Art v 1 IgE epitopes of patients and humanized mice are conformational.

BACKGROUND: Worldwide, pollen of the weed mugwort (Artemisiavulgaris) is a major cause of severe respiratory allergy, with its major allergen, Art v 1, being the key pathogenic molecule for millions of patients. Humanized mice transgenic for a human T-cell receptor specific for the major Art v 1 T-cell epitope and the corresponding HLA have been made. OBJECTIVE: We sought to characterize IgE epitopes of Art v 1-sensitized patients and humanized mice for molecular immunotherapy of mugwort allergy. METHODS: Four overlapping peptides incorporating surface-exposed amino acids representing the full-length Art v 1 sequence were synthesized and used to search for IgE reactivity to sequential epitopes. For indirect mapping, peptide-specific rabbit antibodies were raised to block IgE against surface-exposed epitopes on folded Art v 1. IgE reactivity and basophil activation studies were performed in clinically defined mugwort-allergic patients. Secondary structure of recombinant (r) Art v 1 and peptides was determined by circular dichroism spectroscopy. RESULTS: Mugwort-allergic patients and humanized mice sensitized by allergen inhalation showed IgE reactivity and/or basophil activation mainly to folded, complete Art v 1 but not to unfolded, sequential peptide epitopes. Blocking of allergic patients' IgE with peptide-specific rabbit antisera identified a hitherto unknown major conformational IgE binding site in the C-terminal Art v 1 domain. CONCLUSIONS: Identification of the new major conformational IgE binding site on Art v 1, which can be blocked with IgG raised against non-IgE reactive Art v 1 peptides, is an important basis for the development of a hypoallergenic peptide vaccine for mugwort allergy.

4-1BB costimulation promotes bystander activation of human CD8 T cells.

Costimulatory signals potently promote T-cell proliferation and effector function. Agonistic antibodies targeting costimulatory receptors of the TNFR family, such as 4-1BB and CD27, have entered clinical trials in cancer patients. Currently there is limited information how costimulatory signals regulate antigen-specific but also bystander activation of human CD8 T cells. Engineered antigen presenting cells (eAPC) efficiently presenting several common viral epitopes on HLA-A2 in combination with MHC class I tetramer staining were used to investigate the impact of costimulatory signals on human CD8 T-cell responses. CD28 costimulation potently augmented the percentage and number of antigen-reactive CD8 T cells, whereas eAPC expressing 4-1BB-ligand induced bystander proliferation of CD8 T cells and massive expansion of NK cells. Moreover, the 4-1BB agonist urelumab similarly induced bystander proliferation of CD8 T cells and NK cells in a dose-dependent manner. However, the promotion of bystander CD8 T-cell responses is not a general attribute of costimulatory TNF receptor superfamily (TNFRSF) members, since CD27 signals enhanced antigen-specific CD8 T cells responses without promoting significant bystander activation. Thus, the differential effects of costimulatory signals on the activation of human bystander CD8 T cells should be taken into account when costimulatory pathways are harnessed for cancer immunotherapy.

IgE-cross-blocking antibodies to Fagales following sublingual immunotherapy with recombinant Bet v 1.

BACKGROUND: Evidence has accumulated that birch pollen immunotherapy reduces rhinoconjunctivitis to pollen of birch homologous trees. Therapeutic efficacy has been associated with IgE-blocking IgG antibodies. We have recently shown that sera collected after 16 weeks of sublingual immunotherapy with recombinant Bet v 1 (rBet v 1-SLIT) display strong IgE-blocking bioactivity for Bet v 1. Here, we assessed whether rBet v 1-SLIT-induced IgG antibodies display cross-blocking activity to related allergens in Fagales pollen. METHODS: IgE, IgG1 and IgG4 reactivity to recombinant Bet v 1, Aln g 1, Car b 1, Ost c 1, Cor a 1, Fag s 1, Cas s 1 and Que a 1 were assessed in pre- and post-SLIT samples of 17 individuals by ELISA. A basophil inhibition assay using stripped basophils re-sensitized with a serum pool containing high Bet v 1-specific IgE levels was established and used to assess CD63 expression in response to allergens after incubation with pre-SLIT or post-SLIT samples. IgG1 and IgG4 were depleted from post-SLIT samples to assess its contribution to IgE-cross-blocking. RESULTS: Sublingual immunotherapy with recombinant Bet v 1 boosted cross-reactive IgE antibodies and induced IgG1 and IgG4 antibodies with inter- and intra-individually differing reactivity to the homologs. Highly variable cross-blocking activities of post-SLIT samples to the different allergens were found. IgG1 and IgG4 antibodies displayed cross-blocking activity with individual variance. CONCLUSIONS: Our mechanistic approach suggested that immunotherapy with the reference allergen Bet v 1 induces individual repertoires of cross-reactive IgG1 and IgG4 antibodies. The cross-blocking bioactivity of these antibodies was also highly variable and neither predictable from protein homology nor IgE-cross-reactivity.

