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The Journal of Immunology, 2007, 179, 45-52
Copyright © 2007 by The American Association of Immunologists, Inc.
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Control of In Vitro Immune Responses by Regulatory Oligodeoxynucleotides through Inhibition of pIII Promoter Directed Expression of MHC Class II Transactivator in Human Primary Monocytes

Jinhai Wang1,*, Gregory Roderiquez*, Taneishia Jones*, Peter McPhie{dagger} and Michael A. Norcross*

* Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration and {dagger} National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ag presentation is a key step in the initiation of adaptive immune responses that depends on the expression of MHC Ags and costimulatory molecules. Immune-enhancing CpG and non-CPG oligodeoxynucleotides (ODNs) stimulate Ag presentation by stimulating the expression of these molecules and by promoting dendritic cell maturation. In this report, we identify immunoregulatory orthophosphorothioate non-CpG molecules, referred to as regulatory ODNs (rODNs), by their ability to inhibit allogeneic monocyte-stimulated T cell responses and down-regulate HLA-DR in human primary monocytes. The rODNs promoted the survival of macrophages and were able to activate IL-8 secretion through a chloroquine-resistant pathway. Messenger RNAs for HLA-DR {alpha} and beta and the MHC CIITA were reduced by rODNs but not by stimulatory CpG ODN2006 and non-CpG ODN2006a. CIITA transcription in monocytes was controlled primarily by promoter III and not by promoter I or IV. rODNs blocked promoter III-directed transcription of CIITA in these cells. Under conditions that induced dendritic cell differentiation, rODNs also reduced HLA-DR expression. The activity of rODNs is phosphorothioate chemistry and G stretch dependent but TLR9 independent. G tetrads were detected by circular dichroism in active rODNs and associated with high m.w. multimers on nondenaturing gels. Heat treatment of rODNs disrupted G tetrads, the high m.w. aggregates, and the HLA-DR inhibitory activity of the ODNs. The inhibition of immune responses by regulatory oligodeoxynucleotides may be useful for the treatment of immune-mediated disorders including autoimmune diseases and graft rejection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Polymorphic HLA-DR proteins are encoded by MHC class II genes and expressed as {alpha}beta-heterodimers on the cell surface. These molecules play essential roles in the initiation of adaptive cellular and humoral immune responses. MHC class II molecules are involved in autoimmune disorders and transplant rejection. The constitutive expression of MHC class II proteins, unlike the MHC class I molecules that are universally expressed on most nucleated cells, is limited to APCs including macrophages, dendritic cells (DCs),2 B lymphocytes, and thymic epithelial cells.

Expression of class II MHC genes necessary for exogenous Ag presentation is controlled by the CIITA. CIITA is the so-called "master control factor" for the expression of MHC class II genes (1, 2). The gene encoding CIITA is MHC2TA. Transcription of MHC2TA gene is driven by one of three alternative promoters, pI, pIII, or pIV (3). Alternative splicing of transcripts produces three isoform-specific mRNAs that differ in the first exon. These three isoforms differ in N-terminal sequences but share four common domains (acidic domain, proline/serine/threonine-rich domain, GTP-binding domain, and leucine-rich repeats) (2). MHC class II molecules are not detected on the surfaces of splenic B cells and DCs in CIITA knockout mice (2), indicating tissue-specific impairment of MHC class II expression.

CpG oligodeoxynucleotides (ODNs) have multiple effects on immune cells, including B cells, DCs, and monocytes/macrophages. Several different types of immune-enhancing ODNs have been described. Type A ODNs, such as ODN2216, are potent type I IFN inducers, whereas type B ODNs, such as ODN2006, are potent B cell stimulators with weaker IFN inducing ability (4). We previously observed that non-CpG ODNs based on type B CpG ODN2006, when mixed with GM-CSF, induced chemokine secretion and DC maturation with high levels of HLA-DR, CD86, CD40, and CD83 expression in human primary monocytes (5).

In contrast to immune-enhancing CpG ODNs, inhibitory CpG ODNs have also been reported that down-modulated MHC expression on ConA-activated murine macrophages (6). Agents that can down-modulate HLA-DR and thereby inhibit adaptive immune responses may provide novel treatments for graft rejection, graft-vs-host disease, autoimmune disease, and diabetes.

