title: Identification of isoform-specific T-cell epitopes in the major timothy grass pollen allergen, Phl p 5.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: ALK Abelló, Horsholm, Denmark.
authors.forename: P A
authors.initials: PA
authors.lastname: Würtzen
authors.forename: A
authors.initials: A
authors.lastname: Bufe
authors.forename: M
authors.initials: M
authors.lastname: Wissenbach
authors.forename: H O
authors.initials: HO
authors.lastname: Madsen
authors.forename: H
authors.initials: H
authors.lastname: Ipsen
authors.forename: J
authors.initials: J
authors.lastname: Arnved
authors.forename: R J
authors.initials: RJ
authors.lastname: Van Neerven
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D001782:Blood Donors; D002478:Cells, Cultured; D002999:Clone Cells; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D006680:HLA Antigens / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006109:Poaceae / Q000276:immunology*; D011058:Pollen / Q000276:immunology*; D020033:Protein Isoforms; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000485:Allergens; D018984:Epitopes, T-Lymphocyte; D006680:HLA Antigens; C076049:Phl p V protein, Phleum pratense; D010940:Plant Proteins; D020033:Protein Isoforms; D016693:Receptors, Antigen, T-Cell, alpha-beta