Sequence requirements for oligodeoxyribonucleotide inhibitory activity

Robert F. Ashman1,2, J. Adam Goeken1, Jennifer Drahos1 and Petar Lenert1

1 Department of Internal Medicine, Division of Rheumatology, University of Iowa, 200 Hawkins Drive C31 GH, Iowa City, IA 52242, USA
2 VA Medical Center, Iowa City, IA 52242, USA

Correspondence to: R. F. Ashman; E-mail: robert-ashman{at}uiowa.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Inhibitory oligonucleotides (IN-ODN) differing from stimulatory CpG ODN (ST-ODN) by as few as two bases can block ST-ODN-induced proliferation, apoptosis protection and IL-6 secretion in B lymphocytes and the production of IL-12p40 by non-B cells. The main objective of this study was to determine the ODN sequence requirements for inhibition in mice. Starting with a strongly inhibitory 15-mer prototype phosphorothioate sequence, we tested the 60 sequences that differed from the prototype by one base, revealing the three areas that are critical for activity. Between these areas were the spacer sequences where base composition mattered little, but the number of bases was important. Truncation of three bases at the 3' end of the 15-mer and one at the 5' end was tolerated with minimal loss of activity. This approach yielded an ‘optimal’ sequence of 5' CC x notC notC xxGGGx or CC x notC notC xGGGxx 3', where x is any base. The sequence requirements for optimal inhibition of B cell responses to Type B (K) ODN and mixed splenocyte IL-12p40 responses to Type A (D) ODN were strikingly similar. Inhibition of ST-ODN by IN-ODN was competitive. A hypothetical model of the ODN-binding site is proposed. Synthetic IN-ODN with the sequence characteristics defined here should provide antidotes for excessive innate reactions to DNA.

Keywords: apoptosis, B lymphocytes, cellular activation, cytokines, rodent


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
The response of B cells to bacterial DNA (1, 2) is an important element in our natural immune response to bacteria. The optimal stimulatory motif in bacterial DNA for mouse B cells centers on a sequence described as ‘purine, purine, C, G, pyrimidine, pyrimidine’ (1) or as ‘not C, unmethylated C, G, not G’ (3), which is present at much less than the chance frequency in mammalian DNA (2). Synthetic oligonucleotides (ODN) with this motif possess all the mitogenic properties of bacterial DNA. Mouse B cells proliferate, secrete IgM and secrete cytokines, all without either engagement of the B cell receptor or application of T help (2, 3). The ODN that work best on B cells are called Type B (K) (4, 5) to distinguish them from Type A (D) ODN which are more effective in driving cytokine secretion from dendritic cells or macrophages. Type A (D) and B (K) ODN may share a central ACGTT motif, except that in Type B (K), greatest potency is shown by ODN with a nuclease-resistant phosphorothioate backbone throughout (6), whereas in Type A (D), the central motif must be in a palindromic phosphodiester form, perhaps allowing the formation of secondary structure. In Type B (K) ODN there is no evidence that secondary structure is needed (7). An intermediate form Type C has been described with features of both Types A (D) and B (K) (8).

Recently, we have shown that changing as few as two bases (TT to GG) in the optimal stimulatory CpG ODN (ST-ODN) converts it into an inhibitor (7). So far, all downstream effects of ST-ODN on B cells tested are inhibited, from nuclear factor {kappa}B (NF{kappa}B) (9) and activation protein-1 (AP-1) activation (10), to induction of gene products active in proliferation and apoptosis protection, specifically BclXL (7).

Currently, the best candidate for the ODN receptor is Toll-like receptor (TLR) 9. Cells deficient in TLR9 (11) or its adapter molecule MyD88 (12) are refractory to Type A (D) or B (K) ODN stimulation, but still respond to LPS. Transfecting human or mouse TLR9 into such cells confers the ODN-base sequence preference of its species on the recipient cells (13). There is evidence that at 5 µM, TLR9 can bind both ST-ODNs and their less-active GpC congener (14), and contrasting evidence using Biacore technology that the CpG sequence binds better (15, 16). Further study is required to conclude that the extreme differences in ODN activity in the nanomolar range resulting from single base changes derive solely from differences in the direct binding avidity of ODN for TLR9, and there is as yet no evidence that inhibitory ODN (IN-ODN) act by binding TLR9. It is entirely possible that sequence-dependent activation by IN-ODN may involve other molecules that react secondarily with TLR9. In contrast to the complex sequence-non-specific suppressive effects of single base phosphorothioate polymers on immune cell activation seen at micromolar concentrations (17, 18), we have shown that the potency of IN-ODN in the nanomolar range is sequence specific (7), as is that of ST-ODN (3).

In this paper we further define the sequence requirements of the optimal IN-ODN motif for Type B (K) ODN in mouse B cells. Sequence variations that affect activity the most are confined to three pairs of bases whose spacing is critical. Two of these can be shared with ST-ODN and thus are postulated to affect receptor binding, whereas the third pair determines whether a signal is sent. Truncation showed an 11-mer to be the shortest ODN with near-maximal activity and a 10-mer to be the shortest with detectable activity. Inhibition by IN-ODN was competitive and reversible. Sequence requirements for inhibition of Type A (D) ODN on non-B cells and Type B (K) ODN on B cells were strikingly similar, but not quite identical.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Cell purification and culture
To increase the yield of resting B cells, specific pathogen-free 8- to 12-week-old B6D2F1 mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and housed in a barrier facility with autoclaved bedding and water and high efficiency particulate air (HEPA)-filtered air. B cells were prepared by RBC lysis, followed by negative selection with anti-CD43-coated magnetic Midi-Macs beads (Miltenyi Biotec, Auburn, CA, USA), typically yielding 25 x 106 B cells per spleen which were >97% CD19+. By acridine orange (AO) flow cytometry, 1% of these were apoptotic, 1% in G1 and 98% in G0. Purified B cells were cultured at 2 x 106 ml–1 in 5% FCS–RPMI 1640. For IL-12p40 experiments, unseparated splenocytes were used.

