Claudia Kitzmüller

Human blood basophils do not act as antigen-presenting cells for the major birch pollen allergen Bet v 1

Several studies in mice have recently shown that basophils can act as antigen‐presenting cells (APC) inducing Th2‐mediated immune responses against parasites or protease allergens. The aim of this study was to investigate whether human basophils function as APC for the major birch pollen allergen Bet v 1.

Assessing protein immunogenicity with a dendritic cell line-derived endolysosomal degradome.

Background The growing number of novel candidate molecules for the treatment of allergic diseases imposed a dramatic increase in the demand for animal experiments to select immunogenic vaccines, a pre-requisite for efficacy. Because no in vitro methods to predict the immunogenicity of a protein are currently available, we developed an in vitro assay that exploits the link between a proteins immunogenicity and its susceptibility to endolysosomal proteolysis. Methodology We compared protein composition and proteolytic activity of endolysosomal fractions isolated from murine bone marrow- and human blood- derived dendritic cells, and from the dendritic cell line JAWS II. Three groups of structurally related antigen variants differing in their ability to elicit immune responses in vivo (Bet v 1.0101 and Bet v 1.0401, RNases A and S, holo- and apo-HRP) were subjected to in vitro simulated endolysosomal degradation. Kinetics and patterns of generated proteolytic peptides were evaluated by gel electrophoresis and mass spectrometry. Results Antigens displaying weak capacity of T cell priming in vivo were highly susceptible to endolysosomal proteases in vitro. As proteolytic composition, activity, and specificity of endolysosomal fractions derived from human and murine dendritic cells were comparable, the JAWS II cell line could be used as a substitute for freshly isolated human or murine cells in in vitro degradation assays. Conclusions Endolysosomal fractions prepared from the JAWS II cell line provide a reliable tool for in vitro estimation of protein immunogenicity. The rapid and simple assay described here is very useful to study the immunogenic properties of a protein, and can help to replace, reduce, and refine animal experiments in allergy research and vaccine development in general.

Correlation of sensitizing capacity and T-cell recognition within the Bet v 1 family

Background Bet v 1 is the main sensitizing allergen in birch pollen. Like many other major allergens, it contains an immunodominant T cell–activating region (Bet v 1142-156). Api g 1, the Bet v 1 homolog in celery, lacks the ability to sensitize and is devoid of major T-cell epitopes. Objective We analyzed the T-cell epitopes of Mal d 1, the nonsensitizing Bet v 1 homolog in apple, and assessed possible differences in uptake and antigen processing of Bet v 1, Api g 1, and Mal d 1. Methods For epitope mapping, Mal d 1–specific T-cell lines were stimulated with overlapping synthetic 12-mer peptides. The surface binding, internalization, and intracellular degradation of Bet v 1, Api g 1, and Mal d 1 by antigen-presenting cells were compared by using flow cytometry. All proteins were digested with endolysosomal extracts, and the resulting peptides were identified by means of mass spectrometry. The binding of Bet v 1142-156 and the homologous region in Mal d 1 by HLA class II molecules was analyzed in silico. Results Like Api g 1, Mal d 1 lacked dominant T-cell epitopes. The degree of surface binding and the kinetics of uptake and endolysosomal degradation of Bet v 1, Api g 1, and Mal d 1 were comparable. Endolysosomal degradation of Bet v 1 and Mal d 1 resulted in very similar fragments. The Bet v 1142-156 and Mal d 1141-155 regions showed no striking difference in their binding affinities to the most frequent HLA-DR alleles. Conclusion The sensitizing activity of different Bet v 1 homologs correlates with the presence of immunodominant T-cell epitopes. However, the presence of Bet v 1142-156 is not conferred by differential antigen processing.

Differential activation of dendritic cells by toll-like receptors causes diverse differentiation of naive CD4+ T cells from allergic patients.

To avert the differentiation of allergen‐specific Th2 cells in atopic individuals is a major goal in the prevention and therapy of IgE‐mediated allergy. We aimed to compare different toll‐like receptor (TLR) agonists regarding their effects on antigen‐presenting cells and the differentiation of naïve T cells from allergic patients.

A hypoallergenic variant of the major birch pollen allergen shows distinct characteristics in antigen processing and T‐cell activation

BM4 is a novel genetically engineered variant of the major birch pollen allergen Bet v 1 that lacks the typical Bet v 1‐like fold and displays negligible IgE‐binding but strong T cell–activating capacity. The aim of this study was to elucidate possible differences between BM4 and Bet v 1 in internalization, antigen processing, and presentation.

