Peptide-induced immune regulation by a promiscuous and immunodominant CD4T-cell epitope of Timothy grass pollen: a role of Cbl-b and Itch in regulation

Abstract

Background: T-cell targeted peptide epitope tolerogens from grass pollen allergens may be useful in treating seasonal allergic rhinitis, but there is urgent need for optimisation of approaches from improved understanding of mechanism.

Objective: We sought to identify human leukocyte antigen (HLA)-DR1-restricted epitopes from the Timothy grass pollen allergen, Phleum pratense, and characterise T-cell immune regulation following intranasal administration of a single, immunodominant epitope.

Methods: T-cell epitopes within P pratense were identified using HLA-DR1 transgenic mice and tetramer-guided epitope mapping (TGEM) in HLA-DR1-positive individuals with grass allergy. An immunodominant epitope was tested in HLA-DR1 transgenics for impact on responses to whole Phl p5 b or peptide. Microarrays and quantitative PCR were used to characterise T-cell immunity.

Results: Peptide 26 (p26) was identified in HLA-DR1 transgenic mice and by TGEM analysis of HLA-DR1-positive individuals with grass allergy. p26 shows promiscuous binding to a wide range of HLA class II alleles, making it of relevance across immunogenetically diverse patients. The epitope is conserved in rye and velvet grass, making it applicable across a spectrum of grass pollen allergy. Intranasal pretreatment of mice with p26 results in significantly reduced T-cell responses. Transcriptomic array analysis in mice showed T-cell regulation in the intranasal treatment group associated with increased expression of members of the Cbl-b and Itch E3 ubiquitin ligase pathway.

Conclusions: We defined an immunodominant P pratense epitope, p26, with broad binding across multiple HLA class II alleles. Intranasal treatment of mice with p26 results in T-cell regulation to whole allergen, involving the Cbl-b and Itch regulatory pathway.

Keywords: Allergic lung disease; Lymphocyte Biology.

Figure 1

Human leukocyte antigen (HLA)-DR1 restricted T-cell responses to Phl p 5b protein and Phl p 5b 20-mer overlapping peptide panel. HLA-DR1-restricted T-cell lines were made against nPhl p 5 protein. HLA-DR1-Tg/Aβ° mice (n=6) expressing human HLA-DR1 were immunised with nPhl p 5b emulsified in Complete Freund’s Adjuvant. Draining lymph node cells were harvested at day 10 and T-cell lines generated against Phl p 5 protein. T-cell responses of lines were assessed after 72 h in a ³H-thymidine incorporation assay against Phl p 5 protein and 20-mer overlapping peptides spanning Phl p 5b, each at 50 μg/mL. The y-axis indicates T-cell proliferation in response to peptide, measured in Δcpm of ³H-thymidine incorporation (ie, the difference between the mean count from triplicate wells in the presence or absence of peptide). The x-axis indicates the identity of the tested Phl p 5b peptides that were assayed for the ability to elicit a T-cell response. This assay was repeated on three separate occasions. Epitopes p18, p23 and p26 were confirmed by priming with the specific peptides (data not shown).

Figure 2

Tetramer-guided epitope mapping identifies peptide 26 (p26) as a human leukocyte antigen (HLA)-DR1-restricted CD4T-cell epitope of Phl p 5b. A panel of overlapping 20-mer peptides spanning Phl p 5b was synthesised. Peptides were divided into pools and loaded onto PE-conjugated HLA-DR1 tetramers. Isolated CD4 T cells were cultured with adherent cells in the presence of the pooled peptides for 7 days before adding interleukin 2. Between days 12 and 14 the cells were stained with anti-CD4 and the pooled peptide tetramers (A). Positive tetramer staining was identified by flow cytometry. Individual peptide tetramers were generated and staining repeated (B). In this representative example, positive labelling is seen with HLA-DR1 tetramers loaded with p26 of Phl p 5b in patient URM031.

Figure 3

Phl p 5b peptide 26 (p26) human leukocyte antigen (HLA)-DR1 tetramer-positive populations in study participants with and without allergy. Whole peripheral blood mononuclear cells isolated from HLA-DR1-positive individuals with grass allergy and seasonal allergic rhinitis, and healthy controls without allergy were cultured with Phl p 5b p26. At day 14, cells were labelled with anti-CD3-FITC, anti-CD4-PE-Cy5 antibodies and PE-conjugated-HLA-DR1 tetramer loaded with p26 (A, top panel) or control tetramer loaded with irrelevant peptide (A, bottom panel) and analysed by flow cytometry. Cells were gated on live lymphocytes according to forward and side scatter and the CD3 CD4 positive population to detect the p26-specific T-cell population. The percentage of p26 tetramer-positive cells for subjects with and without allergy is shown (B). Statistical analysis was performed using the Mann– Whitney U test (bars indicate median values). Samples were processed during the grass pollen season, 2010 (subjects with allergy, n=6; healthy controls without allergy, n=8).

Figure 4

Effector memory phenotype of Phl p 5 peptide 26 (p26) tetramer-positive CD4 T cells from subjects with allergy. Peripheral blood mononuclear cells isolated from subjects with allergy were labelled immediately ex vivo with Phl p 5b peptide 26 tetramer. This was followed by cell surface staining with FL-1 conjugated cell surface maker antibodies and an anti-CD4PE-Cy5. The expression of a FL-1 conjugated cell surface maker antibody was compared on total CD4^high and CD4 human leukocyte antigen (HLA)-DR1 Phl p 5 p26 tetramer-positive cells from HLA-DR1-positive individuals with grass allergy. The reduction in mean fluorescence intensity for CD62L and CD45RA staining of tetramer-positive cells is indicated in online supplementary figure S1. Statistical analysis was performed using a Wilcoxon signed rank test. Samples were processed during the grass pollen season, summer 2010.

Figure 5

Intranasal treatment with peptide 26 (p26) results in reduced T-cell proliferation to rPhl p 5b and p26. (A, B) Mice were treated intranasally with phosphate-buffered saline (PBS, filled squares) or p26 (filled triangles) at days 1, 2 and 3 and footpad primed at day 21 with (C) rPhl p 5 protein (each group, n=7), (D) p26 (each group, n=7) or (E) whole Phl p 1 protein (each group, n=5). Draining lymph nodes were harvested at day 31 and ^3Hthymidine incorporation measured for protein or peptide as shown. An unpaired t test was used to determine significant differences between groups. Statistically significant differences were defined as p values of less than 0.05 (*p<0.05).

Figure 6

Intranasal treatment of peptide 26 (p26) is associated with increased expression of regulatory genes and decreased expression of T helper 2 inflammatory genes. Mice were treated intranasally with phosphate-buffered saline (PBS) (n=6) or p26 (n=6) before footpad immunisation and in vitro p26 stimulation of draining lymph node (DLN) cells. (A) A heat map shows the key genes differentially expressed following in vitro p26 stimulation of DLN cells where fold change is greater than ±1.4 with a p value of <0.05 by analysis of variance. (B) Fold change values and p values for each of these differentially expressed genes are shown.

Figure 7

Intranasal treatment of peptide 26 (p26) results in increased expression of key targets in the Itch-Foxp3-transforming growth factor β inducible early gene 1 (TIEG-1) pathway. (A) Cbl-b, (B) Itch, (C) TIEG-1, (D) Foxp3 and (E) Egr3 are upregulated in p26-treated (n=3) compared with phosphate-buffered saline (PBS)-treated (n=3) mouse draining lymph node cells by real-time PCR. Student’s t test was used to determine significant differences between groups. Statistically significant differences were defined as p values less than 0.05 (*p<0.05, ***p<0.0005).