Allergen-specific CD4+ T lymphocytes are activated at extremely low allergen concentrations in vivo as a result of serum-facilitated allergen presentation (S-FAP). It is not clear at present if specific allergy vaccination (SAV) has an effect on this mechanism. Here we show that birch allergen-specific serum-IgE facilitates the presentation of Bet v 1, the major birch pollen allergen, to Bet v 1-specific CD4+ T lymphocytes by a factor of >100. This process is CD23 mediated, could be detected in sera from the majority of birch-allergic patients, and was clearly dose dependent. S-FAP of Bet v 1 was inhibited in patients undergoing long-term birch SAV, but not by sera from patients undergoing grass SAV, indicating that birch-specific Abs are involved. This resulted in decreased proliferation and IL-4, IL-5, IL-10, and IFN-γ production of Bet v 1-specific T cells. The inhibition was already noted after 3–9 mo of SAV and could not be solely explained by increased serum levels of birch-specific IgG4. When IgG- and IgA/IgM-containing fractions of long-term SAV sera were used to inhibit S-FAP, only IgG-containing fractions were shown to inhibit S-FAP. These results indicate that blocking IgG Abs induced by SAV inhibits the occurrence of S-FAP at very low allergen concentrations, resulting in significantly higher allergen threshold levels to obtain T cell proliferation and cytokine production and thus allergen-induced late-phase responses.

Atopic allergy is characterized by elevated levels of allergen-specific serum-IgE and tissue eosinophilia, two parameters that are responsible for the immediate and late-phase allergic responses, respectively. It is well established that both IgE production and eosinophilia are dependent on the presence and activation of allergen-specific CD4+ T lymphocytes with a bias toward the production of Th2 cytokines (1, 2). These Th2-like cells produce IL-4, which induces B lymphocytes to switch to the production of IgE, and IL-5, which plays an important role in the differentiation and maturation of eosinophilic granulocytes. Allergen-specific Th2 cells have been identified in the peripheral blood (1, 2) as well as in affected tissues of allergic patients (3, 4, 5), underlining the role that allergen-specific T cells play in atopic allergies.

In addition, atopic allergic patients have been shown to have an increased expression of CD23 on B cells and macrophages (6, 7) and FcεRI on monocytes and dendritic cells (8, 9). The expression of IgE Fc receptors on APC suggests that they may play a functional role in the presentation of allergens to specific T cells. Indeed, both IgE receptors have been shown to facilitate the presentation of allergens in the presence of specific serum-IgE, resulting in T cell activation at 100- to 1000-fold lower allergen concentrations than are required when control sera are used (10, 11, 12, 13, 14). This is a crucial step in the activation of allergen-specific Th2-like cells, because the factual exposure to airborne allergens is extremely low, i.e., in the range of several micrograms per year. Therefore, inhibition of this mechanism would theoretically result in increasing the allergen concentrations required for in vivo T cell activation and Th2 cytokine production.

Specific immunotherapy with allergen extracts, recently termed therapeutic allergy vaccines in the World Health Organization position paper on allergen immunotherapy (15), has been practiced since its introduction by Noon in 1911 (16). Specific allergy vaccination (SAV)2 is currently the only allergy treatment that not only has an antisymptomatic effects but also influences the course of the allergic disease itself (15). The clinical efficacy of SAV is well documented (reviewed in Ref. 15), and clear effects are noted with respect to symptom/medication scores, early- and late-phase skin responses, and increased allergen threshold levels in provocation tests. Most importantly, SAV has been shown to have long-term efficacy (17, 18), prevent the development of new sensitizations (19), and even inhibits the progression from rhinitis to asthma (20).

However, the precise immunological mechanisms underlying SAV are not entirely clear. Several immunological effects have been described. There is a decreased influx of activated eosinophils and activated CD4+ T cells into target tissues after topic allergen provocation, as well as during the pollen season (21, 22); a sharp rise in allergen-specific IgG4 Abs (23, 24), and a decreased number of circulating basophils (25). However, specific serum-IgE levels do not decrease after SAV but rather show a transient increase (26). In addition, a shift in the production of T cell cytokines from Th2 to a more moderate Th0 type (27, 28, 29, 30), a decreased proliferative response of peripheral T cells (28, 31), an increased production of IL-10 by T cells (32), as well as an increased production of IL-12 (33) and IL-10 (32) by APC has been reported. However, these immunological changes that SAV induces cannot completely explain the clinical observations that much higher allergen threshold levels are required to obtain a positive late-phase response (LPR). As inhibition of serum-facilitated allergen presentation (S-FAP) would offer a valid explanation of these clinical observations, the present study addresses the question whether SAV interferes with S-FAP, thereby explaining why SAV is clinically efficacious without changes in serum levels of allergen-specific IgE.

Anti-CD23 mAb MHM6, anti-CD19 mAb HD37, FITC-labeled F(ab′)2 of goat anti-mouse Igs, rabbit anti-human IgG, and rabbit anti-human IgE were obtained from Dako (Glostrup, Denmark). Anti-FcεRI mAb 15-1 was a kind gift of Dr. J. P. Kinet (Boston, MA), anti-CD80 mAb BB1 and anti-CD86 mAb FUN-1 were obtained from PharMingen (San Diego, CA), anti-CD40 mAb 89 was purchased from Immunotech (Marseille, France), and anti-CD3 mAb OKT3 and anti-HLA-DR mAb L243 were obtained from American Type Culture Collection (Manassas, VA).