Glutathione-S-Transferase: A Minor Allergen in Birch Pollen due to Limited Release from Hydrated Pollen

Background Recently, a protein homologous to glutathione-S-transferases (GST) was detected in prominent amounts in birch pollen by proteomic profiling. As members of the GST family are relevant allergens in mites, cockroach and fungi we investigated the allergenic relevance of GST from birch (bGST). Methodology bGST was expressed in Escherichia coli, purified and characterized by mass spectrometry. Sera from 217 birch pollen-allergic patients were tested for IgE-reactivity to bGST by ELISA. The mediator-releasing activity of bGST was analysed with IgE-loaded rat basophil leukaemia cells (RBL) expressing human FcεRI. BALB/c mice were immunized with bGST or Bet v 1. Antibody and T cell responses to either protein were assessed. IgE-cross-reactivity between bGST with GST from house dust mite, Der p 8, was studied with murine and human sera in ELISA. The release kinetics of bGST and Bet v 1 from birch pollen were assessed in water, simulated lung fluid, 0.9% NaCl and PBS. Eluted proteins were quantified by ELISA and analysed by immunoblotting. Principle findings Only 13% of 217 birch pollen-allergic patients showed IgE-reactivity to bGST. In RBL assays bGST induced mediator release. Immunization of mice with bGST induced specific IgE and a Th2-dominated cellular immune response comparably to immunization with Bet v 1. bGST did not cross-react with Der p 8. In contrast to Bet v 1, only low amounts of bGST were released from pollen grains upon incubation in water and the different physiological solutions. Conclusion/Significance Although bGST is abundant in birch pollen, immunogenic in mice and able to induce mediator release from effector cells passively loaded with specific IgE, it is a minor allergen for birch pollen-allergic patients. We refer this discrepancy to its limited release from hydrated pollen. Hence, bGST is an example demonstrating that allergenicity depends mainly on rapid elution from airborne particles.

The quantity and quality of α‐gal‐specific antibodies differ in individuals with and without delayed red meat allergy

IgG to galactose‐α‐1,3‐galactose (α‐gal) are highly abundant natural antibodies (Ab) in humans. α‐Gal‐specific IgE Ab cause a special form of meat allergy characterized by severe systemic reactions 3–7 h after consumption of red meat. We investigated 20 patients who experienced such reactions and characterized their α‐gal‐specific IgE and IgG responses in more detail.

A novel role for neutrophils in IgE-mediated allergy: evidence for antigen-presentation in late-phase reactions

Background Neutrophils and allergen‐specific T cells accumulate in patients with allergic late‐phase reactions (LPRs). Their presence is associated with severe inflammation. Cytokines, such as GM‐CSF, IFN‐&ggr;, and IL‐3, which are typically found in patients with allergic LPRs, have been proposed to convert neutrophils into antigen‐presenting cells (APCs). Objective We sought to assess the antigen‐processing and antigen‐presenting capacities of neutrophils from allergic patients. Methods Neutrophils were isolated from peripheral blood of donors with birch pollen allergy and stimulated with GM‐CSF, IFN‐&ggr;, and IL‐3. The viability and expression of HLA‐DR, CD80, and CD86 were assessed by using flow cytometry. HLA‐DM expression was analyzed by means of immunoblotting. Allergen uptake was studied after fluorescence labeling of the major birch pollen allergen Bet v 1. Bet v 1 was digested with neutrophilic endolysosomal extracts, and the resulting fragments were sequenced by using mass spectrometry. Neutrophils were used as APCs in coculture experiments with autologous HLA‐DR–restricted and Bet v 1–specific T‐cell clones reactive with epitopes in different regions of the allergen. In all experiments monocytes were used for comparison. Fluids from suction blisters formed on top of LPRs induced by using intradermal allergen injection were assessed for HLA‐DR+ neutrophils by using flow cytometry. Results The cytokines significantly enhanced the survival, allergen uptake, and expression of HLA‐DM and HLA‐DR on neutrophils. Neutrophils rapidly degraded Bet v 1 into fragments containing all relevant T‐cell epitopes. Cytokine‐activated, allergen‐pulsed neutrophils induced proliferative and cytokine responses of Bet v 1–specific T cells irrespective of epitope specificity, confirming that they fully processed and presented the allergen. HLA‐DR+ neutrophils were detected in patients with cutaneous allergic LPRs. Conclusion Neutrophils can serve as APCs for local allergen‐specific effector T cells in patients with allergic LPRs. Graphical abstract Figure. No Caption available.

Glutathione-s-transferase is a minor allergen in birch pollen because of restricted release from hydrated pollen grains

Methods bGST was expressed in Escherichia coli, purified and characterized by mass spectrometry. BALB/c mice were immunized with bGST or Bet v 1. Antibody and T cell responses were assessed. 217 sera from birch pollen-allergic patients were tested for IgE-reactivity to bGST by ELISA. The allergenicity of bGST was evaluated with IgE-loaded rat basophil leukaemia cells (RBL) expressing the a-chain of the human receptor FceRI. Cross-reactivity of IgE between bGST and GST from house dust mite, Der p 8, was assessed with murine and human sera in ELISA. The release kinetics of bGST and Bet v 1 from birch pollen upon hydration were studied by immunoblotting.