Ara h 1-reactive T cells in individuals with peanut allergy

Abstract

Background: Effective immunotherapy for peanut allergy is hampered by a lack of understanding of peanut-reactive CD4(+) T cells.

Objective: To identify, characterize, and track Ara h 1-reactive cells in subjects with peanut allergy by using Ara h 1-specific class II tetramers.

Methods: Tetramer-guided epitope mapping was used to identify the antigenic peptides within the peanut allergen Ara h 1. Subsequently, HLA class II/Ara h 1-specific tetramers were used to determine the frequency and phenotype of Ara h 1-reactive T cells in subjects with peanut allergy. Cytokine profiles of Ara h 1-reactive T cells were also determined.

Results: Multiple Ara h 1 epitopes with defined HLA restriction were identified. Ara h 1-specific CD4(+) T cells were detected in all of the subjects with peanut allergy tested. Ara h 1-reactive T cells in subjects with allergy expressed CCR4 but did not express CRTH2. The percentage of Ara h1-reactive cells that expressed the β7 integrin was low compared with total CD4(+) T cells. Ara h 1- reactive cells that secreted IFN-γ, IL-4, IL-5, IL-10, and IL-17 were detected.

Conclusion: In individuals with peanut allergy, Ara h 1-reactive T cells occurred at moderate frequencies, were predominantly CCR4(+) memory cells, and produced IL-4. Class II tetramers can be readily used to detect Ara h 1-reactive T cells in the peripheral blood of subjects with peanut allergy without in vitro expansion and would be effective for tracking peanut-reactive CD4(+) T cells during immunotherapy.

Figure 1

Frequencies of Ara h 1 epitope-reactive T cells. A. Frequencies of Ara h 1_321–340-specific T cells in a DR1101 allergic subject and a DR1101 non-atopic subject. The frequencies of Ara h 1-specific T cells per million CD4+ T cells are as indicated. B. Frequencies of Ara h 1 epitope-reactive T cells in 11 peanut allergic subjects, 6 non-atopic subjects and 5 peanut non-allergic atopic subjects. Each data point represents the frequency of T cells specific for a single epitope in Ara h 1. A Student t test was used in the statistical analysis. * P < 0.05.

Figure 2

Phenotype of Ara h 1-reactive T cells. A. PBMC of a DR1101 subject with peanut allergy were stained with PE-labeled DR1101/Ara h 1_321–340 tetramers and a panel of antibodies. B. Comparison of ex vivo phenotypes of Ara h 1-reactive and total CD4+ T cells for multiple peanut-allergic subjects. Ara h 1-reactive cells are denoted by circles, total CD4+ T cells by triangles. A Student t test was used in the statistical analysis. * P < 0.05.

Figure 3

Intracellular cytokine staining of Ara h 1-reactive cell lines. First row: an IL-4 and IL-5 producing DR1101- restricted Ara h 1_169–188-stimulated cell line. Second row: an IL-4 and IL-10 producing DR0401-restricted Ara h 1_329–348-stimulated cell line. Third row: an IFN-γ producing DR1401-restricted Ara h 1_321–340-stimulated cell line. Fourth row: an IL-4 and IL-17 producing DR0404-restricted Ara h 1_329–348 -stimulated cell line. Fifth row: an Il-4 and IL-5 producing DR0401-restricted Ara h 1_577–596-specific T cell clone. Each percentage shown indicates the percentage of cytokine producing cells from the tetramer positive population.

Figure 4

Dual cytokine analysis of Ara h 1-reactive T cell lines. PBMC were stimulated with Ara h 1 peptide for 2 weeks and stained with tetramers. Tetramer positive cells were gated and cytokine profiles were analyzed by ICS staining. A. IL-4 and IL-5 producing DR1101-restricted Ara h 1_169–188-reactive T cells from Allergic Subject #5 (Table I). B. IL-4 and IL-10 producing DR0401-restricted Ara h 1_329–348-reactive T cells from Allergic Subject #1. C. IL-4 and IL-17 producing DR0404-restricted Ara h 1_329–348-reactive T cells from Allergic Subject #2. Each percentage shown indicates the percentage of cytokine producing cells from the tetramer positive population. Data shown is representative of at least three independent experiments.