Sera from healthy controls, birch-allergic patients, birch-allergic patients undergoing SAV (birch Alutard SQ, ALK-Abelló, Hørsholm, Denmark), and grass-allergic patients undergoing SAV (grass Alutard SQ) were obtained from our internal serum bank. The duration of SAV is defined as months of therapy after initial updosing. The study was approved by the local ethics committee. All sera were blinded, and clinical data were not available. Allergen-specific IgE and IgG4 were measured using the Magic Lite SQ specific IgE system (ALK-Abelló) (34).

Two long-term SAV sera (1598 and 1490) were separated into IgG- and IgM/IgA-containing fractions by separating the sera on a γ Bind Plus protein G column (Pharmacia, Uppsala, Sweden). The column was washed with PBS, pH 7.2, followed by 2 ml of serum. The run through was collected, and the IgG bound to the column was eluted using a Pierce elution buffer (Pierce, Rockford, IL). All fractions were extensively dialized against PBS, and volumes were adjusted to the starting volume.

The presence of IgM, IgG, and IgA was measured using a human IgG, IgA, and IgM “NL” bind a rid kit according to the manufacturer’s description (The Binding Site, Birmingham, U.K.).

Bet v 1 was enriched from an aqueous birch pollen extract as described by Ipsen (35, 36). In brief, size-exclusion chromatography of 150 mg (dry weight) of the pollen extract was performed on a G-75 superfine (Pharmacia) 90 cm × 2 cm2 (K15/100; Pharmacia) column, equilibrated with 0.125 NH4HCO3, pH 8.3. The fractions containing Bet v 1 (assayed by fused rocket immunoelectrophoresis and fused rocket radio-immunoelectrophoresis) were pooled and lyophilized. Typically, 15–30 mg of dry weight material was obtained.

T cells were cultured in RPMI 1640 medium (Life Technologies, Paisley, U.K.) supplemented with 2 mM l-glutamine (Life Technologies), 100 IU/ml of penicillin-streptomycin (Life Technologies), and 5% heat-inactivated, screened human AB+ serum (BioWhittaker, Walkersville, MD) (later referred to as complete medium). All cultures were grown under sterile conditions at 37°C in a humidified atmosphere of 5% CO2. EBV-transformed B cell lines were cultured in RPMI 1640, 2 mM l-glutamine, 100 IU penicillin-streptomycin, and 10% heat-inactivated FCS (Life Technologies) under the same culture conditions as for the T cells.

Bet v 1-specific T cell line and clones were generated from the peripheral blood of birch-allergic patient AF, who is clinically allergic to birch pollen (37). The CD4+ T cell clone (TCC) AF28 reacted with aa 21–33, CD4+ TCC AF19 and AF24 with aa 37–51, and AF line (which contains both CD4+ and CD8+ T cells) reacted with several T cell epitopes simultaneously.

Serum of birch-allergic patients and control donors was incubated for 1 h at 37°C with serial dilutions of Bet v 1. Then EBV-B cells (irradiated with 5000 rad) were added at a concentration of 3.105/ml to the allergen-complexed serum and incubated for 1 h at 37°C. After incubation, the cells were washed two times in RPMI 1640 and used as APC in T cell stimulation assays.

In blocking experiments with polyclonal anti-IgG or anti-IgE, the sera were incubated with these antisera for 1 h at 37°C before adding Bet v 1. In blocking experiments with anti-CD23, EBV-B cells were incubated for 1 h at 4°C with this Ab before adding the cells to the sera. In experiments in which S-FAP induced by a patient serum was inhibited by SAV sera or fractions thereof, a mixture of the two sera (both at 40%) was preincubated for 1 h at 37°C before B cells were added.

Cells were Ficoll-separated, washed twice in Cellwash (Becton Dickinson, San Jose, CA), and labeled on ice for 30 min with mAb diluted in Cellwash. Subsequently, cells were washed three times and incubated with FITC-labeled F(ab′)2 fragments of rabbit anti-mouse Ig (Dako). Cells were washed three times again, and fluorescence was analyzed on a FACScalibur flow cytometer (Becton Dickinson). For each sample, 10,000 cells were analyzed. No gates were set.

Bet v 1-specific T cells were washed twice in RPMI 1640 medium to remove excess PHA and IL-2. Ag-specific proliferation was assessed by stimulation of the T cells, 2–4 × 104 cells/well, by Ag using irradiated (5000 rad) autologous EBV-transformed B cells as APC (1–2 × 104 cells/well). The cells were cultured for 24 h, followed by a 16-h pulse in the presence of tritiated methyl thymidine ([3H]TdR) (NEN-DuPont, Wilmington, DE), 0.5 μCi/well. Proliferation was expressed as mean cpm of [3H]TdR incorporation of triplicate cultures. All experiments shown were performed at least two times with AF line and at least one of the CD4+ Bet v 1-specific TCC.

Bet v 1-specific T cells (2–4 × 104/well) were activated for 24 h in the presence or absence of Bet v 1 in the presence of autologous, irradiated EBV-transformed B cells (1–2 × 104cells/well) as described above. Supernatants were collected for cytokine measurements after a 24-h culture period.

The presence of IL-4, IL-5, IFN-γ, and IL-10 in the TCC supernatants was measured using the Duoset (IL-4, IL-5, and IFN-γ) or Predicta (IL-10) ELISA kits according to the manufacturer’s description (Genzyme, Cambridge, MA).