Inhibitory non-CpG ODNs that are able to down-modulate HLA-DR on human cells have not been reported. In this study we identify a class of phosphorothioate (PS) non-CpG ODNs, referred to as regulatory ODNs (rODNs), that inhibits the expression of CIITA and cell surface HLA-DR on monocytes and reduces monocyte-stimulated, allogeneic T cell proliferative responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and reagents

Human monocytes and PBLs were isolated by countercurrent centrifugal elutriation from single-donor preparations of peripheral blood leukocytes as described previously (7, 8). Monocyte preparations contained <1% of plasmacytoid DCs as detected by anti-BDCA-4 Ab staining. Cells were cultured in macrophage serum-free medium (Invitrogen Life Technologies).

Oligodeoxynucleotides

The following ODNs were synthesized by the Center for Biologics Evaluation and Research Core Facility (Table I).


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  		Table I. Regulatory ODNs and stimulatory ODNs

 
Cell surface immunofluorescence staining

Cells were stained with the indicated Abs to cell surface Ags and then subject to flow cytometry analysis as described previously (7, 9). Anti-HLA-DR, anti-CD86, and isotype controls were purchased from BD Pharmingen.

Chemokine secretion

The amounts of IL-8 secreted by monocytes into culture supernatants were measured using ELISA kits purchased from BioSource International. Data were analyzed with SoftMax Pro software.

RT-PCR analysis

Total cellular RNA of human monocytes was isolated using TRIzol reagent and then treated with DNase I. Equal amounts of total RNA were subject to first-strand cDNA synthesis using RNase H minus SuperScript II reverse transcriptase (Invitrogen Life Technologies). cDNA was amplified by 30 cycles of PCR with the following human specific primers (10): DRA, 5'-GTCTGGCGGCTTGAAGAATT-3' and 5'-ACCTTGAGCCTCAAAGCTGG-3'; DRB, 5'-CGTGACAAGCCCTCTCACAG-3' and 5'-TGTGCAGATTCAGACCGTGC-3'; CIITA, 5'-GCTCTGAGTGGCGAAATCAAG-3' and 5'-CAATGCTAGGTACTGCGGGAG-3'; type I CIITA, 5'-GGAGACCTGGATTTGGCCCT-3' and 5'-GAAGCTCCAGGTAGCCACCTTCTA-3'; type III CIITA, 5'-GGGGAAGCTGAGGGCACG-3' and 5'-GAAGCTCAGGTAGCCACCTTCTA-3'; type IV CIITA, 5'-GCGGCCCCAGAGCTGG-3' and 5'-GAAGCTCCAGGTAGCCACCTTCTA-3'; and GAPDH, 5'-CACCACCATGGAGAAGGCTGG-3' and 5'-GCCATGCCAGTGAGCTTCCCG-3'. PCR products were separated and stained with SYBR Green 1 (Molecular Probes) and analyzed with a FluorImager scanning densitometer and ImageQuant software (Amersham Biosciences).

Proliferation assay

PBLs in RPMI 1640 with 10% FBS at 1 x 106/ml were mixed with monocytes at a 2:1 ratio and then incubated in 96-well plates for 3 days, followed by the addition of [3H]TdR for 16 h. Cells were harvested using a semiautomatic cell harvesting apparatus (Skatron) and counted in a Wallac LKB 1205 Betaplate liquid scintillation counter.

Multimeric oligonucleotide analysis on native acrylamide gels

Oligonucleotides were either untreated or heated at 90°C for 10 min and placed on ice and then samples (150 ng each) were resolved by electrophoresis in native 20% (19:1) acrylamide/TBE-buffered gels. Oligos were visualized by staining gel with SYBR Gold (catalog no. S11494; Invitrogen Life Technologies) nucleic acid gel stain and analyzed with a FluorImager scanning densitometer and ImageQuant software.