Reagents
Integrated DNA Technologies (Coralville, IA, USA) and Coley Pharmaceutical Group (Wellesley, MA, USA) were the sources of our synthetic ODN, all of which had a nuclease-resistant phosphorothioate backbone. The prototype inhibitory sequence, 2114, was previously shown to be a strong inhibitor of ODN action (7). The ST-ODN 2084 was routinely included at 100 nM where inhibitory activity was measured (7). Agonist anti-mouse CD40 was clone 1C10 rat IgG2a from BioLegend (San Diego, CA, USA). LPS is from Sigma (St. Louis, MO, USA).

Flow cytometry
AO staining allowed simultaneous detection of cell cycle entry and apoptosis protection by flow cytometry by a modification of the method of Traganos (19) as previously described. The G0/G1 boundary was set with freshly isolated G0 B cells. The term ‘cell cycle entry’ applied to cells in G1, G2, S and M phases of the cell cycle, but the majority was in G1.

IL-12p40 ELISA
Splenocyte supernatants were stored at –80°C before assay. Complementary specific antibodies for IL-12p40 from hybridoma clones C15.6 and C17.8 and recombinant mouse IL-12 were purchased from eBiosciences (San Diego, CA, USA). Assay sensitivity was <16 pg ml–1 (20).

IL-6 ELISA
Culture supernatants were assayed on ELISA plates using paired anti-IL-6 antibodies from BioLegend, one to coat the plate and the other to develop it after the addition of supernate and washing. Clones MP5-32C11-biotin and MP5-20F3 anti-IL-6 were used. The assay sensitivity was also 16 pg ml–1.

Data analysis
The potency of an IN-ODN in B cells was calculated as the concentration producing half-maximal inhibition of cell cycle entry, apoptosis protection or IL-6 production stimulated by 100 nM ODN 2084, as determined from the mean result of three experiments. Since, on average, the potencies for a given ODN in the three B cell assays differed by a factor of ~2.7, a single ‘potency index’ for each ODN was calculated as the geometric mean of the potencies determined independently in each of the three assays. Potencies of IN-ODN were greatest for IL-6 and least for apoptosis protection. The ‘percent activity’ of an IN-ODN was defined as the potency of the prototype ODN divided by the potency of the test ODN. For example, if 15 nM of the prototype IN-ODN 2114 or 1500 nM test ODN each produce 50% inhibition, the percent activity of the test ODN is 15 ÷ 1500 or 1%. To estimate the statistical significance of differences in potency index, we calculated means and standard errors from the logs of the individual determinations in a sample of the data.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Inhibition by IN-ODN is competitive
ODN 1826 has two stimulatory motifs, whereas ODN 2084 has one. On a molar basis, 1826 is about twice as potent in B cells as 2084 (data not shown). In Fig. 1(A), IN-ODN 2088 was titrated against 100 nM of these two ST-ODN, showing that inhibition of 1826 required about twice as much IN-ODN as inhibition of 2084. In Fig. 1(B), titration curves for ST-ODN 2084 are shown, with and without 100 nM of IN-ODN 2088. Not only can full activation be achieved by adding enough 2084 to 2088 but the symmetrical titration curves have the same slope. In other experiments not shown, weaker ST-ODN were shown to require less IN-ODN for equivalent inhibition.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Inhibition by IN-ODN is competitive. (A) B cells were cultured with 100 nM of ST-ODN 1826 (filled square) which has two ACGTT motifs, or strong one-motif ST-ODN 2084 (filled diamond), with a range of concentrations of IN-ODN 2088. By AO flow cytometry G1 entry was measured at 18 h. (B) B cells were cultured with 100 nM of strong IN-ODN 2088 alone (filled square) and with a range of concentrations of the strong ST-ODN 2084 (filled diamond 7). The percent of cells in G1 by AO flow cytometry was determined at 18 h. Sequences: ST-ODN 2084 T C C T G A C G T T G A A G T, 1826 T C C A T G A C G T T C C T G A C G T T, IN-ODN 2088 T C C T G G C G G G G A A G T. Also used in this experiment, but omitted for clarity, were IN-ODN 2114 T C C T G G A G G G G A A G T, which gave results identical to 2088, and ODN 2310 T C C T G C A G G T T A A G T, which was inactive in this concentration range.

 
Defining the optimal motif: single base changes
All the ODN that differ by one base from the 15-mer prototype IN-ODN 2114 were titrated into 100 nM of the strong Type B (K) ST-ODN 2084, in order to determine the bases that contribute most to inhibitory activity in B cells. The three readouts were apoptosis protection, G1 entry and IL-6 secretion. Positions in the ODN 2114 were numbered according to the following scheme:


View this table:
[in this window]
[in a new window]
 
 
The ‘potency index’ (defined in Methods) provides a single number for comparison among ODN but does not allow statistical analysis. To estimate the precision of our data, we determined the standard errors of the logs of the means of the potencies calculated in the three individual experiments on each ODN at positions –7 to –5, converted to antilogs and performed t-tests (Table 1). From these data (details available as Supplementary Figure 1 at International Immunology Online), we conclude that the potency of none of the single base variants at position –7 was significantly different from IN-ODN 2114, whereas each of the variants at positions –6 and –5 was significantly different from 2114. In determining potencies for the rest of the 60 single base changes, we adopted the less-laborious method of calculating the geometric means of data from the three assays for each concentration and determining the potency of an ODN from one titration curve. The mean of 19 independent determinations of the potency index of 2114 (each an average of three experiments) was 18 ± 2 (SEM) nM, identical to that determined from our sample of individual experiments at position –7, –6 and –5: 17 ± 4 nM. Thus, it was legitimate to use the data from positions –7, –6 and –5 to estimate the potency index necessary to achieve a conservative P < 0.005 in a two-tailed t-test, which was ~50 nM.


