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 (α and γ) 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.

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-α, IFN-γ, 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-γ 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.

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.

The medium used throughout was RPMI 1640 (Seromed, Berlin, Germany) supplemented with 2 mM l-glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 2 × 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-γ (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).

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.

Table I.

Sequence, length, and source of ODNs used in the study

NameSequenceaLengthSource
3Da GAG AAC GCTCGA CCT TCC AT 20 mer  
DSP30 TCG TCG CTG TCT CCG CTT CTT CTT GCC 27 mer Antisense of HIV-rev 
DSP30/GC TGC TGC CTG TCT GCC CTT CTT CTT GCC 27 mer  
DSP17 GCC GAG GTC CAT GTC GTA CG21 mer Antisense of HSV-orf 
2105 TTG CTT CCA TCT TCC TCG TC 20 mer From Papilloma virus 
Myco GTG CTC GAG GAT GCG CTT CG21 mer From Mycobacterium tuberculosis 
1326 CAG CGC TCC CGT CGC TCT CTC 21 mer From CD30 gene 
Poly CG CGC GCG CGC GCG CGC GCG CGC G 22 mer  
NameSequenceaLengthSource
3Da GAG AAC GCTCGA CCT TCC AT 20 mer  
DSP30 TCG TCG CTG TCT CCG CTT CTT CTT GCC 27 mer Antisense of HIV-rev 
DSP30/GC TGC TGC CTG TCT GCC CTT CTT CTT GCC 27 mer  
DSP17 GCC GAG GTC CAT GTC GTA CG21 mer Antisense of HSV-orf 
2105 TTG CTT CCA TCT TCC TCG TC 20 mer From Papilloma virus 
Myco GTG CTC GAG GAT GCG CTT CG21 mer From Mycobacterium tuberculosis 
1326 CAG CGC TCC CGT CGC TCT CTC 21 mer From CD30 gene 
Poly CG CGC GCG CGC GCG CGC GCG CGC G 22 mer  
a

Italics indicates “CpG motifs” (5′-pur-pur-CpG-pyr-pyr-3′). Underlining indicates CpG dinucleotides.

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 × 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.

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 × 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-α, 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.

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-α (Biosource, Camarillo, CA), and anti-IFN-γ (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 × 104 T cell blasts were incubated in the presence of 5 × 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 IFN-γ and IL-4 synthesis at the single-cell level was performed as described (31, 32). Briefly, 1 × 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 CD3CD16+ cells, for each sample, were acquired.

To evaluate the percentages of NK cells in Der p 1-specific T cell lines obtained in the absence or presence of ODNs, 2 × 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.

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-γ (Endogen) concentrations were measured into cell-free supernatants by homemade ELISAs using commercial pairs of mAbs, as previously described (31).

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

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.

Table II.

PS-, but neither PE- nor PS/met-, ODNs stimulate human B cell proliferationa

Reagent Added in Bulk Culture (10 μg/ml)B Cell Proliferation (MI)b
Expt. 1Expt. 2
PEPSPS/metPEPSPS/met
DSP30 1.1 10.0 2.1 0.9 27.0 4.0 
DSP17 1.3 13.0 2.0 0.7 21.0 3.5 
3Da 0.9 6.5 1.8 1.1 27.8 4.2 
2105 1.0 6.0 3.0 1.1 17.0 5.0 
Myco 0.8 10.4 2.3 1.3 25.0 7.5 
1326 1.0 7.0 1.4 0.9 22.5 4.0 
Poly CG 1.1 1.2 1.0 0.7 1.1 0.9 
Reagent Added in Bulk Culture (10 μg/ml)B Cell Proliferation (MI)b
Expt. 1Expt. 2
PEPSPS/metPEPSPS/met
DSP30 1.1 10.0 2.1 0.9 27.0 4.0 
DSP17 1.3 13.0 2.0 0.7 21.0 3.5 
3Da 0.9 6.5 1.8 1.1 27.8 4.2 
2105 1.0 6.0 3.0 1.1 17.0 5.0 
Myco 0.8 10.4 2.3 1.3 25.0 7.5 
1326 1.0 7.0 1.4 0.9 22.5 4.0 
Poly CG 1.1 1.2 1.0 0.7 1.1 0.9 
a

Highly purified B cells from two healthy volunteers were stimulated for 2 days in the presence or absence of PE-, PS-, or PS/met-ODNs (10 μg/ml). After 16 h pulsing with [3H]TdR, cultures were harvested and radionuclide uptake measured by scintillation counting, as described in Materials and Methods.

b

MI was calculated as the ratio between the mean values of cpm obtained in ODN-stimulated cultures and cpm obtained in the presence of medium alone.

