HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Parronchi, P. Articles by Maggi, E. Search for Related Content PubMed PubMed Citation Articles by Parronchi, P. Articles by Maggi, E.

Phosphorothioate Oligodeoxynucleotides Promote the In Vitro Development of Human Allergen-Specific CD4⁺ T Cells into Th1 Effectors¹

Department of Internal Medicine, Immunoallergology and Respiratory Disease Unit, University of Florence, Florence, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
DNA vaccination is an effective approach in inducing the switch of murine immune responses from a Th2 to a Th1 profile of cytokine production that has been related to the activity of unmethylated CpG motifs present in bacterial, but not mammalian, DNA. We report here that some synthetic phosphorothioate, but not phosphodiester, oligodeoxynucleotides (ODNs) were able to induce B cell proliferation and to shift the in vitro differentiation of Dermatophagoides pteronyssinus group 1-specific human CD4⁺ T cells from atopic donors into Th cell effectors showing a prevalent Th1, instead of Th2, cytokine profile. This latter effect was completely blocked by the neutralization of IL-12 and IFN ( and ) in bulk culture, suggesting that the Th1-inducing activity of phosphorothioate ODNs was mediated by their ability to stimulate the production of these cytokines by monocytes, dendritic, and NK cells. Cytosine methylation abolished the Th1-inducing activity of ODNs; however, CpG dinucleotide-containing ODNs exhibited the Th1-shifting effect independently of the presence or the absence of CpG motifs (5'-pur-pur-CpG-pyr-pyr-3'). Moreover, the inversion of CpG to GpC resulted only in a partial reduction of this activity, suggesting that the motif responsible for the Th1-skewing effect in humans is at least partially different from that previously defined in mice. These results support the concept that the injection of allergens mixed to, or conjugated with, appropriate ODNs may provide a novel allergen-specific immunotherapeutic regimen for the treatment of allergic disorders.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Allergic disorders in humans are characterized by a prevalent Th2 effector response to "innocuous" environmental Ags (allergens) (1, 2, 3). Allergen-specific Th2 cells produce IL-4 and IL-13, which are responsible for the IgE isotype switching (4, 5, 6), IL-10, which, like as IL-4, favors the growth of mast cells (7, 8), and IL-5, which promotes the differentiation, activation, and in situ survival of eosinophils (9, 10). Thus, allergen-specific Th2 responses account for the joint involvement of IgE-producing B cells, mast cells, and eosinophils in the allergic inflammation (reviewed in Ref. 11).

Immunization with Ag-encoding plasmid DNA has generated great interest, especially for its ability to provide a highly favorable microenvironment for the preferential development of Ag-reactive T cells into Th1 effector cells (12). Therefore, DNA vaccination has been attempted, and proved to be an effective approach, in altering the allergen-specific IgE responses in mice (13, 14). However, the effects of DNA allergen gene vaccination in humans are presently unknown, as factors contributing to DNA vaccine immunogenicity have not yet been fully clarified and the possible side effects of this type of vaccination remain, at least partially, to be defined. Recently, several reports have shown that the ability of DNA vaccines to favor the development of Th1 responses in experimental animal models is mainly due to unmethylated "CpG motifs" (5'-pur-pur-CpG-pyr-pyr-3'), which are present in bacterial, but not mammalian, DNA (12, 15). These compounds are indeed able to stimulate APC and NK cells to produce a series of immunomodulatory cytokines, including IL-12, IFN-, IFN-, and IL-18 (16, 17, 18, 19), which induce the development of both Th1 cells and Th1-dependent cytotoxic T cell responses (20, 21, 22, 23), as well as IL-6 (24), which promotes B cell activation and Ab secretion (25). Accordingly, coadministration with the Ag of oligodeoxynucleotides (ODNs)³ containing the CpG-motif (CpG-ODNs) prevented airway eosinophilia, Th2 cytokine induction, IgE production, and bronchial hyperreactivity in murine models of asthma (26, 27, 28, 29).

In this study, we have tested the activity of a series of synthetic ODNs on both human B cell proliferation and in vitro development of Dermatophagoides pteronyssinus group 1 (Der p 1)-specific T cells from atopic Der p-sensitive donors. Phosphorothioate (PS), but not phosphodiester (PE), ODNs induced not only B cell proliferation but also a dose-dependent switch from a prevalent Th2 to a prevalent Th1 cytokine profile in allergen-specific short-term T cell lines, as detected by both the cytofluorometric analysis of intracellular cytokine synthesis at single-cell level and the measurement of IFN- and IL-4 concentrations released in their supernatants. In agreement with the results reported in experimental animal models (19, 20), the Th1-inducing activity of CpG-containing PS-ODNs on allergen-specific human T cells was due to their ability to induce the production of Th1-inducing cytokines, as their effect was completely inhibited by the addition in culture of a mixture of anti-IL-12 and anti-IFNs Abs. Although cytosine methylation abolished the ODN-mediated activity, ODNs lacking the entire CpG motif also exerted a maximal effect, whereas the inversion of CpG dinucleotides to GpC resulted only in a partial reduction of the Th1-inducing activity. These findings suggest that, differently from mouse models, CpG motifs and CpG dinucleotides may not be necessarily required to evoke the ODN-mediated effects in humans.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Subjects

Blood samples used in this study were obtained from 10 informed adult atopic Der p-sensitive volunteers and four healthy volunteers in accordance with the ethical standards of the responsible regional committee on human experimentation.