View this table:
[in this window]
[in a new window]
 
Table 1. Statistical data derived from sample experiments

 


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 2. Inhibitory potency for single base variants of IN-ODN 2114 in B cells. B cells were cultured with 100 nM strong Type B (K) ODN 2084 which produced >90% of maximal apoptosis protection, G1 entry and IL-6 secretion by 18 h. Each of the 60 possible single-base variants of prototype IN-ODN 2114 were also tested over a two-log range of concentrations. The concentration of each IN-ODN providing 50% inhibition was determined, and these values were averaged for three experiments to give a mean potency for each assay. Values >1000 nM or <10 nM could usually be determined by extrapolation, though some concentrations outside this range were tested when needed. Symbols of the base that differed from the prototype (A, adenosine; C, cytosine; T, thymidine; G, guanidine) indicate the potency index (geometric mean of the average potencies for each of the three assays). The backbone is deoxyphosphorothioate. Sequence of the prototype 2114 is shown as hollow letters. Based on the log of the standard deviation of the potency indices for the data from positions –7 to –5, it was possible to estimate the potency index required to differ from that of ODN 2114 with a P < 0.005 to be ~50 nM.

 
Figure 2 plots the potency index for each of the 60 ODN differing by one base from the prototype IN-ODN 2114. Remarkably, single base substitutions changed potency radically at only three pairs of positions, designated ‘active areas’: area 1 at positions –6 and –5, area 2 at positions –3 and –2 and area 3 at positions +2 and +3. Base changes had little or no effect (four potencies within a 3-fold range) at –7, –4, –1, +1 and +5 to +8, although the G at –4, T at +1 and A at +4 and +7 were slightly less potent than the prototype. Table 2 presents the t-test results for these positions, showing that only the G to A change at +4 was significant at the P < 0.005 level. The potency indices closely approximated the mean calculated from individual data points in each instance (Table 2). As noted earlier (7), CG was not required for inhibitory activity.


View this table:
[in this window]
[in a new window]
 
Table 2. t-Tests on selected IN-ODN comparisons

 


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3. Inhibitory potency for single base variants of IN-ODN 2114 in non-B cells. Splenocytes were cultured with 100 nM Type A ST-ODN 1585 to which the single base variants of IN-ODN 2114 were added at a range of concentrations, as in Fig. 2. IL-12p40 secretion was measured at 18 h by ELISA. Positions +5 to +8 were omitted because omitting these bases did not alter IN-ODN activity for splenocytes (Table 4). Mean potencies for three experiments are shown with the variant bases identified as in Fig. 2. Sequence for ODN 1585 is GGggtcaacgttgagGGGGG, where capitals represent phosphorothioate backbone and lower case represents phosphodiester backbone. Sequence of the prototype IN-ODN 2114 is shown as hollow letters.

 
Remarkably, none of the variants was more potent than the prototype 2114 and its CG-containing partner 2088, which were derived from a strong ST-ODN by changes in area 3. The ODN with the worst choice at all six ‘active’ positions had <2% of the activity of 2114. An ODN with the 2114 base sequence arranged in the reverse direction was only 10% as potent as 2114 (potency index 195 nM).

Cell type and ODN type comparison
Believing that the marked ODN sequence preferences revealed in Fig. 2 could be interpreted as revealing the arrangement of ODN-binding regions on a putative ODN receptor, we wondered whether ODN inhibiting Type B (K) ST-ODN in B cells would show the same sequence preferences when inhibiting Type A (D) ST-ODN in non-B cells or indeed whether any inhibition would be observed. B cell depletion experiments showed that <10% of IL-12p40 produced in ODN-stimulated cultures of unseparated splenocytes was derived from B cells, implying that the other two ODN-responsive cell types (macrophages and dendritic cells) were the main source (data not shown). Figure 3 shows a plot of the effect of single base changes from position –7 to +4 on the potency of IN-ODN for inhibiting IL-12p40 secretion in spleen cells responding to 100 nM Type A (D) ODN 1585 (6). We did not study positions +5 to +8 because our previous study showed that they were not contributory (19).


View this table:
[in this window]
[in a new window]
 
Table 4. Truncation experiment

 
In general, 2114 and the other sequences which were strong inhibitors in B cells responding to Type B (K) ST-ODN in Fig. 2 were also strong inhibitors in splenocytes making IL-12p40 in response to Type A (D) ST-ODN. As in B cells (Fig. 2), active areas appeared at –6, –5, at –2 and at +2, +3 (Fig. 3). The best bases were C at –6, C at –5, G at –3, G or T at –2, G at +2 and G at +3 in both B cells (Fig. 2) and splenocytes (Fig. 3). The worst base was T at –6, G at –5, C at –3, C at +2 and C at +3 in both cell types. Positions –7, –4, –1 and +4 were inactive in both cell types. An alternative method for displaying how sharply the potency differences associated with single base changes varied from position to position was to simply calculate the standard deviation of the four potency values (Supplementary data are available at International Immunology Online). The same three active areas stand out in B cells as in mixed splenocytes. Despite the remarkable similarity in sequence requirements for inhibition between cell types and ODN types, there was a 10-fold greater range of potency changes caused by single base differences in splenocytes (contrast Figs 2 and 3). Variation at –3 was significant only in B cells. The ODN with C at +1 was significantly less potent (P ~ 0.002) only for splenocytes.

Result of changing both bases at active areas
Substituting CC with AA in active area 1 (–6, –5) produced a profound loss of activity to 6% in B cells (Table 3), but no greater than that seen with a single A at –5. Similarly, the CC substitution at area 2 (–3, –2) or area 3 (+2, +3) produced a loss of activity similar to that caused by a C at –2 or at +2, respectively. Substitutions at +7, +8 (4137) were inconsequential, like the single base changes, marking this as an area that does not interact with the receptor (Table 3). When area 3 was changed to meet neither the requirements for ST-ODN (CGTT at –1 to +3) or for IN-ODN (GGG either at +1 to +3 or +2 to +4), a ‘neutral’ ODN 2310 resulted with <0.5% inhibitory activity. GG at –5, –6 partially compensated for the effect of CC at –2, –3 (4234 versus 4141), suggesting a possible contribution from CC to GG association.