To address the role of serum IgE in the presentation of Bet v 1 to specific T cells, two CD4+ Bet v 1-specific T cell clones and a polyclonal Bet v 1-specific T cell line from donor AF were studied in an in vitro culture system model using autologous EBV-transformed B cells (CD23+, CD19+, CD20+, HLA-DR+, CD40+, CD80+, CD86+, CD3, and FcεRI) as APC. This culture system has been used previously to show S-FAP of the house dust mite allergen Der p 2 (10). Six sera containing very high levels of birch-specific IgE and three control sera that did not contain any detectible IgE (Table I) were preincubated with serial dilutions of Bet v 1 for 1 h at 37°C, AF EBV-B cells were added for 1 h at 4°C to prevent fluid phase endocytosis, and the B cells were washed twice to remove any free allergen not complexed with IgE and bound to CD23. T cells were added, and proliferation was measured after a 24-h culture period. Fig. 1 shows that the control sera could not facilitate the presentation of Bet v 1 to the Bet v 1-specific T cells. All sera containing high levels of birch-specific IgE could facilitate the presentation of Bet v 1 to the T cells, and significant proliferation could be measured at 100 and even at 10 ng/ml, indicating that >100-fold lower allergen concentrations are sufficient to activate Bet v 1-specific T cells in the presence of high levels of specific serum IgE.

Table I.

Specific IgE measurements of sera of birch-allergic patients

Serum No.Birch-Specific IgEa (SU/ml)Grass-Specific IgE (SU/ml)Total IgE (IU/ml)
Control Sera    
734 <1 <1 <30 
745 <1 <1 <30 
981 <1 <1 <30 
    
Patient Sera    
1151 182 282 
831 92 
1446 39 148 
1547 14 48 177 
1531 23 82 >3,000 
1327 50 118 805 
1370 80 151 
920 143 29 >3,000 
1372 241 262 
1463 310 119 >3,000 
1374 373 279 
1008 795 94 571 
1464 >800 60 2,826 
102 >800 34 19,000 
894 >800 290 1,283 
1083 >800 94 NTb 
Serum No.Birch-Specific IgEa (SU/ml)Grass-Specific IgE (SU/ml)Total IgE (IU/ml)
Control Sera    
734 <1 <1 <30 
745 <1 <1 <30 
981 <1 <1 <30 
    
Patient Sera    
1151 182 282 
831 92 
1446 39 148 
1547 14 48 177 
1531 23 82 >3,000 
1327 50 118 805 
1370 80 151 
920 143 29 >3,000 
1372 241 262 
1463 310 119 >3,000 
1374 373 279 
1008 795 94 571 
1464 >800 60 2,826 
102 >800 34 19,000 
894 >800 290 1,283 
1083 >800 94 NTb 
a

IgE levels present in sera were measured using the Magic Lite system as described in Materials and Methods. Values given for specific IgE levels are expressed as standardized units of specific IgE per ml (SU/ml), and total IgE levels are expressed as international units per ml (IU/ml).

b

NT, not tested.

FIGURE 1.

Allergen presentation of Bet v 1 is facilitated by patient sera containing high levels of birch-specific IgE. Serial dilutions of Bet v 1 were preincubated with 80% serum from birch-allergic patients (1463, 1464, 1374, 102, 894, 1008, 1083) or control donors (734, 745, 981). AF EBV-B cells were added, incubated for 1 h at 37°C, and were cocultured (1–2 × 104/well) with Bet v 1-specific T cell line AF (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

FIGURE 1.

Allergen presentation of Bet v 1 is facilitated by patient sera containing high levels of birch-specific IgE. Serial dilutions of Bet v 1 were preincubated with 80% serum from birch-allergic patients (1463, 1464, 1374, 102, 894, 1008, 1083) or control donors (734, 745, 981). AF EBV-B cells were added, incubated for 1 h at 37°C, and were cocultured (1–2 × 104/well) with Bet v 1-specific T cell line AF (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

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As allergen presentation studied at 4°C may not be relevant to the in vivo situation, similar experiments were performed with B cell incubation at 4°C, 37°C, and 37°C without washing away excess Bet v 1 and serum. As shown in Fig. 2, S-FAP by patient serum occurred at 4°C and, more efficiently, at 37°C. When excess allergen was not washed away after the incubation with APC at 37°C, an even more efficient presentation of Bet v 1 was observed, which may be explained by additional allergen presentation via non-IgE-mediated routes. Control sera were not able to induce T cell proliferation when the incubation with APC was performed at 4°C, but could be detected at the highest allergen concentrations when the incubation was performed at 37°C. This is probably the result of Ag uptake via fluid-phase endocytosis. These results indicate that S-FAP occurs very efficiently at physiological temperature, and therefore additional experiments were performed at 37°C, unless stated otherwise.

FIGURE 2.

S-FAP of Bet v 1 is more effective at 37°C than at 4°C. Serial dilutions of Bet v 1 were preincubated with 80% patient serum 894 or nonallergic control serum 745 for 1 h at 4°C or 37°C. AF EBV-B cells were added and incubated for 1 h at 37°C. The AF EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific T cell line AF (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

FIGURE 2.

S-FAP of Bet v 1 is more effective at 37°C than at 4°C. Serial dilutions of Bet v 1 were preincubated with 80% patient serum 894 or nonallergic control serum 745 for 1 h at 4°C or 37°C. AF EBV-B cells were added and incubated for 1 h at 37°C. The AF EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific T cell line AF (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

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S-FAP of Bet v 1 occurred when sera containing high levels of birch-specific IgE were used. To show that binding of IgE/allergen complexes to CD23 mediated to this effect, two sets of experiments were performed. First, patient and control sera were preincubated for 1 h at 37°C with serial dilutions of polyclonal IgE- or IgG-specific rabbit antiserum before allergen was added. Fig. 3 clearly shows that anti-IgE Abs completely inhibited the serum-facilitated presentation of Bet v 1, whereas anti-IgG Abs had no effect. To evaluate the role of CD23 in our culture model, experiments were performed by blocking CD23 or CD19 as a control B cell marker by preincubating the B cells for 1 h at 4°C with anti-CD23 or anti-CD19 mAb. Table II shows that preicubation of AF EBV-B cells with anti-CD23 mAb, but not anti-CD19 mAb, prevented the occurrence of S-FAP of Bet v 1 to Bet v 1-specific T cells. These experiments demonstrate that S-FAP of Bet v 1 is IgE dependent and is mediated via binding to CD23.

