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Originally published online as doi:10.1189/jlb.0104034 on June 24, 2004

Published online before print June 24, 2004
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(Journal of Leukocyte Biology. 2004;76:585-593.)
© 2004 by Society for Leukocyte Biology

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

Jörg Vollmer*,1, Risini D. Weeratna{dagger}, Marion Jurk*, Heather L. Davis{dagger}, Christian Schetter*, Meike Wüllner*, Tanja Wader*, Ming Liu{dagger}, Andrea Kritzler* and Arthur M. Krieg{ddagger}

Coley Pharmaceutical,
* Langenfeld, Germany,
{dagger} Ottawa, Canada, and
{ddagger} Wellesley, Massachusetts

1 Correspondence: Coley Pharmaceutical GmbH, Elisabeth-Selbert-Str. 9, 40764 Langenfeld, Germany. E-mail: jvollmer{at}coleypharma.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

Key Words: oligodeoxynucleotides • immune stimulation • Toll-like receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial DNA has stimulatory effects on the vertebrate immune system, but vertebrate DNA itself does not [1 ]. It is now understood that these immune-stimulatory effects result from the presence of unmethylated CpG dinucleotides, in particular, base contexts (CpG motifs), which are common in bacterial DNA but methylated and under-represented in vertebrate DNA [2 , 3 ]. Vertebrates have evolved an innate immune system to recognize the pattern of foreign DNA. Thus, bacterial or viral DNA containing CpG dinucleotides is a "danger signal" to vertebrate immune cells, indicating invasion by a pathogen. The immune-stimulatory effects of bacterial DNA can be mimicked with synthetic phosphorothioate-stabilized oligodeoxynucleotides (ODN) containing CpG motifs [4 ], which have been described to optimally and differentially induce the stimulation of human (optimal motif 5'-GTCGTT-3') or murine (optimal motif 5'-GACGTT-3') immune cells [2 , 5 ]. These sequence-specific differences could also be observed with nonresponsive cells after transfecting them with human or murine Toll-like receptor 9 (TLR9) and a nuclear factor (NF)-{kappa}B-luciferase reporter [6 7 8 ].

The receptor responsible for the recognition of bacterial DNA or CpG ODN was recently identified using mice genetically deficient in TLR9 [9 ], which is a member of a family of at least 11 different receptors. Other TLR subtypes are essential for the recognition of specific pathogen-associated molecular patterns such as lipopolysaccharide (TLR4), double-stranded RNA (TLR3), or small, antiviral compounds, guanosine nucleoside analogs, or viral single-stranded RNA (TLR7 or -8) [10 11 12 13 14 15 ].

CpG ODN are highly stimulatory for human and murine leukocytes, inducing B cell proliferation, cytokine and immunoglobulin secretion, natural killer cell lytic activity, and activation of dendritic cells (DC) and other antigen-presenting cells (APC) to express costimulatory molecules and secrete cytokines or chemokines, especially T helper cell type 1 (Th1)-like cytokines such as interferon (IFN)-{alpha}, which are important in promoting the development of Th1-like T cell responses [4 ]. Three classes of CpG ODN were identified so far: the A-, B-, and C-Classes, which differ in their immune-stimulatory activities [16 , 17 ]. The human TLR9 is expressed in B cells and plasmacytoid DC (pDC), whereas the murine TLR9 is also expressed in the myeloid compartment [9 , 18 , 19 ]. Recent reports also have observed low levels of TLR9 mRNA in human eosinophils and neutrophils but could not detect CpG-mediated stimulation of these cell types without additional stimuli [20 , 21 ].

The minimal sequence motif of most immune-stimulatory ODN contains a 5'-CpG dinucleotide. Simple inversion to 5'-GpC reduces the immune-stimulatory properties [2 ]. Similarly, replacement of cytosine by 5-methylcytosine results in reduction or abolishment of immune stimulation [22 , 23 ]. As this structural difference is the basis for the pattern recognition between bacterial and vertebrate DNA, it is comprehensible that modifications of deoxycytosine are usually not very well tolerated [4 ]. In addition, murine studies suggested that most deoxyguanosine modifications, except for 7-deaza deoxyguanosine, result in decrease or loss of immune-stimulatory activity [23 , 24 ]. Similar findings were reported for deletion of the guanine or cytosine base through incorporation of an abasic residue or dSpacer [25 ].