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-γ 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-γ 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).

Table III.

Effects of PS-ODNs on the cytokine profile of human Der p 1-specific short-term T cell linesa

Reagents Added in Bulk CultureConcentration (μg/ml)% of Cells Expressing the Indicated Cytokine
Expt. 1Expt. 2
IL-4+IFN-γ+IL-4+ IFN-γ+IL-4+IFN-γ+IL-4+ IFN-γ+
Medium  34.2 12.6 10.1 20.9 21.4 15.4 
rIL-12  6.1 35.7 19.1 6.7 34.1 16.1 
PS-DSP30 0.5 34.3 13.7 9.4 24.5 22.0 25.7 
 5.0 14.9 29.7 17.9 6.3 31.5 10.2 
 10.0 18.5 24.6 23.9 5.9 34.6 8.2 
PS-DSP17 0.5 32.2 16.7 11.9 17.9 27.4 14.4 
 5.0 18.5 20.8 12.1 5.0 30.5 4.4 
 10.0 21.5 19.9 15.6 7.0 30.9 5.5 
PS-3Da 10.0 16.3 27.1 18.3 6.5 30.8 8.2 
PS-2105 10.0 22.2 24.4 16.5 6.4 33.2 5.4 
PS-Myco 10.0 21.5 19.8 16.0 5.4 35.8 6.6 
PS-1326 10.0 18.0 25.0 22.0 8.1 38.7 17.8 
Reagents Added in Bulk CultureConcentration (μg/ml)% of Cells Expressing the Indicated Cytokine
Expt. 1Expt. 2
IL-4+IFN-γ+IL-4+ IFN-γ+IL-4+IFN-γ+IL-4+ IFN-γ+
Medium  34.2 12.6 10.1 20.9 21.4 15.4 
rIL-12  6.1 35.7 19.1 6.7 34.1 16.1 
PS-DSP30 0.5 34.3 13.7 9.4 24.5 22.0 25.7 
 5.0 14.9 29.7 17.9 6.3 31.5 10.2 
 10.0 18.5 24.6 23.9 5.9 34.6 8.2 
PS-DSP17 0.5 32.2 16.7 11.9 17.9 27.4 14.4 
 5.0 18.5 20.8 12.1 5.0 30.5 4.4 
 10.0 21.5 19.9 15.6 7.0 30.9 5.5 
PS-3Da 10.0 16.3 27.1 18.3 6.5 30.8 8.2 
PS-2105 10.0 22.2 24.4 16.5 6.4 33.2 5.4 
PS-Myco 10.0 21.5 19.8 16.0 5.4 35.8 6.6 
PS-1326 10.0 18.0 25.0 22.0 8.1 38.7 17.8 
a

PBMC from two atopic Der p-sensitive donors were stimulated for 6 days with Der p 1 (10 μg/ml) in the presence of medium alone, IL-12 (100 U/ml), or different concentrations of PS-ODNs, as indicated. T cell blasts were then expanded for subsequent 8 days with IL-2 and then assessed for their IL-4 and IFN-γ intracellular content by flow cytometry, following stimulation with PMA and ionomycin for 4 h, the last two being in the presence of brefeldin A, as described in Materials and Methods.

In subsequent experiments, the activity of PS-DSP30, used at a fixed concentration (10 μg/ml), was assessed on Der p 1-specific T cells from a higher number of atopic donors (eight subjects total). As additional controls, parallel Der p 1-stimulated cultures were also established in the presence of PE-DSP30 (10 μg/ml) or IL-12 (100 U/ml). The results of these experiments are summarized in Fig. 1 (upper panel). A significant increase in the proportion of IFN-γ-producing T cells and a significant decrease in the proportion of IL-4-producing T cells were observed when cultures were conditioned with PS-DSP30 (p < 0.0001 and <0.005, respectively) or IL-12 (p < 0.005) in comparison with either unconditioned cultures or cultures conditioned with PE-DSP30. By contrast, the proportion of cells able to produce both IFN-γ and IL-4 was not significantly different in all types of cultures. The cytofluorometric profile observed in one representative experiment is shown in Fig. 1 (lower panel).

FIGURE 1.

Effect of PS-ODN on intracellular IL-4 and IFN-γ 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-γ alone (▪), or both IL-4 and IFN-γ (▦) (∗∗, 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.

FIGURE 1.