View this table:
[in this window]
[in a new window]
 
Table 3. Two-base changes and IN-ODN potency

 
Substitution of Cs for Gs at both +1 and +4, two inactive positions, produced an informative result, a loss of almost 99% of activity in B cells (Table 3), in sharp contrast to the slight effect of single base substitutions at those positions (Fig. 2), and similar to the result of CC at +2, +3. An alternative approach leading to the same conclusion was performed with IL-12p40 production driven by Type A ST-ODN 1585 in mixed spleen cells, showing that if there were G at +4, it did not matter whether +1 had G like 2114, or T (100 and 114% of 2114's activity, respectively). But if +4 had A or C, then +1 must have G, not T, to retain activity (26% activity for A +4, G +1, 4% for A +4, T +1, 28% for C +4, G +1 and 1% for C +4, T +1). Taken together, these experiments suggested that full inhibitory activity required three consecutive Gs somewhere in the +1 to +4 interval, so area 3 is actually three bases wide.

Truncation experiments: what is the shortest active IN-ODN?
Trimming the T at –7 and the GT at +7, +8 from the ends of 2114 made no difference to the ability to inhibit ST-ODN 2084 in B cells (4104 versus 2114 in Table 4). Loss of the A at +6, an inactive position, reduced activity 40% or less (compare 4177 with 4104 and 4178 with 4105). Loss of the A at +5 produced a further moderate loss of activity (compare 4171 with 4177 and 4178 with 4203). In contrast, the loss of C at –6 at the 5' end (part of active area 1) caused >99% of activity to be lost (4103 versus 4177, or 4172 versus 4171). The shortest ODN with activity not significantly <2114 was the 11-mer 4203. Similar experiments were run with ODN 1585-treated splenocytes (Table 4). Truncating +5 to +8 from the 3' end or T from the 5' end had no significant effect on inhibitory potency. But as with B cells, loss of C at –6 caused profound activity loss in splenocytes.

Duplication and spacing of active areas
Using ODN 4105 as the prototype (equipotent with 2114 in Table 4), we showed that covalent linkage of two 4105 sequences in cis was twice as active as one on a molar basis (Fig. 4). Duplication of area 1 in 2114 with one A as spacer also made an IN-ODN twice as potent on a molar basis (Fig. 4). Duplication of areas 2 or 3 with one spacer greatly reduced activity (Fig. 4). Additional data posted on the web show that increasing the spacing between 4105 sequences or the duplicated area 1 sequences to five spaces had no effect on activity, whereas increased spacing between duplicated areas 2 or 3 tended to restore activity. Increasing the space between areas 1 and 2 or between 2 and 3 reduced activity (Fig. 4), and increased spacing to three spacers had no further effect. Thus, the spacing between areas 1 and 3 appeared to be most critical for B cells, as previously noted for splenocytes (20).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4. Effects of active area duplication and spacing on activity of IN-ODN 2114. B cells were cultured with 100 nM ST-ODN 2084 with and without a concentration range of the IN-ODN variants of 4105. (The potency of 4105 is equal to 2114, as shown in Table 4.) Mean of three experiments. Error bars represent SEM. Vertical line is the potency index. Dotted line is the potency index for the prototype 4105. Active areas are indicated by boxes and numbered.

 

View this table:
[in this window]
[in a new window]
 
Table 5. Re-examination of published inhibitory sequences

 


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. Hypothetical model of competition between ST-ODN and IN-ODN. The ODN receptor (TLR9 ?) is drawn so as to present two binding loops a critical distance apart, as might occur at acid pH in the lysosomal compartment (14). One loop engages area 1 of both ST- and IN-ODN at site {alpha}. The other recognizes unmethylated CGTT in area 3 of ST-ODN at site ß, creating the {varepsilon} binding site for MyD88, and generates the signal continuously over time as long as the complex remains assembled. However, if GGG is present in area 3 in place of GTT or TTx, the ODN is inhibitory, engaging the receptor at site {gamma} so as to prevent the formation of the MyD88-binding site {varepsilon}. However, a C at –2 or –2, –3 causes the ODN to bind at another site {delta}, which prevents it from signaling, inhibiting or competing with ST-ODN, as in neutral ODN 2310. If ODN and MyD88 bind on opposite sides of the endosomal membrane, a more complex model involving aggregation of TLR9 with itself or other molecules would be favored. In this ‘aggregation version,’ the {alpha} site would be on one TKR9 molecule, and the ß and {gamma} sites on another molecule (R. F. Ashman and P. Lenert, manuscript in preparation).

