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Keywords:

  • allergen;
  • allergoid;
  • basophils;
  • dendritic cells;
  • T helper type 1/T helper type 2

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Although allergen-specific immunotherapy is a clinically effective therapy for IgE-mediated allergic diseases, the risk of IgE-mediated adverse effects still exists. For this reason, chemically modified allergoids have been introduced, which may destroy IgE-binding sites while T-cell activation should be retained. The aim of the study was to analyse the differences between intact allergens and differently modified/aggregated allergoids concerning their internalization as well as T-cell and basophil activation. For this purpose human monocyte-derived immature dendritic cells (DC) were incubated with Phleum pratense or Betula verrucosa pollen extract or with the corresponding allergoids, modified with formaldehyde or glutaraldehyde. After an additional maturation process, the antigen-loaded mature DC were co-cultured with autologous CD4+ T cells. Allergenicity was tested by leukotriene release from basophils. In addition, the uptake of intact allergens and allergoids by immature DC was analysed. The proliferation of, as well as the interleukin-4 (IL-4), IL-10, IL-13 and interferon-γ production by, CD4+ T cells which had been stimulated with glutaraldehyde allergoid-treated DC was reduced compared with CD4+ T cells stimulated with intact allergen-treated or formaldehyde allergoid-treated DC. In line with this, glutaraldehyde-modified allergoids were more aggregated and were internalized more slowly. Furthermore, only the allergoids modified with glutaraldehyde induced a decreased leukotriene release by activated basophils. These findings suggest that IgE-reactive epitopes were destroyed more efficiently by modification with glutaraldehyde than with formaldehyde under the conditions chosen for these investigations. Glutaraldehyde-modified allergoids also displayed lower T-cell stimulatory capacity, which is mainly the result of greater modification/aggregation and diminished uptake by DC.


Abbreviations:
DC

dendritic cells

F

formaldehyde

G

glutaraldehyde

TT

tetanus toxoid

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

The prevalence of allergic diseases like allergic rhinitis and asthma has increased dramatically in the last decades. Atopic allergic diseases are characterized by allergen specific T helper type 2-dominated immune responses to otherwise innocuous antigens (e.g. allergens from grass and tree pollen).1 T helper type 2 cells provide help for strong antibody responses including IgE synthesis by B lymphocytes and support the growth and differentiation of eosinophils and mast cells.2–4 Specific immunotherapy (SIT) is the only disease-modifying therapy known.5 During subcutaneous SIT, increasing amounts of allergens are injected to achieve tolerance. The T helper type 2-dominated immune response is modified by SIT to an immune response characterized by a shift towards T helper type 1 cytokine production and immunoregulation with enhanced production of interleukin-10 (IL-10) and transforming growth factor-β.6–9 These cellular changes are accompanied by induction of allergen-specific non-IgE antibodies competing with IgE for allergen binding.10

To reduce the possible clinical side-effects of SIT, e.g. the risk of anaphylactic shock, allergoids have been introduced for SIT by Marsh et al.,11 who hypothesized that allergen preparations with a reduced allergenicity and retained or even increased immunogenicity would be beneficial to the treatment effect of SIT. Allergoids are allergen extracts chemically modified by formaldehyde or glutaraldehyde that are intended to have reduced allergenicity while maintaining immunogenicity. Formaldehyde and glutaraldehyde react with primary amino groups in the polypeptide chain of the allergen leading to intramolecular and intermolecular cross-linked high-molecular-weight allergen polymers; in this way conformational IgE epitopes should be destroyed while the linear T-cell epitopes remain unaffected.12–15 However, some publications have demonstrated that allergoids also display a reduced T-cell stimulatory capacity.16–19 Additionally, the stimulation of T cells by the allergoid seems to be dependent on the type of antigen-presenting cell: whereas dendritic cells (DC) and macrophages are highly effective cells for the presentation of allergoids, peripheral blood mononuclear cells (PBMC) and B cells are less effective.17,20 As DC are not only critical for the induction of primary immune responses, but are also important for tolerance induction during specific immunotherapy21 we used this type of antigen-presenting cell in our study to investigate in more detail the in vitro T-cell reactivity of intact timothy grass pollen and birch pollen allergens in comparison with differently modified/aggregated allergoids induced by formaldehyde or glutaraldehyde. Furthermore, we measured the allergenicity of intact allergens and allergoids by basophil activation tests and analysed their internalization by immature DC by sequential flow cytometry and confocal laser scanning microscopy.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Allergoid preparation and SDS–PAGE