In this study, several chemical modifications of deoxyguanosine were evaluated, and only ODN with deoxyinosine-containing CpI dinucleotides were found to be relatively strong immune stimulators, although they were still inferior to unmodified CpG ODN for induction of Th1-like cytokines or chemokines from human peripheral blood mononuclear cells (PBMC) or stimulation of TLR9-dependent signaling. Murine in vitro and in vivo studies indicate that CpI-containing ODN induce the same effects that have been described previously for CpG ODN, although with considerably lower efficiency.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
ODN
ODN were purchased from Biospring (Frankfurt, Germany) or Sigma-ARK (Darmstadt, Germany) and controlled for identity (matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry) and purity (capillary gel electrophoresis) by Coley Pharmaceutical GmbH (Langenfeld, Germany). ODN were suspended in sterile, endotoxin-free Tris-EDTA (Sigma, Deisenhofen, Germany) and stored and handled under aseptic conditions to prevent microbial and endotoxin contamination. All dilutions were carried out using pyrogen-free phosphate-buffered saline (PBS; Life Technologies, Eggenstein, Germany).

TLR9 and TLR7 assays
Stably transfected human embryonic kidney (HEK)293 cells expressing the human or murine TLR9 or the human TLR7 were described before [12 , 16 ]. Briefly, HEK293 cells were transfected by electroporation with vectors expressing the murine or human TLR9 or the human TLR7 and a 6x NF-{kappa}B-luciferase reporter plasmid. Stable transfectants (3x104 cells/well) were incubated with ODN for 16 h at 37°C in a humidified incubator. Each data point was done in triplicate. Cells were lysed and assayed for luciferase gene activity (using the BriteLite kit from Perkin-Elmer, Zaventem, Belgium). Stimulation indices (SI) were calculated in reference to reporter gene activity of medium without addition of ODN.

Cell purification
Peripheral blood buffy coat preparations from healthy human volunteers were obtained from the German Red Cross (Breitscheid, Germany) or the Blood Bank of the University of Düsseldorf (Germany), and PBMC were purified by centrifugation over Ficoll-Hypaque (Sigma, Deisenhofen, Germany). Cells were cultured in a humidified 5% CO2 incubator at 37°C in RPMI-1640 medium supplemented with 5% (v/v) heat-inactivated human AB serum (BioWhittaker, Walkersville, MD) or 10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Sigma).

Cytokine detection and flow cytometric analysis
PBMC were resuspended at a concentration of 3 x 106–5 x 106 cells/ml and added to 48-well, flat-bottomed plates (1 ml/well) or 96-well, round-bottomed plates (200 µl/well). ODN were added, and cells were cultured at 37°C in a humidified incubator. Culture supernatants (SN) were collected after the indicated time-points. If not used immediately, SN were stored at –20°C until required. Amounts of cytokines in the SN were assessed using commercially available enzyme-linked immunosorbent assay (ELISA) kits [IFN-inducible protein 10 (IP-10), interleukin (IL)-6, or IL-10; from Diaclone, Besançon, France] or an in-house ELISA for IFN-{alpha}, developed using commercially available antibodies (from PBL, New Brunswick, NJ, for detection of multiple IFN-{alpha} species).

For detection of CD80 expression on CD14-positive monocytes, human PBMC (3x106cells/ml) were incubated for 24 h with ODN. Cells were harvested and stained with monoclonal antibodies (mAb) to CD14 and CD80 (from Becton Dickinson, San Jose, CA), and results were acquired by flow cytometry on a FACSCalibur (Becton Dickinson).

