Interestingly, SAT05f could up-regulate CD80, CD86, and HLA-DR on the pDCs in vitro, implying that SAT05f-mediated inhibition on IFN-α production could be related to the activation of pDCs. The data suggest that SAT05f could be developed as a candidate medicament for the treatment of TLR7/9 activation-associated diseases by inhibiting TLR7/9 signaling pathways.     

Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12

Human plasmacytoid dendritic cells (DC) (PDC, CD123+) and myeloid DC (MDC, CD11c+) may be able to discriminate between distinct classes of microbial molecules based on a different pattern of Toll-like receptor (TLR) expression. TLR1-TLR9 were examined in purified PDC and MDC. TLR9, which is critically involved in the recognition of CpG motifs in mice, was present in PDC but not in MDC. TLR4, which is required for the response to LPS, was selectively expressed on MDC. Consistent with TLR expression, PDC were susceptible to stimulation by CpG oligodeoxynucleotide (ODN) but not by LPS, while MDC responded to LPS but not to CpG ODN. In PDC, CpG ODN supported survival, activation (CD80, CD86, CD40, MHC class II), chemokine production (IL-8, IP-10) and maturation (CD83). CD40 ligand (CD40L) and CpG ODN synergized to activate PDC and to stimulate the production of IFN-alpha and IL-12 including bioactive IL-12 p70. Previous incubation of PDC with IL-3 decreased the amount of CpG-induced IFN-alpha and shifted the cytokine response in favor of IL-12. CpG ODN-activated PDC showed an increased ability to stimulate proliferation of naive allogeneic CD4 T cells, butTh1 polarization of developing T cells required simultaneous activation of PDC by CD40 ligation and CpG ODN. CpG ODN-stimulated PDC expressed CCR7, which mediates homing to lymph nodes. In conclusion, our studies reveal that IL-12 p70 production by PDC is under strict control of two signals, an adequate exogenous microbial stimulus such as CpG ODN, and CD40L provided endogenously by activated T cells. Thus, CpG ODN acts as an enhancer of T cell help, while T cell-controlled restriction to foreign antigens is maintained.     

Bacterial DNA and CpG-containing oligodeoxynucleotides activate cutaneous dendritic cells and induce IL-12 production: implications for the augmentation of Th1 responses

BACKGROUND: Unmethylated CpG sequences in bacterial DNA act as adjuvants selectively inducing Th1 predominant immune responses during genetic vaccination or when used in conjunction with protein Ag. The precise mechanism of this adjuvant effect is unknown. Because dendritic cells (DC) are thought to be crucially involved in T cell priming and Th1/Th2 education during vaccination via skin, we characterized the effects of bacterial DNA and CpG-containing oligodeoxynucleotides (CpG ODN) on cutaneous DC. METHODS AND RESULTS: Stimulation with CpG ODN 1826 (6 micrograms/ml) induced activation of immature Langerhans cell (LC)-like DC as determined by an increased expression of MHC class II and costimulatory molecules, loss of E-cadherin-mediated adhesion and increased ability to stimulate allogeneic T cells. Composition-matched control ODN 1911 lacking CpG sequences at equal concentrations was without effect. In comparison to LPS and ODN 1911, CpG ODN 1826 selectively stimulated DC to release large amounts of IL-12 (p40) and little IL-6 or TNF-alpha within 18 h and detectable levels of IL-12 p70 within 72 h. Stimulation with Escherichia coli DNA, but not calf thymus DNA, similarly induced DC maturation and IL-12 p40 production. Injection of CpG ODN into murine dermis induced enhanced expression of MHC class II and CD86 by LC in the overlying epidermis and intracytoplasmic IL-12 p40 accumulation in a subpopulation of activated LC. CONCLUSION: Bacterial DNA and CpG ODN stimulate DC in vitro and in vivo and may preferentially elicit Th1-predominant immune responses because they can activate and mobilize DC, inducing them to produce IL-12.     

