Gernot Achatz

AllergoOncology: the role of IgE-mediated allergy in cancer

Epidemiological studies have suggested inverse associations between allergic diseases and malignancies. As a proof of concept for the capability of immunoglobulin E (IgE) to destruct tumor cells, several experimental strategies have evolved to specifically target this antibody class towards relevant tumor antigens. It could be demonstrated that IgE antibodies specific to overexpressed tumor antigens have been superior to any other immunoglobulin class with respect to antibody‐dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) reactions. In an alternative approach, IgE nonspecifically attached to tumor cells proved to be a powerful adjuvant establishing tumor‐specific immune memory. Active Th2 immunity could also be achieved by applying an oral immunization regimen using mimotopes, i.e. epitope mimics of tumor antigens. The induced IgE antibodies could be cross‐linked by live tumor cells leading to tumoricidic mediator release. Thus, IgE antibodies may not only act in natural tumor surveillance, but could possibly also be exploited for tumor control in active and passive immunotherapy settings. Thereby, eosinophils, mast cells and macrophages can be armed with the cytophilic IgE and become potent anti‐tumor effectors, able to trace viable tumor cells in the tissues. It is strongly suggested that the evolving new field AllergoOncology will give new insights into the role of IgE‐mediated allergy in malignancies, possibly opening new avenues for tumor therapy.

Induction of long-lived allergen-specific plasma cells by mucosal allergen challenge

BACKGROUND Allergen-specific IgE antibodies are responsible for the pathogenesis of type I hypersensitivity. In patients with allergy, IgE titers can persist in the apparent absence of allergen for years. Seasonal allergen exposure triggers clinical symptoms and enhances allergen-specific IgE. Whether allergen-specific plasma cells originating from seasonal allergen exposures can survive and become long-lived is so far unclear. OBJECTIVE We analyzed the localization and lifetimes of allergen-specific IgE-secreting, IgA-secreting, and IgG(1)-secreting plasma cells after allergen inhalation in an ovalbumin-induced murine model of allergic asthma. METHODS Ovalbumin-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting cells in lungs, spleen, and bone marrow were isolated and tested for antibody secretion by the ELISpot technique. Longevity of ovalbumin-specific plasma cells was determined by cyclophosphamide treatment, which depletes proliferating plasmablasts but leaves plasma cells untouched. Ovalbumin aerosol-induced infiltrates in lungs were localized by confocal microscopy. RESULTS Long-lived ovalbumin-specific plasma cells were generated by systemic sensitization and survived in bone marrow and spleen, maintaining systemic ovalbumin-specific titers of IgG, IgA, and IgE. On inhalation of ovalbumin-containing aerosol, sensitized mice developed airway inflammation and more ovalbumin-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting cells in the lungs and in secondary lymphoid organs. These plasma cells joined the pool of ovalbumin-specific plasma cells in the bone marrow and became long-lived-that is, they are resistant to cyclophosphamide. Termination of ovalbumin inhalation depleted ovalbumin-specific plasma cells from the lungs, but they persisted in spleen and bone marrow. CONCLUSION Our results show that inhalation of aerosolized allergen generates long-lived, allergen-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting plasma cells that survive cytostatic treatment.

The riddle of the dual expression of IgM and IgD.

Signalling through the B cell antigen receptor (BCR) is required for peripheral B lymphocyte maturation, maintenance, activation and silencing. In mature B cells, the antigen receptor normally consists of two isotypes, membrane IgM and IgD (mIgM, mIgD). Although the signals initiated from both isotypes differ in kinetics and intensity, in vivo, the BCR of either isotype seems to be able to compensate for the loss of the other, reflected by the mild phenotypes of mice deficient for mIgM or mIgD. Thus, it is still unclear why mature B cells need expression of mIgD in addition to mIgM. In the current review we suggest that the view that IgD has a simpIy definable function centred around the basic signalling function should be replaced by the assumption that IgD fine tunes humoral responses, modulates B cell selection and homeostasis and thus shapes the B cell repertoire, defining IgD to be a key modulator of the humoral immune response.

Enolases are highly conserved fungal allergens.