Isolation of human B cells or pDC
Human B cells were isolated from whole PBMC with the CD19 B cell isolation kit as described by the manufacturer (Miltenyi, Bergisch-Gladbach, Germany). To determine purity, cells were stained with mAb to CD20 and CD14 and identified by flow cytometry. In all experiments, B cells were more than 95% pure. Purified B cells (2x105–5x105 cells/ml) were incubated with 0.25 µM ODN for 24 h, and IL-6 was measured as described above.

pDC were enriched with the blood DC antigen-4 pDC isolation kit as described by the manufacturer (Miltenyi). pDC enrichment was confirmed by staining with mAb to CD123, CD11c, and human leukocyte antigen-DR, and cells (5x105 cells/ml) were cultured in the presence of IL-3 (10 ng/ml; R&D Systems GmbH, Wiesbaden, Germany) for 48 h with 0.25 µM ODN. IFN-{alpha} in the SN was measured as described above.

Animals
Female BALB/c mice (6–8 weeks of age) were used for all experiments and were purchased from Charles River Canada (Quebec, Canada) and housed at the animal care facility of the Ottawa Loeb Health Research Institute (Ontario, Canada). TLR9-deficient mice (TLR9–/–) were described before [9 , 16 ] and obtained from Dr. Shizuo Akira (Osaka University, Osaka, Japan). The animals were back-crossed to BALB/c for several generations.

In vitro murine assays
Naïve BALB/c or TLR9–/– mouse splenocytes were used for all in vitro assays. Animals were anesthetized with isofluorane and killed by cervical dislocation. Spleens were removed under aseptic conditions and placed in PBS + 0.2% bovine serum albumin (Sigma, St. Louis, MO). Spleens were then homogenized, and splenocytes were resuspended in RPMI-1640 (Life Technologies) medium supplemented with 2% normal mouse serum (Cedarlane Laboratories, Ontario, Canada), 2 mM L-glutamine, penicillin-streptomycin solution (final concentration of 1000 U/ml and 1 mg/ml, respectively), and 5 x 10–5 M ß-mercaptoethanol (all from Sigma, St. Louis, MO). Splenocytes were plated in 96-well, round-bottomed plates (5x106 cells/ml). Each splenocyte sample was plated in quadruplicate, and the cells were incubated in a humidified 5% CO2 incubator at 37°C for 24 h. SN were harvested, and a commercially available assay kit for IL-10 or IL-12 (mouse OptEIA kit, PharMingen, Mississauga, ON, Canada) was used according to the manufacturer’s instructions to assay cytokine levels.

In vivo murine assays
Naïve BALB/c were injected subcutaneously (s.c.) with the indicated amounts of ODN and were bled after 3 h or 6 h postinjection. Cytokines in the plasma were measured by ELISA with commercially available assay kits for IP-10, tumor necrosis factor {alpha} (TNF-{alpha}), and IL-12 (mouse OptEIA kit, PharMingen) according to the manufacturer’s instructions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CpG dinucleotides with deoxyguanosine modifications can stimulate TLR9-dependent signaling
We wanted to determine the effect of a variety of CpG deoxyguanosine substitutions on the immune-stimulatory activity of CpG-containing ODN. TLR9-dependent NF-{kappa}B activation was induced efficiently by B-Class CpG ODN 2006 (Fig. 1A ). ODN 5331 with the same sequence as ODN 2006 but with CpI (I, deoxyinosine) instead of CpG dinucleotides induced TLR9-mediated signaling with lower potency and efficacy (for sequences, see Table 1 ). Even lower NF-{kappa}B activation was stimulated by ODN 21905 with CpR (R, 2-aminopurine) dinucleotides.