Alum triggers infiltration of human neutrophils ex vivo and causes lysosomal destabilization and mitochondrial membrane potential-dependent NET-formation.

Aluminium salts have been used in vaccines for decades. However, the mechanisms underlying their adjuvant effect are still unclear. Neutrophils, the first immune cells at the injection site, can release cellular DNA together with granular material, so-called neutrophil extracellular traps (NETs). In mice, NETs apparently play a role in aluminium hydroxide (alum)-adjuvant immune response to vaccines. Although no experimental data exist, this effect is assumed to be operative also in humans. As a first step to verify this knowledge in humans, we demonstrate that the injection of alum particles into human skin biopsies ex vivo leads to similar tissue infiltration of neutrophils and NET-formation. Moreover, we characterized the mechanism leading to alum-induced NET-release in human neutrophils as rapid, NADPH oxidase-independent process involving charge, phagocytosis, phagolysosomal rupture, Ca2+ -flux, hyperpolarization of the mitochondrial membrane, and mitochondrial ROS. Extracellular flow and inhibition experiments suggested that no additional energy from oxidative phosphorylation or glycolysis is required for NET-release. This study suggests a so far unappreciated role for neutrophils in the initial phase of immune responses to alum-containing vaccines in humans and provides novel insights into bioenergetic requirements of NET-formation.

T-cell-derived cytokines enhance the antigen-presenting capacity of human neutrophils.

Activated allergen-specific Th2 and Th1 cells release cytokines that transform neutrophils into functional APCs characterized by the expression of HLA-DR and CD58 as well as enhanced survival and antigen uptake, irrespectively of the presence of IL-10, which reduces allergen uptake by neutrophils.

The cytokine environment influence on human skin-derived T cells.

Skin resident T cells provide immediate immunologic responses at their specific location and play a role in the pathogenesis of skin diseases such as psoriasis. Recently, IL-9-producing T cells were described as a major T-cell subtype present in the skin, but knowledge on the biology and in situ regulation of this T-cell subtype is scarce. Here, we investigated the cytokine influence on skin T cells with focus on IL-9-producing T cells because a better understanding of their biology may identify novel therapeutic approaches. Healthy human skin biopsies were cultured either in the presence of IL-2, IL-4, and TGF-ß [T helper (Th)9-promoting condition (Th9-PC)] or IL-2 and IL-15 [standard condition (SC)]. Paired analysis of enzymatically isolated skin T cells and emigrated T cells after 4 wk of skin culture showed significant alterations of T-cell phenotypes, cytokine production, and IL-9-producing T-cell frequency. RNA sequencing analysis revealed differentially regulated pathways and identified CXCL8 and CXCL13 as top up-regulated genes in Th9-PC compared with SC. Functionally supernatant of stimulated skin-derived T cells, CXCL8 and CXCL13 increased neutrophil survival. We report that the cytokine environment alters skin-derived T-cell phenotype and functional properties.-Kienzl, P., Polacek, R., Reithofer, M., Reitermaier, R., Hagenbach, P., Tajpara, P., Vierhapper, M., Gschwandtner, M., Mildner, M. Jahn-Schmid, B., Elbe-Bürger, A. The cytokine environment influence on human skin-derived T cells.

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-γ, 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-γ, 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.