Intact allergen extracts (10 mg/ml in PBS) of Betula verrucosa (Bet v) and Phleum pratense (Phl p) (ALK-Abello, Hørsholm, Denmark) were used for modification with formaldehyde or glutaraldehyde. The procedures were optimized based on Marsh et al.11 for formaldehyde and Lee et al.22 for glutaraldehyde, as previously described.19 First, allergen extracts were filtered through a 0·45-μm filter. Then, 0·031 ml 0·5% formaldehyde/mg allergen extract (0·155% formaldehyde/ml allergen extract) (Sigma-Aldrich, Steinheim, Germany) or 0·0625 ml 0·5% glutaraldehyde/mg allergen extract (0·313% glutaraldehyde/ml allergen extract) (Merck, Darmstadt, Germany) was added drop-wise and the mixture was left at room temperature with magnetic stirring for 4 hr. Next, an additional 0·0625 ml 0·5% formaldehyde/mg allergen extract (final formaldehyde concentration of 0·31%/ml original allergen extract) or 0·125 ml 0·5% glutaraldehyde/mg allergen extract (final glutaraldehyde concentration of 0·625%/ml original allergen extract) was added drop-wise and the solution was left at room temperature with magnetic stirring for 18 hr. The reaction was stopped by adding 2·12 ml of 2·5 m glycine per ml of 0·5% formaldehyde or 1·06 ml of 2·5 m glycine per ml of 0·5% glutaraldehyde and the solution was kept at room temperature with magnetic stirring for 1 hr.

To remove excess aldehyde reagents, the samples were applied to a size exclusion column (superdex 75 16/60) (GE Healthcare, Uppsala, Sweden). Fractions containing protein from this size exclusion column were pooled on the basis of the size exclusion column chromatogram, Fused Rocket Immuno Electrophoresis and SDS–PAGE analysis. The pooled fractions were then concentrated on an Amicon ultra (Millipore, Billerica, MA) centrifuge tube.

SDS–PAGE was performed on a NuPAGE 10% bis–tris gel (Invitrogen, Carlsbad, CA). Samples were pre-treated with sample buffer containing SDS (Invitrogen). A pre-stained protein standard (Invitrogen) was run in parallel for quantification of the molecular weights. Proteins were stained with silver staining.

Blood samples

Buffy coats were obtained from 34 atopic donors sensitized to Phleum pratense and/or Betula verrucosa with an ImmunoCAP class ≥ 2 (Transfusion Centre, Mainz, Germany). Specific sensitization was verified by detection of allergen-specific IgE in the sera (ImmunoCAP® specific IgE blood test; Phadia AB, Uppsala, Sweden). The study was approved by the local ethics committee. Informed consent was obtained from all subjects before the study.

Generation of monocyte-derived DC

Peripheral blood mononuclear cells and autologous plasma were isolated by Ficoll Paque 1·077 g/ml (PAA Laboratories GmbH, Cölbe, Germany) density centrifugation from heparinized blood. CD14+ cells were enriched by incubation of 5 × 106 PBMC in a 12-well-plate (Greiner, Frickenhausen, Germany) with 1 ml/well Iscove’s modified Dulbecco’s medium containing l-glutamine and 25 mm HEPES (IMDM; PAA Laboratories GmbH) and 3% autologous plasma, which was collected from the upper phase after Ficoll density centrifugation and which was heat-inactivated for 30 min at 56°, at 37° for 45 min. Non-adherent cells were washed twice with pre-warmed PBS. The remaining monocytes were incubated with 1·5 ml/well IMDM, 1% autologous plasma, 1000 U/ml IL-4 (Miltenyi Biotec, Bergisch Gladbach, Germany) and 200 U/ml granulocyte–macrophage colony-stimulating factor (GM-CSF, Leukine®; Immunex Corp., Seattle, WA). On day 6 the immature DC were pulsed with intact allergen and allergoids in different concentrations (0·8–20 μg/ml) and stimulated with 1000 U/ml tumour necrosis factor-α, 2000 U/ml IL-1β (Miltenyi Biotec) and 1 μg/ml prostaglandin E2 (Cayman Chemical, Ann Arbor, MI) to reach final maturation.23,24 After 48 hr, the mature DC were harvested, washed twice with PBS, and used for T-cell stimulation assays. Mature DC expressed high levels (> 90%) of CD80, CD83, CD86 and MHC-class II molecules as controlled by flow cytometry. Viability and surface marker expression of DC was not affected by allergen and allergoids.