 
Testing other published inhibitory sequences
Using mouse splenocyte IL-12p40, IL-6 and IFN-{gamma} as readouts, Yamada et al. (21) showed inhibition at a 1 : 1 molar ratio of two inhibitors to two ST-ODN containing ACGTT (like 2084). Table 5 shows that their two best inhibitors (H154 and 1502) also have substantial activity for inhibiting B cell cycle entry and apoptosis resistance (78 and 67% of 2114's activity, respectively). We confirmed that H154 also has activity in mixed spleen cells comparable to 2114. H154 has area 1 (CC at the 5' end, –6 –5) and a 4G area 3 (+7 to 10), but these were 10 bases apart. In our series, increasing the distance between most 3' areas 1 and 3 by even one or two bases decreased activity (4179, 4183, 4194 and 4199 in Fig. 4), but a further increase to 13 bases restored activity (4202 in Fig. 4). When we gave H154 areas 1 and 3 at the right interval (4248), activity improved substantially, whereas if we removed a potential area 1 at –6–5 (4250) or destroyed a potential area 3 at +7 to 10 (4251), the activity was reduced. Providing an extra ‘area 1’ in the middle (+1+2) did not increase activity (4249) nor was activity decreased when we inserted CC in the area 2 position (+4+5) to go with an area 1 at +1+2 and area 3 at +7 to 10 (4252), suggesting that the area 1 at +1+2 was acting together with the 3' area 3 (+7 to 10) in H154. Likewise with IN-ODN 1502 (21), which lacks area 1 and 3, providing them at the right interval (–6–5 and +1 to +3) enhanced activity (4244), and deleting two bases –8–7 so area 1 was at the 5' end was even better (4245). Placing CC in the ‘area 2’ position (–3–2) cut activity in half (4259) and also deleting the 5' area 1 (–6–5) cut it in half again (4258), as predicted from our experience with 2114. Another sequence shown to be inhibitory (22) is the triple telomeric hexamer (4000), which had ~59% of 2114's activity in B cells (Table 5). It has the GGG sequences of three area 3s, and ‘not C, not C’ for area 2, but no obvious area 1. When we gave it area 1 in the form of two Cs at the right interval (five bases at –6–5 or six bases at –7–6) from one area 3 (+1+2+3 in 4240, 4241), or when we gave it two area 1s (4243), the activity increased to 83–93% (though not with statistical significance), whereas, when we put the two Cs in the three positions for area 2 determined by the three area 3s (4242), the activity dropped to 10% (significant). Thus, even with sequences that do not appear to share all the features of our model, increasing conformity with our model increased activity, and decreasing conformity had the opposite effect.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
TLR9 is a strong candidate to be the ODN-recognizing molecule. TLR9-deficient cells fail to respond to stimulation by CpG ODN, but can respond to LPS, which utilizes TLR4 (11). Replacing TLR9 by transfection into such TLR9-deficient cells restores ODN responsiveness (11, 13, 23). Thus, TLR9 is necessary for response to ODN. TLR9 also binds DNA (14, 15) and confers species-specific preferences in ODN sequence recognition (13), all of which are consistent with the concept that TLR9 is the ODN receptor. However, it remains possible that mouse and human TLR9, with their adapter MyD88, interact with another molecule that recognizes detailed ODN sequence differences and that the conformation of this ODN-receptor complex accounts for species specificity. One need look no further than LPS for an example of a ligand whose recognition by other proteins precedes engagement of its TLR (24).

This paper defines the ODN-base sequence preferences of the molecule that binds IN-ODN in mouse cells, which may or may not be TLR9. As with ST-ODN, the titration curves are generally symmetrical sigmoid curves (Fig. 1; Supplementory data are available at International Immunology Online), indicating that the biologic effects measured (apoptosis protection, cell cycle entry and IL-6 secretion) are controlled by a homogeneous population of IN-ODN-recognizing sites. Figure 1 demonstrates that inhibition by IN-ODN is fully competitive and reversible. When an ST-ODN is twice as potent as another ST-ODN, twice as much IN-ODN was required to overcome it (Fig. 1A). Adding more ST-ODN to IN-ODN restored full activity, with a titration curve parallel to the curve seen with ST-ODN alone (Fig. 1B). Several other similar examples have been observed (not shown). These parallel symmetrical sigmoid curves, which shift according to the relative potency of IN-ODN and ST-ODN, behave as if ST-ODN and IN-ODN are engaged in affinity-driven competition for the same set of sites, in accord with the Mass Action Law. The ST-ODN-receptor complex would then initiate a signal cascade, but the IN-ODN-receptor complex would not. However, it is also possible that the competition occurs between two downstream signal intermediates generated by ST-ODN and IN-ODN through different receptors. Excluded are models where IN-ODN leads to inactivation or degradation of the ODN receptor, or act at more than one site with different sensitivities.

Other evidence also favors a proximal site of action for IN-ODN. Apoptosis protection, cell cycle entry and IL-6 secretion showed striking fidelity to the same relative potency of the various stimulatory and inhibitory sequences (7; Fig. 4; Supplementary data are available at International Immunology Online), suggesting that these biologic effects are controlled by the same proximal signal. The nuclear accumulation of relevant transcription factors NF{kappa}B, AP-1 and NF-IL-6 in ST-ODN-activated B cells was prevented by IN-ODN (25; P. Lenert, unpublished data) pointing to a site proximal to the generation of these factors. Furthermore, IN-ODN failed to inhibit the induction of NF{kappa}B and AP-1 by LPS or anti-CD40 (25). Confocal microscopic evidence shows that IN-ODN prevents MyD88 from being recruited to endosomal vesicles by ST-ODN (23).

Whichever molecule is finally proven to mediate sequence-specific recognition of IN-ODN, the experiments reported here provide much detail about its ODN-binding properties. Beginning with the prototype sequence of the strong 15-mer IN-ODN 2114 (7), we compared every ODN sequence one base different from the prototype for their ability to inhibit 100 nM of the strong stimulator ODN 2084.

Potency indices of ~50 nM were significantly different from 2114 at the P = 0.005 level chosen as appropriate for the large number of comparisons. By this criterion, only three pairs of positions had single base variants with potencies significantly different from the prototype (Fig. 3), which we called active areas 1 (–6, –5), 2 (–3, –2) and 3 (+2, +3). We hypothesize that these are the areas of IN-ODN which interact with the ‘ODN receptor’. All the other positions contributed little to IN-ODN activity, with rare exceptions (Table 2). None of the 60 variants was significantly more active than the prototype 2114. The CG variant ODN 2088, often used previously as an IN-ODN (9, 26, 27), was among the 33 variants (out of 60) with activity equivalent to 2114, which lacks a CG. The most active IN-ODN sequences had the fact that they were derived by minimal changes from the strongest ST-ODN, including a GG for TT substitution at area 3 (positions +2, +3) in common. Thus, we learn that active area 3 determines whether a signal is sent or not by the ODN-receptor complex. The other two active areas appear to determine potency and can be identical for strong ST-ODN and strong IN-ODN (7). We suggest that they may determine avidity of ODN-receptor binding. It is possible that this binding step determines the secondary structure or configuration of molecular aggregates that is necessary for activity.