Reagents

The medium used throughout was RPMI 1640 (Seromed, Berlin, Germany) supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 2 x 10^-5 M 2-ME (all from Life Technologies, Grand Island, NY), 100 µg/ml kanamycin, and 10 µg/ml gentamicin (Sigma, St. Louis, MO) (complete medium). Der p 1 allergen was kindly provided by Lofarma SpA (Milan, Italy). PMA, ionomycin, brefeldin A, and saponin were all purchased from Sigma. IL-2 was a kind gift from Eurocetus (Milan, Italy), and IL-12 was a kind gift from G. Trinchieri (Wistar Institute, Philadelphia, PA). Purified and fluorochrome-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD16, anti-CD20, and anti-CD56 mAbs were purchased from Becton Dickinson (San Jose, CA). Phycoerythrin-conjugated anti-IL-4 (3010.211, IgG1) and FITC-conjugated anti-IFN- (25723.11, IgG2b) were purchased from Becton Dickinson (San Diego, CA). Purified and fluorochrome-conjugated isotype control mAbs were purchased from Southern Biotechnology Associates (Birmingham, AL).

Synthetic ODNs

The eight ODNs used in this study, listed in Table I, were obtained from Genset (Paris, France). They were all HPLC-purified and free for endotoxin contamination. PE- and PS-ODNs, as well as PS-ODNs in which all cytosines were methylated (PS/met-ODNs), with identical base sequence, were used. The sequences 3Da, DSP30, DSP17, and 2105 have been already described and used by others (25, 30). The sequences Myco, 1326, and poly CG were assessed for the first time in this study. The sequence PS-DSP30, in which all the CpG dinucleotides were inverted in GpC (PS-DSP30/GC), was also used. CpG motifs present in the different ODNs are indicated in italic, whereas CpG dinucleotides are underlined.

PBMC from healthy volunteers were isolated by Ficoll-Hypaque gradient centrifugation and depleted of T cells, NK cells, and macrophages by treatment with anti-CD3, anti-CD16, and anti-CD14 mAbs (2 µg/10⁷ cells) followed by the addition of microbead-conjugated goat anti-mouse IgG (Miltenyi Biotec, Bisley, Germany). Cells were then separated on magnetic cell separation system columns (Miltenyi Biotec) following manifacturer’s instructions. Highly purified B cell populations (containing <1% non-B cells) were seeded at the concentration of 1 x 10⁶/ml in triplicate in U-bottomed well plates (Nunc, Nunclon, Denmark) in complete medium and 10% heat-inactivated FCS (HyClone, Logan, UT) in a 0.2-ml volume for 48 h in the absence or presence of different concentrations of ODNs (10, 5, and 0.5 µg/ml). After 16 h pulsing with 0.5 µCi [³H]TdR per well (Amersham, Little Chalfont, U.K.), cultures were harvested and radionuclide uptake measured by scintillation counting.

Production of proinflammatory cytokines by non-T cells

PBMC from healthy volunteers were isolated by Ficoll-Hypaque and then enriched for non-T cells by a rosetting technique using neuroaminidase-treated SRBC. Non-T cells (1 x 10⁶) were then seeded in 48 flat-bottom well plates (Costar, Corning, NY) in 1 ml complete medium plus 10% heat-inactivated FCS in the absence or presence of PS- or PE-DSP30 (10 µg/ml). After 72 h incubation, supernatants were collected, centrifuged, and stored at -20°C. The amounts of IL-12, IFN-, IL-6, and IL-1 receptor antagonist were evaluated by specific commercial ELISAs (Endogen, Woburn, MA and R&D Systems, Minneapolis, MN) according to manufacturer’s instructions.

Generation of short-term Der p 1-specific T cell lines

Der p 1-specific CD4⁺ T cell lines were generated as previously described (31). Briefly, PBMC were obtained from 10 atopic Der p-sensitive donors by centrifugation on Ficoll-Hypaque gradient and stimulated with Der p 1 (10 µg/ml) for 6 days in complete RPMI 1640, containing 5% heat-inactivated autologous serum, in the absence or presence of different concentrations of PS- or PE-, or PS/met-ODNs, or rIL-12 (100 U/ml). On day 6, activated T cells were expanded for subsequent 8 days by the addition of IL-2 (20 U/ml). In the inhibition experiments, neutralizing anti-IL-12 mAb alone (R&D Systems) or a mixture of anti-IL-12, anti-IFN- (Biosource, Camarillo, CA), and anti-IFN- (R&D Systems) mAbs were added at the beginning of the culture, at a concentration of 10 µg/ml. In parallel cultures, control isotype mAbs were used (Southern Biotechnology Associates).

The specificity of short-term T cell lines was assessed as already described (31). Briefly, 5 x 10⁴ T cell blasts were incubated in the presence of 5 x 10⁴ autologous irradiated PBMC, as APC, and allergen (Der p 1, 10 µg/ml) for 48 h in a 0.2-ml volume in duplicate. After a 16-h pulse with 0.5 µCi [³H]TdR (Amersham), cultures were harvested and radionuclide uptake was measured by scintillation counting. T cell lines were considered as specific when mitogenic index (MI) was 5.

Intracytofluorometric analysis of cytokine production

Intracytofluorometric analysis of IFN- and IL-4 synthesis at the single-cell level was performed as described (31, 32). Briefly, 1 x 10⁶ T cell blasts were stimulated with PMA (10 ng/ml) plus ionomycin (1 µM) for 4 h, the last two of which was in the presence of brefeldin A (5 µg/ml). After incubation, cells were washed twice with PBS, pH 7.2, fixed 15 min with formaldehyde (2% in PBS, pH 7.2), washed twice with 0.5% BSA in PBS, pH 7.2, permeabilized with PBS, pH 7.2, containing 0.5% BSA and 0.5% saponin, and then incubated with the specific mAbs. Cells were analyzed on a FACScalibur cytofluorometer using the CellQuest software (Becton Dickinson). The area of positivity was determined using an isotype-matched mAb. In all cytofluorometric analyses, a total of 10⁴ events, gated as CD3⁺CD4⁺ or CD3^-CD16⁺ cells, for each sample, were acquired.