Purification of T cells

Autologous CD4+ T cells were obtained from PBMC using antibody-coated paramagnetic MicroBeads (MACS, Miltenyi Biotec) according to the protocol of the manufacturer. Separation was controlled by flow cytometry (purity > 98% CD4+ T cells).

Co-culture of T cells and autologous intact allergen-pulsed or allergoid-pulsed DC

For the proliferation assay 1 × 105 CD4+ T cells were co-cultured in triplicates with 1 × 104 DC, pulsed with intact allergen or allergoid, in 200 μl IMDM supplemented with 5% heat-inactivated autologous plasma. After 5 days the cells were pulsed with 37 kBq/well of [methyl-3H]thymidine ([3H]TdR; ICN, Irvine, CA) for 6 hr. Incorporation of [3H]TdR was measured in a beta-counter (1205 Betaplate; LKB Wallac, Turku, Finland).

For the cytokine assay 5 × 105 CD4+ T cells and 5 × 104 intact allergen-pulsed or allergoid-pulsed DC were co-cultured in a 48-well-plate in 1 ml IMDM supplemented with 5% heat-inactivated autologous plasma. On day 7 the T cells were re-stimulated with freshly generated intact allergen-pulsed or allergoid-pulsed DC. After 24 hr the supernatants were collected.

Quantification of cytokine production by ELISA

Human IL-4, IL-10, IL-13, interferon-γ (IFN-γ) (BD Biosciences, Heidelberg, Germany), IL-17A and IL-22 (R&D Systems, Wiesbaden, Germany) were measured by ELISA according to the instructions of the manufacturers of the employed pairs of antibodies.

Determination of leukotriene release from activated basophils

Allergenicity was tested via leukotriene release of activated basophils using the cellular antigen stimulation test (CAST®-2000 ELISA; Bühlmann Laboratories AG, Schönenbuch, Switzerland) according to the protocol of the manufacturer. Briefly, dextran (250 μl) was added to 1 ml EDTA–blood of 10 atopic donors sensitized to Phleum pratense or Betula verrucosa with an ImmunoCAP class ≥ 2 and incubated for 90 min at room temperature to allow sedimentation of erythrocytes. Then, peripheral blood leucocytes were centrifuged at 130 g for 15 min and resuspended in 1 ml stimulation buffer containing IL-3. Two hundred microlitres of peripheral blood leucocytes were stimulated with 50 μl intact allergen or allergoid at different concentrations or mouse anti-human IgE receptor antibody as positive control for 40 min at 37°. Finally, the cells were centrifuged and supernatants were stored at −20° until analysis of leukotriene concentration.

Flow cytometric analysis and confocal laser microscopy

For analysis of allergen uptake by immature DC, intact allergen and allergoid extracts were labelled with Alexa Fluor® 488 Protein Labeling Kit or with pHrodo™ according to the manufacturer’s protocol (Molecular Probes, Eugene, OR). Labelled intact allergen or allergoid was added to DC on day 6 of culture and internalization was analysed after 10 min up to 24 hr in a FACSCalibur (Becton Dickinson, Mountain View, CA) equipped with cellquest software (Becton Dickinson). In some experiments, mannan (200 μg/ml) or dimethylamiloride (300 μm) (both from Sigma-Aldrich) were added 30 min before intact allergen or allergoid.