FIGURE 3.

S-FAP of Bet v 1 is inhibited by αIgE, but not by αIgG Abs. Bet v 1 (0.1 μg/ml) was preincubated for 1 h at 37°C with patient serum 1464 (at a final serum concentration of 40%) in the absence or presence of serial dilutions of polyclonal anti-IgE or anti-IgG Abs. Control serum 734 was used as a negative control. AF EBV-B cells were added and incubated for 1 h at 37°C. AF EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific TCC AF24 (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

FIGURE 3.

S-FAP of Bet v 1 is inhibited by αIgE, but not by αIgG Abs. Bet v 1 (0.1 μg/ml) was preincubated for 1 h at 37°C with patient serum 1464 (at a final serum concentration of 40%) in the absence or presence of serial dilutions of polyclonal anti-IgE or anti-IgG Abs. Control serum 734 was used as a negative control. AF EBV-B cells were added and incubated for 1 h at 37°C. AF EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific TCC AF24 (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

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Table II.

Inhibition of serum IgE-mediated allergen presentation by anti-CD23a

mAb (μg/ml)Patient Serum 1464Control Serum 734
+ Anti-CD19+ Anti-CD23+ Anti-CD19+ Anti-CD23
2.5 ± 0.3 3.7 ± 0.2 0.3 ± 0.0 0.3 ± 0.1 
0.6 4.2 ± 0.4 4.1 ± 0.7 0.5 ± 0.1 0.7 ± 0.1 
2.5 2.9 ± 0.7 3.4 ± 0.6 0.6 ± 0.0 0.7 ± 0.1 
10 5.3 ± 0.2 0.8 ± 0.1 0.7 ± 0.1 0.8 ± 0.1 
40 4.5 ± 0.6 0.9 ± 0.1 0.5 ± 0.1 1.0 ± 0.0 
mAb (μg/ml)Patient Serum 1464Control Serum 734
+ Anti-CD19+ Anti-CD23+ Anti-CD19+ Anti-CD23
2.5 ± 0.3 3.7 ± 0.2 0.3 ± 0.0 0.3 ± 0.1 
0.6 4.2 ± 0.4 4.1 ± 0.7 0.5 ± 0.1 0.7 ± 0.1 
2.5 2.9 ± 0.7 3.4 ± 0.6 0.6 ± 0.0 0.7 ± 0.1 
10 5.3 ± 0.2 0.8 ± 0.1 0.7 ± 0.1 0.8 ± 0.1 
40 4.5 ± 0.6 0.9 ± 0.1 0.5 ± 0.1 1.0 ± 0.0 
a

Bet v 1 (0.1 μg/ml) was preincubated for 1 h at 37°C with control serum 734 or with birch-allergic patient serum 1464 (>800 SU/ml birch-specific IgE in MagicLite assay) at a serum concentration of 33%. AF EBV-B cells were incubated for 1 h at 4°C with no Ab, with serial dilutions of anti-CD23 mAb MHM6, or with anti-CD19 mAb HD37, followed by two wash steps. The EBV-B cells were added to the preincubated serum fractions for 1 h at 37°C, resulting in a final cell concentration of 4 × 106/ml and a serum concentration of 16.7%, followed by two washes to remove excess allergen. The EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific TCC AF24 (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm (×10−3) of triplicate cultures ± SD.

To further study the relevance of S-FAP in birch-allergic patients, sera were collected from a panel of patients with different levels of birch-specific IgE (Table I). The sera that were used initially were sera containing >300 SU of birch-specific IgE, representing only a small percentage of all birch-allergic patients. As is shown in Fig. 4, S-FAP of Bet v 1 was observed in a serum-IgE-dependent fashion. Sera containing >100 SU birch-specific IgE were very efficient in mediating presentation of Bet v 1, but even sera containing 50–100 SU were able to mediate the effect. All other sera with <50 SU birch-specific IgE tested could not facilitate the presentation of Bet v 1. This indicates that the effect is relevant to the majority of clinically allergic patients.

FIGURE 4.

S-FAP is detectable in sera containing ≥50 SU/ml birch-specific IgE. Bet v 1 was preincubated with sera from birch-allergic patients sera (at a final serum concentration of 80%) containing varying amounts of birch-specific IgE for 1 h at 37°C. AF EBV-B cells were added and incubated for 1 h at 37°C. The AF EBV-B cells were cocultured with Bet v 1-specific AF cell line (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

FIGURE 4.

S-FAP is detectable in sera containing ≥50 SU/ml birch-specific IgE. Bet v 1 was preincubated with sera from birch-allergic patients sera (at a final serum concentration of 80%) containing varying amounts of birch-specific IgE for 1 h at 37°C. AF EBV-B cells were added and incubated for 1 h at 37°C. The AF EBV-B cells were cocultured with Bet v 1-specific AF cell line (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

Close modal

As sera from patients undergoing SAV contained high levels of allergen-specific IgG4 Abs, we wanted to address the question whether sera from patients undergoing birch SAV could inhibit the observed facilitated presentation of Bet v 1. To study this, Bet v 1 was preincubated for 1 h at 37°C with a patient serum containing high levels of birch-specific IgE in the presence of medium, nonallergic control sera, long-term birch SAV sera, or long-term grass SAV sera. Fig. 5 shows that all three long-term birch SAV sera inhibited S-FAP of Bet v 1 to the Bet v 1-specific T cells. Similar results were obtained when the experiments were performed at 4°C (data not shown). The effect was specific for birch SAV, because neither control sera nor long-term grass SAV sera could inhibit the effect. This effect was noted in all such experiments. In addition, IgE-mediated allergen presentation of the grass allergen Phl p 5 to a Phl p 5-specific T cell clone could be inhibited with long-term grass SAV but not birch SAV sera, further supporting the relevance of this finding (data not shown).

