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Whole extract or allergen-specific IgE testing has become increasingly popular in the diagnosis of peanut allergy. However, much less is known about T cell responses in peanut allergy and how it relates to different clinical phenotypes. CD4+ T cells play a major role in the pathophysiology of peanut allergy as well as tolerance induction during oral desensitization regimens. We set out to characterize and phenotype the T cell responses and their targets in peanut sensitized patients. Using PBMC from peanut-allergic and non-allergic patients, we mapped T cell epitopes for three major peanut allergens, Ara h 1, 2 and 3 (27 from Ara h 1, 4 from Ara h 2 and 43 from Ara h 3) associated with release of IFNγ (representative Th1 cytokine) and IL5 (representative Th2 cytokine). A pool containing 19 immunodominant peptides, selected to account for 60% of the total Ara h 1-3-specific T cell response in allergics, but only 20% in non-allergics, was shown to discriminate T cell responses in peanut-sensitized, symptomatic vs non-symptomatic individuals more effectively than peanut extract. This pool elicited positive T cell responses above a defined threshold in 12/15 sensitized, symptomatic patients, whereas in the sensitized but non-symptomatic cohort only, 4/14 reacted. The reactivity against this peptide pool in symptomatic patients was dominated by IL-10, IL-17 and to a lesser extend IL-5. For four distinct epitopes, HLA class II restrictions were determined, enabling production of tetrameric reagents. Tetramer staining in four donors (2 symptomatic, 2 non-symptomatic) revealed a trend for increased numbers of peanut epitope-specific T cells in symptomatic patients compared to non-symptomatic patients, which was associated with elevated CRTh2 expression whereas cells from non-symptomatic patients exhibited higher levels of Integrin β7 expression. Our results demonstrate differences in T cell response magnitude, epitope specificity and phenotype between symptomatic and non-symptomatic peanut-sensitized patients. In addition to IgE reactivity, analysis of peanut-specific T cells may be useful to improve our understanding of different clinical manifestations in peanut allergy., Competing Interests: The authors have declared that no competing interests exist.

Background: Japanese cedar (JC) pollen is a common trigger for allergic rhinitis in Japan. Pollen proteins targeted by IgE, including Cry j 1 and Cry j 2, and isoflavone reductase (IFR) have been identified., Objective: To compare antigen-specific IgE titers and T-cell responses to JC pollen-derived extract and peptides in cohorts with high and low pollen exposure., Methods: Peripheral blood mononuclear cells from JC pollen allergic or nonallergic patients who have lived in Japan for at least 1 year and JC pollen allergic patients who have never been to Japan were tested for T-cell responses against JC pollen extract and peptide pools derived from Cry j 1, Cry j 2, or IFR. T-cell reactivity was assessed by interleukin 5 and interferon γ production by ELISPOT., Results: JC pollen-specific T-cell reactivity and IgE titers were significantly higher in the allergic compared with the nonallergic Japanese cohort, which was also associated with different patterns of polysensitization. Interestingly, a significant overlap was observed in the hierarchy of the T-cell epitopes in the allergic Japanese cohort compared with the allergic non-Japanese cohort. In all 3 cohorts, T-cell reactivity was dominantly directed against peptides from the major allergens Cry j 1 and 2, with few T-cell responses detected against IFR., Conclusion: Our studies identify common denominators of T-cell reactivity in patient populations with different sensitization patterns, suggesting that generally applicable immunotherapeutic approaches might be developed irrespective of exposure modality., (Published by Elsevier Inc.)

Background: Different populations of T cells are involved in the pathogenesis of allergic diseases., Objective: We investigated changes in TH-cell populations in patients with allergies after specific immunotherapy (SIT)., Methods: PBMCs were isolated from patients with allergies who received SIT and those who did not (controls). We tested the ability of peptides from 93 timothy grass (TG) proteins to induce T-cell responses (cytokine production). We used ELISPOT and staining assays for intracellular cytokines to measure the production of IL-4, IL-5, IL-13, IFN-γ, and IL-10., Results: Compared with PBMCs from controls, PBMCs from patients who received SIT produced lower levels of TH2 cytokines on incubation with several different TG peptides. These data were used to select 20 peptides to be tested in an independent cohort of 20 patients with allergies who received SIT and 20 controls. We again observed a significant decrease in the production of TH2 cytokines, and an increase in the production of the TH1 cytokine IFN-γ, in PBMCs from the validation groups. These changes correlated with improved symptoms after SIT. Immunization with this selected pool of peptides (or their associated antigens) could protect a substantial proportion of the population from TG allergy., Conclusions: We observed a significant decrease in the production of TH2 cytokines by PBMCs from patients who received SIT for TG allergy compared to those who did not. These changes might be used to monitor response to therapy. The decrease occurred in response to antigens that elicit little (if any) IgE responses; these antigens might be developed for use in immunotherapy., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