Other conclusions regarding structural requirements are as follows: (i) changing both bases at an active area did not reduce activity much more than the most influential single base change (Table 3). (ii) The distance between active areas was important for activity (Fig. 4), and so was the sequence direction (5' to 3' versus 3' to 5'). (iii) Two IN-ODN sequences linked in cis were both active (Fig. 4), suggesting that ODN-binding sites may be close together. (iv) Evidence from base changes and truncation indicates that the sequence length and spacing requirements for inhibition were extremely similar regardless of cell type, ST-ODN type or readout assay (Figs 3 and 4, data not shown; 20). A more careful contrast of dendritic cells with macrophages is needed to confirm this point. (v) Minimum requirements for measurable IN-ODN activity are areas 1 and 3 with a length of 10 bases, though 12 is better (Table 4).

Another purpose for defining the optimal IN-ODN sequence is to develop a possible antidote for the symptoms of excessive ODN-induced cytokine effects which might be encountered in clinical trials. IN-ODN block ODN-driven cytokine secretion in vivo (22). ST-ODN injected into mouse joints elicits an acute synovitis (28), which can be prevented if IN-ODN or mammalian DNA is injected together with the ST-ODN (29).

Before this study, our knowledge of the structural requirements for inhibition was rudimentary. The concept of inhibitory sequences originated with the discovery that adenovirus type 2 with abundant CG-rich sequences inhibited the response to other adenovirus vectors used in DNA vaccines which contained CpG motifs (30). But several CG-rich ODN only partially suppressed IL-12 secretion by mouse splenocytes at a 10 : 1 molar excess (31), no better than poly-T (31) and far weaker than 2114 or 2088. This sequence-non-specific inhibitory ‘phosphorothioate backbone effect’ occurs in the micromolar range (32, 33), at a 20 to 100-fold weight ratio of inhibitor to stimulator (17, 18), outside the range we use (7, 9, 20). The extended titration curves support the authors' hypothesis that several mechanisms are at work in the micromolar range, including interference with DNA uptake and a downstream block which also affects LPS-induced activation (17, 18). So far, no B cell data using these conditions have been published, but we normally do not titrate our IN-ODN >20-fold molar excess over ST-ODN because we also observe sequence-non-specific inhibition in that range. With 100 nm ST-ODN 2084 as our optimal reference stimulus, typically, 50% inhibition of G1 entry required 15–20 nm 2114, whereas inhibition of IL-6 production required 5–7 nm and apoptosis protection 40–50 nm (Supplementary data are available at International Immunology Online). Under these conditions, stimulation by LPS, anti-CD40 or anti-µ +IL-4 is not inhibited, and neither is ODN uptake (7, 25; R. Ashman and P. Lenert).

How can we account for the activity of other published IN-ODN (21, 22) which share some but not all of the active areas of our prototype IN-ODN 2114? (i) Table 5 shows numerous examples where making three of these ODN more like our prototype increased activity and removing the features they already shared with our prototype decreased activity. (ii) These ODN may be using a different set of contact residues on the ODN receptor, perhaps involving backbone interactions (32, 33), like unrelated epitopes which bind the same antibody site. (iii) ODN secondary and tertiary structure may be important for binding. Oligo dG sequences may undergo hydrogen-bonded multimer formation, whose shape may be altered by other sequence features in unpredictable ways (34). 7-Deazaguanosine (7dG) substitution for G disrupts this configuration. We have shown that 7dG substitution at +1 has no effect on ST-ODN activity (7), but Gursel et al. showed that 7dG in a poly-G region disrupts IN-ODN activity (22). (iv) One lesson from the high proportion of sequence variants of 2114 that inhibit as well as 2114 itself (Figs 3 and 4) is that inhibitory sequences may be common. Several octamers from the center of IN-ODN have been shown to be more frequent in mouse than in Escherichia coli DNA (6-fold for 2114 –35). Probably, the abundance of inhibitory sequences and scarcity of stimulatory sequences contributes, along with CpG methylation, and the high frequency of telomere repeats to the net inhibitory activity of mammalian DNA (35).

Figure 5 presents a hypothetical model of the binding site of the ODN receptor, as an aid to future investigation, incorporating these features derived from our data: (i) evidence for affinity-driven competition and the monotonic sigmoid titration curves (Fig. 1) suggests that ST-ODN and IN-ODN compete for binding to a single species of site on the ODN receptor. Since the 5' half of strong ST-ODN and IN-ODN can be the same (7), and since IN-ODN must have two Cs at area 1, we have drawn a site {alpha} for area 1 on one loop of the receptor. (ii) Whereas area 3 of IN-ODN must have (G) GG (Fig. 3, Table 4), and ST-ODN have TT preceded by CG (2, 35), the receptor must have a separate site for each of these ({gamma} and ß, respectively). (iii) When ST-ODN bind one loop of the receptor by area 3 and another by area 1, a docking site {varepsilon} for MyD88 is created, which is missing in the unbound conformation. The 100-fold increased potency of ST-ODN with phosphorothioate backbones over those with more flexible phosphodiester backbones (36, 37) is explained by the need for a stable MyD88-binding site. The MyD88-ODN-receptor complex then generates the signal. (iv) This two-loop model explains how ODN possessing only area 1 or only area 3 fail to inhibit because of their valence disadvantage relative to ODN with both areas (Figs 2 and 3, Table 4), and also the importance of proper spacing between areas 1 and 3 (Fig. 4). (v) When position –2 has C, the ODN neither stimulates nor inhibits (2310 in Table 3), even if it has areas 1 and 3. This prohibition applies to ST-ODN in the phosphodiester format as well (3). C at –2 must make the ODN bind elsewhere ({delta}) so it cannot reach the sites {alpha} and ß, and also CC at –2, –3 must have an environment that keeps it from binding directly to {alpha}. (vi) ODN receptors must be able to approach each other closely to explain how the ‘2 motif’ (4186) and ‘double area 1’ (4191) ODN inhibit better than the prototype. (vii) With recent evidence that TLR binds ODN (14, 15), the likelihood is that the ‘ODN receptor’ in this scheme is TLR9.