Effect of PS-ODN on intracellular IL-4 and IFN-γ 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-γ alone (▪), or both IL-4 and IFN-γ (▦) (∗∗, 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.

Close modal

To provide undoubtable evidence that the Th1-shifting effect of ODNs was indeed exerted on allergen-specific T cells, Der p 1-specific short-term T cell lines, generated in the presence of medium alone, PS-DSP30, PE-DSP30, or IL-12, were stimulated for 72 h with Der p 1 and APC under MHC-restricted conditions. IL-4 and IFN-γ concentrations were then measured by appropriate ELISAs into cell-free supernatants. Even under conditions of specific Ag-stimulation, a lower IL-4 production in PS-DSP30-conditioned than unconditioned, or PE-DSP30-conditioned, T cell cultures was observed (p < 0.05). Moreover, the addition to the cultures of PS-DSP30 resulted in a highly significant increase in the IFN-γ production by Der p 1-stimulated T cells in comparison with unconditioned cultures, or cultures conditioned with PE-DSP30 (p = 0.007) (Fig. 2).

FIGURE 2.

Effect of PS-ODN on Der p 1-stimulated production of IL-4 and IFN-γ 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-γ (▪) (±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)

FIGURE 2.

Effect of PS-ODN on Der p 1-stimulated production of IL-4 and IFN-γ 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-γ (▪) (±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)

Close modal

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-α, 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. CD3CD16+ 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 CD3CD16+ cells able to synthesize intracellular IFN-γ 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).

FIGURE 3.

PS-ODNs expand higher proportions of CD3CD16+ IFN-γ-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 CD3CD16+ 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 CD3CD16+ cells expressing IFN-γ 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)

FIGURE 3.

PS-ODNs expand higher proportions of CD3CD16+ IFN-γ-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 CD3CD16+ 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 CD3CD16+ cells expressing IFN-γ 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)

Close modal

To provide further evidence that the Th1-shifting activity of PS-ODNs was mediated by their ability to induce the production of IL-12 and IFN (α and γ) by cells of the innate immunity (monocytes, dendritic cells, and NK cells), short-term T cell lines were generated from two atopic Der p-sensitive donors by stimulating their PBMC with Der p 1 and PS-DSP30 in the presence of anti-IL-12 neutralizing mAb alone, a mixture of anti-IL-12 and anti-IFN (α and γ) neutralizing mAbs, or isotype-matched control mAbs, respectively. The cytokine profile of Der p 1-specific T cell lines derived under these different experimental conditions was then compared by analyzing the intracellular IL-4 and IFN-γ synthesis at the single-cell level following polyclonal stimulation with PMA and ionomycin. As shown in Fig. 4, the Th1-shifting effect of PS-DSP30 was partially inhibited by the addition of anti-IL-12 mAb, but completely blocked by the mixture of anti-IL-12 and anti-IFNs mAbs. Similar results were obtained when cytokine concentrations were measured in culture supernatants following stimulation with Der p 1 under MHC-restricted conditions (data not shown).

FIGURE 4.

Blocking of the Th1-inducing effect of PS-ODN by neutralization of IL-12, IFN-γ, and IFN-α 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-γ, and anti-IFN-α 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-γ synthesis was evaluated by cytofluorometric analysis on CD3+CD4+ T cell blasts, as described in Materials and Methods.

FIGURE 4.

Blocking of the Th1-inducing effect of PS-ODN by neutralization of IL-12, IFN-γ, and IFN-α 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-γ, and anti-IFN-α 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-γ synthesis was evaluated by cytofluorometric analysis on CD3+CD4+ T cell blasts, as described in Materials and Methods.

Close modal

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).

Table IV.

PS-ODNs with methylated cytosine (PS/met ODNs) are ineffective in inducing the Th1 shifting of allergen-specific T cellsa