Circular dichroism (CD) analysis

Solutions of oligonucleotides (A260 = 1) were made up in PBS. CD spectra were measured by a Jasco J-715 spectropolarimeter with the solutions in a 1-cm pathlength quartz cuvette in a cell holder thermostated by a Neslab RTE-111 circulating water bath. Spectra were scanned four times, from 330 to 210 nm and averaged (speed 50 nm/min, time constant 1s). Spectra were obtained at 5°, 25°, and 70°. To study the effect of potassium ions, 1 M KCl solution was added to give a final concentration of 50 mM. After baseline correction, the measured ellipticities were converted into mean residue {Delta}{epsilon} values using the formula {Delta}{epsilon} = (100 x mdegs) ÷ (3300 x c), where mdegs is the measured ellipticity, and c is the concentration of nucleotide residues (mM).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Suppression of monocyte alloantigen-induced proliferation of T lymphocytes by rODNs

During the course of studies to identify immune modulatory effects of ODNs, we observed that some non-CpG oligonucleotides had suppressive effects on allogeneic T cell responses. We therefore synthesized a series of non-CpG ODNs (all listed in Table I) and tested their activity on monocyte-activated allogeneic responses. Monocytes were treated overnight with this set of ODNs, mixed with allo-PBLs at a 1:2 ratio for 3 days, and cell proliferation was measured. As shown in Fig. 1, rODNs 0001 and 0002 inhibited T cell proliferation by 50% compared with the untreated controls. rODN0009 with GG and rODN0012 with GG, AA, and CC at the marked dinucleotides also showed inhibition of >50% (Table I, rODN0001, boldface). rODN0010 with two GG and one CC at the marked dinucleotides also showed inhibitory activity (Table I). rODN0005 and rODN0006, with replacement of the G-rich regions at both sides, showed weak inhibitory activity. Oligos rODN0003 and rODN0004, which retained one G stretch, remained active. rODN0007 and rODN0008, the phosphodiester (PO) versions of rODN0001 and rODN0002, respectively, did not show inhibition of the proliferative response. These results indicate that the marked sequences internal to the flanking G stretches were not critical to the activity and that at least one G stretch was required for inhibition. PS linkages were required for activity under these conditions.


Figure 1
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  		FIGURE 1. Inhibition of alloresponses by rODNs. Human monocytes were treated with indicated rODNs (10 µg/ml) overnight and then mixed with T cells as described in Materials and Methods. Data are mean ± SD of triplicates. Med, Medium.

 
Down-modulation of HLA-DR by rODNs

Trimolecular interactions of MHC peptide-TCR are the key steps in induction of Ag-specific immune responses. HLA-DR presents exogenous peptides to CD4+ T lymphocytes. We examined the effects of these rODNs on cell surface expression of MHC II molecules. As shown in Fig. 2, HLA-DR expression was reduced by 80% with rODN0001, rODN0009, and rODN0012, but not by rODN0006, which has C-substituted flanking G stretches. HLA-DR was also down-modulated by rODN0012 at 1 µg/ml (data not shown).


Figure 2
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  		FIGURE 2. Down-modulation of HLA-DR in monocytes by rODNs. Monocytes were treated with indicated rODNs (10 µg/ml) overnight. Cells were fixed and stained with FITC-labeled HLA-DR Ab and subject to FACS analysis. Data shown are from one representative experiment of three from different cell donors. Med, Medium.

 
Transcriptional inhibition of DRA and DRB by rODN

HLA-DR is formed by products of the DRA and DRB genes. We next examined the impact of rODN on DRA and DRB messages in monocytes. Cells were treated with rODN00012 with and without stimulatory ODNs 2006 and 2006a. As shown in Fig. 3, DRA and DRB messages were highly expressed in untreated monocytes, but their expression was reduced in rODN0012-treated cultures. ODN 2006 and 2006a did not inhibit DR messages directly or interfere with rODN0012 inhibitory activity.


Figure 3
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  		FIGURE 3. Transcriptional regulation of DRA, DRB, and CIITA in monocytes by rODNs. Monocytes were treated with indicated ODNs (10 µg/ml) for 5 h and subject to sample preparation and PCR analysis as described in Materials and Methods. Data are from one experiment of two from separate donors. Med, Medium.

 
Transcriptional blocking of CIITA

The lack of CIITA in cells from the bare lymphocyte syndrome leads to the absence of MHC class II transcription by interfering with the function of the MHC enhancesome (11). To investigate whether rODN regulates HLA-DRA and HLA-DRB gene expression through the control of CIITA expression, the levels of CIITA mRNA in monocytes treated with rODN were examined 5 h after exposure to rODNs, a time point chosen based on the fact that the half-life for CIITA mRNA is 3.5 h for type I and 7.8 h for type IV (12). CIITA RNA was detected in cultured control monocytes and did not change with immune stimulatory CpG ODN2006 and non-CpG ODN2006a treatment. CIITA RNA was almost completely blocked in rODN0012-treated monocytes, indicating that rODN may regulate MHC class II molecules by limiting CIITA levels. Again, ODN2006 and ODN2006a did not induce CIITA directly or interfere with rODN0012 inhibition of CIITA when cocultured with the inhibitory oligonucleotides.