FIGURE 5.

S-FAP of Bet v 1 is inhibited by birch SAV sera, but not by grass SAV sera. Bet v 1 (0.1 μg/ml) was preincubated for 1 h at 37°C with patient serum 894 at a serum concentration of 40% in the presence of nonallergic control sera (734, ▪; 745, •), long-term birch SAV sera (1598, □; 1490, ○; 808840, ▵), or long-term grass SAV sera (885, closed lozenge; 808945, ▴) all at 40%. Control serum 734 (at 40%) without patient serum was used as a negative control (stippled line). AF EBV-B cells were added and incubated for 1 h at 37°C. The EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific TCC AF19 (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

FIGURE 5.

S-FAP of Bet v 1 is inhibited by birch SAV sera, but not by grass SAV sera. Bet v 1 (0.1 μg/ml) was preincubated for 1 h at 37°C with patient serum 894 at a serum concentration of 40% in the presence of nonallergic control sera (734, ▪; 745, •), long-term birch SAV sera (1598, □; 1490, ○; 808840, ▵), or long-term grass SAV sera (885, closed lozenge; 808945, ▴) all at 40%. Control serum 734 (at 40%) without patient serum was used as a negative control (stippled line). AF EBV-B cells were added and incubated for 1 h at 37°C. The EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific TCC AF19 (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

Close modal

To address the effect of the inhibition of T cell activation with SAV sera on cytokine production, supernatants were removed from cultures that had been set up under S-FAP conditions with a control serum, long-term birch SAV serum, and a long-term grass SAV serum. Table III shows that not only T cell proliferation, but also the production of the cytokines IL-4, IL-5, IL-10, and IFN-γ, was inhibited by the presence of birch SAV serum, but not by the presence of the control sera.

Table III.

Inhibition of S-FAP measured as T cell proliferation and cytokine production by birch SAV sera

T CellAg/Incubationcpma (×10−3)IL-5 (pg/ml)IL-4 (pg/ml)IFN-γ (pg/ml)IL-10 (pg/ml)
AF28 No Ag 0.4 
 Bet v 1 in well (10 μg/ml) 4.3 >3000 1746 
 + 920+ 734 (control serum) 6.6 798 228 
0.01 μg/ml + 920+ 1598 (>30m birch SAV) 1.3 
 + 920+ 885 (>30m grass SAV) 7.4 1876 299 175 
AF line No Ag 1.0 
 Bet v 1 in well (10 μg/ml) 35.6 >3000 416 308 1010 
 + 920+ 734 (control serum) 30.6 1907 72 124 
0.01 μg/ml + 920+ 1598 (>30m birch SAV) 6.9 93 
 + 920+ 885 (>30m grass SAV) 24.7 1368 79 121 
T CellAg/Incubationcpma (×10−3)IL-5 (pg/ml)IL-4 (pg/ml)IFN-γ (pg/ml)IL-10 (pg/ml)
AF28 No Ag 0.4 
 Bet v 1 in well (10 μg/ml) 4.3 >3000 1746 
 + 920+ 734 (control serum) 6.6 798 228 
0.01 μg/ml + 920+ 1598 (>30m birch SAV) 1.3 
 + 920+ 885 (>30m grass SAV) 7.4 1876 299 175 
AF line No Ag 1.0 
 Bet v 1 in well (10 μg/ml) 35.6 >3000 416 308 1010 
 + 920+ 734 (control serum) 30.6 1907 72 124 
0.01 μg/ml + 920+ 1598 (>30m birch SAV) 6.9 93 
 + 920+ 885 (>30m grass SAV) 24.7 1368 79 121 
a

Bet v 1 (0.01 μg/ml) was preincubated for 1 h at 37°C with patient serum 920 in the presence of nonallergic control serum 734, long-term birch SAV serum 1598, or long-term grass SAV serum. AF EBV-B cells were added to a final concentration of 2.5 × 106/ml and incubated for 1 h at 37°C, followed by washing away excess Ab and allergen. The EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific T cell line AF (2–4 × 104/well). After 24 h, supernatant was removed for cytokine measurements, and the cells were pulsed for 16 h with [3H]TdR and liquid scintillation counting.

Clinical efficacy of SAV is already noticeable after short-term SAV. To evaluate if the inhibitory effect of birch SAV sera on presentation of Bet v 1 could be demonstrated with short-term birch SAV sera, inhibition experiments were performed with a range of birch SAV sera (shown in Table IV). With these sera, inhibition could only be demonstrated after >30 mo of birch SAV (data not shown). However, inhibition experiments are performed by mixing an IgE-containing serum with a SAV serum, which may not completely represent the in vivo situation.

Table IV.