Prevention of allergy by virus-like nanoparticles (VNP) delivering shielded versions of major allergens in a humanized murine allergy model.

BACKGROUND: In high-risk populations, allergen-specific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of virus-like nanoparticles (VNP) expressing surface-exposed or shielded allergens. METHODS: Full-length major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. RESULTS: Virus-like nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surface-exposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1-specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergen-specific T cells in vitro. Upon intranasal application in mice, VNP expressing surface-exposed but not shielded allergen induced allergen-specific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergen-protected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Treg-dominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. CONCLUSION: Virus-like nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization.

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.

Allergens with Protease Activity from House Dust Mites.

Globally, house dust mites (HDM) are one of the main sources of allergens causing Type I allergy, which has a high risk of progressing into a severe disabling disease manifestation such as allergic asthma. The strong protease activities of a number of these allergens are thought to be involved in several steps of the pathophysiology of this allergic disease. It has been a common notion that protease activity may be one of the properties that confers allergenicity to proteins. In this review we summarize and discuss the roles of the different HDM proteases in the development of Type I allergy.

Humoral and cellular cross-reactivity between Amb a 1, the major ragweed pollen allergen, and its mugwort homolog Art v 6.

Ragweed and mugwort are closely related weeds that represent the major cause of pollen allergy in late summer. Concomitant sensitization and clinical cross-reactivity frequently occur in subjects who are coexposed to both pollen species, and have implications for diagnosis and specific immunotherapy. Molecules involved in this cross-reactivity might be Amb a 1, the major ragweed pollen allergen, and Art v 6, a highly homologous allergen from mugwort. Therefore, we investigated the IgE and T cell response to Art v 6 of 60 weed pollen-allergic patients and assessed its immunological cross-reactivity with Amb a 1. Results of ELISA inhibition experiments suggested that both allergens are largely cross-reactive, but Amb a 1 possesses more IgE epitopes than Art v 6. In patients with IgE to both allergens, Amb a 1-induced T cell lines and clones responded weakly to Art v 6. Moreover, Art v 6-induced T cell lines responded stronger to Amb a 1. T cell epitope mapping of Art v 6 revealed that it contains only a few cross-reactive epitopes, which is opposed to the multiple T cell-activating regions present in Amb a 1. In summary, Amb a 1 can elicit more diverse allergen-specific IgE and T cell responses than Art v 6 and dominates the cross-reactivity with its homolog. Nevertheless, Art v 6 can act as a primary sensitizing allergen in areas with high mugwort pollen exposure, and consequently may facilitate sensitization to Amb a 1 by epitope cross-recognition of T and B cells.

Oral exposure to Mal d 1 affects the immune response in patients with birch pollen allergy.

BACKGROUND: Antibodies and T cells specific for the major birch pollen allergen Bet v 1 cross-react with structurally related food allergens, such as Mal d 1 in apple. OBJECTIVE: We sought to evaluate the effects of oral uptake of Mal d 1 on the allergen-specific immune response in patients with birch pollen allergy. METHODS: Patients received 50 µg of rBet v 1 sublingually on 2 consecutive days outside of the birch pollen season. One year later, equal amounts of rMal d 1 were administered. Blood samples were collected before and after oral exposure, as well as before and after the intermediate birch pollen season. Allergen-specific IgE levels were determined by using ImmunoCAP. Proliferation of allergen-stimulated PBMCs was assessed, as well as the expression of IL-5, IL-13, IL-10, IFN-γ, and forkhead box protein 3 (Foxp3) in isolated T cells (real-time PCR). Allergen-specific T-cell lines were analyzed for epitope recognition. RESULTS: Orally administered Bet v 1 transiently reduced Bet v 1-specific serum IgE levels, as well as Bet v 1- and Mal d 1-induced T-cell proliferation, and enhanced the expression of IL-5, IL-10, and Foxp3. Orally applied Mal d 1 significantly decreased Bet v 1- and Mal d 1-specific IgE levels and induced IL-5 and IL-10 but no Foxp3 expression. In contrast to Bet v 1, Mal d 1 triggered IFN-γ production and T cells with a different epitope repertoire. Inhalation of birch pollen significantly enhanced allergen-specific IgE levels, T-cell proliferation, and IL-5, IL-10, IL-13, and Foxp3 expression. CONCLUSION: Two sublingual administrations of 50 µg of Mal d 1 were well tolerated and induced transient immune responses seen during peripheral tolerance development. Thus recombinant Mal d 1 might be suitable and relevant for sublingual treatment of birch pollen-related apple allergy.