For confocal laser scanning microscopy, internalization of labelled intact allergen or allergoid was filmed for 90 min or several layers of the cells were shot after 6 hr using the Zeiss Laser Scanning System LSM 710 (Zeiss, Göttingen, Germany) equipped with a CO2 and temperature controlled chamber (H. Saur, Reutlingen, Germany). In some experiments, DC were labelled with 60 nm LysoTracker® Green DND-26 (Molecular Probes) 10 min before analysis.

Statistics

Analysis of variance was used between different experimental groups. Student’s paired t-test was used to test the statistical significance of the results; P ≤ 0·05 was considered to be significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Modification of intact allergen extracts with glutaraldehyde is more pronounced than modification with formaldehyde

First, we compared modification of intact allergen extracts with formaldehyde and glutaraldehyde by SDS–PAGE. In Fig. 1(a) it is shown that treatment of intact allergen extracts with glutaraldehyde resulted in protein aggregation as a high-molecular-weight smear is observed in the gel and no distinct bands were visible, whereas for formaldehyde treatment a smear with less high molecular weight is observed compared with glutaraldehyde treatment. In addition, broader bands were observed compared with the unmodified extracts. The larger amount of aggregates in the glutaraldehyde modification was also confirmed by Fused Rocket Immuno Electrophoresis. For both modified extracts all cathodic proteins were missing (data not shown) indicating that the charge on the proteins was changed by the modification.

image

Figure 1.  Modification of intact allergen extracts with glutaraldehyde resulted in protein aggregation and a reduced stimulatory capacity to induce leukotriene release by basophils. (a) SDS–PAGE on a 10% polyacrylamide gel of different intact allergen and allergoid preparations (where G is glutaraldehyde-modified and F is formaldehyde-modified) (5 μg per lane). Bet v: Lane 1: marker, lane 2: Bet v G allergoid, lane 3: Bet v F allergoid, lane 4: Bet v intact allergen extract; Phl p: lane 1: marker, lane 2: Phl p G allergoid, lane 3: Phl p F allergoid, lane 4: Phl p intact allergen extract. (b) Peripheral blood leucocytes were stimulated with different concentrations of intact allergen extracts or the corresponding allergoids modified with formaldehyde (F) or glutaraldehyde (G), and the supernatants were analysed for leukotriene release by ELISA after 40 min. Shown are the means ± SEM from 10 allergic donors. * indicates statistically significant differences (P < 0.05) between intact allergen extracts and allergoids.

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Glutaraldehyde-modified but not formaldehyde-modified allergoids showed decreased leukotriene release by basophils compared with intact allergens

The IgE-binding and basophil activating capacity of intact allergens and allergoids was tested by their capacity to release leukotrienes from basophils from sensitized donors. As shown in Fig. 1(b), stimulation with high concentrations of intact allergen extracts or the corresponding allergoid preparations led to the release of similar leukotriene levels. However, in contrast to the intact allergen extracts and the formaldehyde allergoids, the leukotriene release was significantly reduced when the cells were stimulated with decreasing amounts of glutaraldehyde allergoids.

Glutaraldehyde-modified allergoids induced reduced T-cell proliferative responses compared with formaldehyde-modified allergoids and intact allergens

To analyse the immunogenicity of the intact allergens and allergoids, immature DC were pulsed with different concentrations of intact allergens and allergoids, matured and then co-cultured with autologous CD4+ T cells. Intact allergen-pulsed and allergoid-pulsed DC were both able to activate T cells. Proliferation increased with increasing concentrations of intact allergens or allergoids. Intact allergen-pulsed and formaldehyde allergoid-pulsed DC induced similar proliferative responses. In contrast to this, glutaraldehyde allergoid-pulsed DC led to a significantly reduced T-cell activation (Fig. 2).

image

Figure 2.  Reduced T-cell activation by glutaraldehyde-modified allergoids. Intact allergen-pulsed or allergoid-pulsed mature dendritic cells (DC) were co-cultured with autologous CD4+ T cells and proliferation was analysed by [3H]thymidine incorporation. Results are expressed as means ± SEM from 34 allergic donors. * indicates statistically significant differences (P < 0.05) between intact allergen-pulsed and allergoid-pulsed DC.

