Antigen dose‐dependent predominance of either direct or sequential switch‘qc in IgE antibody responses

Abstract

Priming of CBA/J mice with minute doses of protein antigens (Ag) leads to high IgE antibody (Ab) titres in the immune sera of these animals. In contrast priming with large doses elicits only a marginal production of IgE Ab. In vitro restimulation of spleen cells from animals primed with large doses and lacking in vivo IgE Ab leads to a burst of IgE Ab-forming cells. This in vitro anamnestic response is lacking in mice primed with minute doses of Ag. In order to trace the cellular basis of the in vitro IgE memory response we have extended the analysis of the distribution of Ab isotypes to Ag-primed IgG1-deficient delta 5'S gamma 1 mice. The data presented here must be interpreted as followed. Priming of mice with minute doses of Ag leads to a direct switch from IgM to IgE Ab expression in both strains. These animals have high IgE Ab titres without establishing an IgE memory. The direct switch was verified by polymerase chain reaction and Southern blot analysis of switch circle DNA isolated from Ag-specific B cells of CBA/J mice primed with minute doses of Ag. In contrast to immunization with minute doses, priming with large doses of Ag fails to induce in vivo IgE Ab production in CBA/J and delta 5'S gamma 1 mice but establishes a B epsilon memory in CBA/J mice which involves IgG1-bearing intermediate B cells. In vivo these B epsilon memory cells do not enter the status of IgE Ab-producing cells. In vitro they can be released from this anergy and presumed suppression and develop in an anamnestic response into a large population of IgE Ab-forming B cells. This increase in the number of IgE Ab-producing cells after restimulation in vitro is lacking in delta 5'S gamma 1 mice, apparently because of their inability to generate IgG1-expressing precursor cells. The notion of a sequential switch and an IgG1 intermediate B epsilon memory status is also supported by depletion and inhibition experiments. Elimination of IgG1-expressing B cells in CBA/J mice primed with high doses of Ag prevents the IgE Ab burst after in vitro challenge with Ag. The data further suggest that the two switch pathways are not mutually exclusive and that the Ag dose can decide which pathway is preferentially used.

Phl p 1–Specific Human Monoclonal IgE and Design of a Hypoallergenic Group 1 Grass Pollen Allergen Fragment

“…It is thus not unlikely that immunization with a fully or partially denatured allergen in an emulsified adjuvant would give rise to Abs targeting epitopes other than the conformational epitopes bound by IgE. Furthermore, natural sensitization in allergy most likely occurs via a very weak Ag stimulation, favoring a direct class switch from IgM to IgE (48), whereas the much stronger stimulation in immunization would mainly trigger a second pathway leading to an initial switch to IgG (48). Because indications that there are differences in epitopes targeted by IgE and IgG have been presented (49), it is possible that Abs developed via these two different class-switch pathways would then also target different epitopes.…”

Transcriptional targeting of dendritic cells for gene therapy using the promoter of the cytoskeletal protein fascin

“…Using cytofluorometry, Detection of antigen-specific IgG antibodies. Sera were recovered from the retro-orbital plexus of immunized mice and EGFP-specific or b-galactosidase-specific IgG was measured by ELISA as described 25 with the modification that recombinant EGFP (2 mg/ml; BD Biosciences CLONTECH) or b-galactosidase (5 mg/ml; Sigma-Aldrich) was used as antigen. The antibody titer was defined as the reciprocal serum dilution, yielding an absorbance value of OD¼0.2 after linear regression analysis.…”

Efficacy of recombinant adenovirus as vector for allergen gene therapy in a mouse model of type I allergy

“…27 All reagents except the blocking buffer and the substrate solution were used in volumes of 100 l/well. After each incubation step, wells were washed three times with PBS (pH 7.2) containing 0.1% Tween 20 (Sigma-Aldrich).…”

“…26 These characteristics lead us to speculate that recombinant Ad might represent an efficient vehicle for allergen gene transfer. We tested this notion in a mouse model of type I allergy where we took advantage of the fact that repeated immunization with low doses of protein antigen induces considerable IgE Ab formation 27 and thus resembles the main hallmark of type I allergy. In this setting, we evaluated the efficacy of preventive or therapeutic vaccination with replication-defective recombinant Ad expressing the model antigen ␤-galactosidase (␤gal) under the control of the CMV promoter (AdCMV-␤gal) to modulate the immune response following immunization with ␤gal-protein.…”