Inhibition of pIII promoter activity by rODN

There are at least three independent promoters controlling human CIITA transcription (3). pI directs the constitutive expression of CIITA in DCs and B lymphocytes. pIII is used for the constitutive expression of CIITA in DCs and B cells and for the induction of CIITA in activated T cells (3, 13, 14). pIV is predominantly induced by IFN-{gamma} (15). We first determined which promoter was used in monocytes by RT-PCR amplification of the first exon of each promoter and found that CIITA expression was exclusively driven by the pIII promoter in human primary monocytes (Fig. 4A). We then examined the impact of rODNs on pIII promoter-directed CIITA RNA expression and found that pIII-directed expression of CIITA was dramatically blocked by rODN0012 (Fig. 4B).


Figure 4
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  		FIGURE 4. Control pIII promoter directed gene expression by rODN. A, Constitutive activity of CIITA promoters in monocytes. B, Monocytes were treated with the indicated rODN for 5 h and subject to sample preparation and PCR analysis as described in Materials and Methods. Med, Medium.

 
Reduction of HLA-DR expression in DCs

Monocyte-derived DCs that express TLR9 are generated in the presence of IL-4 and GM-CSF (16). We have reported that elutriated monocytes differentiate into mature DCs after stimulation with GM-CSF and PS non-CpG and CpG ODNs, but not with PO ODNs as seen by induced expression of CD83, HLA-DR, and CD86 (5). To study the impact of rODN on DC maturation, we cultured monocytes with rODN in the presence of the DC maturation stimulators GM-CSF and non-CpG ODN 2006a and then measured the expression of HLA-DR and CD86. Adding rODN0001 at 10 µg/ml reduced HLA-DR levels without substantially affecting CD86 expression (Fig. 5A) in activated monocytes, indicating that rODN affects DC differentiation and maturation by selectively reducing class II molecules. A 10-fold lower dose of rODN also inhibited HLA-DR expression under these conditions (Fig. 5B).


Figure 5
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  		FIGURE 5. Impact of rODN on HLA-DR and CD86 expression. Monocytes were treated overnight with rODN0001 (10 µg/ml) (A) and rODN0012 (B) in the presence of GM-CSF (20 ng/ml) and ODN2006a (10 µg/ml) and then stained with the indicated Ab and subjected to FACS analysis. Med, Medium.

 
G-stretch dependent down-modulation of HLA-DR

To test whether the sequences between the two G-stretches are important for down-modulation of HLA-DR, eight new oligos were synthesized and tested. As shown in Table II, rODN0013, 0014, and 0015 were inhibitory in cells treated with GM-CSF and ODN2006a. rODN0016 and 0017, in which all sequences between the two G stretches were replaced with either A or T, were also inhibitory, indicating that the inhibitory activity is not dependent on the sequences between the two G stretches. We then determined whether the spacing between the two G stretches is essential for the inhibitory activity. Both rODN0018 and 0019 have shorter spacing between the two G stretches, but both maintained down-modulating activity. It was further shown that rODN0020, which has the same base composition as 0012 with one G stretch at the left end as in 0012 but with the other G stretch placed in the middle of the oligo (Table II), and 0021, which only has one five G stretch in the middle of the rODN (AGGATGGGGGAATCC), also down-modulated HLA-DR (data not shown). These results indicate a dependence on G-stretches for HLA suppression and are consistent with the G dependence of rODN inhibitory effects on allo-responses.


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  		Table II. Inhibition of HLA-DR expression by rODNsa

 
To test whether a G stretch is the principal activity domain, PS ODNs with 20 G and five G nucleotides were synthesized and tested on human monocytes. As shown in Fig. 6A, both 20-G ODNs and 5-G ODNS down-modulated HLA-DR expression in monocytes in the absence or presence of the DC differentiation/maturation factors GM-CSF and non-CpG ODN2006a, indicating that G stretches are the major activity domain of regulatory ODNs. To define the minimal number of G nucleotides in a sequence required for inhibition we tested GGGGG, GGGGGC, and GGGGCC oligonucleotides on HLA-DR expression on resting and activated human monocytes. HLA-DR was inhibited in both stimulated and unstimulated cells by GGGGG and GGGGGC, but not by GGGGCC (Fig. 6B), thus supporting the notion that a five G nucleotide sequence is the minimal functional domain. In the contrast, HLA-DR was not down-modulated by the PS ODNs TTTTT, CCCCC, and AAAAA (data not shown), indicating that the HLA-DR down-modulating activity is poly(G) specific.