BACKGROUND Lack of knowledge of the identity of fungal allergens still is a major obstacle for improvement of diagnosis and therapy of allergies to moulds. We have therefore further analyzed the allergens of the two moulds, Alternaria alternata and Cladosporium herbarum and found that enolases (EC 4.2.1.11) are major allergens, at least of the two fungal species just mentioned. METHODS The enolases of Alternaria and Cladosporium were cloned from cDNA libraries constructed from vegetative cells of the two moulds by immunological screening with sera from selected patients allergic to the moulds. The two enolases were expressed as recombinant nonfusion proteins and used for determination of the incidence of allergy to enolase among a cohort of patients. RESULTS Sequencing of the two enolases showed very close relationships with other known fungal enolase sequences. Competition experiments using immunoblots of the recombinant nonfusion proteins showed nearly complete identity of the epitopes on both enolases. Serum from a patient reactive to Cladosporium enolase reacted equally well with the enolases of Alternaria, Saccharomyces and Candida. About 50% each of the sera from patients reactive to Cladosporium and Alternaria were strongly reactive to the recombinant enolases. CONCLUSIONS Enolases are therefore considered to be highly conserved major fungal allergens.

Inefficient processing of mRNA for the membraneform of IgE is a genetic mechanism to limit recruitment of IgE-secreting cells

Immunoglobulin E (IgE) is the key effector element in allergic diseases ranging from innocuous hay fever to life‐threatening anaphylactic shock. Compared to other Ig classes, IgE serum levels are very low. In its membrane‐bound form (mIgE), IgE behaves as a classical antigen receptor on B lymphocytes. Expression of mIgE is essential for subsequent recruitment of IgE‐secreting cells. We show that in activated, mIgE‐bearing B cells, mRNA for the membrane forms of both murine and human epsilon (ϵ) heavy chains (HC) are poorly expressed compared to mRNA for the secreted forms. In contrast, in mIgG‐bearing B cells, mRNA for the membrane forms of murine gamma‐1 (γ1) and the corresponding human γ4 HC are expressed at a much higher level than mRNA for the respective secreted forms. We show that these findings correlate with the presence of deviant polyadenylation signal hexamers in the 3′ untranslated region (UTR) of both murine and human ϵ genes, causing inefficient processing of primary transcripts and thus poor expression of the proteins and poor recruitment of IgE‐producing cells in the immune response. Thus, we have identified a genetic steering mechanism in the regulation of IgE synthesis that represents a further means to restrain potentially dangerous, high serum IgE levels.

Molecular and cellular targets of anti-IgE antibodies

Immunoglobulin E (IgE) was the last of the immunoglobulins discovered. It is present in very low amounts (nano‐ to micro‐gram per ml range) in the serum of normal healthy individuals and normal laboratory mouse strains and has a very short half‐life. This contrasts with the other immunoglobulin classes, which are present in much higher concentrations (micro‐ to milligram per ml range) and form a substantial component of serum proteins. Immunoglobulins play a role in homeostatic mechanisms and they represent the humoral arm of defence against pathogenic organisms. Since IgE antibodies play a key role in allergic disorders, a number of approaches to inhibit IgE antibody production are currently being explored. In the recent past the use of nonanaphylactic, humanized anti‐IgE antibodies became a new therapeutic strategy for allergic diseases. The therapeutic rational beyond the idea derives from the ability of the anti‐IgE antibodies to bind to the same domains on the IgE molecule that interact with the high‐affinity IgE receptor, thereby interfering with the binding of IgE to this receptor without cross‐linking the IgE on the receptor (nonanaphylactic anti‐IgE antibodies). Treatment with anti‐IgE antibodies leads primarily to a decrease in serum IgE levels. As a consequence thereof, the number of high‐affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells reducing the release of inflammatory mediator such as histamine, prostaglandins and leukotrienes. Experimental studies in mice indicate that injection of some monoclonal anti‐IgE antibodies also inhibited IgE production in vivo. The biological mechanism behind this reduction remains speculative. A possible explanation may be that these antibodies can also interact with membrane bound IgE on B cells, which could interfere the IgE production.

Antigen aggregation decides the fate of the allergic immune response.

