Identification of isoform-specific T-cell epitopes in the major timothy grass pollen allergen, Phl p 5

Background The involvement of CD4(+) T cells in the pathophysiology of atop ic disease is well established. Objective To gain further insight into the activation requirements for alle rgen-specific T cells, we characterized epitope specificity, HLA restrictio n and T-cell receptor (TCR) usage for T cells specific to Phl p 5, the grou p 5 major allergen of the grass Phleum pratense. Methods To identify the T-cell epitopes of Phl p 5, three Phl p 5-specific T-cell lines (TCLs) and 15 T-cell clones (TCCs) generated from the peripher al blood of three grass-allergic patients were tested with recombinant trun cated Phl p 5a fragments and synthetic Phl p 5b peptides representing these two different recombinant Phl p 5 isoallergens. Additional activation expe riments with HLA-subtyped antigen-presenting cells and flow cytometry analy sis with TCR V-specific mAb were performed to further characterize the acti vation requirements for Phl p 5-specific TCCs. Results At least nine distinct T-cell specificities were identified and the T-cell epitopes recognized differed considerably among the three patients. Most of the epitopes found were isoform-specific, whereas three epitopes w ere shared between Phl p 5a and 5b. Several human leucocyte antigen (HLA) c lass II molecules were involved in the recognition of Phl p 5. Different HL A restriction specificities were even found among TCCs specific to the same epitope region. All TCCs were TCR-alpha/beta positive, and an overrepresen tation of TCR V beta 3.1(+) clones among TCCs specific to Phl p 5 appear to exists as 31% (4/13) of the TCCs expressed TCR V beta 3.1 (compared with 5 % TCR V beta 3.1(+) T cells in human peripheral blood) with no correlation with epitope specificity or HLA restriction. Conclusion The T-cell reactivity of the three grass-allergic patients inves tigated shows that isoallergen-specific T-cell epitopes are found throughou t the peptide backbone of Phl p 5a and Phl p 5b, and dominant T-cell epitop es of Phl p 5 were not identified. This indicates that a mixture of at leas t full-length rPhl p 5a and rPhl p 5b may be required to target the total P hl p 5-specific T-cell response of atopic patients.