An abundance of alternative more complex models exist involving accessory molecules as well as an ODN receptor. Since TLR9 is an endosomal membrane protein, ODN act while inside the endosomal vesicle, and MyD88 appears to interact outside the vesicle, an aggregation model is attractive. Most cases where the consequences of molecular interaction appear to cross a membrane involve aggregation. The site {gamma} for area 3 of IN-ODN could be on a second molecule that blocks the MyD88 site. The site {delta} that sequesters ODN with C at –2 could be on an accessory molecule. The MyD88-binding site {varepsilon} could be composed of parts of two molecules instead of one, one containing the area 1 site {alpha} and the other the CGTT site B in Fig. 5. Both of these molecules could be TLR9, or one could be TLR9 and the other an accessory molecule. The cell-type-specific preference for Type A or B ST-ODN also calls for an accessory molecule, in view of the lack of evidence for cell-type-related TLR9 isoforms. The presence of the palindrome within the flexible CGTT Type A (D) ODN 1585 has led to the conjecture that it forms a loop displaying the central CGTT. ODN 1585 does not have CC at –6, –5, yet the IN-ODN that block its action in non-B cells still need this feature (19 and Fig. 3). Perhaps the phosphorothioate poly-G ends of 1585, with or without aggregation, substitute for area 1 by binding another site on the same loop with the same conformational effect. It will be of great interest to study the affinity of various ODN for recombinant TLR9 protein, which may be expected to be acid pH-dependent, with or without accessory molecules, and to apply quantitative physical chemistry and crystallography to understanding this important interaction.


    Supplementary data
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Supplementary data are available at International Immunology Online.


    Acknowledgements
 
The authors are grateful to Jill Kinnaird and Deanna Ollendick for their expert manuscript preparation, Rachel Brummel for technical assistance and Bridget Zimmerman for statistical consultation. The work was supported by National Institutes of Health grant R01AI/GM 47374-01A2 to R.F.A. and equipment from the Department of Veterans Affairs.


    Abbreviations
 
AO   acridine orange
AP-1   activation protein-1
HEPA   high efficiency particulate air
7dG   7-deazaquanosine
IN-ODN   inhibitory oligonucleotide
NF{kappa}B   nuclear factor {kappa}B
ODN   oligonucleotide
ST-ODN   stimulatory oligonucleotide
TLR   Toll-like receptor

    Notes
 
Transmitting editor: H. Kikutani

Received 1 November 2004, accepted 14 January 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