Cytofluorometric analysis of cell-surface Ags

To evaluate the percentages of NK cells in Der p 1-specific T cell lines obtained in the absence or presence of ODNs, 2 x 10⁵ cells were collected at the time of the cytokine assessment and stained with anti-CD3, anti-CD16, anti-CD20, anti-CD14, and anti-CD56 fluorochrome-conjugated mAbs (Becton Dickinson) or with appropriate isotype control mAbs. After two washings, cells were resuspended and analyzed by flow cytometry with the use of the FACScan system (Becton Dickinson). A total of 10⁴ cells for each sample were acquired.

Measurement of IL-4 and IFN- in the supernatants of Der p 1-specific T cell lines

The ability of Der p 1-specific T cell lines to produce cytokines was evaluated following stimulation of 10⁶/ml viable T cell blasts with 10⁶/ml autologous irradiated PBMC, as APC, and Der p 1 10 µg/ml in a 1-ml volume for 72 h. IL-4 (PharMingen, San Diego, CA) and IFN- (Endogen) concentrations were measured into cell-free supernatants by homemade ELISAs using commercial pairs of mAbs, as previously described (31).

Statistical analysis

Statistical analysis of the results was performed by Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
ODNs induce human B cell proliferation

In a first series of experiments, all ODNs listed in Table I were assessed for their ability to induce the proliferation of human B cells. To this end, purified peripheral blood B cells derived from two healthy donors were incubated for 3 days with different concentrations of PS-ODNs, the corresponding PE- or PS/met-ODNs, and [³H]thymidine incorporation was assessed. As shown in Table II, all PS-ODNs (3Da, DSP30, DSP17, and 2105), already reported to act as powerful activators for murine (3Da) (25) or human (DSP30, DSP17, 2105) (30) B cells, as well as two PS-ODNs not previously assessed (Myco and 1326), induced remarkable B cell proliferation, whereas both the corresponding PE- and PS/met-ODNs did not. Table II also shows that poly CG was ineffective in inducing human B cell proliferation.

To investigate the effects of ODNs on the in vitro development of allergen-specific human T cells, two of the above mentioned PS-ODNs (PS-DSP30 and PS-DSP17) were initially used. To this end, Der p 1-specific short-term T cell lines were generated from PBMC of two atopic Der p-sensitive donors in the absence or presence of three different concentrations (0.5, 5, and 10 µg/ml) of PS-DSP30 and PS-DSP17 and assessed by flow cytometry for intracellular synthesis of IFN- and IL-4 following polyclonal stimulation with PMA and ionomycin. As positive control, the effects exerted on parallel cultures by IL-12 (a powerful Th1 inducer) (33), were evaluated. As shown in Table III, there was a dose-dependent increase in the proportion of cells expressing IFN- and a reduction in the proportion of cells expressing IL-4 in cultures conditioned with either PS-DSP30 or PS-DSP17 in comparison with unconditioned cultures. The shift to the Th1 cytokine profile observed in PS-ODN-conditioned cultures was similar to that observed in IL-12-conditioned cultures. Similar results were obtained by establishing Der p 1-specific T cell lines from PBMC of the same donors in the absence or presence of other PS-ODNs, such as 3Da, 2105, Myco, and 1326, all used at the concentration of 10 µg/ml (Table III).

View larger version (41K): [in this window] [in a new window]   FIGURE 1. Effect of PS-ODN on intracellular IL-4 and IFN- expression by Der p 1-specific T cells. Der p 1-specific short-term T cell lines were generated from PBMC of eight atopic Der p-sensitive donors in the absence or presence of IL-12 (100 U/ml), PE-DSP30, or PS-DSP30 (10 µg/ml). After 14 days, T cell blasts were stimulated with PMA plus ionomycin, as described in Materials and Methods, and intracellular cytokine synthesis was evaluated in CD3+ CD4+ T cells by cytofluorometric analysis. In the upper panel, columns represent the mean values (±SE) of cells expressing IL-4 alone (), IFN- alone (), or both IL-4 and IFN- () (**, p < 0.005; ***, p < 0.0001). In the lower panel, the cytofluorometric patterns of CD3+ CD4+ Der p 1-specific T cell blasts from one representative donor are shown.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Effect of PS-ODN on Der p 1-stimulated production of IL-4 and IFN- by Der p 1-specific T cells. Der p 1-specific short-term T cell lines were generated from PBMC of eight atopic Der p-sensitive donors in the absence or presence of IL-12 (100 U/ml), PE-DSP30, or PS-DSP30 (10 µg/ml). After 14 days, 106 T cell blasts were stimulated for 72 h with Der p 1 (10 µg/ml) in the presence of 106 irradiated autologous PBMC, as APCs. The content of IL-4 () and IFN- () (±SE) was measured into cell-free supernatants by specific ELISAs, as described in Materials and Methods. (*, p < 0.05; **, p < 0.007; ***, p < 0.005)

