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Background: Ragweed frequently causes seasonal allergies in North America and Europe. In the United States, several related ragweed species with diverse geographical distribution cause allergic symptoms. Cross-reactivity towards related ragweed species of IgE and treatment-induced IgG 4 has been demonstrated previously. However, less is known about the underlying T-cell cross-reactivity., Methods: The allergen content of ragweed extracts was determined by mass spectrometry and related to T-cell epitopes of Amb a allergens (group 1, 3, 4, 5, 8 and 11) in 20 American ragweed allergic patients determined by FluoroSpot and proliferation assays. T-cell responses to 50 frequently recognized Amb a-derived T-cell epitopes and homologous peptides from western ragweed (Amb p), giant ragweed (Amb t) and mugwort (Art v) were investigated in an additional 11 American and 14 Slovakian ragweed allergic donors., Results: Ragweed extracts contained all known allergens and isoallergens thereof. Donor T-cell responses were diverse and directed against all Amb a 1 isoallergens and to most minor allergens investigated. Similar response patterns were seen in American and Slovakia donors. Several epitopes were cross-reactive between isoallergens and ragweed species, some even including mugwort. T-cell cross-reactivity generally correlated with allergen sequence homology., Conclusion: T-cell epitopes of multiple allergens/isoallergens are involved in the diverse T-cell responses in ragweed allergic individuals. T-cell lines were highly cross-reactive to epitopes of related ragweed species without any apparent geographical response bias. These data support that different ragweed species can be considered an allergen homology group with Amb a as the representative species regarding diagnosis as well as allergy immunotherapy., (© 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: Profilins are dominant pan-allergens known to cause cross-sensitization, leading to clinical symptoms such as pollen-food syndrome. This study aimed to determine the T-cell response to Phl p 12 in profilin-sensitized patients, by measuring the prevalence, strength and cross-reactivity to clinically relevant profilins., Methods: The release of Phl p allergens from pollen was determined by mass spectrometry and immunochemistry. T-cell responses, epitope mapping and cross-reactivity to profilins (Phl p 12, Ole e 2, Bet v 2 and Mal d 4) were measured in vitro using PBMCs from 26 Spanish grass-allergic donors IgE-sensitized to profilin. Cross-reactivity was addressed in vivo using 2 different mouse strains (BALB/c and C3H)., Results: Phl p 12 and Phl p 1 are released from pollen simultaneously and in similar amounts. Both T-cell response frequency (17/26 donors) and strength were comparable between Phl p 12 and Phl p 1. T-cell cross-reactivity to other profilins correlated with overall sequence homology, and 2 immunodominant epitope regions of Phl p 12 were identified. Data from mice immunized with Phl p 12 showed that cross-reactivity to Bet v 2 was mediated by conserved epitopes and further influenced by additional genetic factors, likely to be MHC II., Conclusion: The strength, prevalence and cross-reactivity of T-cell responses towards Phl p 12 are comparable to the major allergen Phl p 1, which supports the hypothesis that T cells to Phl p 12 can play an important role in development of allergic symptoms, such as those associated with pollen-food syndrome., (© 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: House dust mite (HDM) allergens are a common cause of allergy and allergic asthma. A comprehensive analysis of proteins targeted by T cells, which are implicated in the development and regulation of allergic disease independent of their antibody reactivity, is still lacking., Objective: To comprehensively analyse the HDM-derived protein targets of T cell responses in HDM-allergic individuals, and investigate their correlation with IgE/IgG responses and protein function., Methods: Proteomic analysis (liquid chromatography-tandem mass spectrometry) of HDM extracts identified 90 distinct protein clusters, corresponding to 29 known allergens and 61 novel proteins. Peripheral blood mononuclear cells (PBMC) from 20 HDM-allergic individuals were stimulated with HDM extracts and assayed with a set of ~2500 peptides derived from these 90 protein clusters and predicted to bind the most common HLA class II types. 2D immunoblots were made in parallel to elucidate IgE and IgG reactivity, and putative function analyses were performed in silico according to Gene Ontology annotations., Results: Analysis of T cell reactivity revealed a large number of T cell epitopes. Overall response magnitude and frequency was comparable for known and novel proteins, with 15 antigens (nine of which were novel) dominating the total T cell response. Most of the known allergens that were dominant at the T cell level were also IgE reactive, as expected, while few novel dominant T cell antigens were IgE reactive. Among known allergens, hydrolase activity and detectable IgE/IgG reactivity are strongly correlated, while no protein function correlates with immunogenicity of novel proteins. A total of 106 epitopes accounted for half of the total T cell response, underlining the heterogeneity of T cell responses to HDM allergens., Conclusions and Clinical Relevance: Herein, we define the T cell targets for both known allergens and novel proteins, which may inform future diagnostics and immunotherapeutics for allergy to HDM., (© 2016 John Wiley & Sons Ltd.)