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Glutaraldehyde-modified allergoid-pulsed DC induced lower IL-4, IL-10, IL-13 and IFN-γ production in CD4+ T cells compared with formaldehyde-modified allergoid-pulsed DC and intact allergen-pulsed DC

In addition to proliferation, the ability of intact allergen-pulsed and allergoid-pulsed DC to induce cytokine production in CD4+ T cells was investigated. For this, T cells co-cultured with mature intact allergen-pulsed or allergoid-pulsed DC were re-stimulated with newly generated autologous DC after 7 days pulsed in the same way to achieve measurable amounts of cytokine production. Interleukin-13 was predominantly produced by T cells in all conditions (Fig. 3). The T cells stimulated with glutaraldehyde allergoid-pulsed DC produced lower amounts of IL-13 compared with cells stimulated with intact allergen-pulsed or formaldehyde allergoid-pulsed DC. Interleukin-4, IL-10 and IFN-γ were secreted to a lesser extent. Again, glutaraldehyde-stimulated cells showed a reduced production of these cytokines (Fig. 3). Although these reductions were not significant, they reflect and support the observations made for the proliferation. Furthermore, IL-17 and IL-22 were analysed: while IL-17 production was very low or even undetectable (data not shown), no differences in IL-22 production were observed between T cells stimulated with intact allergen-pulsed or allergoid-pulsed DC (Fig. 3).

image

Figure 3.  CD4+ T cells activated by glutaraldehyde allergoid-pulsed dendritic cells (DC) show decreased production of interleukin-4 (IL-4), IL-10, IL-13 and interferon-γ (IFN-γ), but not decreased IL-22 production. CD4+ T cells were stimulated twice with intact allergen-pulsed or allergoid-pulsed DC and cytokine production was quantified by ELISA. Results are expressed as means ± SEM from 32 (for IL-4, IL-10, IL-13 and IFN-γ) or 11 (for IL-22) allergic donors.

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Internalization of glutaraldehyde-modified allergoids by immature DC was retarded compared with formaldehyde-modified allergoids and intact allergens

For internalization experiments intact allergen extracts and modified allergens were first labelled with AlexaFluor® 488 (Molecular Probes) and added to immature DC. The uptake was analysed by flow cytometry and confocal laser scanning microscopy. Both, intact allergen extracts and allergoids were internalized by DC. Allergens modified with glutaraldehyde were taken up about fourfold slower than intact allergens and formaldehyde allergoids (Fig. 4a). This finding was consistent with observations obtained by laser scanning microscopy where DC were filmed for 90 min (Fig. 4b).

image

Figure 4.  Internalization of AlexaFluor® 488 (Molecular Probes) or pHrodo™-labelled intact allergen and allergoid into immature dendritic cells (DC). Intact allergen extract and the corresponding allergoids were labelled with AlexaFluor® 488 (Molecular Probes) (a, b and e) or pHrodo™ (c and d). The labelled antigen was added to immature DC at 10 μg/ml unless otherwise indicated on day 6 of culture, and the uptake was investigated by flow cytometry at different time-points (a, c and e) or by confocal laser scanning microscopy filmed for 90 min (b) or several layers of the cells were shot after 6 hr (d) with additional labelling of DC with 60 nm LysoTracker® Green 10 min before analysis. (e) The inhibitors mannan (200 μg/ml), and dimethylamiloride (DMA; 300 μm) were added to the cells 30 min before the addition of 0.5 μg/ml AlexaFluor® 488-labelled intact allergen or allergoids. Shown are the means ± SEM from four allergic donors (a, c and e), a sequential view of the films (b, scale bar 20 μm), and different layers of the DC from one representative donor [d, scale bar 20 μm (left panels) and 5 μm (right panels)]. * indicates statistically significant differences (P < 0.05) between intact allergen-pulsed and allergoid-pulsed DC (a, c). MFI, mean fluorescence intensity.