Figure 6
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  		FIGURE 6. Regulation HLA-DR expression by PS ODNs consist of pure G stretches. Monocytes were treated overnight with indicated ODNs in the absence or presence of GM-CSF and ODN2006a (A) or in the absence or presence of GM-CSF and ODN2006a or ODN2006 (B) and then stained with the indicated Ab and subjected to FACS analysis. Med, Medium.

 
rODNs stimulate chemokine CXCL8/IL-8 production in monocytes through a chloroquine-insensitive pathway

Although rODNs suppressed cell surface HLA-DR, it was not clear whether these rODNs were globally suppressive or had activating effects on other functions. We next tested whether rODNs had direct effects on another inflammatory secretory product, the chemokine IL-8. Monocytes were treated with rODN0001 or with the non-CpG ODN2006a in the presence or absence of chloroquine, an endosome maturation/acidification inhibitor. Endosome acidification is required for CpG ODN activity (17) and for the optimal induction of CCL3 by non-CpG ODN derived from type B CpG ODN2006 (5). Cells were precultured with chloroquine and then stimulated with rODN and ODNs. We found that IL-8 was induced by rODN0001 and ODN2006a. However, only ODN2006a-induced but not rODN0001-induced IL-8 production was inhibited by chloroquine (Fig. 7), indicating that the mechanisms of induction of IL-8 by rODN and ODN2006a are different.


Figure 7
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  		FIGURE 7. Induction of IL-8 by ODNs. Elutriated monocytes were cultured in the presence or absence of chloroquine (2.5 µg/ml) for 30 min and then the indicated ODNs (10 µg/ml) were added overnight and cell-free supernatants were subjected to ELISA for IL-8. Data from two donors are shown.

 
Multimer formation of active regulatory ODNs

To examine whether regulatory ODNs exist as multimers, rODNs were analyzed by using PAGE with a highly sensitive staining method for DNA. In addition to the main single-stranded bands, all rODNs formed heterogenous high m.w. forms or multimers except for rODN0005 (Fig. 8A) and rODN0006 (data not shown), indicating that the G stretch is essential for the formation of multimers.


Figure 8
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  		FIGURE 8. Multimeric oligonucleotide analysis performed on native acrylamide gels. A, Oligonucleotides were either untreated (C, control) or heated (H) at 90°C for 10 min and then placed on ice and samples (150 ng each) were resolved by electrophoresis in native 20% (19:1) acrylamide/TBE-buffered gels and visualized by gel staining with SYBR Gold. B, rODN0012 with/without heating at 90°C for 10 min was added to monocyte cultures overnight and treated monocytes were subjected to flow cytometry analysis.

 
Multimers and regulatory activities are heat-sensitive

Previous studies showed that aggregated formation and functional activities of these G-rich ODNs are heat sensitive (18). To test whether the multimers of rODNs are labile to heat, rODN0005, 0009, 00012, and 0017 were heated for 10 min at 90°C and analyzed by using PAGE. rODNs 0009, 0012, and 0017 all formed multimers that were disrupted with heating (Fig. 8A).

We then examined whether the activity of rODNs was sensitive to heat. As shown in Fig. 8B, after heating rODN 0012 lost its activity in the down-modulation of HLA-DR.

G stretch-dependent formation of G tetrads

G-rich sequences can form several kinds of secondary structures, including unimolecular hairpins or multistranded structures that may involve non-Watson-Crick G-G base pairs. Potassium ions can stabilize these multistranded G quadruplex structures. These structures can be identified by their characteristic CD spectra (19).

