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).