Toll-like receptor 9-dependent activation of bone marrow-derived dendritic cells by URA5 DNA from Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis in immunocompromised patients. Recently, we reported that Toll-like receptor 9 (TLR9) is involved in host defense against C. neoformans: specifically, it detects the pathogen's DNA. In the present study, we aimed to elucidate the mechanisms underlying TLR9-mediated activation of innate immune responses by using the URA5 gene, which encodes a virulent component of this fungal pathogen. A PCR-amplified 345-bp URA5 gene fragment induced interleukin-12 p40 (IL-12p40) production by bone marrow-derived dendritic cells (BM-DCs) in a TLR9-dependent manner. Similar activity was detected in the 5' 129-bp DNA fragment of URA5 and in a synthesized oligodeoxynucleotide (ODN) with the same sequence. Shorter ODN fragments, which contained GTCGGT or GACGAT but had only 24 or 21 bases, induced IL-12p40 production and CD40 expression by BM-DCs, but this activity vanished when the CG sequence was replaced by GC or when a phosphorothioate modification was introduced. IL-12p40 production caused by active ODN was strikingly enhanced by treatment with DOTAP, a cationic lipid that increases the uptake of DNA by BM-DCs, though DOTAP failed to induce IL-12p40 production by inactive ODN and did not affect the activity of an ODN-containing canonical CpG motif. There was no apparent difference in intracellular trafficking between active and inactive ODNs. Finally, an extremely high dose of inactive ODN suppressed IL-12p40 production by BM-DCs that had been stimulated with active ODN. These results suggest that the C. neoformans URA5 gene activates BM-DCs through a TLR9-mediated signaling pathway, using a mechanism possibly independent of the canonical CpG motif.     

Immunogenicity and safety of liposome-vaccine encapsulating hepatitis B surface antigen and phosphodiester CpG oligodeoxynucleotides

CpG oligodeoxynucleotides (CpG ODN) as adjuvant have been extensively studied in recent years. Phosphodiester CpG ODN (PO CpG ODN) can perfectly mimic bacterial DNA in enhancing immune response but are vulnerable to nucleases in vivo . This study aimed to evaluate the immunostimulatory potential and safety of phosphodiester CpG ODN encapsulated in nonphospholipid liposomes. BALB/c mice were immunized intramuscularly with different formulations of liposomes,CpG ODN and hepatitis B surface antigen (HBsAg). The results demonstrated that the encapsulated PO CpG ODN were protected against rapid degradation in vivo and retained their adjuvant activity. PO CpG ODN encapsulated with HBsAg in liposomes induced strong Th1-biased or Th1/Th2 mixed humoral immune response in mice with the magnitude similar to their phosphothioate equivalent in the same formulation. High IFN-gamma production induced by this formulation confirmed the generation of strong cellular immune response. Additionally, co-delivery of HBsAg and PO CpG ODN improved the immune response over that obtained with separate delivery. Safety experiment showed that liposome-encapsulaed PO CpG ODN and HBsAg caused mild systemic and moderate local adverse reaction. In conclusion, our data shows that PO CpG ODN encapsulated in liposomes fully exhibit their Th1-type adjuvant activity and act as a potential adjuvant for vaccines.     

Stimulation via Toll-like receptor 9 reduces Cryptococcus neoformans-induced pulmonary inflammation in an IL-12-dependent manner

Cytosine-phosphate-guanosine-containing oligodeoxynucleotides (CpG ODN) are important vaccine adjuvants that promote Th1-type immune responses. Cryptococcus neoformans is a serious human pathogen that replicates in the lung but may disseminate systemically leading to meningitis, particularly in immunocompromised individuals. Immunization of susceptible C57BL/6 mice with CpG ODN deviates the immune response from a Th2- toward a Th1-type response following infection with C. neoformans. CpG also induces IL-12, TNF, MCP-1 and macrophage nitric oxide production. CD4(+) and CD8(+) T cells producing IFN-gamma increase in frequency, while those producing IL-5 decrease. More importantly, pulmonary eosinophilia is significantly reduced, an effect that depends on IL-12 and CD8(+) T cells but not NK cells. CpG treatment also reduces the burden of C. neoformans in the lung, an effect that is IL-12-, NK cell- and T cell-independent and probably reflects a direct effect of CpG on pathogen opsonization or an enhancement of macrophage antimicrobial activity. An equivalent beneficial effect is also observed when CpG ODN treatment is delivered during established cryptococcal disease. This is the first study documenting that promotion of lung TLR9 signaling using synthetic agonists enhances host defense. Activation of innate immunity has clear therapeutic potential and may even be beneficial in patients with acquired immune deficiency.     