Background: Allergen-specific immunotherapy is the only curative treatment for type I allergy. It can be administered subcutaneously (SCIT) or sublingually (SLIT). The clinical efficacy of these two treatment modalities appears to be similar, but potential differences in the immunological mechanisms involved have not been fully explored., Objective: To compare changes in the allergen-specific T cell response induced by subcutaneous vs. sublingual administration of allergen-specific immunotherapy (AIT)., Methods: Grass pollen-allergic patients were randomized into groups receiving either SCIT injections or SLIT tablets or neither. PBMCs were tested for Timothy grass (TG)-specific cytokine production by ELISPOT after in vitro expansion with TG-peptide pools. Phenotypic characterization of cytokine-producing cells was performed by FACS., Results: In the SCIT group, decreased IL-5 production was observed starting 10 months after treatment commenced. At 24 months, T cell responses showed IL-5 levels significantly below the before-treatment baseline. No significant reduction of IL-5 was observed in the SLIT or untreated group. However, a significant transient increase in IL-10 production after 10 months of treatment compared to baseline was detected in both treatment groups. FACS analysis revealed that IL-10 production was associated with CD4(+) T cells that also produced IFNγ and therefore may be associated with an IL-10-secreting type 1 cell phenotype., Conclusion and Clinical Relevance: The most dominant immunological changes on a cellular level were a decrease in IL-5 in the SCIT group and a significant, transient increase of IL-10 observed after 10 months of treatment in both treated groups. The distinct routes of AIT administration may induce different immunomodulatory mechanisms at the cellular level., (© 2015 John Wiley & Sons Ltd.)

Airway epithelial cells (AECs) form polarized barriers that interact with inhaled allergens and are involved in immune homeostasis. We examined how monocyte-derived dendritic cells (MDDCs) are affected by contact with the airway epithelium. In traditional setups, bronchial epithelial cell lines were allowed to polarize on filter inserts, and MDDCs were allowed to adhere to the epithelial basal side. In an optimized setup, the cell application was reversed, and the culture conditions were modified to preserve cellular polarization and integrity. These two parameters were crucial for the MDDCs' immunoregulatory properties; thus, previous observations obtained using traditional setups should be considered with caution. Using the optimized setup, AEC conditioning of MDDCs led to increased expression of programmed death 1 ligand 1, immunoglobulin-like transcript 3, CD40, CD80, and CD23. This increased expression was accompanied by decreased secretion of monocyte chemotactic protein 1 and eotaxin and donor-variable effects on IL-12 and IL-10 secretion. Conditioning varied between maturation states and depended partly on direct contact between AECs and MDDCs. The setup allowed MDDCs on the basal side of the epithelium to sample allergens administered to the apical side. Allergen uptake depended on polarization and the nature of the allergen. AEC conditioning led to decreased birch allergen-specific proliferation of autologous T cells and a trend toward decreased secretion of the Th2-specific cytokines IL-5 and IL-13. In conclusion, we determined that AEC conditioning favoring cellular integrity leads to a tolerogenic MDDC phenotype, which is likely to be important in regulating immune responses against commonly inhaled allergens.