  1. Messina, J. P., Gilkeson, G. S. and Pisetsky, S. D. 1991. Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA. J. Immunol. 147:1759.[Abstract/Free Full Text]
  2. Krieg, A. M., Yi, A.-K., Matson, S. et al. 1995. CpG motifs in bacterial DNA trigger direct B cell activation. Nature 374:546.[CrossRef][ISI][Medline]
  3. Yi, A.-K., Chang, M., Peckham, D. W., Krieg, A. M. and Ashman, R. F. 1998. CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry. J. Immunol. 160:5898.[Abstract/Free Full Text]
  4. Verthelyi, D., Ishii, K. J., Gursel, M., Takeshita, F. and Klinman, D. 2001. Human peripheral blood cells differentially recognize and respond to two distinct CpG motifs. J. Immunol. 166:2372.[Abstract/Free Full Text]
  5. Krug, A., Rothenfusser, S., Hornung, V. et al. 2001. Identification of CpG oligonucleotide sequences with high induction of INF-a/ß in plasmacytoid dendritic cells. Eur. J. Immunol. 31:2154.[CrossRef][ISI][Medline]
  6. Ballas, Z. A., Rasmussen, W. L. and Krieg, A. M. 1996. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J. Immunol. 157:1840.[Abstract]
  7. Stunz, L. L., Lenert, P., Peckham, D. et al. 2002. Inhibitory oligonucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells. Eur. J. Immunol. 32:1212.[CrossRef][ISI][Medline]
  8. Hartmann, G., Battiany, J., Poeck, H. et al. 2003. Rational design of new CpG oligonucleotides that combine B cell activation with high IFN-{alpha} induction in plasmacytoid dendritic cells. Eur. J. Immunol. 33:1633.[CrossRef][ISI][Medline]
  9. Lenert, P., Stunz, L., Yi, A.-K., Krieg, A. M. and Ashman, R. F. 2001. CpG stimulation of primary mouse B cells is blocked by inhibitory oligodeoxyribonucleotides at a site proximal to NF-kappaB activation. Antisense Nucleic Acid Drug Dev. 11:247.[CrossRef][ISI][Medline]
  10. Lenert, P., Yi, A.-K., Krieg, A. M. Stunz, L. L. and Ashman, R. F. 2003. Inhibitory oligonucleotides block the induction of AP-1 transcription factor by stimulatory CpG oligonucleotides in B cells. Antisense Nucleic Acid Drug Dev. 13:143.[CrossRef][ISI][Medline]
  11. Hemmi, H., Takeuchi, O., Kawal, T. et al. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740.[CrossRef][ISI][Medline]
  12. Schnare, M., Holt, A. C., Takeda, K., Akira, S. and Medzhitov, R. 2000. Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88. Curr. Biol. 10:1139.[CrossRef][ISI][Medline]
  13. Bauer, S., Kirschning, C. J., Häcker, H. et al. 2001. Human TLR-9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl Acad. Sci. USA 98:9237.[Abstract/Free Full Text]
  14. Latz, E., Schoenemeyer, A., Visintin, A. et al. 2004. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5:190.[CrossRef][ISI][Medline]
  15. Rutz, M., Metzger, J., Gellert, T. et al. 2004. Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur. J. Immunol. 34:2541.[CrossRef][ISI][Medline]
  16. Cornélie, S., Hoebeke, J., Schacht, A. et al. 2004. Direct evidence that Toll-like receptor 9 (TLR9) functionally binds plasmid DNA by specific cytosine-phosphate-quanine motif recognition. J. Biol. Chem. 279:15124.[Abstract/Free Full Text]
  17. Zhu, F. G., Reich, C. F. and Pisetisky, D. S. 2002. Inhibition of murine dendritic cell activation by synthetic phosphorothioate oligodeoxynucleotides. J. Leukoc. Biol. 72:1154.[Abstract/Free Full Text]
  18. Zhu, F. G., Reich, C. F. and Pisetsky, D. S. 2002. Inhibition of murine macrophage nitric oxide production by synthetic oligonucleotides. J. Leukoc. Biol. 71:686.[Abstract/Free Full Text]
  19. Traganos, F., Darzynkiewicz, Z., Sharpless, T. and Melamed, M. R. 1977. Simultaneous staining of ribonucleic acid and deoxyribonucleic acid in unfixed cells using acridine orange in a flow cytometric system. J. Histochem. Cytochem. 25:46.[Abstract]
  20. Lenert, P., Rasmussen, W., Ashman, R. F. and Ballas, Z. K. 2003. Structural characterization of the inhibitory DNA motif for the type A D-CpG-induced cytokine secretion and NK-cell lytic activity in mouse spleen cells. DNA Cell Biol. 22:621.[CrossRef][ISI][Medline]
  21. Yamada, H., Gursel, I., Takeshita, F. et al. 2002. Effect of suppressive DNA on CpG-induced immune activation. J. Immunol. 169:5590.[Abstract/Free Full Text]
  22. Gursel, I., Gursel, M., Yamada, H., Ishii, K. J., Takeshita, F. and Klinman, D. M. 2003. Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation. J. Immunol. 171:1393.[Abstract/Free Full Text]
  23. Takeshita, F., Leifer, C. A., Gursel, I. et al. 2001. Role of Toll-like receptor 9 in CpG DNA-induced activation of human cells. J. Immunol. 167:3555.[Abstract/Free Full Text]
  24. Shimazu, R., Akashi, S., Ogata, H. et al. 1999. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J. Exp. Med. 189:1777.[Abstract/Free Full Text]
  25. Ashman, R. F., Goeken, J. A., Drahos, J. and Lenert, P. 2004. Sequence preferences for inhibitory oligodeoxyribonucleotides. Immunology 2004, Collection of Free Papers presented at the 12th Int. Congress of Immunology. Medimond S.r.l. Bologna Italy, pp. 125–128.
  26. Leadbetter, E. A., Rifkin, I. R., Hohlbaum, A. H., Beaudette, B., Shlomchik, M. J. and Marshak-Rothstein, A. 2002. Immune complexes activate autoreactive B cells by co-engagement of sIgM and Toll-like receptors. Nature 419:603.[CrossRef][ISI][Medline]
  27. Viglianti, G. A., Lau, C. M., Hanley, T. M., Mido, B. A., Shlomchik, M. J. and Marshak-Rothstein, A. 2003. Activation of autoreactive B cells by CpG dsDNA. Immunity 19:837.[ISI][Medline]
  28. Deng, G.-M., Nilsson, I.-M., Vergrengh, M., Collins, L. V. and Tarkowski, A. 1999. Intra-articularly localized bacterial DNA containing CpG motifs induces arthritis. Nat. Med. 5:702.[CrossRef][ISI][Medline]
  29. Zeuner, R. A., Ishii, K. J., Lizak, M. J. et al. 2002. Reduction of CpG-induced arthritis by suppressive oligodeoxynucleotides. Arthritis Rheum. 46:2219.[CrossRef][ISI][Medline]
  30. Krieg, A. M., Wu, T., Weeratna, R. et al. 1998. Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. Proc. Natl Acad. Sci. USA 95:12631.[Abstract/Free Full Text]
  31. Zhao, H., Cheng, S. H. and Yew, N. S. 2000. Requirements for effective inhibition of immunostimulatory CpG motifs by neutralizing motifs. Antisense Nucleic Acid Drug Dev. 10:381.[ISI][Medline]
  32. Sester, D. P., Naik, S., Beasley, S. J., Hume, D. A. and Stacey, K. J. 2000. Phosphorothioate backbone modification modulates macrophage activation by CpG DNA. J. Immunol. 165:4165.[Abstract/Free Full Text]
  33. Pisetsky, D. S. and Reich, C. F. 1999. Influence of backbone chemistry on immune activation by synthetic oligonucleotides. Biochem. Pharmacol. 58:1981.[CrossRef][ISI][Medline]
  34. Williamson, J. R. 1994. G-Quartet structures in telomeric DNA. Annu. Rev. Biophys. Biomol. Struct. 23:703.[CrossRef][ISI][Medline]
  35. Stacey, K. J., Young, G. R., Clark, F. et al. 2003. The molecular basis for the lack of immunostimulatory activity of vertebrate DNA. J. Immunol. 170:3614.[Abstract/Free Full Text]
  36. Yi, A., Chang, M., Peckham, D. W., Krieg, A. M. and Ashman, R. F. 1998. CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry. J. Immunol. 160:5898.[Abstract/Free Full Text]
  37. Stein, C. A., Subasinghe, C., Shinozuka, K. and Cohen, J. S. 1988. Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res. 16:3209.[Abstract]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
17/4/411    most recent
dxh222v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Request Permissions
Google Scholar
Articles by Ashman, R. F.
Articles by Lenert, P.
PubMed
PubMed Citation
Articles by Ashman, R. F.
Articles by Lenert, P.