The effect of combined IL10 siRNA and CpG ODN as pathogen-mimicking microparticles on Th1/Th2 cytokine balance in dendritic cells and protective immunity against B cell lymphoma

Success of an immunotherapy for cancer often depends on the critical balance of T helper 1 (Th1) and T helper 2 (Th2) responses driven by antigen presenting cells, specifically dendritic cells (DCs). Th1-driven cytotoxic T cell (CTL) responses are key to eliminating tumor cells. It is well established that CpG oligonucleotides (ODN), a widely studied Toll-like receptor 9 (TLR9) agonist, used to enhance Th1 response, also induces high levels of the anti-inflammatory, Th2-promoting cytokine IL10, which could dampen the resulting Th1 response. Biomaterials-based immunomodulatory strategies that can reduce IL10 production while maintaining IL12 levels during CpG delivery could further enhance the Th1/Th2 cytokine balance and improve anti-tumor immune response. Here we report that dual-delivery of IL10-silencing siRNA along with CpG ODN to the same DCs using pathogen-mimicking microparticles (PMPs), significantly enhances their Th1/Th2 cytokine ratio through concurrent inhibition of CpG-induced IL10 production. Co-delivery of poly(I:C), a TLR3 agonist had only minor effects on IL10 levels. Further, simultaneous immunotherapy with CpG ODN and IL10 siRNA enhanced immune protection of an idiotype DNA vaccine in a prophylactic murine model of B cell lymphoma whereas co-delivery of poly(I:C) and CpG did not enhance protection. These results suggest that PMPs can be used to precisely modulate TLR ligand-mediated immune-stimulation in DCs, through co-delivery of cytokine-silencing siRNAs and thereby boost antitumor immunity.     

Immunostimulatory DNA as an Adjuvant in Vaccination against Leishmania major

Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote T helper 1 (Th1) responses, an adjuvant activity that is desirable for vaccination against leishmaniasis. To test this, susceptible BALB/c mice were vaccinated with soluble leishmanial antigen (SLA) with or without CpG ODN as adjuvant and then challenged with Leishmania major metacyclic promastigotes. CpG ODN alone gave partial protection when injected up to 5 weeks prior to infection, and longer if the ODN was bound to alum. To demonstrate an antigen-specific adjuvant effect, a minimum of 6 weeks between vaccination and infection was required. Subcutaneous administration of SLA alone, SLA plus alum, or SLA plus non-CpG ODN resulted in exacerbated disease compared to unvaccinated mice. Mice receiving SLA plus CpG ODN showed a highly significant (P < 5 x 10(-5)) reduction in swelling compared to SLA-vaccinated mice and enhanced survival compared to unvaccinated mice. The modulation of the response to SLA by CpG ODN was maintained even when mice were infected 6 months after vaccination. CpG ODN was not an effective adjuvant for antibody production in response to SLA unless given together with alum, when it promoted production of immunoglobulin G2a, a Th1-associated isotype. Our results suggest that with an appropriate antigen, CpG ODN would provide a stable, cost-effective adjuvant for use in vaccination against leishmaniasis.     

CpG oligodeoxynucleotide treatment enhances innate resistance and acquired immunity to African trypanosomes

Relative resistance to African trypanosomiasis is based on the development of a type I cytokine response, which is partially dependent on innate immune responses generated through MyD88 and Toll-like receptor 9 (TLR9). Therefore, we asked whether enhancement of the immune response by artificial stimulation with CpG oligodeoxynucleotide (ODN), a TLR9 agonist, would result in enhanced protection against trypanosomes. In susceptible BALB/c mice, relative resistance to infection was significantly enhanced by CpG ODN treatment and was associated with decreased parasite burden, increased cytokine production, and elevated parasite-specific B- and T-cell responses. In relatively resistant C57BL/6 mice, survival was not enhanced but early parasitemia levels were reduced 100-fold and the majority of the parasites were nondividing, short stumpy (SS) forms. CpG ODN treatment of lymphocyte-deficient C57BL/6-scid and BALB/cByJ-scid mice also enhanced survival and reduced parasitemia, indicating that innate resistance to trypanosome infection can be enhanced. In C57BL/6-scid and BALB/cByJ-scid mice, the parasites were also predominantly SS forms during the outgrowth of parasitemia. However, the effect of CpG ODN treatment on parasite morphology was not as marked in gamma interferon (IFN-gamma)-knockout mice, suggesting that downstream effects of IFN-gamma production may play a discrete role in parasite cell differentiation. Overall, these studies provide the first evidence that enhancement of resistance to African trypanosomes can be induced in susceptible animals in a TLR9-dependent manner and that CpG ODN treatment may influence the developmental life cycle of the parasites.