Effector cell activation and T-cell activation, the latter mediated by facilitated antigen presentation, are immunological mechanisms that play crucial roles in the manifestation and maintenance of allergic disease. In addition to their relevance for the pathogenesis of allergy in-vivo, in-vitro assays based on these immunological mechanisms have been established and used for diagnostics, for monitoring the progression of disease and for the effect of specific immunotherapy as well as for basic research purposes. Here we review different parameters that affect effector cell activation and facilitated antigen uptake and presentation, including assay designs, readout parameters and critical experimental conditions. Central to the two immunological mechanisms is complex formation between allergen-specific IgE, allergen, and cell surface-anchored immunoglobulin receptor; the high affinity IgE-receptor FcεRI on basophils and mast cells, and the low affinity IgE-receptor FcεRII (CD23) on B-cells. Accordingly, the effect of IgE repertoire complexity and allergen diversity on effector cell and facilitated antigen presentation is discussed in detail., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The incidence of allergic diseases is increasing in industrialized countries and the immunological mechanisms leading to tolerance or allergy are poorly understood. Cytokines with suppressive abilities and CD4(+)CD25(+) regulatory T cells have been suggested to play a central role in allergen-specific responses. The aim was to determine whether major grass allergens induce production of suppressive cytokines in allergic and healthy subjects and to examine the inhibitory effect of these cytokines on allergic responses., Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy and grass-allergic donors and stimulated with the major grass allergens Phl p 1 or Phl p 5. The effects of endogenous IL-10 and/or TGF-beta on proliferation and cytokine production were determined by use of blocking antibodies. In addition, the number of CD4(+)CD25(+) T cells and their expression of chemokine receptors were investigated by flow cytometry., Results: Phl p 1 and Phl p 5 induced IL-10 production, which down-regulated proliferation and cytokine production, in PBMC cultures from atopic but not from non-atopic donors. Comparable frequencies of CD4(+)CD25(+) T cells were present in PBMCs in the two groups, but fewer cells from atopic donors were CD4(+)CD25(+)CCR4(+) and more cells were CD4(+)CD25(+)CLA(+) compared to healthy donors., Conclusion: Allergen-specific responses of grass allergic patients but not in non-atopic subjects are influenced by regulatory cytokines produced in response to the important allergens. Differences in CD4(+)CD25(+) T cell expression of chemokine receptors in allergic compared to non-atopic donors could suggest that the homing of CD4(+)CD25(+) T cells is important for the regulation of allergen-specific responses., (2010 S. Karger AG, Basel.)

It is well established that both the production of IgE by B lymphocytes and the maturation and recruitment of eosinophils in late-phase reactions are dependent on the activation of allergen-specific type-2 T-helper cells. What is less well known is the fact that efficient activation of allergen-specific T cells upon low-dose exposure to allergens is critically dependent on IgE-mediated or -facilitated allergen presentation. In fact, changes in the level of IgE-mediated allergen presentation may account for many of the immunological effects described for specific immunotherapy or anti-IgE treatment. This review aims to summarize the current knowledge, and will discuss the clinical relevance of blocking IgG antibodies induced by specific immunotherapy and anti-IgE monoclonal antibodies that both interfere with IgE-mediated allergen presentation., (Copyright (c) 2006 S. Karger AG, Basel.)

The prevalence of atopic diseases has increased dramatically in the Western world over the last 20 years. Further insight into the mechanisms of the allergic reaction may help in the development of new more efficient protocols for the specific treatment of allergic diseases. Until a decade ago the major focus in allergy research was on the IgE reactivity of atopic patients and the description of major allergens responsible for the allergic reactions. More recently, the important role of CD4+ T-cells in orchestrating the production of IgE and the maturation of eosinophils through the production of IL-4 (or IL-13) and IL-5 has been clearly established. This has led to the characterization of T-cell responsiveness to numerous major allergens, demonstrating the importance of T-cells for the allergic response of atopic patients and for the specific treatment of allergic diseases. One of the major objectives of allergy research is the development of improved treatment protocols for specific therapy. In this review our results on the T-cell reactivity of grass allergic patients to the group 5 major allergen of the grass Phleum pratense (Phl p 5) are discussed in relation to the literature on allergen-specific T-cell reactivity and the specific treatment of allergic disease. Our investigations focused on three subjects: 1) Grass allergen-specific T-cell reactivity of atopic patients in freshly isolated PBMC cultures, 2) Phl p 5 isoallergen recognition of T-cell lines (TCL) and clones (TCC) established from PBMC of grass allergic patients, and 3) Identification of T-cell epitopes on Phl p 5.