Fig. 9A shows the CD spectra of ODN0005 and ODN0006, which do not contain G stretches and are inactive in decreasing HLA-DR expression. Positive peaks at 280 nm and small negative ellipticities below 260 nm indicate that these oligonucleotides are single stranded. In contrast, the spectra shown in Fig. 9B show that the G stretch-containing ODNs rODN0001, rODN0002, and rODN0012 all form multistranded structures. The intense positive peak at 260 nm, shown by all of these nucleotides, indicates the presence of extensive amounts of parallel G quadruplexes. The variable shoulders near 300 nm (rODN0001, and rODN0002) are compatible with the presence of some antiparallel quadruplex structures also. rODN0007 and 0008 also form G tetrads (data not shown) but are not functionally active at the dose tested (10 µg/ml), demonstrating that the inhibitory activity was dependent on both G tetrads and PS chemistry. The spectra of active rODNs were not changed by the addition of potassium or overnight incubation at 5°C, indicating that folding of the oligonucleotides is complete at 25°C. All of the structures were partially unfolded at 70°C, as shown in Fig. 9C for rODN0012. After cooling back to 25°C, recovery of the positive CD bands was very slow, suggesting that the quadruplexes are formed by intermolecular association (data not shown).


Figure 9
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  		FIGURE 9. Structure analysis of rODNs by CD. CD analysis of regulatory ODNs in solution was performed as described in Materials and Methods. A, rODN0005, and rODN0006. B, rODN0001, rODN0002, and rODN0012. C, rODN0012 at 25°C and 70°C.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we found that non-CpG PS ODNs containing five G nucleotide stretches inhibited CIITA expression and down-modulated HLA-DR mRNA and cell surface expression in human primary monocytes in a G tetrad- and PS chemistry-dependent manner. The suppression of CIITA was associated with the restriction of CIITA promoter III-directed gene expression. Inhibition of HLA-DR was associated with reduced allostimulatory activity of rODN-treated monocytes. rODNs also inhibited HLA-DR expression in monocytes induced to differentiate into DCs with GM-CSF and ODN but did not inhibit expression of the CD86 costimulatory molecule.

Inhibitory effects of ODNs on innate and adaptive immune responses have been reported in several models. In the mouse, modulation of the class II molecule has been observed in murine macrophages by CpG ODN (6). Suppressive ODNs block the effects of stimulatory CpG ODNs in mouse B cells (20, 21). Suppressive ODNs containing the TTAGGG motif suppress inflammatory cytokine production by interacting with STAT1 and STAT4 and also reduce CpG-induced arthritis (22, 23, 24, 25).

Some suppressive ODNs contain G-rich regions. Early studies found that G-rich antisense oligonucleotides could function by a nonantisense mechanism to inhibit proliferation and IFN-{gamma} signaling (26, 27, 28). NK cell activity and DNA synthesis induced by ODNs containing palindromic sequences that act through the scavenger receptor are inhibited by poly(G) ODNs (29). Poly(G) ODNs suppress CpG ODN activity in murine DCs (30). Alterations in the cell surface markers MHC class I and ICAM-1 were reported for some G-rich ODNs (31, 32). Suppressive ODNs containing GGAGGGGTTGT have TLR9 inhibitory activity (33). IFN-{gamma} production by murine splenocytes was inhibited by an ODN with only five G nucleotides (34). Suppressive ODNs also inhibit CpG-induced inflammatory TNF-{alpha} and chemokine MIP-2 secretion (35).

In this report, we show that rODNs containing G stretches with pure PS linkage inhibit alloresponses to human monocytes and that this activity is associated with the down-modulation of HLA-DR in the target cells. Replacing G nucleotides with C nucleotides in G stretches, as in rODN0005 and rODN0006 in Fig. 1, attenuates the rODN blocking activity on the alloresponse, thus indicating a dependence on G stretches. The direct down-modulation of HLA-DR by 20 G nucleotide and five G nucleotide ODNs, but not with PS ODN GGGGCC, TTTTT, CCCCC, and AAAAA, indicates that the five G stretch is the principle activity domain in regulatory ODNs.

Poly(G) ODNs and other G-rich ODNs with suppressive effects on cytokine production in the mouse have been shown to exhibit G tetrad formation. Poly(G) binding to the macrophage scavenger receptor requires a minimum of five G nucleotides in the ODN, which was shown to correlate with four stranded helices stabilized by G quartets (G tetrads) as detected by CD spectra (18). G tetrad-containing ODNs also formed large m.w. heterogeneous populations by size exclusion analysis. In this report we show that MHC class II inhibition by rODNs is dependent on the PS chemistry and formation of stable G tetrad polymeric structures that are heat sensitive.