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  		Figure 1. ODN with chemical modifications of the CpG deoxyguanosine stimulate human TLR9-dependent NF-{kappa}B signaling. HEK293 cells stably transfected with vectors expressing human TLR9 and a NF-{kappa}B-luciferase reporter were incubated for 16 h with ODN. NF-{kappa}B stimulation was measured through luciferase activity. SI were calculated in reference to luciferase activity of medium (Med) without addition of ODN. (A) Comparison of B-Class CpG (CG) ODN 2006, ODN 5331 (2006 with the CpG deoxyguanosines replaced by deoxyinosine=I), and 21905 (2006 with the CpG deoxyguanosines replaced by 2-aminopurine=R). (B) Comparison of ODN 2006 (CpG), 5249 [2006 with all deoxyguanosines (G) replaced by deoxyinosine (I)], 5279 [2006 with all deoxyguanosines replaced by 2-aminopurine (R)], 5274 [IpG (IG) dinucleotides], and 5343 [IpI (II) dinucleotides]. (C) Comparison of ODN 2006, 5286 (CpE dinucleotides; E=7-deaza-guanine), 5290 (CpW dinucleotides; W=nebularine), 5389 (CpM dinucleotides; M=2,6-diaminopurine), and 5388 [EpG (EG) dinucleotides]. (D) Comparison of ODN 2006, 5280 (CpD dinucleotides; D=dSpacer), 5281 [DpG (DG) dinucleotides], and 5282 [DpD (DD) dinucleotides]. Shown is one representative out of two similar experiments.

 

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  		Table 1. ODN Used in This Study

 
ODN 5249 (with deoxyinosine substituted for all deoxyguanosines in ODN 2006) and ODN 5279 (with 2-aminopurine substituted for all deoxyguanosines in ODN 2006) stimulated weak TLR9-dependent signaling comparable with the results with ODN 5331 and 21905 (Fig. 1B) . Therefore, modification of all deoxyguanosines in ODN 2006 did not further influence TLR9-mediated signaling. ODN 5274 with deoxycytosine replaced by deoxyinosine (IpG) appeared to be nonstimulatory, as was ODN 5343 with IpI instead of CpG dinucleotides.

The guanine bases of the CpG dinculeotides of ODN 2006 were also substituted by 7-deaza-guanine (ODN 5286; CpE), nebularine (ODN 5290; CpW), or 2,6-diaminopurine (ODN 5389; CpM). The cytosine bases in ODN 5388 were substituted by 7-deaza-guanine (EpG). All ODN with guanine-base modifications were still capable of inducing NF-{kappa}B stimulation (Fig. 1C) ; however, their stimulatory activity was markedly less than with unmodified CpG ODN 2006. ODN 5388 (EpG) did not mediate TLR9-dependent NF-{kappa}B activation.

A sugar-phosphate moiety without a nucleotide base (an abasic residue or dSpacer) can be incorporated into nucleic acid backbones like normal nucleotides. ODN 5280, with the same sequence as ODN 2006, with the exception that the deoxyguanosines of the CpG dinucleotides are substituted by dSpacers, still induced NF-{kappa}B stimulation (Fig. 1D) . In contrast, ODN 5281 (with deoxycytosines substituted by dSpacers) or ODN 5282 (with CpG substituted by two dSpacers) was inactive.

Deoxyguanosine-to-deoxyinosine exchange retains the ability to stimulate cytokine secretion from human immune cells
The exchange of all CpG dinucleotides in the optimal human CpG ODN 2006 with CpI dinucleotides resulted in an ODN (5331) that retained most of its immune-modulatory capacities upon culture with human PBMC. No strong difference between ODN 2006 and 5331 was apparent for IL-10 or IL-6 secretion (Fig. 2 ). The non-CpG ODN 1982 in these experiments stimulated the secretion of IL-10 as well as IL-6, although with lower potency compared with the CpG or CpI ODN.



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  		Figure 2. ODN with deoxyinosine-modified CpG dinucleotides induce cytokine secretion from human PBMC, which were cultured for 24 h (for IL-6) or 48 h (for IL-10) with the indicated concentrations of CpG ODN 2006, ODN 5331 (ODN 2006 with CpI dinucleotides), or ODN 1982 (non-CpG control). SN were harvested, and amounts of secreted IL-10 (A) or IL-6 (B) were measured by ELISA. Shown is the mean ± SEM of three donors.