Background: At present, recombinant allergens are considered for the diagnosis and treatment of atopic allergies. To evaluate the theoretical impact of isoallergen variation on the selection of isoallergens for specific allergy vaccination, we characterized the T-cell response of allergic patients to the Phleum pratense major allergen, Phl p 5, and five of its recombinant isoallergens., Methods: Phl p 5-specific T cell lines (TCLs) were isolated from the peripheral blood of 3 allergic rhinitis patients, and their reactivity patterns were studied in detail., Results: The TCLs were highly crossreactive with related grasses. The crossreactivity with Poa pratensis was more extensive than with Lolium perenne, directly reflecting the sequence identity between Phl p 5, Poa p 5, and Lol p 5. The TCLs produced IFN-gamma and IL-4 simultaneously, resembling a Th0-like cytokine production profile. Interestingly, when T cell clones were tested with natural Phl p 5 and five rPhl p 5 isoallergens, a differential recognition pattern was found. One of the TCLs exclusively reacted with Phl p 5b, another reacted with all isoforms tested, and the third reacted strongly with native purified Phl p 5, but only weakly with all five recombinant isoallergens., Conclusion: These results indicate that Phl p 5-specific T cells are highly heterogeneous, and that they differentially recognize isoallergens. This indicates that when recombinant Phl p 5 is considered for allergy vaccination, a mixture of isoallergens representing the different isoallergen groups may clinically prove to be more effective than single isoallergens.

Background: Pollinosis, caused by grasses of the Poaceae family, is a problem worldwide. The relative importance of grass groups 1 and 5 major allergens is well established. However, not much is known about the recognition of these allergens by T cells and whether this T-cell reactivity correlates with skin reactivity and serum IgE levels., Objectives: The aim of this study was to characterize the cross-reactive, grass allergen-specific T-cell responses from patients allergic to grass and nonatopic individuals and to investigate whether these responses correlate with grass-specific IgE and skin reactivity., Methods: Skin prick test wheal areas and grass-specific serum IgE levels were determined in all patients (n = 21) and nonallergic control donors (n = 20). Peripheral blood mononuclear cells were isolated and stimulated with grass allergen extracts (Phleum pratense, Poa pratensis, Lolium perenne) and immunoaffinity-purified group 5 allergens, and the production of type 1 and type 2 cytokines was determined in the patient group., Results: Donors allergic to grass showed increased T-cell-proliferative responses to grass allergens compared with nonatopic control subjects. We find it interesting that the magnitude of the patients' T-cell responses could not be correlated with the individual skin prick test areas and specific serum IgE levels, and several patients with allergies to grass had group 5-specific T-cell responses in the absence of group 5-specific IgE. The absence of a correlation between T-cell proliferation and IgE levels or skin prick test results may in part be explained by the finding that patients predominantly produced IL-5 in response to Phl p 5, the major allergen, and predominantly IFN-gamma in response to Phl p extract. In general, all donors responded equally well to all three grasses. Additional experiments with Phl p 5-specific T-cell lines indicated that the equal proliferation of peripheral blood mononuclear cells to all three species is the direct result of cross-reactivity., Conclusions: Grass allergen-specific T-cell responses are highly cross-reactive, and patients with allergies exhibit higher responses than nonallergic donors, suggesting that T cells are involved in the allergic reaction to grass group 5 allergens. However, group 5-specific T-cell responses are also found in donors without group 5-specific IgE, and the patients' grass-specific T-cell responses and cytokine production do not correlate to skin reactivity or to concentrations of grass-specific IgE.