The possibility that the Th1 shift in the development of Der p 1-specific T cells induced by PS-ODNs was due to their ability to promote the production of Th1-inducing cytokines was then investigated. To this end, in a first series of experiments, T cell-depleted PBMC suspensions from two healthy subjects were incubated for 3 days with medium alone, PS-DSP30, or PE-DSP30 (10 µg/ml), and concentrations of IFN-, IL-12, IL-6, and IL-1 receptor antagonist released in their supernatants were measured. Only non-T cells incubated in the presence of PS-DSP30 showed a detectable increase in the production of these cytokines (data not shown). Subsequently, to determine whether an expansion of non-T cells could contribute to the Th1-shift observed in PS-ODN-modulated cultures, Der p 1-specific T cell lines generated in the presence of either PS- or PE-DSP30 were assessed for both the proportions and cytokine profile of non-T cells. As shown in Fig. 3 (upper panel), a significantly higher proportion of CD3^- CD16⁺ cells (NK cells) was observed in cultures generated in the presence of PS-DSP30 in comparison with those generated in the presence of PE-DSP30 (p < 0.005), whereas the proportions of CD3^- CD20⁺ cells (B cells) into the same cultures were comparable and low, probably due to the poor viability of B lymphocytes after 14 days of culture. CD3^-CD16⁺ cells also showed CD56 expression, which confirms their belonging to the NK cell population (data not shown). More importantly, PS-DSP30-conditioned cultures showed significantly higher proportions of CD3^-CD16⁺ cells able to synthesize intracellular IFN- in response to the stimulation with PMA plus ionomycin than PE-DSP30-conditioned cultures (Fig. 3, lower panel). No significant difference in the proliferation of PS-DSP30-conditioned cultures in response to Der p 1 plus autologous irradiated PBMC (Ag-specific proliferation) was observed, despite the increased numbers of NK cells (data not shown).

View larger version (32K): [in this window] [in a new window]   FIGURE 3. PS-ODNs expand higher proportions of CD3-CD16+ IFN--expressing cells in Der p 1-stimulated cultures. Der p 1-specific short-term T cell lines were generated from eight atopic Der p-sensitive donors and, at the time of cytokine assessement, cells were stained with anti-CD3, anti-CD16, and anti-CD20 mAbs. Mean percentages (±SE) of CD3-CD16+ cells present in PE-DSP30-conditioned () or PS-DSP30-conditioned cultures () are reported in the left of the upper panel, whereas the cytofluorometric analysis from a representative experiment is shown on the right. Mean percentages (±SE) of CD3-CD16+ cells expressing IFN- after stimulation with PMA plus ionomycin in cultures derived from the same atopic donors and conditioned with PE-DSP30 () or PS-DSP30 () are reported in the left of the lower panel, whereas hystograms from a representative experiment are shown on the right. (*, p = 0.008; **, p < 0.005)

View larger version (44K): [in this window] [in a new window]   FIGURE 4. Blocking of the Th1-inducing effect of PS-ODN by neutralization of IL-12, IFN-, and IFN- activity. Der p 1-specific short-term T cell lines were generated from two atopic Der p-sensitive donors and conditioned with PS-DSP30 in the presence of neutralizing anti-IL-12 mAb, a mixture of anti-IL-12, anti-IFN-, and anti-IFN- mAbs or isotype control mAbs (10 µg/ml), respectively. After 14 days, T cell blasts were stimulated with PMA plus ionomycin, and intracellular IL-4 and IFN- synthesis was evaluated by cytofluorometric analysis on CD3+CD4+ T cell blasts, as described in Materials and Methods.

The data reported above (Tables II and III) demonstrate that not only CpG motif-containing (3Da and 1326), but also CpG motif-lacking (DSP30, DSP17, 2105, and Myco) ODNs were able to induce both the proliferation of human B cells and the Th1-shift of allergen-specific T cells. This suggests that the presence of particular bases flanking CpG dinucleotides may not be necessarily required to promote these effects in humans. Therefore, to better clarify the nature of the molecular structures responsible for the activity of ODNs, the effect of cytosine methylation on the Th1-shifting activity of PS-DSP30 and PS-DSP17 was first assessed. In agreement with the results already reported in experimental animal models (25, 34), methylation completely abolished not only the activity of PS-ODNs on human B cell proliferation (already shown in Table II), but also their Th1-inducing ability (Table IV). In contrast, a PS-repetitive sequence of CpG, (CpG)₁₁, such as poly CG, did not affect neither human B cell proliferation (Table II) nor the differentiation of Der p 1-specific T cells (data not shown). Finally, the actual role of CpG dinucleotides as responsible for the effects of PS-ODNs was further investigated, comparing the ability of PS-DSP30 and the same ODN in which CpG dinucleotides were inverted to GpC (PS-DSP30/GC), to induce B cell proliferation and to shift the differentiation of Der p 1-specific T cells toward the Th1 profile. As shown in Fig. 5, the modified ODN retained a remarkable, even if lower, activity on both B cell proliferation (upper panel) and induction of Th1 shift in Der p 1-specific T cell lines (lower panel).