Human TCR transgenic Bet v 1-specific Th1 cells suppress the effector function of Bet v 1-specific Th2 cells.

Pollinosis to birch pollen is a common type I allergy in the Northern Hemisphere. Moreover, birch pollen-allergic individuals sensitized to the major birch pollen allergen Bet v 1 frequently develop allergic reactions to stone fruits, hazelnuts, and certain vegetables due to immunological cross-reactivity. The major T cell epitope Bet v 1(142-153) plays an important role in cross-reactivity between the respiratory allergen Bet v 1 and its homologous food allergens. In this study, we cloned and functionally analyzed a human αß TCR specific for the immunodominant epitope Bet v 1(142-153). cDNAs encoding TCR α- and ß-chains were amplified from a Bet v 1(142-153)-specific T cell clone, introduced into Jurkat T cells and peripheral blood T lymphocytes of allergic and nonallergic individuals, and evaluated functionally. The resulting TCR transgenic (TCRtg) T cells responded in an allergen-specific and costimulation-dependent manner to APCs either pulsed with Bet v 1(142-153) peptide or coexpressing invariant chain::Bet v 1(142-153) fusion proteins. TCRtg T cells responded to Bet v 1-related food and tree pollen allergens that were processed and presented by monocyte-derived dendritic cells. Bet v 1(142-153)-presenting but not Bet v 1(4-15)-presenting artificial APCs coexpressing membrane-bound IL-12 polarized allergen-specific TCRtg T cells toward a Th1 phenotype, producing high levels of IFN-γ. Coculture of such Th1-polarized T cells with allergen-specific Th2-differentiated T cells significantly suppressed Th2 effector cytokine production. These data suggest that human allergen-specific TCR can transfer the fine specificity of the original T cell clone to heterologous T cells, which in turn can be instructed to modulate the effector function of the disease initiating/perpetuating allergen-specific Th2-differentiated T cells.

T-cell subset changes during the first year of pre-seasonal allergoid allergen-specific immunotherapy.

Allergen-specific immunotherapy (AIT) is the only treatment for type I allergy, which achieves long-lasting effects. Repeated subcutaneous applications of allergen extracts cause a protective antibody response and an immune deviation of T cells. In AIT with allergoids, chemically modified allergen extracts are injected. During a so-called special pre-seasonal application scheme, after the initial phase of applying increased doses of allergoids is followed by natural allergen exposure as a maintenance phase. The effectiveness of allergoid vaccines has been described regarding the improvement of clinical symptoms and the development of protective humoral responses. In this longitudinal observational study, we sought to investigate changes at the T cell level in pre-seasonal AIT with allergoid. Different subsets within CD4+ and CD8+ T cells were monitored by flow cytometry in PBMC of patients known to possess protective antibody responses. Compared to before treatment, a small early boost among allergenic Th cells was observed after 4 months of AIT. In line, a slight Th2 bias was observed after 4 months within circulating T follicular T cells, Tfh and Tfc, representing pre-existing memory Th2 cells. Furthermore, it was demonstrated that responsiveness of CD8+ T cells to allergen stimulation decreased during the course of treatment. Apart from that, we found an influence of the meteorological season on the activation profile of Tfh and Tfc over the course of the treatment. Together, this is the first study investigating changes of different T cell subsets over the course of an allergoid AIT against airborne allergens. Our findings match previous reports on conventional AIT, especially the initial increase of Th2 responses. However, the observed changes were less pronounced which may be either due to the modification of allergens or to the reduced maintenance dose provided by natural allergen exposure compared to a perennial protocol.

Association of HLA-DR1 with the allergic response to the major mugwort pollen allergen: molecular background.