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However, the fluorescence intensity (labelling) of glutaraldehyde-modified allergoids was also about two to three times lower than the intensity of intact allergen extracts and formaldehyde-modified allergoids despite equal protein contents, probably because the AlexaFluor® 488 (Molecular Probes) reactive dye reacts with primary amines of proteins, which may be less accessible in the glutaraldehyde-modified allergoid showing higher aggregate formation. Therefore, we used another dye for labelling, the rhodamine-based, fluorogenic dye pHrodo™, with which we achieved equal labelling of intact allergens and allergoids. Additionally, this dye is non-fluorescent at neutral pH and has a bright red fluorescence in acidic environments making it ideal for studying endocytosis. The addition of pHrodo™-labelled intact allergen or allergoids to immature DC confirmed our results obtained in Fig. 4(a,b), as we also observed a retarded uptake and internalization of glutaraldehyde-modified allergoid into the acid lysosomes compared with formaldedyde-modified allergoid or intact allergen (Fig. 4c). Furthermore, confocal laser-scanning microscopy revealed a good internalization of the intact allergen and the formaldehyde-treated allergoid into the lysosomes after 6 hr, whereas the glutaraldehyde-treated allergoid still stacked on the surface of the DC and was not yet internalized at this time-point (Fig. 4d). Co-staining of DC with LysoTracker® Green led to a co-localization of the red allergens and the green lysosomes resulting in a yellow staining of the DC lysosomes.

To investigate and characterize the mechanisms of internalization of the allergens/allergoids, mannan, a polymer of mannose that competitively blocks endocytosis of mannose-rich structures present in grass pollen allergens,25 was used to block the mannose receptor-mediated antigen uptake or dimethylamiloride was used to block macropinocytosis.26,27 All inhibitors were added 30 min before application of the allergens/allergoids. Blockade of macropinocytosis completely inhibited internalization, and blockade of the mannose receptor reduced antigen uptake of allergens and formaldehyde-treated allergoids by about 50–75%, whereas uptake of glutaraldehyde-treated allergoids was only reduced by about 20–50% (Fig. 4e).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

The aim of this study was to investigate the differences in immunogenicity and allergenicity between intact Phleum pratense and Betula verrucosa allergen extracts and the corresponding allergoids modified with formaldehyde or glutaraldehyde. We could demonstrate that glutaraldehyde modified the allergen extracts to a higher degree than formaldehyde (less distinct bands, larger high-molecular-weight aggregates) in the conditions investigated. Furthermore, glutaraldehyde-modified allergoids, but not formaldehyde-modified allergoids, led to a reduced leukotriene release by basophils of sensitized donors compared with the intact allergen extracts. This finding suggests that IgE-binding epitopes of modified allergoids were destroyed most efficiently by modification with glutaraldehyde. Besides the destroyed IgE-binding epitopes, glutaraldehyde modified allergoid-pulsed DC also exhibited lower T-cell stimulatory capacity leading to a significantly reduced proliferation and also a tendency to reduced cytokine production (IL-4, IL-10, IL-13 and IFN-γ) of CD4+ T cells co-cultured with glutaraldehyde allergoid-pulsed DC compared with cells stimulated with formaldehyde allergoid-pulsed or intact allergen-pulsed DC. Glutaraldehyde allergoid-pulsed mature DC differed neither morphologically from formaldehyde allergoid-pulsed or intact allergen-pulsed DC nor concerning their expression of surface markers including MHC class II and co-stimulatory molecules (data not shown). However, we could show by flow cytometry and confocal laser scanning microscopy that glutaraldehyde allergoid was taken up into immature DC more slowly than formaldehyde allergoid and intact allergen.