View larger version (29K): [in this window] [in a new window]   FIGURE 5. Effect of CpG inversion to GpC on the ability of PS-DSP30 to stimulate B cell proliferation and to shift the cytokine profile of Der p 1-specific T cells. Highly purified human B cells were obtained from two healthy donors and stimulated in the absence (medium) or presence of different concentrations (10, 5, and 0.5 µg/ml) of PS-DSP30 in which CpG dinucleotides were inverted to GpC (PS-DSP30/GC), as described in Materials and Methods. PE- and PS-DSP30 (10 µg/ml) were used in the same experiments as negative or positive controls, respectively (upper panel). Der p 1-specific short-term T cell lines were derived from two atopic Der p-sensitive donors in the presence of medium alone, IL-12 (100 U/ml), PE-DSP30, PS-DSP30, or PS-DSP30/GC (10 µg/ml), as described in Materials and Methods. After 14 days, T cell blasts were stimulated with PMA plus ionomycin, and intracellular cytokine synthesis was evaluated by flow cytometry (lower panel).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The results of our study first confirm those previously reported in humans, showing that certain synthetic ODNs were able to induce strong proliferative response by purified peripheral blood B cells. Such an immunostimulatory activity was usually associated with nuclease-resistant PS, but not PE, compounds and was abolished by cytosine methylation (30). More importantly, the results of our study provide the first evidence that the same synthetic ODNs active on B cells are able, at least in vitro, to shift the differentiation of allergen-specific human CD4⁺ T cells of atopic donors from a prevalent Th0/Th2-like to a prevalent Th1-like profile of cytokine production, whereas the corresponding PE-ODNs are not. The lack of effects of diester ODNs could be due to the shorter half-life in culture (4–6 h) of these compounds because of the rapid degradation by cell nucleases (30). Moreover, the interpretation of the absence of any mitogenic effects on B cells by PE-ODNs could be complicated by the fact that thymidine released by degraded ODNs may compete with [³H]thymidine used in standard proliferation assays. Nevertheless, 20-fold higher concentrations of PE-ODNs (as high as 200 µg/ml) were completely inactive on both B cell proliferation and T cell differentiation. In addition, even when a different enzymatic cell proliferation assay was used, no mitogenic effect on B cells by diester compounds could be observed (data not shown).

The Th1-inducing effect of ODNs appeared to be related to their ability to induce the production of IL-12 and IFN- by monocytes and/or dendritic cells and possibly the production of IFN- by NK cells. First, PS-ODNs, but not the corresponding PE-compounds, induced the activation of monocytes and/or dendritic cells, as indicated by the production of small, but detectable over the background, concentrations of IL-12 and IFN-, by human non-T cells. Second, at the time of cytokine assessment, a significant expansion of CD3^-CD16⁺CD56⁺, IFN--producing cells (NK cells) was observed in PS-ODN-conditioned in comparison with PE-ODN-conditioned or unconditioned cultures. Finally, and more importantly, the Th1-shifting effect of ODNs was partially inhibited by the addition in bulk culture of anti-IL-12 mAb and completely blocked by a mixture of anti-IL-12, anti-IFN-, and anti-IFN- mAbs. These findings indicate that the ability of PS-ODNs to induce the Th1-shift in allergen-specific T cells mainly resides in the stimulation of IL-12 and IFN- production by monocytes and/or dendritic cells, as well as of IFN- production by NK cells. This is consistent with our previous observations showing that cytokines produced by cells involved in the natural immune response against intracellular bacteria and some viruses (IFN-, IFN-, IL-12) may indeed favor the development of the subsequent response of human T cells toward the Th1 effector profile (33, 36, 37). In another study, we showed that the Th1-inducing activity of synthetic dsRNA (poly:I-poly:C) was mediated by its ability to induce the production of both IL-12 and IFN- by non-T cells (38). IFN- is indeed critical in the development of human Th1 cells (36, 39) by up-regulating the IL-12 receptor ß₂ chain (40). These data are also in agreement with the results obtained in several animal models showing that CpG-containing ODNs are able to directly stimulate the production of IL-12, IFN-, IL-18, and IFN- by dendritic cells, macrophages, and/or NK cells, which then act as Th1-inducing cytokines (16, 17, 18, 19). In contrast, differently from murine models (23), in preliminary experiments no direct effect of PS-ODNs on human T cells could be observed (data not shown).

The characterization of sequences responsible for the effects on cells of both innate and adaptive immune response in humans is less clear. A large body of evidence, obtained from animals models, indicates that DNA motifs consisting of an unmethylated CpG dinucleotide flanked by two 5' purines (optimally a GpA) and two 3' pyrimidines (optimally a TpC or TpT) (CpG motifs) play a fundamental role in conferring the immunostimulatory ability. Indeed, CpG motif-lacking, GpC-inverted, or cytosine-methylated ODNs were ineffective (15, 25). In agreement with the results reported in mice, our findings demonstrate that the Th1-shifting effect of ODNs requires phosphorothioation and is abolished by cytosine methylation. However, differently from the results obtained in the murine experimental models, several sequences were able to stimulate B cell proliferation and to modulate the functional profile of allergen-specific T cells. We found indeed that all the PS-nonrepetitive sequences were effective, irrespective of the presence (3Da and 1326) or absence (DSP30, DSP17, 2105, and Myco) of the CpG-motifs previously defined in mice. Actually, a CpG motif-lacking ODN, such as DSP30, exerted the maximal activity. Moreover, CpG dinucleotides per se did not appear to be sufficient, because a PS-repetitive sequence of CpG (poly CG), of similar length to the other ODNs, was completely ineffective. Of note, this type of repeated CG sequence has recently been reported to constitute an inhibitory motif (41). Finally, the PS-DSP30 ODN, in which CpG dinucleotides were inverted to GpC (PS-DSP30/GC), retained its immunomodulatory activity, even if to a lower extent. This suggests that CpG dinucleotides may be important to obtain the maximal effect, but they are not necessarily required to induce an immunomodulatory activity in human T cells.