Measurement of birch and grass allergen-specific IgG4 and IgE

SerumBirch IgE (SU/ml)Grass IgE (SU/ml)Total IgE (IU/ml)SAV (mo)Birch IgG4 (RLU)Grass IgG4 (RLU)
Controls       
734 <30 No SAV 3,369 2,236 
745 <30 No SAV 7,510 5,636 
Birch allergics       
1327 50 118 805 No SAV 17,732 16,771 
1370 80 151 No SAV 13,984 28,065 
1568 56 880 No SAV 3,871 1,654 
1372 241 262 No SAV NTa NT 
1451 164 66 No SAV 6,983 3,577 
1334 133 29 218 No SAV 11,975 395,914 
920 143 29 >3,000 No SAV 18,130 10,621 
Birch SAV       
1430 61 34 3–9b 293,792 437,325 
809218 85 209 NT 3–9c 271,105 654,830 
808926 123 445 251 9–18b 114,544 367,286 
1459 157 125 1,035 9–18b 211,815 477,977 
1293 108 30 9–18 190,292 8,643 
809622 203 225 18–30 254,874 25,180 
809038 140 166 340 18–30c 5,035 416,457 
939 130 85 310 18–30c 141,947 437,288 
809978 62 79 18–30 165,063 5,133 
1598 67 29 >30 379,707 3,933 
1490 88 124 >30 4,643 1,874 
1713 113 73 >30 102,049 6,254 
809652 280 252 >30c 187,027 191,878 
808840 114 NT >30 189,814 3,387 
Grass SAV       
885 150 871 >30d 6,046 399,663 
808954 137 NT >30d 10,272 410,198 
SerumBirch IgE (SU/ml)Grass IgE (SU/ml)Total IgE (IU/ml)SAV (mo)Birch IgG4 (RLU)Grass IgG4 (RLU)
Controls       
734 <30 No SAV 3,369 2,236 
745 <30 No SAV 7,510 5,636 
Birch allergics       
1327 50 118 805 No SAV 17,732 16,771 
1370 80 151 No SAV 13,984 28,065 
1568 56 880 No SAV 3,871 1,654 
1372 241 262 No SAV NTa NT 
1451 164 66 No SAV 6,983 3,577 
1334 133 29 218 No SAV 11,975 395,914 
920 143 29 >3,000 No SAV 18,130 10,621 
Birch SAV       
1430 61 34 3–9b 293,792 437,325 
809218 85 209 NT 3–9c 271,105 654,830 
808926 123 445 251 9–18b 114,544 367,286 
1459 157 125 1,035 9–18b 211,815 477,977 
1293 108 30 9–18 190,292 8,643 
809622 203 225 18–30 254,874 25,180 
809038 140 166 340 18–30c 5,035 416,457 
939 130 85 310 18–30c 141,947 437,288 
809978 62 79 18–30 165,063 5,133 
1598 67 29 >30 379,707 3,933 
1490 88 124 >30 4,643 1,874 
1713 113 73 >30 102,049 6,254 
809652 280 252 >30c 187,027 191,878 
808840 114 NT >30 189,814 3,387 
Grass SAV       
885 150 871 >30d 6,046 399,663 
808954 137 NT >30d 10,272 410,198 
a

NT, not tested.

b

Birch, grass, and other SAV.

c

Birch and grass SAV.

d

Grass, but not birch, SAV.

Therefore, control sera, sera from birch-allergic patients receiving no treatment, and birch-allergic patients receiving birch SAV were matched for birch-specific serum IgE, and their ability to facilitate the presentation of Bet v 1 to specific T cells was measured in a direct S-FAP assay (Fig. 6,A for sera containing 100–300 SU/ml birch-specific IgE and Fig. 6,B for sera containing 50–100 SU/ml birch-specific IgE). These experiments clearly demonstrated that sera from untreated patients facilitate Bet v 1 presentation and that sera from the majority of patients undergoing birch SAV have a decreased ability to do so. This effect was already noted after 3–9 mo of birch SAV. The fact that some patients did not have a decreased ability to mediate S-FAP (809038, 809652) may indicate that these patients did not have optimal clinical efficacy. In line with the data presented in Table III, IL-5 production was also decreased in culture supernatants of these experiments, with the exception of the same two sera that did not have a decreased ability to mediate S-FAP (data not shown).

FIGURE 6.

Inhibition of S-FAP by SAV sera occurs rapidly after the start of treatment. Bet v 1 (0.01 μg/ml) was preincubated for 1 h at 37°C with sera from birch-allergic patients, nonallergic control sera, birch SAV sera, or grass SAV sera (all at 80%). The birch-specific serum IgE levels in the birch-allergic sera and the birch SAV sera were matched (i.e., 100–300 SU/ml birch-specific IgE (A) or 50–100 SU/ml birch-specific IgE (B)). AF EBV-B cells were added and incubated for 1 h at 37°C. The EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific T cell line AF (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

FIGURE 6.

Inhibition of S-FAP by SAV sera occurs rapidly after the start of treatment. Bet v 1 (0.01 μg/ml) was preincubated for 1 h at 37°C with sera from birch-allergic patients, nonallergic control sera, birch SAV sera, or grass SAV sera (all at 80%). The birch-specific serum IgE levels in the birch-allergic sera and the birch SAV sera were matched (i.e., 100–300 SU/ml birch-specific IgE (A) or 50–100 SU/ml birch-specific IgE (B)). AF EBV-B cells were added and incubated for 1 h at 37°C. The EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific T cell line AF (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

Close modal

As serum levels of allergen-specific IgG4 have been documented to increase after SAV, birch-specific IgG4 Ab levels were determined in the SAV sera (Table IV). Subjects were divided into several groups: nonallergic controls, birch-allergic controls that did not undergo SAV, birch-allergic patients that underwent 3–9 mo of SAV, 9–18 mo SAV, 18–30 mo of SAV, and >30 mo SAV. In addition, sera from grass allergic patients that had no sensitization to birch and had >30 mo of grass SAV were included as control sera. The levels of birch- and grass-specific IgE and IgG4 were measured in all these sera. Table IV shows that nonallergic controls and allergic patients that did not undergo SAV had very low levels of birch- or grass-specific IgG4. In contrast, sera from patients undergoing birch- or grass-specific SAV had high levels of specific IgG4 Abs, independent on the duration of treatment. Only one serum long-term birch SAV (serum 1490) did not have high serum levels of birch-specific IgG4. However, this serum was as potent as the other SAV sera in inhibiting S-FAP (see Figs. 5 and 6). Another serum (809652) did have increased birch allergen-specific IgG4 but showed no inhibition of S-FAP (Table IV, Fig. 6 A). In addition, as stated above, we have only been able to measure the inhibition of S-FAP in indirect assays with long-term SAV sera. Short-term SAV sera that contained similar or even higher levels of birch-specific IgG4 could not inhibit S-FAP (data not shown), strongly indicating that the inhibition of S-FAP noted cannot solely be explained by increased serum-levels of birch allergen-specific IgG4.