Our observations are in line with several other studies also demonstrating reduced T-cell stimulation by chemically modified allergens without interfering with the cytokine production profile.16–19 However, there are also reports showing only reduced IgE-binding activity but no difference when comparing the allergen and allergoid-induced PBMC proliferation.14,28 The degree of T-cell stimulation certainly depends on the type of antigen-presenting cell employed. Kahlert et al.20 have observed the same stimulatory capacity of allergoid-pulsed DC compared with allergen-pulsed DC, whereas other APC (i.e. PBMC and B cells) induced less proliferation after stimulation with allergoids. In contrast to B cells and PBMC, DC may process the allergoid more efficiently by macropinocytosis or phagocytosis rather than by an Fc-receptor-mediated mechanism used for antigen recognition and uptake.26,29 According to this statement, allergoids should be taken up by immature DC in a manner similar to allergens. To answer this question we labelled formaldehyde-modified and glutaraldehyde-modified allergoids and intact allergens with a fluorescence dye and analysed the uptake by immature DC of all three antigens via confocal laser microscopy and by flow cytometry. We observed a much slower internalization of glutaraldehyde-modified allergoid compared with formaldehyde-modified allergoid or intact allergen by immature DC from allergic donors and a later co-localization in lysosomal compartments. To analyse uptake of allergen/allergoid in further detail, we blocked the two pathways DC use for antigen capture, i.e. macropinocytosis and the mannose receptor. In accordance with the studies by Sallusto et al.26 and Noirey et al.,30 we found that internalization of both allergen and allergoid was strongly or even completely inhibited after blockade of the mannose receptor or pinocytosis pathway, respectively. The observation that the sensitivity to blockade with mannan seems to be lower for glutaraldehyde-treated allergoids may be explained by destroyed mannose receptor binding sites because of aggregate formation during glutaraldehyde treatment. As monocyte-derived DC not only express the mannose receptor but also the high-affinity receptor for IgE, FcεRI,31 the reduced responsiveness of T cells to grass or birch pollen glutaraldehyde-modified allergoid-pulsed DC in our study and to house dust mite or birch pollen allergoid-pulsed DC in other studies16–18,32 may also be the result of impaired FcεRI-mediated uptake because of destroyed IgE-binding epitopes, especially when DC from sensitized donors are used in cultures with autologous serum. Indeed, in some allergic donors we observed a slightly reduced uptake of allergen and allergoid after blockade of the Fcε receptor (data not shown). As we have shown in a recent publication that dissociated monomers of the cockroach allergen Per a III were internalized faster by immature DC than the intact hexameric molecule, also leading to enhanced T-cell proliferation,33 the different uptake of formaldehyde-modified compared with glutaraldehyde-modified allergoid may be because of differences in polymerization. In any case, slower uptake and processing of glutaraldehyde-modified allergoids seems to be one central reason for reduced proliferation and cytokine production of DC-stimulated CD4+ T cells. Another possibility may be that the decreased stimulatory activity of allergoid-pulsed DC primarily results from a selective loss or degradation of certain T-cell epitopes because of the glutaraldehyde treatment as suggested by studies employing T-cell clones with distinct specificities. Despite this loss of reactivity to allergoid in some allergen-specific T-cell clones, several clinical studies have shown the efficacy of allergoid therapy, especially in combination with adjuvants such as aluminium hydroxide or monophosphoryl lipid A.34–39 This leads to the presumption that the remaining allergoid-responsive clones are sufficient to induce similar detectable immunological changes as observed for immunotherapy with intact allergens, i.e. down-regulation of allergen-specific IgE, induction of IgG/IgG4, and induction of IFN-γ and IL-10 production.35,36,40–42 However, clinical studies directly comparing treatment effect of and immunological responses to intact allergens versus allergoids are missing. Dose–efficacy assessments are also hampered by difficulties in standardization of allergoid preparations (measurement of protein content of the major allergen).

In summary, the glutaraldehyde-modified allergoids used in this study had disrupted IgE-binding sites and induced a lower T-cell stimulatory capacity in antigen-presenting DC, which is mainly the result of diminished uptake but probably also of a lower frequency of responding T cells. In contrast, formaldehyde-modified allergoids retained both IgE-binding and T-cell epitopes. Hence, reduced allergenicity seems to be at least partially linked to reduced in vitro T-cell reactivity, which must be taken into account concerning the clinical use of allergoids for immunotherapy as discussed above.

Disclosures

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

H.H. and P.A.W are employed by ALK-Abelló A/S. The rest of the authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References