 
Nevertheless, IFN-{alpha} induced by CpG ODN 2006 appeared to be superior to ODN 5331 (Fig. 3A ). In addition, CpI ODN 5331 appeared to induce inferior amounts of IP-10 as well as stimulated up-regulation of CD80 expression on monocytes less efficiently (Fig. 3B and 3C) . Activation of APC such as monocytes induced by CpG ODN is mediated by pDC IFN-{alpha} and is characterized by efficient IP-10 production and up-regulation of cell-surface molecules such as costimulatory molecules [26 27 28 ]. The non-CpG ODN 1982 stimulated no IFN-{alpha} or IP-10 production. To further investigate the immune-stimulatory effects of other CpI ODN, we choose A-Class CpG ODN 2216. A-Class CpG ODN are characterized by their palindromic CpG phosphodiester sequences with phosphorothioate G-rich ends and stimulate high IFN-{alpha} secretion from pDC [29 ]. Similar to B-Class CpG ODN 2006 and CpI ODN 5331, the IL-10 production, stimulated by ODN 21253 with the same sequence as ODN 2216 but with CpI dinucleotides, was slightly decreased (data not shown). Nevertheless, the IFN-{alpha}-inducing capacity of ODN 2216 was strongly reduced (Fig. 3D) .



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  		Figure 3. Th1-like effects are decreased upon culture with CpI ODN. (A–C) Human PBMC were cultured with the indicated concentrations of CpG ODN 2006, 5331, or 1982. SN were harvested after 48 h, and amounts of secreted IFN-{alpha} (A) or IP-10 (B) were measured by ELISA. (C) Cells were harvested after 24 h, and CD80 expression on CD14-positive monocytes was measured by flow cytometry. (D) Human PBMC were cultured with A-Class CpG ODN 2216 or CpI ODN 21253 (2216 with the CpG deoxyguanosines replaced by deoxyinosine) and IFN-{alpha} measured in the SN after 48 h. Shown is the mean ± SEM of five (A) or three (B–D) donors.

 
Human TLR9 is expressed in B cells as well as pDC, and CpG ODN induce direct stimulation of these cell types [4 ]. As demonstrated in whole PBMC, CpI ODN 5331 was similar to CpG ODN 2006 in terms of the stimulation of IL-6 secretion from purified B cells but induced inferior levels of IFN-{alpha} secretion from pDC-enriched cell cultures (Fig. 4A and 4B ). Non-CpG ODN 1982 stimulated in these experiments much lower levels of IL-6 from purified B cells (IL-6 levels observed in an additional experiment were with 0.25 µM ODN 1982: 28.7±5.3 pg/ml; ODN 2006: 109.5±40.6 pg/ml; or medium control: 10.5±2.3 pg/ml; mean±SEM of two donors) but no IFN-{alpha} from pDC.



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  		Figure 4. ODN with CpI dinucleotides stimulate B cells and pDC. (A) Human B cells were isolated from whole PBMC and cultured with 0.25 µM ODN 2006, 5331, or 1982 for 24 h. SN were harvested, and IL-6 was measured by ELISA. (B) Human pDC were enriched, and cells were stimulated with 0.25 µM ODN 2006, 5331, or 1982 for 48 h. SN were harvested, and IFN-{alpha} was measured by ELISA. Shown is the mean ± SEM of two donors each (mean±SD for one donor with ODN 1982 in A).

 
TLR9 is necessary and sufficient for signaling mediated by ODN with CpI dinucleotides
TLR9–/– mice are unresponsive to stimulation with CpG ODN [9 , 30 ]. We cultured splenocytes from wild-type and TLR9–/– mice with phosphorothioate ODN 1826, which is optimal to stimulate murine TLR9 [31 ]. Stimulation was compared with the same sequence except with CpI (ODN 21103) or IpG dinucleotides (ODN 21104; for sequences, see Table 1 ). The CpI ODN 21103 induced IL-10 as well as IL-12 production from splenocytes of wild-type mice (Fig. 5A and 5C ), although the magnitude of cytokine expression was inferior to that induced by CpG ODN 1826. ODN 21104 (IpG) did not stimulate cytokine production. None of these ODN was capable of stimulating IL-10 or IL-12 production from TLR9–/– mice (Fig. 5B and 5D) .