Reagents Added in Bulk CulturePreparation% of Cells Expressing the Indicated Cytokine
Expt. 1Expt. 2
IL-4+IFN-γ+IL-4+ IFN-γ+IL-4+IFN-γ+IL-4+ IFN-γ+
Medium  34.2 12.6 10.1 14.6 20.1 4.7 
IL-12  6.1 35.7 19.1 4.4 43.9 7.1 
DSP30 PS 18.5 24.6 23.9 4.7 38.8 17.8 
 PS/met 31.8 20.0 13.3 8.4 18.9 1.1 
DSP17 PS 21.5 19.9 15.6 5.4 35.9 6.6 
 PS/met 35.5 22.2 19.8 8.1 27.5 3.1 
Reagents Added in Bulk CulturePreparation% of Cells Expressing the Indicated Cytokine
Expt. 1Expt. 2
IL-4+IFN-γ+IL-4+ IFN-γ+IL-4+IFN-γ+IL-4+ IFN-γ+
Medium  34.2 12.6 10.1 14.6 20.1 4.7 
IL-12  6.1 35.7 19.1 4.4 43.9 7.1 
DSP30 PS 18.5 24.6 23.9 4.7 38.8 17.8 
 PS/met 31.8 20.0 13.3 8.4 18.9 1.1 
DSP17 PS 21.5 19.9 15.6 5.4 35.9 6.6 
 PS/met 35.5 22.2 19.8 8.1 27.5 3.1 
a

PBMC from two atopic Der p-sensitive donors were stimulated for 6 days with Der p 1 (10 μg/ml) in the presence of medium alone, IL-12 (100 U/ml), or PS- or PS/met ODNs (10 μg/ml). T cell blasts were then expanded for additional 8 days with IL-2 and then assessed for their IL-4 and IFN-γ intracellular content by flow cytometry, following stimulation with PMA and ionomycin for 4 h, the last two of which in the presence of brefeldin A, as described in Materials and Methods.

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).

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).

Close modal

The immunomodulatory activity of bacterial DNA, originally recognized by examining the effect of CFA, has been extensively investigated in the mouse (12). This activity on the immune system has been ascribed to unmethylated CpG dinucleotides, which are present at a high degree of frequency in bacterial, but not mammalian, DNA (12). More recently, synthetic ODNs containing unmethylated CpG dinucleotides flanked by particular bases (CpG motifs) have been shown to be as powerful as bacterial DNA in triggering the proliferation of B cells and polyclonal Ig secretion in a T cell- and Ag-independent fashion (21, 25), the activation of NK cells (20), and the production of several proinflammatory cytokines (16, 19, 24). In addition, synthetic ODNs have been found to be able to bias the specific immune response to a Th1-dominated cytokine pattern, both in vitro, inasmuch as they are able to stimulate the production of Th1-inducing cytokines, such as IL-12, IFNs, and IL-18 (22, 34), and in vivo, because they have been successfully employed in animal models of Th2-dominated diseases, such as leishmaniasis (23, 35) and asthma (26, 27, 28, 29). Thus, these compounds might be used as effective and safe immunomodulators even in established Ag-specific responses.

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-α by monocytes and/or dendritic cells and possibly the production of IFN-γ 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-α, by human non-T cells. Second, at the time of cytokine assessment, a significant expansion of CD3CD16+CD56+, IFN-γ-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-α, and anti-IFN-γ 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-α production by monocytes and/or dendritic cells, as well as of IFN-γ 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-γ, IFN-α, 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-α by non-T cells (38). IFN-α is indeed critical in the development of human Th1 cells (36, 39) by up-regulating the IL-12 receptor β2 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-α, IL-18, and IFN-γ 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.

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).

3

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

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
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
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
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 γ and α and prostaglandin E2.
Proc. Natl. Acad. Sci. USA
85
:
6880
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
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
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
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
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
11
Romagnani, S..
1994
. Regulation of the development of type 2 T-helper cells in allergy.
Curr. Opin. Immunol.
6
:
838
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
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
15
Spiegelberg, H. L., E. M. Orozco, M. Roman, E. Raz.
1997
. DNA immunization: a novel approach to allergen-specific immunotherapy.
Allergy
52
:
964
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 γ.
Proc. Natl. Acad. Sci. USA
93
:
2879
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
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
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
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
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
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
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
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
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
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
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
28
Broide, D., E. Raz.
1999
. DNA-based immunization for asthma.
Int. Arch. Allergy Immunol.
118
:
453
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
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
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
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 γ and interleukin 4 expressing cells.
Eur. J. Immunol.
24
:
1097
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
34
Klinman, D. M., G. Yamshchikov, Y. Ishigatsubo.
1997
. Contribution of CpG motifs to the immunogenicity of DNA vaccines.
J. Immunol.
158
:
3635
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-γ-dependent mechanisms.
Proc. Natl. Acad. Sci. USA
96
:
6970
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
37
Romagnani, S..
1992
. Induction of Th1 and Th2 responses: a key role for the ‘natural’ immune response?.
Immunol. Today
13
:
379
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-α 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-α of cytokine profile, T cell receptor repertoire and peptide reactivity of human allergen-specific T cells.
Eur. J. Immunol.
26
:
697
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
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