These findings are consistent with the results reported by Liang et al. (30) with regard to the ability of a variety of ODNs to stimulate the proliferation and differentiation of human B cells. These authors clearly showed that, although CpG-containing ODNs are the best stimulators of the B cell response, both CpG motif- and CpG dinucleotide-lacking ODNs possess the ability to induce human B cell activation. Thus, it can be concluded that the immunostimulatory sequences active on murine or human lymphocytes may be, at least partially, different. At present, the nature of the immunostimulatory sequences active in humans remains unclear. Certainly, nonrepetitive sequences (only one or two bases) are necessary, together with a sufficient length. However, in the absence of a well-recognized sequence, such as the CpG motif in mice, additional studies are required to disclose the ‘motif’ responsible for the immunomodulatory activity on human B and T cells. Despite this still unsolved problem, the results of the present study provide additional support to the possibility, emerged from the murine models of asthma (13, 27, 28, 29), that allergen gene DNA vaccination or injection of allergen mixed to, or modified by the conjugation with, appropriate ODNs may provide a new immunotherapeutic strategy for the treatment of human allergic disorders.

	Footnotes

 
¹ This work was supported by grants provided by Associazione Italiana Ricerca sul Cancro, the Italian Ministery of Health (Project AIDS 1998), and European Community (Projects BIO4-CT96-0246 and BIO4-98-0458).

² Address correspondence and reprint requests to Dr. Sergio Romagnani, Dipartimento di Medicina Interna, Sezione di Immunoallergologia, Policlinico di Careggi, Viale Morgagni 85, 50134, Florence, Italy. E-mail address:

³ Abbreviations used in this paper: ODN, oligodeoxynucleotide; PS, phosphorothioate; PE, phosphodiester; Der p 1, Dermatophagoides pteronyssinus group 1; MI, mitogenic index.

