Previously undescribed grass pollen antigens are the major inducers of T helper 2 cytokine-producing T cells in allergic individuals - PubMed (original) (raw)

. 2013 Feb 26;110(9):3459-64.

doi: 10.1073/pnas.1300512110. Epub 2013 Feb 11.

Jason A Greenbaum, Michael Hauser, Denise M McKinney, John Sidney, Ravi Kolla, Cecilia S Lindestam Arlehamn, Carla Oseroff, Rapheul Alam, David H Broide, Fatima Ferreira, Howard M Grey, Alessandro Sette, Bjoern Peters

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Previously undescribed grass pollen antigens are the major inducers of T helper 2 cytokine-producing T cells in allergic individuals

Véronique Schulten et al. Proc Natl Acad Sci U S A. 2013.

Erratum in

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Known allergens do not account for the total T-cell response against whole pollen extract. Thirty-five pools (average 20 peptides per pool) of overlapping peptides spanning the 10 major TG allergens along with whole TG extract were screened for recognition by PBMCs from allergic donors by using IL-5 ELISPOT assays. The majority of donors who had an IL-5 T-cell response to TG extract of ≥100 SFCs per million input PBMCs showed a response pattern similar to that shown in A, with several pools eliciting strong IL-5 responses. However, some donors showed a response pattern as depicted in B, in which a vigorous response was detected against extract, but no response was detected against peptides from known allergens. In total, as shown in C, 33% of donors reacted to none of the peptide pools despite strong extract responses (n = 21).

Fig. 2.

Fig. 2.

Two-dimensional gel electrophoresis and immunoblot analysis of TG extract using pooled sera from allergic donors. (A) TG pollen extract was run on a 2D gel and stained with Coomassie brilliant blue. (B) A second gel run in parallel was blotted and probed with a serum pool from eight TG-allergic donors to identify proteins reactive with human IgE and IgG. Anti IgE, red; anti IgG, green; dual reactivity, yellow.

Fig. 3.

Fig. 3.

A majority of TG-specific T cells target previously undescribed antigens. (A) Bar chart indicating the IL-5 response rate of PBMCs from allergic and nonallergic donors to peptide pools from known and previously undescribed antigens. Open bars indicate response rates from allergic donors; filled bars indicate responses from normal donors (n = 20 per donor group). (B) Bar chart showing the total number of IL-5–producing T cells targeting previously undescribed antigens (light gray) vs. known allergens (dark gray) in normal and allergic patients.

Fig. 4.

Fig. 4.

Percentage recognition of PUTGAs by TG-allergic donors. Bar chart shows percentage of donors producing IL-5 in response to PUTGA-derived peptide stimulation. PUTGAs are categorized according to antibody reactivity as indicated on the x axis. The dashed line indicates the minimum threshold of ≥20% recognition to be considered an allergen.

Fig. 5.

Fig. 5.

Memory T cells are the source of IL-5 T-cell responses after in vitro expansion. IL-5 production in naïve and memory T cells in response to TG extract (Extract), the dominant known (TG P20), and PUTGA peptide pools (PUTGA P19) were measured after 14 d of expansion following TG stimulation in vitro (n = 6).

Fig. 6.

Fig. 6.

T-cell responses against conventional allergens and PUTGAs can be detected directly ex vivo. (A) FACS plot showing the Th2 T-cell subset sorted based on expression of CXCR3 (Th1) and CCR4 (Th2). (B) IL-5 production by Th2 cells from allergic donors in response to TG extract, the dominant known (TG P20) and previously undescribed (PUTGA P19) peptide pools as measured ex vivo (n = 8).

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