We note that stimulatory oligonucleotides containing poly(G) stretches have been described in the literature. Recent reports suggest that the scavenger receptors CXCL16, a chemokine that functions to bind G-rich regions in D-type CpG ODNs in human cells, and SR-A may mediate the effects of poly(G) CpG ODNs in murine macrophages (36, 37). Scavenger receptor binding may enhance the uptake of ODN and facilitate interactions with TLR9 receptors. Thus, it is likely that the activity of ODNs that contain inhibitory G stretches along with activating motifs such as CpG results in a balance between inhibitory or enhancing functions that may depend on the target cell and the type of ODN binding receptors. The formation of G tetrads may provide multimerizing anionic cores that could potentiate either inhibition or activation, depending on the context of nearby sequences.

The PS backbone is essential for rODN inhibitory activity as demonstrated by the lack of activity of the PO ODNs rODN0007 and rODN0008, which also contain multimers when compared with their active PS homologues rODN0001 and rODN0002. This is consistent with another report in which a poly(G) PO ODN was required at concentrations 10–100 times higher to show inhibitory activity compared with its PS homologue on PS ODN-induced E-selectin promoter activity in a murine macrophage-like cell line (38). In general, PS ODNs are active at concentrations 10 to 100 times lower than their corresponding PO ODNs (39). Weak functional activity of PO rODNs may be secondary to sensitivity to degradation, differences in uptake, or specific structural properties.

Toll-like receptors play important roles in discriminating among distinct classes of microbial molecules. Studies using gene knockout animals and human TLR9 transfection into nonresponder cells have demonstrated the role of TLR9 in CpG-DNA signaling (40, 41). Although some studies indicated that purified monocytes do not react to CpG ODNs, it has been shown that monocyte-derived DCs (cultured in the presence of GM-CSF and IL-4) express TLR9 and react to CpG ODN directly (16). Another study found that CpG ODN directly stimulates purified human monocytes to secrete IL-8 and TNF-{alpha} (42). We reported that GM-CSF to some extent induced the expression of TLR9 in elutriated monocytes (5), which may increase monocyte responses to CpG and non-CpG ODNs.

Recent studies indicate that TLR9 is also the receptor for certain non-CpG ODNs (43, 44). In accordance with these findings, we reported recently that the induction of CCL3 by non-CpG ODN derived from type B CpG ODN2006 is endosome acidification/maturation dependent (5). However, a recent study indicated that suppressive ODNs inhibit LPS-mediated endotoxic shock by direct binding to STAT1 and STAT4 in macrophages (23) and that B-form DNA induces antiviral responses in a TLR-independent manner (45). In the current study, we found that rODNs that contain a G-rich PS backbone with no CpG motifs strongly activate IL-8 secretion while inhibiting HLA expression. Activation of IL-8 production was not sensitive to pretreatment with chloroquine in contrast to non-CpG stimulatory ODNs, supporting a non-TLR9 dependent signaling pathway for rODN effects.

DCs are the most potent APCs (46). Monocytes become immature DCs after migrating from peripheral blood into tissues. After capturing Ags these immature DCs mature and migrate to lymph nodes where they present captured Ags as peptide fragments to T cells and stimulate T cell-dependent immunity. By inhibiting Ag presentation of monocytes and differentiation/maturation of monocytes to DCs, rODNs are possible candidates for attenuating immune responses. Inhibitory strategies incorporating peptides that control monocyte migration (47) along with rODN may be effective in controlling immune responses in vivo. Through the control of CIITA-directed expression of class II molecules, regulatory ODNs could have applications in autoimmune disease, transplantation, and immune tolerance induction.


   Acknowledgments
 
We are grateful to Drs. Kathleen Clouse, C. Lankford, and F. Schwartzkopff for elutriation of human monocytes and Samantha M. Shaw for her technique assistance. We thank Dr. Serge Beaucage for helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Jinhai Wang, Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, National Institutes of Health Building 29B, Room 4E12, 8800 Rockville Pike, Bethesda, MD 20892. E-mail address: jinhai.wang{at}fda.hhs.gov Back

2 Abbreviations used in this paper: DC, dendritic cell; CD, circular dichroism; ODN, oligodeoxynucleotide; PO, phosphodiester; PS, phosphorothioate; rODN, regulatory ODN. Back

Received for publication August 31, 2006. Accepted for publication April 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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