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  		Figure 5. Cytokine secretion stimulated by ODN with CpI dinucleotides is not observed from cells lacking TLR9 expression. Splenocytes from wild-type or TLR9–/– mice were cultured with CpG ODN 1826, ODN 21103 (1826 with CpI), or 21104 (1826 with IpG) for 24 h. SN were harvested, and IL-10 (A and B) or IL-12 (C and D) from wild-type (A and C) or TLR9–/– (B and D) splenocytes was measured by ELISA. Shown is the mean ± SD of one out of two similar experiments.

 
The human and murine TLR9 differ in the recognition of specific CpG motifs [1 ]. As they also could differ in their ability to be stimulated by CpI ODN, we incubated HEK293 transfectants expressing murine TLR9 and a NF-{kappa}B-luciferase reporter with ODN 1826, 21103, and 21104. CpI ODN 21103 still induced TLR9-dependent NF-{kappa}B activation, although with much lower efficacy than ODN 1826 (Fig. 6A ). ODN 21104 (IpG) was inactive.



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  		Figure 6. ODN with CpI dinucleotides stimulate murine TLR9-dependent but not human TLR7-dependent NF-{kappa}B signaling. HEK293 cells stably transfected with vectors expressing murine (m) TLR9 (A) or human (h) TLR7 (B), and a NF-{kappa}B-luciferase construct was incubated for 16 h with ODN or R-848. NF-{kappa}B stimulation was measured through luciferase activity. Shown is a dose-response comparing (A) CpG ODN 1826 with CpI ODN 21103 and IpG ODN 21104 and (B) CpG ODN 2006, CpI ODN 5331, 21103, and R-848.

 
Synthetic, low molecular weight immune modifiers such as imidazoquinolines (R-848) or substituted guanosine nucleosides were reported to mediate TLR7 (substituted guanosine nucleosides or R-848)- or TLR8-mediated (R-848) immune stimulation [12 , 15 ]. It is interesting that previous reports suggested that the inosine analog methyl inosine monophosphate can induce immune-modulatory effects as an adjuvant in vivo [32 , 33 ]. Therefore, we tested the effects of ODN 21103 (CpI of ODN 1826) or 5331 (CpI of ODN 2006) on stable transfectants expressing human TLR7 and compared it to stimulation induced by R-848. No TLR7-mediated NF-{kappa}B stimulation using the CpI ODN 5331 or 21103 could be observed, in contrast to TLR7 signaling induced by R-848 (Fig. 6B) .

Deoxyinosine-modified ODN stimulate in vivo cytokine production
Naïve BALB/c mice (n=5/group) were injected s.c. with increasing doses of CpG ODN 1826, CpI ODN 21103, or IpG ODN 21104 (Fig. 7 ). CpG ODN 1826 induced production of IP-10, TNF-{alpha}, or IL-12. In contrast, CpI ODN 21103 stimulated only moderate production of these cytokines. ODN 21104 did not stimulate cytokine production at the same ODN concentrations.



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  		Figure 7. CpI compared with CpG ODN induce a similar but less efficient cytokine pattern in vivo. BALB/c mice (n=5 each) were injected s.c. with the indicated ODN concentrations or PBS and were bled at 3 h (IP-10 and IL-12) or 6 h (TNF-{alpha}) postinjection. (A) IP-10, (B) TNF-{alpha}, or (C) IL-12 in plasma was measured by ELISA.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Synthetic, unmethylated CpG-containing ODN are strong immune modulators [29 ]. Chemical modifications of CpG ODN are used to modulate their stability against nucleases, their cellular uptake characteristics, or their immune-stimulatory profile [4 ]. Although various chemical modifications are known from the work with antisense ODN [34 ], only relatively few of them have been applied to CpG ODN.

In most studies reported to date, a chemically unmodified CpG dinucleotide was essential for optimal immune modulation [4 , 22 23 24 , 35 ]. In contrast, modification of nucleotides 5' or 3' of the CpG was able to increase or decrease immune stimulation depending on the exact position of the modified nucleotide [36 ]. To our knowledge, only 7-deaza deoxyguanosine substitution for natural deoxyguanosine in the CpG dinucleotide has been reported to show immune-stimulatory activity with similarities to the parent CpG ODN [24 ]. Chemical modifications or analogs of deoxyguanosine or deoxycytosine all might possibly result in ODN with at least some immune-stimulatory capacities, as they, in principle, still contain a pattern that although slightly modified, could be recognized by the receptor TLR9.

