Julia Eckl-Dorna

Basophils are not the key antigen-presenting cells in allergic patients.

Recent data obtained in mouse models have initiated a controversy whether basophils are the key antigen‐presenting cells (APCs) in allergy. Here, we investigate whether basophils are of importance for the presentation of allergen and the induction of T cell proliferation in allergic patients.

The majority of allergen‐specific IgE in the blood of allergic patients does not originate from blood‐derived B cells or plasma cells

The production of allergen‐specific IgE antibodies is a hallmark of IgE‐mediated allergy but the contribution of blood cells to allergen‐specific IgE production in allergic patients has not been studied in detail.

Two types of BCR interactions are positively selected during leukemia development in the Eμ-TCL1 transgenic mouse model of CLL

Chronic lymphocytic leukemia (CLL) is a common B-cell malignancy characterized by a highly variable course and outcome. The disease is believed to be driven by B-cell receptor (BCR) signals generated by external antigens and/or cell-autonomous BCR interactions, but direct in vivo evidence for this is still lacking. To further define the role of the BCR pathway in the development and progression of CLL, we evaluated the capacity of different types of antigen/BCR interactions to induce leukemia in the Eμ-TCL1 transgenic mouse model. We show that cell autonomous signaling capacity is a uniform characteristic of the leukemia-derived BCRs and represents a prerequisite for CLL development. Low-affinity BCR interactions with autoantigens generated during apoptosis are also positively selected, suggesting that they contribute to the pathogenesis of the disease. In contrast, high-affinity BCR interactions are not selected, regardless of antigen form or presentation. We also show that the capacity of the leukemic cells to respond to cognate antigen correlates inversely with time to leukemia development, suggesting that signals induced by external antigen increase the aggressiveness of the disease. Collectively, these findings provide in vivo evidence that the BCR pathway drives the development and can influence the clinical course of CLL.

Staphylococcus aureus fibronectin-binding protein specifically binds IgE from patients with atopic dermatitis and requires antigen presentation for cellular immune responses.

BACKGROUND Staphylococcus aureus superinfections occur in more than 90% of patients with atopic dermatitis (AD) and aggravate skin inflammation. S aureus toxins lead to tissue damage and augment T-cell-mediated skin inflammation by a superantigen effect. OBJECTIVE To characterize IgE-reactive proteins from S aureus. METHODS A genomic S aureus library was screened with IgE from patients with AD for DNA clones coding for IgE-reactive antigens. One was identified as fibronectin-binding protein (FBP). Recombinant FBP was expressed in Escherichia coli, purified, and tested for specific IgE reactivity in patients with AD. Its allergenic activity was studied in basophil activation experiments and T-cell cultures. The in vivo allergenic activity was investigated by sensitizing mice. RESULTS Using IgE from patients with AD for screening of a genomic S aureus library, an IgE-reactive DNA clone was isolated that coded for FBP. Recombinant FBP was expressed in E coli and purified. It reacted specifically with IgE from patients with AD and exhibited allergenic activity in basophil degranulation assays. FBP showed specific T-cell reactivity requiring antigen presentation and induced the secretion of proinflammatory cytokines from PBMCs. Mice sensitized with FBP mounted FBP-specific IgE responses, showed FBP-specific basophil degranulation as well as FBP-specific T-cell proliferation, and mixed T(h)2/T(h)1 cytokine secretion. CONCLUSION Evidence is provided that specific humoral and cellular immune responses to S aureus antigens dependent on antigen presentation represent a novel mechanism for S aureus-induced skin inflammation in AD. Furthermore, FBP may be used for the development of novel diagnostic and therapeutic strategies for S aureus infections.

What Is the Source of Serum Allergen-Specific IgE?

Immunoglobulin E (IgE), the key effector element in the induction and propagation of allergic diseases, is the least abundant antibody class. In allergic patients, class switch recombination to IgE in B cells is induced by allergen contact in conjunction with T cell interaction and a Th2 cytokine environment. With regard to future therapeutic approaches, the sites of IgE production in human subjects and the nature and characteristics of IgE-producing cells are of great interest. In this context, it has been shown that allergen-specific IgE levels can be boosted by contact with allergens via the respiratory mucosa of the nose. Also, it has been proposed that allergy effector organs (e.g., the nasal mucosa and the lung) may be important sites of IgE production in allergic patients. IgE-producing cells have also been found in the blood, but their numbers are extremely low. Transfer of specific sensitization during bone marrow transplantation indicates the presence of IgE-producing B memory cells or plasma cells also in the bone marrow. This review summarizes data on the induction of IgE production, IgE memory and the sites of IgE production in human allergic patients.

CD23 surface density on B cells is associated with IgE levels and determines IgE-facilitated allergen uptake, as well as activation of allergen-specific T cells

Background: Increasing evidence suggests that the low‐affinity receptor for IgE, CD23, plays an important role in controlling the activity of allergen‐specific T cells through IgE‐facilitated allergen presentation. Objective: We sought to determine the number of CD23 molecules on immune cells in allergic patients and to investigate whether the number of CD23 molecules on antigen‐presenting cells is associated with IgE levels and influences allergen uptake and allergen‐specific T‐cell activation. Methods: Numbers of CD23 molecules on immune cells of allergic patients were quantified by using flow cytometry with QuantiBRITE beads and compared with total and allergen‐specific IgE levels, as well as with allergen‐induced immediate skin reactivity. Allergen uptake and allergen‐specific T‐cell activation in relation to CD23 surface density were determined by using flow cytometry in combination with confocal microscopy and T cells transfected with the T‐cell receptor specific for the birch pollen allergen Bet v 1, respectively. Defined IgE‐allergen immune complexes were formed with human monoclonal allergen‐specific IgE and Bet v 1. Results: In allergic patients the vast majority of CD23 molecules were expressed on naive IgD+ B cells. The density of CD23 molecules on B cells but not the number of CD23+ cells correlated with total IgE levels (RS = 0.53, P = .03) and allergen‐induced skin reactions (RS = 0.63, P = .008). Uptake of allergen‐IgE complexes into B cells and activation of allergen‐specific T cells depended on IgE binding to CD23 and were associated with CD23 surface density. Addition of monoclonal IgE to cultured PBMCs significantly (P = .04) increased CD23 expression on B cells. Conclusion: CD23 surface density on B cells of allergic patients is correlated with allergen‐specific IgE levels and determines allergen uptake and subsequent activation of T cells.