To further investigate which type of Ab mediates the blocking of S-FAP after SAV, we separted two long-term SAV sera on a protein G column. As Table V shows, all IgG Abs were present in the eluate of the column, and all IgA and IgM Abs were present in the run through. When the sera and the serum fractions were tested for their ability to inhibit S-FAP, only unfractionated sera and the fractions containing IgG inhibited S-FAP (Fig. 7), demonstrating that blocking IgG Abs but not IgA or IgM Abs inhibit S-FAP. This is supported by our observation that rabbit anti-Bet v 1 antiserum, as well as murine Bet v 1-specific monoclonal IgG Abs, also inhibit S-FAP (data not shown).

Table V.

Fractionation of long-term birch SAV seraa

IgG (μg/ml)IgA (μg/ml)IgM (μg/ml)
1598    
Serum 14,000 5,500 400 
Run through 5,700 400 
Eluate 9,900 
1490    
Serum 13,500 6,500 400 
Run through 6,500 400 
Eluate 9,800 
IgG (μg/ml)IgA (μg/ml)IgM (μg/ml)
1598    
Serum 14,000 5,500 400 
Run through 5,700 400 
Eluate 9,900 
1490    
Serum 13,500 6,500 400 
Run through 6,500 400 
Eluate 9,800 
a

Two long-term birch SAV sera (1598 and 1490) were fractionated on a protein G-Sepharose column. The eluate and run through fractions were adjusted to the original volume of the serum, and contents of IgG, IgA, and IgM were measured (see Materials and Methods).

FIGURE 7.

IgG but not IgA or IgM present in SAV sera inhibit S-FAP. Bet v 1 (0.01 μg/ml) was preincubated for 1 h at 37°C with patient serum 1372 at a serum concentration of 40% in the presence of nonallergic control serum 734, long-term birch SAV sera 1598 and 1490, or fractions of these sera containing IgG or IgA and IgM (all at 40%). Serum 1372 (at 40%) without Ag was used as a negative control. AF EBV-B cells were added to a final concentration of 1.5 × 106/ml for 1 h at 37°C, followed by washing away excess serum and allergen. The EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific TCC AF19 (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

FIGURE 7.

IgG but not IgA or IgM present in SAV sera inhibit S-FAP. Bet v 1 (0.01 μg/ml) was preincubated for 1 h at 37°C with patient serum 1372 at a serum concentration of 40% in the presence of nonallergic control serum 734, long-term birch SAV sera 1598 and 1490, or fractions of these sera containing IgG or IgA and IgM (all at 40%). Serum 1372 (at 40%) without Ag was used as a negative control. AF EBV-B cells were added to a final concentration of 1.5 × 106/ml for 1 h at 37°C, followed by washing away excess serum and allergen. The EBV-B cells (1–2 × 104/well) were used as APC and were cocultured with Bet v 1-specific TCC AF19 (2–4 × 104/well) for 24 h, followed by a 16-h [3H]TdR pulse and liquid scintillation counting. Values shown are mean cpm of triplicate cultures. Error bars represent SDs.

Close modal

The presentation of extremely low doses of allergen to CD4+ T lymphocytes is greatly enhanced by IgE-dependent Ag focussing to CD23 or FcεRI. Here, we demonstrate that this process is inhibited by blocking allergen-specific IgG Abs that are induced by SAV. As a result of this, increased doses of allergens are required to induce T cell proliferation and cytokine production in vivo.

An increasing body of evidence indicates that S-FAP is a mechanism that occurs in vivo. Atopic allergic individuals have been shown to have increased expression of CD23 on B cells and macrophages (6, 7) and FcεRI on monocytes and dendritic cells (8, 9). Both receptors have been shown to be involved in S-FAP in vitro (10, 11, 12, 13, 14). In vivo Th2 responses and specific IgG1 and IgE production in mice are enhanced by allergen-IgE complexes (38, 39) or by allergen-anti-CD23 mAb conjugates (40), whereas CD23 knockout mice lack this ability (41).

In addition, clinical trials with a neutralizing anti-IgE mAb have shown that anti-IgE mAb inhibits bronchial LPR and the number of eosinophils in sputum of allergic asthma patients (42), strongly suggesting that IgE is crucial for the development of LPR (43, 44) and that inhibition of this mechanism results in decreased late-phase allergic reactions. Similarly, the production of IL-4 and IL-5 and the recruitment of eosinophils to the lungs of mice sensitized with house dust mite can be inhibited by treatment with an anti-CD23 or anti-IgE mAb, and the same effect is observed in CD23 knockout mice (45).

All these findings strongly suggest that IgE, as well as IgE receptors, plays an important role in the in vivo activation of CD4+ T cells, the regulation of humoral responses, and most importantly in late-phase allergic responses.