Previously, defined naturally occurring isoforms of allergenic proteins were classified as hypoallergens and therefore suggested as an agent for immunotherapy in the future. In this paper, we report for the first time the molecular background of hypoallergenicity by comparing the immunological behavior of hyperallergenic Betula verrucosa major Ag 1a (Bet v 1a) and hypoallergenic Bet v 1d, two isoforms of the major birch pollen allergen Betula verrucosa 1. Despite their cross-reactivity, Bet v 1a and Bet v 1d differ in their capacity to induce protective Ab responses in BALB/c mice. Both isoforms induced similar specific IgE levels, but only Bet v 1d expressed relevant titers of serum IgGs and IgAs. Interestingly, hypoallergenic Bet v 1d activated dendritic cells more efficiently, followed by the production of increased amounts of Th1- as well as Th2-type cytokines. Surprisingly, compared with Bet v 1a, Bet v 1d-immunized mice showed a decreased proliferation of regulatory T cells. Crystallographic studies and dynamic light scattering revealed that Bet v 1d demonstrated a high tendency to form disulfide-linked aggregates due to a serine to cysteine exchange at residue 113. We conclude that aggregation of Bet v 1d triggers the establishment of a protective Ab titer and supports a rationale for Bet v 1d being a promising candidate for specific immunotherapy of birch pollen allergy.

Modified Recombinant Allergens for Safer Immunotherapy

Molecular cloning and recombinant production of allergens offered new perspectives for the increasing problem of allergies. A variety of preparations are being developed aiming to increase safety and improve efficacy of specific immunotherapy. Recombinant-based approaches are mostly focused on genetic modification of allergens to produce molecules with reduced allergenic activity and conserved antigenicity, i.e. hypoallergens. Studies dealing with genetic modifications of allergen genes reported the production of site-directed mutants, deletion mutants, allergen fragments and oligomers, and allergen chimeras. An alternative to genetic engineering is the chemical modification of pure recombinant allergens. It has been shown that allergens modified with immunostimulatory DNA sequences (allergen-ISS conjugates), which masks IgE epitopes and adds a desirable Th1-inducing character to the allergen molecule. Other chemical modifications include oligomerization by aldehydes (allergoids) and maleylation, which seems to target allergens to particular antigen presenting cells. Several of these modified allergen preparations have been already evaluated for their safety in clinical provocation studies. So far, clinical trials showed the efficacy and safety of immunotherapy with an Amb a 1-ISS conjugate for ragweed pollen-allergic patients. In addition, a preparation consisting of hypoallergenic fragments of Bet v 1 was evaluated for immunotherapy of birch pollen-allergic patients. In parallel, several animal studies have now demonstrated the potential of genetic immunization for allergy treatment in the future.

Targeting the extracellular membrane-proximal domain of membrane-bound IgE by passive immunization blocks IgE synthesis in vivo.

The classical allergic reaction starts seconds or minutes after Ag contact and is committed by Abs produced by a special subset of B lymphocytes. These Abs belong to the IgE subclass and are responsible for Type I hyperreactivity reactions. Treatment of allergic diseases with humanized anti-IgE Abs leads primarily to a decrease of serum IgE levels. As a consequence, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells. The biological mechanism behind anti-IgE therapy remains partly speculative; however, it is likely that these Abs also interact with membrane IgE (mIgE) on B cells and possibly interfere with IgE production. In the present work, we raised a mouse mAb directed exclusively against the extracellular membrane-proximal domain of mIgE. The interaction between the monoclonal anti-mIgE Ab and mIgE induces receptor-mediated apoptosis in vitro. Passive immunization experiments lead to a block of newly synthesized specific IgEs during a parallel application of recombinant Bet v1a, the major birch pollen allergen. The decrease of allergen-specific serum IgE might be related to tolerance-inducing mechanisms stopping mIgE-displaying B cells in their proliferation and differentiation.

Migration of antibody secreting cells towards CXCL12 depends on the isotype that forms the BCR

Truncation of the cytoplasmic tail of membrane‐bound IgE in vivo results in lower serum IgE levels, decreased numbers of IgE‐secreting plasma cells and the abrogation of specific secondary immune responses. Here we present mouse strain KN1 that expresses a chimeric ε‐γ1 BCR, consisting of the extracellular domains of the ε gene and the transmembrane and cytoplasmic domains of the γ1 gene. Thus, differences in the IgE immune response of KN1 mice reflect the influence of the “γ1‐mediated signalling” of mIgE bearing B cells. KN1 mice show an increased serum IgE level, resulting from an elevated number of IgE‐secreting cells. Although the primary IgE immune response in KN1 mice is inconspicuous, the secondary response is far more robust. Most strikingly, IgE‐antibody secreting cells with “γ1‐signalling history” migrate more efficiently towards the chemokine CXCL12, which guides plasmablasts to plasma cell niches, than IgE‐antibody secreting cells with WT “ε‐signalling history”. We conclude that IgE plasmablasts have an intrinsic, lower chance to contribute to the long‐lived plasma cell pool than IgG1 plasmablasts.