BACKGROUND: Mugwort pollen allergens represent the main cause of pollinosis in late summer. The major allergen, Art v 1, contains only one single immunodominant, solely HLA-DR-restricted T cell epitope (Art v 125-36). The frequency of HLA-DRB1*01 is highly increased in mugwort-allergic individuals and HLA-DR1 serves as restriction element for Art v 125-36. However, Art v 125-36 also binds to HLA-DR4 with high affinity and DR1-restricted Art v 125-36 -specific T cell receptors can be activated by HLA-DR4 molecules. To understand the predominance of HLA-DR1 in mugwort allergy in spite of the degeneracy in HLA/peptide-binding and TCR-recognition, we investigated the molecular background of Art v 125-36 /MHC/TCR interactions in the context of HLA-DR1 compared to -DR4. RESULTS: The majority of Art v 125-36 -specific T cell lines and clones from HLA-DR1 carrying, mugwort pollen-allergic donors reacted to synthetic and naturally processed Art v 1-peptides when presented by HLA-DR1 or HLA-DR4 expressing antigen presenting cells. However, at limiting peptide concentrations DR1 was more effective in T cell stimulation. In addition, the minimal epitope for 50% of Art v 125-36 -specific T cells was shorter for DR1 than for DR4. In vitro binding assays of Art v 125-36 mutant peptides to isolated DR1- and DR4-molecules indicated similar binding capacities and use of the same register. In silico simulation of Art v 125-36 binding to HLA-DR1 and -DR4 suggested similar binding of the central part of the peptide to either molecule, but a higher flexibility of the N- and C-terminal amino acids and detachment at the C-terminus in HLA-DR1. CONCLUSIONS: The predominance of HLA-DR1 in the response to Art v 125-36 may be explained by subtle conformation changes of the peptide bound to DR1 compared to DR4. Computer simulation supported our experimental data by demonstrating differences in peptide mobility within the HLA-DR complex that may influence TCR-binding. We suggest that the minor differences observed in vitro may be more relevant in the microenvironment in vivo, so that only presentation by HLA-DR1, but not -DR4 permits successful T cell activation.

Bet v 1-specific T-cell receptor/forkhead box protein 3 transgenic T cells suppress Bet v 1-specific T-cell effector function in an activation-dependent manner.

BACKGROUND: Regulatory T (Treg) cells establish and maintain tolerance to self-antigens and many foreign antigens, such as allergens, by suppressing effector T-cell proliferation and function. We have previously shown that human T-cell receptor (TCR) αß-chains specific for allergen-derived epitopes confer allergen specificity on peripheral blood T cells of individuals with and without allergy. OBJECTIVE: To study the feasibility of generating allergen-specific human Treg cells by retroviral transduction of a transcription unit encoding forkhead box protein 3 (FOXP3) and allergen-specific TCR αß-chains. METHODS: cDNAs encoding the α and ß-chains of a Bet v 1(142-153)-specific TCR (TCR alpha variable region 6/TCR beta variable region 20) and human FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit from an internal ribosomal entry site-green fluorescence protein-containing retroviral vector. Retrovirally transduced peripheral blood T cells were tested for expression of transgenes, Treg phenotype, and regulatory capacity toward allergen-specific effector T cells. RESULTS: Transduced T cells displayed a Treg phenotype with clear-cut upregulation of CD25, CD39, and cytotoxic T-lymphocyte antigen 4. The transduced cells were hyporesponsive in cytokine production and secretion and, like naturally occurring Treg cells, did not proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was inducible upon exposure to exogenous IL-2. In coculture experiments, TRAV6(+)TRBV20(+)FOXP3(+) transgenic T cells, unlike FOXP3(+) single transgenic T cells or naturally occurring Treg cells, highly significantly suppressed T cell cytokine production and proliferation of corresponding allergen-specific effector T cells in an allergen-specific, dose-dependent manner. CONCLUSION: We demonstrate a transgenic approach to engineer human allergen-specific Treg cells that exert their regulatory function in an activation-dependent manner. Customized Treg cells might become useful for tolerance induction therapies in individuals with allergic and other immune-mediated diseases.

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.