We report here that CpG ODN with deoxyinosine for deoxyguanosine substitutions (CpI) in contrast to other deoxyguanosine modifications still induce cytokine secretion from human PBMC or signaling mediated by TLR9, however, less than the parent CpG ODN. A CpI ODN derived from B-Class CpG ODN 2006 retained most of its ability to induce IL-10 or IL-6 secretion from human B cells. In contrast, the secretion of IFN-{alpha} from pDC or of IP-10 from monocytes appeared to be considerably decreased upon culture with CpI ODN. This effect on IFN-{alpha} production was even more pronounced upon CpG deoxyinosine for deoxyguanosine substitution in an A-class CpG ODN that has a central palindromic CpG phosphodiester sequence with flanking phosphorothioate G-rich sequences. Another observation that indicated decreased ability of CpI ODN to activate APC came from experiments demonstrating a less efficient up-regulation of costimulatory molecules on monocytes. In contrast, CpG ODN induce normally strong stimulation or maturation of APC, a central feature of this class of immune modulators, with pDC IFN-{alpha} as one important mediator of these effects [1 ]. We previously demonstrated that the thymidine content of phosphorothioate ODN strongly contributes to immune stimulation [4 , 22 ]. Nevertheless, phosphorothioate, thymidine-rich, non-CpG ODN, which signal via TLR9 (J. Vollmer, C. Schetter, and A. M. Krieg, manuscript in preparation), are inferior to CpG ODN in their stimulatory activities, and although stimulating IL-10 or IL-6 production from B cells, they do not appear to induce IFN-{alpha} production from pDC. These data demonstrate a surprising and unexpected diversity in the possible immune effects of stimulating TLR9. We hypothesize that activation of human B cells by the TLR9 complex can be accomplished by ODN containing modified CpG motifs and even may be achieved by more substantial modifications or phosphorothioate, thymidine-containing, non-CpG ODN. In contrast, strong and efficient activation of the TLR9 complex and IFN-{alpha} production in pDC and the resulting effects such as Th1-like stimulation of APC function would appear to absolutely require an unmodified CpG motif. It is possible that B cells and pDC express different cofactors for TLR9 or that ODN with chemically modified CpG dinucleotides or CpG-free, thymidine-rich phosphorothioate ODN interact with the human TLR9 receptor in a qualitatively different way from unmodified CpG ODN. Although such ODN induce human TLR9-mediated secretion of B cell-related cytokines such as IL-10 or IL-6, powerful induction of type I IFN appears to be dependent on the presence of an unmodified CpG dinucleotide. Further studies will be required to elucidate the molecular basis for these differential effects.

A variety of other guanine-base modifications in CpG ODN, including 2-aminopurine, 2,6-diaminopurine, nebularine, or 7-deaza-guanine, was tested for their ability to stimulate NF-{kappa}B activation in human TLR9-transfected HEK293 cells. Previously, some of these base modifications, such as 2-aminopurine or nebularine, were reported to show little immune-modulatory effects [23 ]. Similar to these results on murine-immune cells, we could detect only limited stimulation of human TLR9-dependent signaling with these guanine-base modifications. It is interesting that we found that 7-deaza-guanine modifications reduced TLR9-mediated NF-{kappa}B activation considerably, although previous murine in vitro and in vivo assays demonstrated activity of such modified ODN similar to the parent CpG ODN [23 , 24 ]. It is now recognized that the human and murine TLR9 receptors have different requirements for efficient recognition of CpG ODN. Although CpG ODN with the optimal murine CpG motif 5'-GACGTT-3' can stimulate TLR9-mediated effects in human immune cells, ODN with the optimal human CpG motif 5'-GTCGTT-3' are superior [6 ]. Therefore, not only sequence but also subtle modifications of the nucleobases, such as replacement of guanine by 7-deaza-guanine, might differentially affect the recognition by human compared with murine TLR9.