Received for publication July 12, 1999. Accepted for publication September 20, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Wierenga, E. A., M. Snoek, C. de Groot, I. Chretien, J. D. Bos, H. M. Jansen, M. L. Kapsenberg. 1990. Evidence for compartmentalization of functional subsets of CD4⁺ T lymphocytes in atopic patients. J. Immunol. 144:4651.[Abstract]
2. Parronchi, P., D. Macchia, M.-P. Piccinni, P. Biswas, C. Simonelli, E. Maggi, M. Ricci, A. A. Ansari, S. Romagnani. 1991. Allergen- and bacterial antigen-specific T-cell clones established from atopic donors show a different profile of cytokine production. Proc. Natl. Acad. Sci. USA 88:4538.[Abstract/Free Full Text]
3. Hamid, Q., M. Azzawi, S. Ying, R. Moqbel, A. J. Wardlaw, C. J. Corrigan, B. Bradley, S. R. Durham, J. V. Collins, P. K. Jeffrey, D. J. Quint, A. B. Kay. 1991. Expression of mRNA for Interleukin-5 in mucosal bronchial biopsies from asthma. J. Clin. Invest. 87:1541.
4. Del Prete, G. F., E. Maggi, P. Parronchi, I. Chretien, A. Tiri, D. Macchia, M. Ricci, J. Banchereau, J. E. de Vries, S. Romagnani. 1988. IL-4 is an essential factor for the IgE synthesis induced in vitro by human T cell clones and their supernatants. J. Immunol. 140:4193.[Abstract]
5. Pene, J., F. Rousset, F. Briere, I. Chretien, J. Y. Bonnefoy, H. Spits, T. Yokota, N. Arai, K. Arai, J. Banchereau, J. E. de Vries. 1988. IgE production by normal human lymphocytes is induced by interleukin-4 and suppressed by interferons and and prostaglandin E2. Proc. Natl. Acad. Sci. USA 85:6880.[Abstract/Free Full Text]
6. Punnonen, J., G. Aversa, B. G. Cocks, A. N. J. MacKenzie, S. Menon, G. Zurawski, R. de Waal Malefyt, J. E. de Vries. 1993. Interleukin 13 induces interleukin 4-independent IgG4 and IgE synthesis and CD23 expression by human B cells. Proc. Natl. Acad. Sci. USA 90:3730.[Abstract/Free Full Text]
7. Thompson-Snipes, L., V. Dhar, M. W. Bond, T. R. Mosmann, K. W. Moore, D. M. Rennick. 1991. Interleukin-10: a novel stimulatory factor for mast cells and their progenitors. J. Exp. Med. 173:507.[Abstract/Free Full Text]
8. Kitamura, Y., T. Kasugai, N. Arizono, H. Matsuda. 1993. Development of mast cells and basophils: processes and regulation mechanisms. Am. J. Med. Sci. 306:185.[Medline]
9. Sanderson, C. J., D. J. Warren, M. Strath. 1985. Identification of a lymphokine that stimulates eosinophil differentiation in vitro: its relationship to interleukin 3, and functional properties of eosinophils produced in cultures. J. Exp. Med. 162:60.[Abstract/Free Full Text]
10. Yamaguchi, Y., Y. Hayashi, Y. Sugama, Y. Miura, T. Kasahara, S. Kitamura, M. Torisu, S. Mita, A. Tominaga, K. Takatsu. 1988. Highly purified murine interleukin 5 (IL-5) stimulates eosinophil function and prolongs in vitro survival: IL-5 as an eosinophil chemotactic factor. J. Exp. Med. 167:1737.[Abstract/Free Full Text]
11. Romagnani, S.. 1994. Regulation of the development of type 2 T-helper cells in allergy. Curr. Opin. Immunol. 6:838.[Medline]
12. Sato, Y., M. Roman, H. Tighe, D. Lee, M. Corr, M.-D. Nguyen, G. J. Silverman, M. Lotz, D. A. Carson, E. Raz. 1996. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 73:352.
13. Raz, E., E. Tighe, Y. Sato, M. Corr, J. A. Dudler, M. Roman, S. L. Swain, H. L. Spiegelberg, D. A. Carson. 1996. Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization. Proc. Natl. Acad. Sci. USA 93:5141.[Abstract/Free Full Text]
14. Hsu, C. H., K. Y. Chua, M. H. Tao, S. K. Huang, K. H. Hsieh. 1996. Inhibition of specific IgE response in vivo by allergen-gene transfer. Int. Immunol. 8:1405.[Abstract/Free Full Text]
15. Spiegelberg, H. L., E. M. Orozco, M. Roman, E. Raz. 1997. DNA immunization: a novel approach to allergen-specific immunotherapy. Allergy 52:964.[Medline]
16. Klinman, D. M., A.-K. Yi, S. L. Beaucage, J. Conover, A. M. Krieg. 1996. CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon . Proc. Natl. Acad. Sci. USA 93:2879.[Abstract/Free Full Text]
17. Krieg, A. M., L. Love-Homan, A-K. Yi, J. T. Harty. 1998. CpG DNA induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge. J. Immunol. 161:2428.[Abstract/Free Full Text]
18. Roman, M., E. Martin-Orozco, J. S. Goodman, M. D. Nguyen, Y. Sato, A. Ronaghy, R. S. Kornbluth, D. D. Richman, D. A. Carson, E. Raz. 1997. Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nat. Med. 3:849.[Medline]
19. Sun, S., X. Zhang, D. F. Tough, J. Sprent. 1998. Type I interferon-mediated stimulation of T cells by CpG DNA. J. Exp. Med. 188:2335.[Abstract/Free Full Text]
20. Ballas, Z. K., W. L. Rasmussen, A. M. Krieg. 1996. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J. Immunol. 157:1840.[Abstract]
21. Lipford, G. B., M. Bauer, C. Blank, R. Reiter, H. Wagner, K. Heeg. 1997. CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: a new class of vaccine adjuvants. Eur. J. Immunol. 27:2340.[Medline]
22. Chu, R. S., O. S. Targoni, A. M. Krieg, P. V. Lehmann, C. V. Harding. 1997. CpG oligodeoxynucleotides act as adjuvants that switch T helper 1 (Th1) immunity. J. Exp. Med. 186:1623.[Abstract/Free Full Text]
23. Zimmermann, S., O. Egeter, S. Hausmann, J. B. Lipford, M. Rocken, H. Wagner, K. Heeg. 1998. CpG oligodeoxynucleotides trigger protective and curative Th1 responses in lethal murine leishmaniasis. J. Immunol. 160:3627.[Abstract/Free Full Text]
24. Yi, A.-K., D. M. Klinman, T. L. Martin, S. Matson, A. M. Krieg. 1996. Rapid immune activation by CpG motifs in bacterial DNA: systemic induction of IL-6 transcription through an antioxidant-sensitive pathway. J. Immunol. 157:5394.[Abstract]
25. Krieg, A. M., A.-K. Yi, S. Matson, T. J. Waldschmidt, G. A Bishop, R. Teasdale, G. A. Koretzky, D. M. Klinman. 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374:546.[Medline]
26. Broide, D., J. Schwarze, H. Tighe, T. Gifford, M. D. Nguyen, S. Malek, J. Van Uden, E. Martin-Orozco, E. W. Gelfand, E. Raz. 1998. Immunostimulatory DNA sequences inhibit IL-5, eosinophilic inflammation, and airway hyperresponsiveness in mice. J. Immunol. 161:7054.[Abstract/Free Full Text]
27. Kline, J. N., J. T. Waldschmidt, T. R. Businga, J. E. Lemish, J. V. Weinstock, P.S. Thorne, A. M. Krieg. 1998. Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma. J. Immunol. 160:2555.[Abstract/Free Full Text]
28. Broide, D., E. Raz. 1999. DNA-based immunization for asthma. Int. Arch. Allergy Immunol. 118:453.[Medline]
29. Sur, S., J. S. Wild, B. K. Choudhury, N. Sur, R. Alam, D. M. Klinman. 1999. Long term prevention of allergic lung inflammation in a mouse model of asthma by CpG oligodeoxynucleotides. J. Immunol. 162:6284.[Abstract/Free Full Text]
30. Liang, H., Y. Nishioka, C. F. Reich, D. S. Pisetsky, P. E. Lipsky. 1996. Activation of human B cells by phosphorothioate oligodeoxynucleotides. J. Clin. Invest. 98:1119.[Medline]
31. Parronchi, P., S. Sampognaro, F. Annunziato, F. Brugnolo, A. Radbruch, F. Di Modugno, A. Ruffilli, S. Romagnani, E. Maggi. 1998. Influence of both T cell receptor repertoire and severity of the atopic status on the cytokine secretion profile of Parietaria officinalis-specific T cells. Eur. J. Immunol. 28:37.[Medline]
32. Assenmacher, M., J. Schimtz, A. Radbruch. 1994. Flow cytometric determination of cytokines in activated murine T helper lymphocytes: expression of interleukin 10 in interferon and interleukin 4 expressing cells. Eur. J. Immunol. 24:1097.[Medline]
33. Manetti, R., P. Parronchi, M. G. Giudizi, M.-P. Piccinni, E. Maggi, G. Trinchieri, S. Romagnani. 1993. Natural killer stimulatory factor (NKSF/IL-12) induces Th1-type specific immune responses and inhibits the development of IL-4-producing Th cells. J. Exp. Med. 177:1199.[Abstract/Free Full Text]
34. Klinman, D. M., G. Yamshchikov, Y. Ishigatsubo. 1997. Contribution of CpG motifs to the immunogenicity of DNA vaccines. J. Immunol. 158:3635.[Abstract]
35. Walker, P. S., T. Scharton-Kersten, A. M. Krieg, L. Love-Homan, E. D. Rowton, M. C. Udey, J. C. Vogel. 1999. Immunostimulatory oligeoxynucleotides promote protective immunity and provide systemic therapy for Leishmaniasis via IL-12- and IFN--dependent mechanisms. Proc. Natl. Acad. Sci. USA 96:6970.[Abstract/Free Full Text]
36. Parronchi, P., M. De Carli, R. Manetti, C. Simonelli, S. Sampognaro, M-P. Piccinni, D. Macchia, E. Maggi, G. F. Del Prete, S. Romagnani. 1992. IL-4 and IFNs exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones. J. Immunol. 149:2977.[Abstract]
37. Romagnani, S.. 1992. Induction of Th1 and Th2 responses: a key role for the ‘natural’ immune response?. Immunol. Today 13:379.[Medline]
38. Manetti, R., F. Annunziato, L. Tomasevic, V. Giannò, P. Parronchi, S. Romagnani, E. Maggi. 1995. Poly-inosinic acid: poly-cytidylic acid promotes T helper type 1-specific immune responses by stimulating macrophage production of IFN- and interleukin-12. Eur. J. Immunol. 25:265.
39. Parronchi, P., S. Mohapatra, S. Sampognaro, L. Giannarini, U. Wahn, P. Chong, S. S. Mohapatra, E. Maggi, H. Renz, S. Romagnani. 1996. Modulation by IFN- of cytokine profile, T cell receptor repertoire and peptide reactivity of human allergen-specific T cells. Eur. J. Immunol. 26:697.[Medline]
40. Rogge, L., L. Barberis, N. Passini, D. H. Presky, U. Gubler, F. Sinigaglia. 1997. Selective expression of an interleukin 12 receptor component by human T helper 1 cells. J. Exp. Med. 185:825.[Abstract/Free Full Text]
41. Krieg, A. M., T. Wu, R. Weeratna, S. M. Efler, L. Love-Homan, L. Yang, A. K. Yi, D. Short, H. L. Davis. 1998. Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. Proc. Natl. Acad. Sci. USA 95:12631.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. Gerstmayr, N. Ilk, I. Schabussova, B. Jahn-Schmid, E. M. Egelseer, U. B. Sleytr, C. Ebner, and B. Bohle A Novel Approach to Specific Allergy Treatment: The Recombinant Allergen-S-Layer Fusion Protein rSbsC-Bet v 1 Matures Dendritic Cells That Prime Th0/Th1 and IL-10-Producing Regulatory T Cells J. Immunol., December 1, 2007; 179(11): 7270 - 7275. [Abstract] [Full Text] [PDF]