S-FAP of Bet v 1, the major birch pollen allergen, was shown to occur at both 4°C and 37°C and was dependent on the levels of birch-specific IgE present in the sera. The effect was demonstrated to occur with sera that contained >50 SU birch-specific IgE, indicating that the effect is relevant for the majority of patients undergoing SAV. The local production of allergen-specific IgE in the nasal mucosa of grass pollen-allergic patients (46) indicates that allergens entering the nasal and possibly also the bronchial mucosa may indeed be captured in the target organ by allergen-specific IgE and be focussed to CD23+ or FcεRI+ APC.

S-FAP induced by patient sera containing >300 SU birch-specific IgE could be inhibited by sera from patients undergoing long-term birch SAV, resulting in a strongly reduced proliferation of Bet v 1-specific CD4+ T cells. In addition, the production of the allergy-associated type 2 cytokines IL-4 and IL-5 was strongly inhibited, as was the production of the cytokines IL-10 and IFN-γ.

The effect of this inhibition was allergen specific as it was shown that sera from patients undergoing long-term grass SAV could not inhibit the effect on Bet v 1-specific T cells.

Additional inhibition experiments showed that S-FAP of Bet v 1 could also not be inhibited by a combination of long-term grass SAV sera with grass extract (data not shown), thereby excluding the possibility that IgG-allergen complexes induce an inhibitory signal in B lymphocytes after engagement of FcγRIIb1, as has been shown in other models (47).

Furthermore, when sera from untreated birch-allergic patients and patients undergoing birch SAV that were matched for birch-specific IgE levels were compared in their ability to induce S-FAP, sera from the untreated patients could induce proliferation of allergen-specific T cells at low allergen concentrations, whereas this ability was inhibited in most sera from patients undergoing birch SAV. This further substantiates that SAV-treated patients may require higher allergen levels to activate allergen-specific T cells in vivo. The inhibition was not seen in all patients undergoing birch SAV. This may indicate that these patients did not have an optimal clinical response to SAV and that the effect may correlate with the clinical response to treatment. As clinical data of the patients were not available, we are currently testing sera from a placebo-controlled double blind birch SAV study to address this question.

One of the long term-birch SAV sera that inhibited S-FAP did not contain birch-specific IgG4 (serum 1490), and only one of the two sera that did not show a clear inhibition of S-FAP had no high birch-specific IgG4 levels (serum 809038 and 809652). Therefore, the inhibition of S-FAP after SAV could not completely be explained by levels of birch-specific IgG4 induced by birch SAV, which may explain why the rise in specific IgG4 Abs after SAV does not correlate with clinical efficacy (15). However, our data show that serum IgG, and not other Ab classes, mediates the inhibition of S-FAP (Table V and Fig. 7). This indicates that the induction of blocking allergen-specific IgG Abs including IgG4, rather than IgG4 Abs alone, prevent the occurrence of S-FAP. These data are supported by the observation that rabbit anti-Bet v 1 antiserum, as well as Bet v 1-specific monoclonal IgG Abs, can also inhibit S-FAP (data not shown).

Late-phase allergic responses have been shown to be dependent on the presence and activation of CD4+ T lymphocytes (48, 49, 50, 51, 52), eosinophils (50, 51), and factors involved in eosinophil differentiation such as IL-5 (43, 53, 54). Allergen threshold levels for immediate hypersensitivity responses and LPR are strongly increased after SAV (reviewed in Refs. 15 and 55). This may be explained by our observation that sera from patients undergoing SAV have a decreased ability to activate allergen-specific CD4+ T cells via S-FAP.

Based on these observations, we would like to propose the following working mechanism of SAV: Initially, SAV induces the production of IL-12 and IL-10 by APC (32, 33). This results in a shift in the production of cytokine-producing CD4+ T cells toward the Th0 phenotype (27, 28, 29, 30). Both the production of more Th0-like cytokines and IL-10 (32) induce the production of all subclasses of allergen-specific IgG, including IgG4 (32, 56, 57, 58).

When these Abs reach sufficiently high levels or affinities to compete with the binding of IgE to allergens (which according to Table IV and Fig. 6 may already occur after 3 mo of SAV), IgE-mediated allergen presentation to CD4+ T cells is inhibited at low allergen concentrations, and even histamine release from mast cells and basophils may be inhibited via a similar process (59). Because LPR are mediated by the costimulation-dependent activation and cytokine production by CD4+ T lymphocytes (48, 49, 50, 51, 52), this results in a much lower LPR as the result of a much higher allergen threshold for T cell activation in the presence of unchanged levels of allergen-specific IgE in the serum of patients undergoing birch SAV.

In addition, it has also been demonstrated that CD23 interacts with CD11b and CD11c on monocytes (60), which results in an increased production of proinflammatory cytokines. Binding of IgE to CD23 inhibits the production of these factors, which include Th1-biasing cytokines. An inhibition of IgE binding to CD23 by SAV as shown in this paper may therefore result in and enhanced production of Th1-biasing cytokines by APC, which results in a feedback inhibition of Th2 responses. This is accompanied by a shift in the use of B cells to the use of dendritic cells or macrophages as APC, which may also promote Th1, rather than Th2, development.

Thus, the results shown here provide evidence that links many of the previous observations on the mechanisms of SAV. Future research should be focused on the further characterization of the inhibitory allergen-specific IgG responses induced by SAV, as well as on studies that can correlate the observed inhibition of IgE-mediated allergen presentation with clinical efficacy of SAV treatment.

We thank V. Nebel for expert technical assistance, Dr. M. L. Kapsenberg for critically reading the manuscript, and Dr. J. P. Kinet for the kind gift of FcεRI-specific mAb 15.1.

2

Abbreviations used in this paper: SAV, specific allergy vaccination; LPR, late phase responses; S-FAP, serum-facilitated allergen presentation; TCC, T cell clone.

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