Background: The involvement of CD4+ T cells in the pathophysiology of atopic disease is well established.

Objective: To gain further insight into the activation requirements for allergen-specific T cells, we characterized epitope specificity, HLA restriction and T-cell receptor (TCR) usage for T cells specific to Phl p 5, the group 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 peripheral blood of three grass-allergic patients were tested with recombinant truncated Phl p 5a fragments and synthetic Phl p 5b peptides representing these two different recombinant Phl p 5 isoallergens. Additional activation experiments with HLA-subtyped antigen-presenting cells and flow cytometry analysis with TCR V-specific mAb were performed to further characterize the activation 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 were shared between Phl p 5a and 5b. Several human leucocyte antigen (HLA) class II molecules were involved in the recognition of Phl p 5. Different HLA restriction specificities were even found among TCCs specific to the same epitope region. All TCCs were TCR-alpha/beta positive, and an overrepresentation of TCR Vbeta 3.1+ clones among TCCs specific to Phl p 5 appear to exists as 31% (4/13) of the TCCs expressed TCR Vbeta 3.1 (compared with 5% TCR Vbeta 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 investigated shows that isoallergen-specific T-cell epitopes are found throughout the peptide backbone of Phl p 5a and Phl p 5b, and dominant T-cell epitopes of Phl p 5 were not identified. This indicates that a mixture of at least full-length rPhl p 5a and rPhl p 5b may be required to target the total Phl p 5-specific T-cell response of atopic patients.