				S. Radhakrishnan, K. R. Wiehagen, V. Pulko, V. Van Keulen, W. A. Faubion, K. L. Knutson, and L. R. Pease Induction of a Th1 Response from Th2-Polarized T Cells by Activated Dendritic Cells: Dependence on TCR:Peptide-MHC Interaction, ICAM-1, IL-12, and IFN-{gamma} J. Immunol., March 15, 2007; 178(6): 3583 - 3592. [Abstract] [Full Text] [PDF]

				G. M. Gauvreau, E. M. Hessel, L.-P. Boulet, R. L. Coffman, and P. M. O'Byrne Immunostimulatory Sequences Regulate Interferon-inducible Genes but not Allergic Airway Responses Am. J. Respir. Crit. Care Med., July 1, 2006; 174(1): 15 - 20. [Abstract] [Full Text] [PDF]

				S.-C. Liao, Y.-C. Cheng, Y.-C. Wang, C.-W. Wang, S.-M. Yang, C.-K. Yu, C.-C. Shieh, K.-C. Cheng, M.-F. Lee, S.-R. Chiang, et al. IL-19 Induced Th2 Cytokines and Was Up-Regulated in Asthma Patients J. Immunol., December 1, 2004; 173(11): 6712 - 6718. [Abstract] [Full Text] [PDF]

				B. Bohle, A. Breitwieser, B. Zwolfer, B. Jahn-Schmid, M. Sara, U. B. Sleytr, and C. Ebner A Novel Approach to Specific Allergy Treatment: The Recombinant Fusion Protein of a Bacterial Cell Surface (S-Layer) Protein and the Major Birch Pollen Allergen Bet v 1 (rSbsC-Bet v 1) Combines Reduced Allergenicity with Immunomodulating Capacity J. Immunol., June 1, 2004; 172(11): 6642 - 6648. [Abstract] [Full Text] [PDF]

				K. H. Baek, S. J. Ha, and Y. C. Sung A Novel Function of Phosphorothioate Oligodeoxynucleotides as Chemoattractants for Primary Macrophages J. Immunol., September 1, 2001; 167(5): 2847 - 2854. [Abstract] [Full Text] [PDF]

				B. Bohle, L. Orel, D. Kraft, and C. Ebner Oligodeoxynucleotides Containing CpG Motifs Induce Low Levels of TNF-{{alpha}} in Human B Lymphocytes: Possible Adjuvants for Th1 Responses J. Immunol., March 15, 2001; 166(6): 3743 - 3748. [Abstract] [Full Text] [PDF]

				Z. Peng, H. Wang, X. Mao, K. T. HayGlass, and F. E. R. Simons CpG oligodeoxynucleotide vaccination suppresses IgE induction but may fail to down-regulate ongoing IgE responses in mice Int. Immunol., January 1, 2001; 13(1): 3 - 11. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Parronchi, P. Articles by Maggi, E. Search for Related Content PubMed PubMed Citation Articles by Parronchi, P. Articles by Maggi, E.