Recombinant allergen-based provocation testing

Over the last 25 years, recombinant allergens from all important allergen sources have been cloned and are now available as recombinant proteins. These molecules can be produced in practically unlimited amounts without biological or batch-to-batch variability. It has been shown in provocation tests that recombinant allergens have similar clinical effects as their natural counterparts. With the help of these tools it is possible to reveal the precise reactivity profiles of patients and to uncover and differentiate cross-reactivity from genuine sensitization to an allergen source. Although it has been shown some time ago that it would be possible to replace crude allergen extracts with recombinant allergens for skin prick testing, and even though the use of allergen components can improve routine diagnosis, these tools are still not available for clinical routine applications. The use of provocation tests is a crucial step in the development of new, hypoallergenic vaccines for therapy of allergic disease. Here we describe important provocation methods (skin prick test, intradermal test, atopy patch test, nasal provocation, colonoscopic provocation test) and give an overview of the clinical provocation studies which have been performed with recombinant allergens so far.

Critical and direct involvement of the CD23 stalk region in IgE binding

Background: The low‐affinity receptor for IgE, Fc&egr;RII (CD23), contributes to allergic inflammation through allergen presentation to T cells, regulation of IgE responses, and enhancement of transepithelial allergen migration. Objective: We sought to investigate the interaction between CD23, chimeric monoclonal human IgE, and the corresponding birch pollen allergen Bet v 1 at a molecular level. Methods: We expressed 4 CD23 variants. One variant comprised the full extracellular portion of CD23, including the stalk and head domain; 1 variant was identical with the first, except for an amino acid exchange in the stalk region abolishing the N‐linked glycosylation site; and 2 variants represented the head domain, 1 complete and 1 truncated. The 4 CD23 variants were purified as monomeric and structurally folded proteins, as demonstrated by gel filtration and circular dichroism. By using a human IgE mAb, the corresponding allergen Bet v 1, and a panel of antibodies specific for peptides spanning the CD23 surface, both binding and inhibition assays and negative stain electron microscopy were performed. Results: A hitherto unknown IgE‐binding site was mapped on the stalk region of CD23, and the non–N‐glycosylated monomeric version of CD23 was superior in IgE binding compared with glycosylated CD23. Furthermore, we demonstrated that a therapeutic anti‐IgE antibody, omalizumab, which inhibits IgE binding to Fc&egr;RI, also inhibited IgE binding to CD23. Conclusion: Our results provide a new model for the CD23‐IgE interaction. We show that the stalk region of CD23 is crucially involved in IgE binding and that the interaction can be blocked by the therapeutic anti‐IgE antibody omalizumab.

Safety and efficacy of immunotherapy with the recombinant B-cell epitope–based grass pollen vaccine BM32

Background: BM32 is a grass pollen allergy vaccine based on recombinant fusion proteins consisting of nonallergenic peptides from the IgE‐binding sites of the 4 major grass pollen allergens and the hepatitis B preS protein. Objective: We sought to study the safety and clinical efficacy of immunotherapy (allergen immunotherapy) with BM32 in patients with grass pollen–induced rhinitis and controlled asthma. Methods: A double‐blind, placebo‐controlled, multicenter allergen immunotherapy field study was conducted for 2 grass pollen seasons. After a baseline season, subjects (n = 181) were randomized and received 3 preseasonal injections of either placebo (n = 58) or a low dose (80 &mgr;g, n = 60) or high dose (160 &mgr;g, n = 63) of BM32 in year 1, respectively, followed by a booster injection in autumn. In the second year, all actively treated subjects received 3 preseasonal injections of the BM32 low dose, and placebo‐treated subjects continued with placebo. Clinical efficacy was assessed by using combined symptom medication scores, visual analog scales, Rhinoconjunctivitis Quality of Life Questionnaires, and asthma symptom scores. Adverse events were graded according to the European Academy of Allergy and Clinical Immunology. Allergen‐specific antibodies were determined by using ELISA, ImmunoCAP, and ImmunoCAP ISAC. Results: Although statistical significance regarding the primary end point was not reached, BM32‐treated subjects, when compared with placebo‐treated subjects, showed an improvement regarding symptom medication, visual analog scale, Rhinoconjunctivitis Quality of Life Questionnaire, and asthma symptom scores in both treatment years. This was accompanied by an induction of allergen‐specific IgG without induction of allergen‐specific IgE and a reduction in the seasonally induced increase in allergen‐specific IgE levels in year 2. In the first year, more grade 2 reactions were observed in the active (n = 6) versus placebo (n = 1) groups, whereas there was almost no difference in the second year. Conclusions: Injections of BM32 induced allergen‐specific IgG, improved clinical symptoms of seasonal grass pollen allergy, and were well tolerated.

An assay that may predict the development of IgG enhancing allergen-specific IgE binding during birch immunotherapy

It has been shown that birch pollen immunotherapy can induce IgG antibodies which enhance IgE binding to Bet v 1. We aimed to develop a serological assay to predict the development of antibodies which enhance IgE binding to Bet v 1 during immunotherapy.