T cells play an important role in the pathogenesis of allergic diseases. However, the proteins considered as potential immunogens of allergenic T-cell responses have traditionally been limited to those that induce IgE responses. Timothy grass (TG) pollen is a well-studied inhaled allergen for which major IgE-reactive allergens have also been shown to trigger T helper 2 (Th2) responses. Here we examined whether other TG pollen proteins are recognized by Th2 responses independently of IgE reactivity. A TG pollen extract was analyzed by 2D gel electrophoresis and IgE/IgG immunoblots using pooled sera from allergic donors. Mass spectrometry of selected protein spots in combination with de novo sequencing of the whole TG pollen transcriptome identified 93 previously undescribed proteins for further study, 64 of which were not targeted by IgE. Predicted MHC binding peptides from the previoulsy undescribed TG proteins were screened for T-cell reactivity in peripheral blood mononuclear cells from allergic donors. Strong IL-5 production was detected in response to peptides from several of the previously undescribed proteins, most of which were not targeted by IgE. Responses against the dominant undescribed epitopes were associated with the memory T-cell subset and could even be detected directly ex vivo after Th2 cell enrichment. These findings demonstrate that a combined unbiased transcriptomic, proteomic, and immunomic approach identifies a greatly broadened repertoire of protein antigens targeted by T cells involved in allergy pathogenesis. The discovery of proteins that induce Th2 cells but are not IgE reactive may allow the development of safer immunotherapeutic strategies.

Background: Nonspecific lipid transfer proteins (nsLTPs) are important food allergens. Often, patients allergic to the nsLTP in peach suffer from allergy to hazelnuts. We aimed to analyse the T-cell response to Cor a 8, the nsLTP in hazelnut and its immunological cross-reactivity with the nsLTP in peach, Pru p 3., Methods: Cor a 8-reactive T-cell lines (TCL) established from patients allergic to hazelnut and peach were stimulated with 12-mer peptides representing the complete amino acid sequence of Cor a 8 to identify its T-cell-activating regions and with Pru p 3 to investigate cellular cross-reactivity. T-cell clones specific for different major T-cell-activating regions of Pru p 3 were stimulated with Cor a 8. Both nsLTPs were subjected to endolysosomal degradation assays. Immunoglobulin E (IgE) cross-reactivity between Cor a 8 and Pru p 3 was assessed in inhibition enzyme-linked immunosorbent assay., Results: No major T-cell-activating region was found among 26 T-cell-reactive peptides identified in Cor a 8. Although generated with Cor a 8, 62% of the TCL responded more strongly to Pru p 3. This cross-reactivity was mediated by T cells specific for the immunodominant region Pru p 3(61-75) . Peptide clusters encompassing this region were generated during lysosomal degradation of both nsLTPs. Cor a 8 was more rapidly degraded by lysosomal proteases than Pru p 3. Pre-incubation of sera with Pru p 3 completely abolished IgE binding to Cor a 8, which was not the case vice versa., Conclusions: T-cell reactivity to Cor a 8 is predominantly based on cross-reactivity with Pru p 3, indicating that the latter initiates sensitisation to its homolog in hazelnut. The limited allergenic potential of Cor a 8 seems to be associated with rapid lysosomal degradation during allergen processing and the lack of major T-cell-activating regions., (© 2011 John Wiley & Sons A/S.)

Background: Pru p 3, the nonspecific lipid transfer protein from peach, is an important plant food allergen that frequently induces systemic reactions., Objective: We sought to analyze the allergic T-cell response to Pru p 3., Methods: PBMCs from Italian and Spanish patients with peach allergy were stimulated with purified natural Pru p 3. Allergen-specific T-cell lines were used to identify T-cell epitopes of Pru p 3. Pru p 3-specific T-cell clones (TCCs) were analyzed for allergen-induced secretion of IL-4, IFN-gamma, and IL-10 and expression of the integrin beta7, a receptor critical for gut homing., Results: No difference in T-cell responses of Italian and Spanish patients was found. Among several T cell-activating regions, Pru p 3(13-27), Pru p 3(34-48), Pru p 3(43-57), and Pru p 3(61-75) were most frequently recognized in 18 Pru p 3-specific T-cell lines. The majority of 32 Pru p 3-specific TCCs belonged to the T(H)2 subset. In contrast to TCCs specific for other plant food and pollen allergens, only a limited number of Pru p 3-specific TCCs produced significant amounts of IL-10. The expression of integrin beta7 on Pru p 3-specific TCCs was comparable with that observed on peanut-specific TCCs and higher compared with that seen in different pollen-specific TCCs., Conclusion: The T-cell response to Pru p 3 is dominated by T(H)2 cells presumably primed in the gut. The identification of relevant T cell-activating regions provides a basis for engineering hypoallergenic variants of Pru p 3 with less IgE binding and retained T-cell stimulatory capacity for safe immunotherapy of peach allergy.