One surprising result was that a nucleobase was not absolutely required in the deoxyguanosine position of CpG. Although it was less active than a CpG ODN, an ODN containing a dSpacer in place of the deoxyguanosine induced substantial activation through the human TLR9. A previous study reported that dSpacers are not allowed in either CpG position, but this study only examined stimulation of murine immune cells, not human [25 ]. Indeed, we found that stimulation of the human TLR9 was only abolished by the substitution of a dSpacer at the deoxycytosine position or at deoxycytosine and deoxyguanosine positions. A similar result was obtained on human TLR9 for ODN with other deoxycytosine substitutions (with deoxyinosine or 7-deaza deoxyguanosine). Therefore, it is tempting to speculate that within a CpG motif, the deoxyguanosine position is more flexible to induce human TLR9-dependent, immune-stimulatory effects than the deoxycytosine position and that the human TLR9 is more forgiving in this regard than the mouse. Indeed, we demonstrated previously that locked nucleic acid monomers, which are conformationally locked nucleotides that structurally mimic standard RNA monomers at the deoxycytosine position of CpG, affect immune stimulation stronger than at the deoxyguanosine position [37 ].

To further elucidate the immune-modulatory capacities of ODN with deoxyinosine substitutions, we used the optimal murine CpG ODN 1826 [31 ] and introduced CpI dinucleotides. The CpI ODN was inferior to the parent CpG ODN 1826 to stimulate NF-{kappa}B signaling in a murine TLR9 transfectant. Although the CpI ODN induced a similar pattern of in vitro and in vivo secretion of Th1-like cytokines and chemokines, such as IL-12 and IP-10, or proinflammatory cytokines, such as TNF-{alpha}, the magnitudes of these responses were inferior to those obtained with the unmodified CpG ODN 1826 at the concentrations tested. Therefore, the CpG ODN appeared to be much more potent to induce efficient cytokine secretion. In contrast to the relatively clear differences in the immune effects observed on human cells, immune-stimulatory ODN with deoxyinosine for deoxyguanosine substitutions appear to induce TLR9-mediated effects in vivo in mice more similar to CpG ODN, although with much lower efficiency and potency.

Substituted guanosine analogs or imidazoquinolines activate immune cells and lead to the production of cytokines [38 39 40 41 ]. From these, 7-allyl-7,8-dihydro-8-oxo-guanosine and the imidazoquinoline R-848 were demonstrated to stimulate intracellular signaling via TLR7 (7-allyl-7,8-dihydro-8-oxo-guanosine and R-848) or TLR8 (R-848) [12 , 15 , 38 ]. It is interesting that another purine derivative that appeared capable of enhancing immune responses, as a vaccine adjuvant, is the inosine-derivative methyl inosine monophosphate [32 , 33 ]. Therefore, it was possible that immune stimulation by ODN containing deoxyinosine might involve another mechanism in addition, independent of TLR9. Nevertheless, the effects induced by CpI ODN depended solely on the presence of TLR9, as stimulation of splenocytes from TLR9–/– mice did not result in production of cytokines. In addition, NF-{kappa}B activation stimulated by CpI ODN could not be observed on transfectants stably expressing the human TLR7.

In summary, this is the first study to show that TLR9 is required for the recognition of immune-stimulatory ODN containing chemically modified deoxynucleotides at the CpG positions. Such ODN are inferior to CpG ODN in most in vitro and in vivo human and murine assays. Although they induce good stimulation of the production of IL-6 or IL-10 from human PBMC, they are inferior to unmodified CpG ODN in the induction of Th1-like cytokines or chemokines such as IFN-{alpha} or IP-10 from pDC and monocytes. Therefore, an unmodified CpG motif appears to be a requirement for optimal Th1-biased immune stimulation.


   ACKNOWLEDGEMENTS
 
We thank Dr. Eugen Uhlmann for carefully reading the manuscript and helpful discussions and Claudia Schmitz for perfect technical assistance.

Received January 22, 2004; revised May 10, 2004; accepted May 26, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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